BACKGROUND OF THE INVENTION This invention relates to a composition comprising a purified mycobacterial lipid celt-wall component or analog or derivative thereof and a pharmaceutically acceptable excipient, medium, carrier or adjuvant and the use of the purified mycobacterial lipid cell-wall component or analog or derivative thereof and the composition containing it in the prevention, treatment and diagnosis of disease. It has been known for a long time that BCG*) vaccination leads to the induction of a positive tuberculin skin test, resulting in delayed type hypersensitivity (DTH) This delayed hypersensitivity in turn has been considered to be indicative of the successful induction of protective immunity against tuberculosis and has led to the almost world-wide .BCG immunization in the 1950s-1970s. This convenient test is in fact the only immunological criterion/parameter on which epidemiological assessments of the affectiveness of the immunization have been based. This view it no longer generally accepted and many immunologists are of the opinion that i) the induction of DTH is not directly related to the degree of protective immunity; ii) the protective efficacy obtained in vaccination with BCG varies between 0 to 80% (Snider, 1994) *) BCG: (Bacillus of Calmena and Guerin) Calmtne and Gutnn aaenuatud a strain of M. bovit by paatagiiuj it 231 time* over a period of 23 years through a meitum containing gtyctrme and ox-bile and in addition iii) BCG vaccination has detrimental side-effects being partially responsible for tissue destruction in patients, withcut offering sufficient protection (Fine, 1994). The unsatisfactory results observed and reported in a number of countries with the BCG vaccine currently used for the prevention of tht spread of tuberculosis (Dolin, Raviglione and Koch, 1994; Snider, 1994) could be explained by: i) variations between BCG vaccines, which could be caused by strain variation or by differences between manufacturing processes, ii) differences in pathogenesis of Mycobacterium tuberculosis; iii) differences in the exposure to the environmental mycobacteria. The environmental mycobacteria may act antagonistically or synergistically with BCG; iv) genetic differences between population groups subjected to vaccination with BCG; v) differences in nutrition and exposure to sunlight between various population groups; vi) differences between designs of various stuthes, vii) inadequacies of the criteria used for the evaluation of protective effects of vaccination with BCG. Efforts directed at finding an effective vaccine capable of inducing long-lasting immunity have centered over the last decade on three main approaches: i) identifying "protective" antigens and epitopes of M. tuberculosis presented by macrophages and recognized by human lymphocytes; ii) developing a DNA-based vaccine with protective aatigen and interleukin genes (Lowrie ex at., 1994); iii) identifying which types of cells of the immune system and which types of cytokines are involved in tuberculosis in order to manipulate their activity towards offering a cure or protection against tuberculosis. SUMMARY OF THE INVENTION According to one aspect of the invention there is provided a conjugate comprising an organic carrier and a purified lipid cell-wall component associated therewith, provided that if the organic carrier is a piotein, it is not bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin or the CD, molecule. The organic carrier may be a protein excluding bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD, molecule. The protein may be a microbial protein. More specifically, it may be a modified bacterial protein and may be derived from a bacterium from the genus Mycobacterium, Corynebacterium, Nocardia or Rhodococcus. It may be a heat-shock protein, such as heat-shock protein 60 (HSP60) or heat- shock protein 65 (HSP65), or it may be a serum protein from an animal The animal may have a mycobacterial infection, such as a Mycobacterium tuberculosis infection. The animal may be a mammal, typically a human Alternatively, the protein may be derived from a mammal, particularly a human, and is preferably a protein which mimics the structure of collagen or a collagen-derived protein or a plasma protein, such as the collagen-like segment of human serum component CI,, Alternatively, the carrier may be a carbohydrate such as galactocnannan or arabinogalactan or a lipopolysaccharide. Further alternatively, the organic earner may be a micelie, such as a liposome According to another aspect of the invention there is provided a diagnostic kit comprising a support containing a conjugate as described above immobilised thereon According to another aspect of the invention rhere is provided a pharmaceutical composition which comprises a therapeutic, prophylactic or tolerogenic amount of a conjugate as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component or analog or derivative thereof associated therewith or a biologic illy active purified lipid cell-wall component or analog or derivative thereof and a pharmaceutically acceptable or compatible pharmaceutical excipient, medium, carrier or adjuvant. The organic carrier may be a protein including bovine seruti albumin (BSA). gelatin, keyhole limpets haemocyanin and the CD 1 molecule, or may be a carbohydrate or may be a micelle The pharmaceutical composition may also contain at least one irnmunomodulator. The immunomodulator may be a cytokine. The cytokine may be an interleukin, such as interleukin 4 (IL4), intcrleukin 10 (IL10) or interleukin 12 (TL12), or may be an interferon. The suitable pharmaceutical carrier or adjuvant which may also be suitable for veterinary applications may be a solid, such as polymer dust, a liquid, such as an oil, typically Marcol 52, or a water-in-oil emuLsior, typically Freund's Incomplete Adjuvant (FIA), or a solution, typically a saline solution or PBS, in which case the composition may be in the form of a suspension or a vapouhsed liquid, typically a neubulisabie physiological saline solution, or a gas. or a transdermal delivery system. The composition may comprise a therapeutic, prophylactic or tolerogenic amount of the purified lipid cell-wall component. The pharmaceutical composition may comprise about 5µg or less, typically 1µg, of the purified lipid cell-wall component per ml of the conposition A unit dose of the pharmaceutical composition for administration to a human subject preferably comprises from about 5 to 10 mg of the purified lipid cell-wail component According to another aspect of the invention mere is provided a vaccine containing a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in precventing an immune disorder or an inflammatory condition in a subject. According to another aspect of the invention there is provided a vaccine containing a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier, except bovine serum albumin (BSA) and a purified lipid cell-wall component associated therewith, for use in preventing a microbial infection in a subject. The organic earner may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haexnocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle. According to another aspect of the invention there is provided an isolated antibody which is capable of forming, separately, an antigen/antibody complex with any two or more of the following antigens: a purified lipid cell-wall component derived from a microorganism: a protein derived from a bacterial species or from a mammal: a conjugate as described above; and a conjugate comprising any organic carrier and a purified my abacterial lipid cell-wall component associated therewith. The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle. According to another aspect of the invention there is provided a method of diagnosing a microbial infection in a subject comprising the step of contacting a sample from the subject with a conjugate as descrbed above or with a support containing a conjugate as described above; and detecting Many reaction between the conjugate and the sample. More preferably, the method of diagnosis may comprise the step of detecting the binding of an antibody present in the sample to the conjugate. According to another aspect of the invention there is provided i. conjugate or a pharmaceutical composition as described above for use in a method of diagnosis of a microbial infection in a subject According to another aspect of the invention there is provided the use of a conjugate or a pharmaceutical composition as described above in a method of making a medicament for use in a method of diagnosis of * microbial infection in a subject. According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use as a tolerogen to enhance resistance and/or reduce susceptibility to a microbial infection in a subject. According to anomer aspect of the invention there is provided the use of a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and purified lipic. cell-wall component associated therewith in a method of making a medicament for use as a tolerogen to enhance resistance and/or reduce susceptbllity to a microbial infection in a subject. According to another aspect of the invention there is provided a purified lipid cell-wallcomponent or analog cr derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of enhancing resistance or towering susceptibility to microbial infections m a subject According to another aspect of the invention there is provided use of a purified lipid cell-wall component or analog or derivative thsreof or a conjugate or pharmaceutical composition as described above or n conjugate comprising any organic earner and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in enhancing resistance or lowering susceptibility to microbial infections in a subject. According to another aspect of the invention there is provided a method of treatment of a microbial infection in a subject comprising tie step of administering to the subject a purified bacterial lipid cell-wall component or analog or derivative thereof or a pharmaceutical composition of the invention to the subject. According to another aspect of the invention there is provided a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall i:omponent associated therewith or a purified lipid cell-wall component or analog or derivative thereof for use in a method of treatment of a microbial infection in a subject. According to another aspect of the invention there is provided the use of a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith or a purified lipid cell-wall component or analog or derivative thereof in a method of making a medicament for use in a matted of treatment of a microbial infection m a subject The organic carrier may be a protein including bovine senim albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle The method of treatment may be a prophylactic and/or therapeutic method and may be a high zone tolerance treatment or may be a low zone tolerance treatment or a treatment aiming at an idiotypic regulation involving the conjugate or the antibody The method may be an immunoregulatory method. The method of treatment may be a prophylactic method which enhances resistance or reduces susceptibility to a microbial infection in a subject. The prophylactic method may promote an inflammatory response in an infected organ, typically the lungs, kidney and/or liver of the subject. The infected organ is usually the lungs. According to another aspect of the invention there is provided a method of treatment of an immune disorder in a subject comprising the step of administering to the subject a purified bacterial lipid cell-wall component or analog or derivative thereof or a pharmaceutical composition as described above According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative mereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of treatment and/or diagnosis of an immune disorder in a subject. According to another aspect of the invention there is provided the use of punned Lipid cell-wall component or analog or derivative tncreof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in a method of treatment and/or diagnosis of an autoimmune disease n a subject. The organic carrier may be a protein including bovine senna albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle. According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of treatment and/or diagnosis of an inflammatory condition in a subject. According to another aspect of the invention there is provided tae use of a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or ly FIA, the rat on the right having received a reagent for the induction of adjuvant arthritis and showing a bleeding nose and arthiitic nodules visible on the front paws; Figure 25b is a photograph of two rats, the rat on the right having been pre-treaied with 1 mg of mycolic acids/serum in FIA before having received a reagent for the induction of adjuvant arthritis and showing minimal signs of arthritis, the rat on the left not having been pre-treated before having received a reagent for the induction of adjuvant arthritis and showing swollen and inflamed arthritic hind legs; Figure 25c is a photograph of the rat showing the typical deformation of the joints in the hind legs, the so-called 'swimming position" and a necrosis developing at the she of the injection; Figure 26a shows X-ray photographs of the hind limbs of rats used in the arthritis experiments. Group 5 of Tables 8a and 8b -a "negative" control treated with Freund's Adjtvantottfy; Figure 26b shows X-ray photographs of the hind limbs of rats used in the arthritis experiments. Group 1 of Tables U and 8b- a positive" control treated with M. tuberculosis H37Rv suspended in Freund's Adjuvant: Figure 26c shows X-ray photographs of the hind limbs of the rats pre-treated with 1 rag mycolic acids (from M. tuberculosis) prior to the induction of arthritis. Group 4 of Tables 8a and 8b; Figure 27 shows the emaciation of a rat with typical induced adjuvant arthritis; Figure 28 shows rat antibody response to mycolic acids suspended in oil after three months' treatment using 1,0, 0,3 and 0,1 mg mycolic acids per immunization, after three months treatment; Figure 29 shows ELISA results of human tuberculosis ps dents' sera in comparison to healthy control on the plates coated with mycolic acids; Figure 30 is a specificity assay of antibothes of human nberculosis patient No. 38 assessed by inhibition of ELISA; Figure 31 is a Western blot of mouse serum with and without exposure to mycolic acids, probed with human tuberculosis patients' sera and a healthy control serum; Figure 32a shows the stimulation by mycolic acids of human T cells by CDl presenting cells; Figure 32b shows the stimulation of human T cells by mycolic acids. DESCRIPTION OF PREFERRED EMBODIMENTS This invention is based on the involvement of isolatec, purified, biologically active mycobacterial lipid cell-wall components and more particularly purified, biologically active mycolic acids or analogs or derivatives thereof, in the regulation of the immune response in a subject or host to infection or abnormal activation. These purified mycobacterial lipid cell-wall components originate from bacteria assigned to the genus M.mycobacteriian and to the genera Corynebactertum, Nocardia and Rhodococcus. In particular, the present invention teaches the application of isolated and purified mycolic acids in: 1. the modulation and knmunoregulation of an immune response towards infection wiih mycobacteria in humans and animals; 2. the development of anti-tuberculosis and other anti-mycobacterial treatment for human and veterinary use. It is envisaged that tolerogenic doses of the purified lipid cell-wall components of M. tuberculosis and other Mycobacteria, on their own or presented on carriers such as proteins, liposomes or carbohydrates or in a medium or pharmaceutical carrier or excipient such as liquid (oil, saline, water) gas or vapour, could be used in the modulation of immune responses to mycobacterial infections; 3. the development and production of diagnostic tests (in vitro and/or in vivo tests) for the confirmatior of the presence of mycobacterial cells in samples such as blood, cersbrospinal fluid, tears and saliva or tests that would be bassd on the identification and detection of auto-reactive antibothes and/or T-cells, and antibothes against the purified lipid cell-wall components with or without host carriers; 4. the prevention and immunotherapy and relation of autoimmune illnesses of mycobacterial and other origin and particularly those associated with infection with mycobacteria. Such a therapy could be achieved by modulating the immune response directed against mycobacterial lipid cell-wall components. It is envisaged that the tolerogenic doses of the purified lipid cell-wall components of M. tuberculosis and other Mycobacteria presented in a supportive medium or in a pharmaceutically acceptable carrier or exctpient or on carriers such as liposomes or proteins or suspended in a:i adjuvant, typically a water-in-oil emulsion such as HA, can be used for the regulation of immune responses to mycobacter.al antigens leading to the prevention of tuberculosis and at to-immune diseases such as arthritis, often associated with tuberculosis. The invention utilises the method of isolation and purification of a mycobacterial lipid cell-wall component described in South African Patent No 96/1412 and is based on the demonstration of the immunogeniciry of my colic acids described in South African Patent No. 95/3077. The invention also provides an improvement on the method of isolation and purification of mycobacterial lipid cell-wall components described in South African Parent No. 96/1412. An improvement involves a preliminary purification of crude mycobacterial extract to remove redundant or unnecessary cellular components prior to the contercurrent purification and to reduce the soap content in the crude extract. A further improvemewnt to the method involves the addition of sodium chloride to the solvent. This allows larger amounts of the extracted mycolic acids to be purified per run of the countercurrent separation The availabtlity of the isolated and purified required components, particularly mycolic acids, is essential for the development and assessment of the potential applications listed above. As indicated above, toe conventional approaches to the prevention of infection with At. tuberculosis by attempting to induce humoral and/or cellular memory leading to a long-term protection against tuberculosis have been unsuccessful. The present invention offers another approach. It is based in part on the use of appropriate tolerogenic doses of mycotic acids in a supportive medium or pharmaceuiically acceptable carriers or excipients or on appropriate carriers, with or without appropriate int<;rleukine(s) (Heath and Playfair, 1992), to modulate/regulate the immune rei,ponse(s) in the human body. Such a treatment would help to prevent or decrease mortality due to tuberculosis and could be used as a potential tniatment for the disease. The presentation of purified mycolic acids in supportive media, conjugates, pharmaceutical compositions comprising phannaceutically acceptable excipients, carriers and adjuvants, uses and methods of the inven:ion should be considered in the light of what is known about the pathology of tuberculosis as described below The encounter between the bacterium Mycobacterium tuberculosis and its human host is exceptionally complex and multifaccted and leads to a number of intertwined, interdependent and interconnected processes, in which mycolic acids appear to be playing a significant role. For the sake of clarity these processes will be discussed under the following heading Outline: Clinical stages of tuberculosis I Primary- pulmonary tuberculosis 2. Post-primary pulmonary/reactivation tuberculosis 3. Immunology/imrnunopathology in tuberculosis 3.1 Overview 3 2 Synthesis of cytokines 3.3 Activation of macrophages and the intracellular fite of U. tuberculosis 3.4 Delayed-Type Hypersensitivity (DTH) 3.5 Activation of T cells and their functions 3.6 Cytokine circuits in tuberculosis 3 7 Cytokine induction by M. tuberculosis 3 8 Cytokine profiles in tuberculosis patients 3.9 Involvement of other T cells in tuberculosis 4 Proposed approach 5. Immunogenicity of mycolic acids OUTLINE: Clinical stages of tuberculosis 1. Primary pulmonary tuberculosis It is generally accepted that the primary infection with M, tuberculosis occurs by inhalation of a very small number of bacilli into the respiratory tract. Usuaily fewer than 10% of the inhaled microorganisms retch file respiratory bronchioles and alveoli, the rest being successfully removed from the upper respiratory epithelium by the nonspecific, external innate immunity, i.e., by mucus and the ciliated epithelium of The upper respiratory tract acting as filters. Mycobacteria that reach alveoli tfe dealt with by the internal innate Immunity mechanisms. In the case of the mycobacterial infection, phagocytosis (engulfment and destruction of the pathogens by mononuclear leucocytes) and Inflammatory reaction play the decisive pan The age of an individual, general state of his/hur health, conditions of living, nutrition as well as ethnic and geographic differences affect the susceptibility to the disease and its severity (Fine, 1994). The inhaled cells of M. tuberculosis are ingested by alveolar ma:rophages (see section 3 3 on the activation of macrophages), which have a predilection for this function. Some of the bacilli are destroyed within the phagolysosomes of the macrophages and some remain in the microphagal vacuoles for undetermined periods of time. In some cases, even replication of the bacilli may occur in the macrophage's vacuoles. Within the period of 2 to 3 weeks some of the surviving bacteria destroy their host macrophages and, after being released, can infect additional macrophages fFenron and Vermeulen. 1996). The chemotactic factors released by the destroyed macrophages artract other leucocytes: monocytes, lymphocytes and neutrophils, which are not capable of destroying the released mycobacteria. The accumulation of lymphocytes and the formation of macrophage-dcrived epitheloid giant cells constitute the beginning of the inflammation process (see section 3.1 and 3.3) and lead to the formation of nodules called granulomas, which in the case of tuberculosis are referred to as tubercles. The degree of success of granuloma formation depends on the initial number of mycobacteria present in the tissue and on the number of macrophages present at the site of infection (Fetiton and Vermeulen, 1996). The formation of granulomas is a result of an interaction between the macrophages and T lymphocytes and the secretion of proteins stimalated crlls of both types. It constitutes the beginning of the developing and mediated immunity (for a detailed description see section 3 4). Although the cells of ingested At. tuberculosis are not always completely destroyed, the formation of granulomas is an effective defense mechanism of "containing'/walling-off the pathogens, which stops the infection from spreading further and retains it in a subclinical state If, however, the resistance of the infected person is low, the formation of granulomas may not stop the growth and spread of the mycobacteria and the disease progresses further to primary clinical tuberculosis. During this stage of the disease more tubercles are formed and they become larger. A hypersensitivity reaction (see section 3.4) accompanied by tissue necrosis and fusion of me dead macrophages constitute the characteristic response. The fusion of macrophages leads to the formation of caseation necrosis widun the granulomas. In the majority of cases, in crder to seal off the necrotic site, lymphocytes and other cells collect at the site and form a fibrous tissue. The walled-off caseated granulomas frequendy heal over time, shrink and calcify. The disease frequendy stops at this stige and the damaged lung tissue, detected by X-ray, remains the proof of me successfully combated disease. In me majority of cases the encounter between mycobacteria and the human immune system stops he-e and as a result of this exposure, most individuals successfully control the focus of infection and develop some degree of immunity. If, however, the healing process is impaired, the lesions do not calcify but expand, eroding adjacent bronchi and leading to the formation of cavities, m which live cells of M. tuberculosis can multiply freely, sometimes reaching numbers exceeding 10* per cavity (Nardell, 1993]. living mycobacteria can leak from such open cavities divecdy into bronchia leading to even further spread of the disease and a continuous discharge of these bacilli into the sputum. The leaked bacilli can be inhaled into other parts 32 of the hosts lungs resulting in tuberculous bronchopneumonia. As the caseation necrosis develops further and spreads, the patient starts to display the clinical symptoms of tuberculosis such as loss of weight, night sweats, persistent cough, loss of appetite and fatigue. At this stage an intensive therapy is required (Fenton and Vermeulen, 1996). If mycobacteria spread to the blood, they can localise in any otgan forming new tubercles and the disease becomes a generalised form of liberculosis, referred to as miliary tuberculosis. 2. Post-primary pulmonary/reactivation tuberculosis Post-primary pulmonary tuberculosis or reactivation tubei'culasis can develop following either inhalation of additional mycobacteria or by reactivation of a dormant primary lesion, in which bacilli can survive in a dormant state for decades. The post-primary tuberculosis progresses despite the existing immunity developed during the primary exposure (Fenton and Vermeulen, 1996) and is considered to be the most prevalent form of the disease (Elgert, 1996a). It usually occurs concomitantly with a period of excessive environmental stress, malnutrition or lowering of immune competence of the body. The pathogen, no longer held in check within the tubercles, multiplies vigorously and leads to the development of symptoms observed in the primary clinical tuberculosis. Generalized clinical tuberculosis causes damage to various vital organs and can result in death of the patient. 3. Immunology/immunopathology in tuberculosis 3.1 Overview Although the breaking down and digestion of mycobacteria by macrophages is considered to be essentially a non-antiger.-specific process, it initiates the development of the adaptive, i.e., antigen-specific imnum; responses, which develop after about 3 weeks since the initial exposure to M. tuberculosis and significantly contribute to the combating of toe infection. Certain components of the macrophage-digested bacilli, after being released from the phagolysosomes, are transported by specialised proteins referred to as major histocompatibility molecules (MHC molecules) to the surface of details see section 3.3). If any of the presented components of M. tuberculosis is recognised by a T cell, certain proteins called cytokines*) (see section 3.2) capable of initiating the proliferation of the T cell into a clone of cells recognising this particular antigen, are synthesized and secreted. The mycobacterial antigens presented by the infected macrophages attract specific T ce'.ls and the process of inflammation leading to the formation of granulomas is initiated. The antigen recognition which takes place is due to the complementarity of the combining sites between the {MHC-M. tuberculosis component}-compIex presented on the surface of the macrophage and the receptors present on the T cell The interaction between the T cells and macrophages ieads to the stimulation/activation of macrophages (for details see section 3.3) which start secreting various cytokines. One of the cytokines produced by the activated macrophage, iiiicficukin i (IL-i), induces the proliferation of neighbouring T cells *) Small protein molecules via which me communication bttwera vancos parts tad various cells of me immune system Is accomplished. (inflammatory CD4 T cells), which in aim start secreting interferon  (IFN-). IFN- acting as a chemotaxin, attracts monocytes to the site of infection and converts resting monocytes into activated macropliages (Fenton and Vermeulen, 1996; Elgert, 1996b) The further course of the disease depends to a large degree upon the type of the activated lymphocyte, i.e., whether it is a cytotoxic (CD8) or a helper (CD4) T cell, or whether T-helper cell is a T-helper 1 (Thl) or a T-faeiper-2 (Th2) type. The stimulation of CD8 or CD4 T cells depends on whether an antigen presenting tell (APC) presented the antigen in association with a major histocompatibility complex (MHC) molecule of class 1 or MHC class II, respectively. The mechanism of the selection between Thl or Th2 type T-cell response is less clearly understood. On the basis of the accumulated evidence it appears that the activated macrophages and Thl CD4 cells play the most important role in the immune response to tuberculosis, leading to the development of acute phase inflammatory response i.e.. the development of delayed-type hypersensitivity (DTH) (see section 3.4) and the formation of granulomas. The Thl cells are known to secrete, apart from DL-2, interferon y, in response to IL-12 secretion by activated macrophages. INF- as the principal cytokine activating macrophages is of critical importance in combating intracellular mycobacterial infection (Toossi, 1996; Kaufmann, 1995a and 1995b). The mvolvement of CD4 cells of the Thl type, which activate B cells and induce the production of antibothes, has also been confirmed in tuberculosis (.Grange, 1984; Fine, 1994). Although a number of various antibothes reactive with different types of mycobacterial antigens have been detected in the sera of tuberculosis patients (Grange, 1984; Dolin, Raviglione and Koch, 1994; Snider, 1994), their protective role has not been demonstrated. For a long time the activities of Tbl sells in tuberculosis have been associated with the "good" immunity resulting in the "containment" of the mycobacterial infection and leading to an optimistic prognosis, whereas the Th2 cells activities have been held responsible for the development of allergic reactions and consequently were considered to be a "bad" type of immunity in this disease. The current approach considers the activities of Th2 cells as more positive and assigns to them a regulating function, which allows them to terminate the defence reactions initiated by Thl cells which, it left unchecked, could cause serious tissue damage (Kaufmann, 1995a). Furthermore, the importance of the initial innate immunity anc the decisive role it plays in the differentiation of T helper cells (ThO) to Thl and Th2 cells has become better understood. The balance between macrophage-activating and -deactivating cytokines determines the outcome of the infection (Toossi, 1996; Kaufmann, 1995a and 1995b). Secrevkm of IL-12 by the infected macrophages and the production of IFN- by NK cells leads to the development of Thl cells which initially offer protection against the pathogen. On the other hand, mycobacteria are potent inducers of IL-10 which inhibits the Thl response to this pathogen, probably by uihibiring the synthesis of UFN-7 by NK cells, and has a wide range of effects on antigen-presenting cells. IL-10 has suppressive function, downregulates the major histocompatibility complex molecules and inhibits the production of monokines (Gong et al> 1996), The intracellular bacteria within the macrophage also appear to suppress the early production of EL-4 by CD4+ NK1" cells. Therefore if, at the early stage of the infection when innate immunity plays a critical pan, the production of EL-IO by macrophages and of IL-4 by CD4+ NXl* takes place, the differentiation of T cells will be biased towards the premature development of Th2 cells with detrimental effects on the prognosis of the illness (Kaufmann. 1995a). 3.2 Synthesis of cytokines Cytokines - general Cytokines .ire low molecular weight, soluble proteins which are transiently produced by some cells upon their activation and which spec ficaily affect the behaviour of other cells. They act at picomolar to nanomolar concentrations on cytokine receptors inducing significant effects on the proliferation and function of a variety of cells involved in eliciting an adaptive immune response (Elgert, 1996b). Cytokines are retaliatory and effector molecules which induce signal transduction, activaton of genes responsible for growth, differentiation and cell activity. Cytokines can act as regulators of cell functions by binding to specific, high-affinity receptors on the cells they affect. Cytokines are produced by T and B lymphocytes, NK cells, macrophages and granulocyte;. The term interleukins is used for the molecules secreted by lymphoid cells which allow them to communicate with each other. Currently some 60 cytokines are known. A list of the more important of them. I.e., those involved in me "cytokine cascades" and in the "cytokine regulatory network" in tuberculosis, is given in Table 1 A large panel of monoclonal antibothes specific for human and mouse cytokines, cytokine receptors and cell-surface differentiation antigens has been established. These antibothes constitute powerful probes for analyzing the roles played by different cytokines as intercellular regulators and effector molecules used by lymphocyte populations to mediau: protective natural and specific immune responses to foreign antigens, e.j;. bacteria, viruses and neoplasms. Cytokine production by lymphocytes is restricted due to the instability of their respective mRNA. The mRNA instability is caused by tte presence of an "instability sequence" in their 3' untranslated region Janeway and Travers, 1994a). This allows the strict control of cytokine production and release. The stabilization of mRNA increases the synthesis of the cytokines by 20 to 30-fold. The production ot IL-2 is a decisive factor determining whether a T cell will proliferate and become a functional effector cell. The interaction of IL-2 with its receptor enhances clonal expansion of CD4 memory T cells at the site of active infections. These cells, in contrast to naive CD4 T cells are capable of producing and secreting IFN-. The production of IL-2 and other cytokines depends on the induction of several transcription factors (Janeway and Travers, 1994a). The induction of these factors is in turn initiated by the recognition of a specific antigen by the T cell. One of these factors, nuclear factor of activation (NF-AT), activates IL-2 transcription by binding to the promoter region of the IL-2 gene. However, this event on its own will not result in the production of IL-2, for which in additional binding of CD28 (a receptor molecule of Th cells) by B7 pro ein or APC is required. A signal which stabilizes IL-2 MRNA is passed through CD28. The stabilization of IL-2 mRNA together with the ligand binding of CD28 leads to the increase in the production of DL-2 by 100-fold (Jineway and Travers, 1994a). However, if the recognition of a specific antigen by a T cell is not accompanied by co-stimulation through CD28 molecule, the amount of IL-2 produced is rriinimal and T cells are not stimulated. Table 1Major cytokines (based on Elgert, 1996b) (Table Removed) e central role played by IL-2 in the adaptive immunity s reflected by the number of drugs designee to suppress undesirable immune response* and interfere with the synthesis of this interleukin. The examples of such drugs ate: cyclosporin and rapamycin, administered in order to prevent tissue grafts rejection. Another interleukin having a profound influence on the development of adaptive immunity is interleukin 12 (IL-12), It is a product of monoruclaar phagocytes (macrophages) and plays a crucial role in the differentiation of T-helper cells, inducing the development of Thl cells in vitro and in vivo (Flynn et al., 1995; Toossi, 1996), thus increasing the resistance to several intracellular pathogens in experimental mice (Flynn et al., 1995). IL-12 also enhances the production of IFN-7 from T and NK cells (D'Andrea et al., 1992; Kobay.iahi et al., 1989, Toossi, 19%), increases their proliferation (Flynn et al.. 1995) and enhances cytolytic activity of CD8 T and NK cells (Flynn et al., 1995; Kobayashi et al., 1989; Gately et ai, 1994). On the other hand, cytokines such as TGF-ß and IL-10 inhibit the production and activity of IL-12, suppress the Thl response and stimulate the development of intracellular infections (D'Andres a al., 1992). 3.3 Activation of macrophages and the intracellular fate of M. tuberculosis Following phagocytosis the intracellular growth of mycobacteria depends on their ability to avoid destruction by lysosomal enzymes and reactive nitrogen intermediates. Sufficient evidence has been accumulated to support the observation that U. tuberculosis bacilli have the ability to block Hie fusion of mycobacterium-containing phagosomes (acidic phagocytic vacuoles) with lysosomes (organelles containing hydrolytic enzymes) I(Mc Doesmol masss and Bloom, 1993) and the ability to disrupt me normal functioning (Rastogi, Bschcict and Carvalho de Sousa, 1992). It is generally accepted that the ability ot 14. tuberculosis to survive and multiply within ihe macrophages is linked 10 the unusual physigochemical properties of the mycobacterial cell wall, attributed mainly to the lipid and Jipid-associated components Normally, activated macrophages have the ability to process the mycobacterial antigens and u> transport them to the cell surface where they are presented in association with major histocompatibility complex (MHC) proteins. In this process, macrophages become antigen presenting cells (APCs). Macrophage activation i.e., the induction of antibacterial mechanisms in macrophages, is initiated by the contact of the macrophage conhtining ingested 14. tuberculosis with an inflammatory T cell. Macrophages require two signals before becoming activated: the macrophage-activating cytokine, interfcron-7 (IFN-7] (Interferons are cytokines that can induce cells to resist viral replication) and membrane-bound form of tissue necrotic factor α. (TNF-α) or a small amount of bacterial lipopolysacharide (Janeway and Travers, 1994a). The first signal, which sensitizes the macrophage to the second signal, is delivered by inflammatory CD4 T cells. The second signal is delivered by membrane-bound molecules induced on effector CD4 T cells As the process of cytokine synthesis and of the synthesis of cell-surface molecules mediating their effects requires several hours, the inflammatory CD4 T cells must adhere to tbofcr target macrophages for this period. The newly synthesisecl cytokines are• transferred through the microvcsicles to the site of contact between the CD4 t cell and the macrophage. The activation ot a macrophage through the stimulation with EFN-7 and the contact with CD4 T cell results in a series of biochemical responses, which enable the macrophage to become highly bactericidal. The activated macrophages: 1) ruse their lysosomes more efficiently to phagosomes, which leads to the exposure of the intracellular bacteria to a variety of destjuctive/bactericidal enzymes; ii) secrete IL-12 (activating Thl cells), IL-6, 11-8 and TNF-α iii) produce oxygen radicals - antibacterial agents; iv> produce nitric oxide - an antibacterial agent; v) produce antibacterial peptides; vi) amplify the immune response by increasing the number of major histocompatibility molecules class II (MCH class Ii; and of TNF-α receptors on their surface; vn) recruit other immune cells to the site of infection (Janeway and Travers, 1994a and 1994b; Tizard, 1995a; Fenton and Vermeulen, 1996). The TNF-α further contributes to the INF-y-activation of the macrophage particularly in the induction of nitric oxide. These functions ami at the destruction of the mycobacteria. However, if antigenic sumuktkm persists, the macrophages become chronically activated and produce addidaotl eytpkmes, growth factors and lysosomal enzymes. The latter can attack and destroy the surrounding tissue (Janeway and Travers. 1994a) leading to the formation of pulmonary cavities. The enhancement of the macrophage microbicidal activities can be brought about by T cells, especially CD4 αß T cells which secrete IFN-7 and IL-2 fOrme, 1993). As the activities of Thl CD4 cells are antigen specific, their involvement in the activation of macrophages serves as one of the numerous examples of the mterovining between the innate and specific adaptive immunity. In addition, the activated macrophages produce and secrete other interleukins such as IL-6, 11-8, and tumour necrosis factor a (TNF-α). 11-8 attracts T cells and neutrophils, while IL-6 and IL-8 initiate the acute phase irtflanunatory response. TNF-α plays a particularly prominent part in the immune response to mycobacteria. The production of TNF-α is induced by the presence of lipo-arabinomannan, a constituent of the mycobacterial cell wall ("oossi, 1996). By attracting monocytes to the site of infection, TNF-ot is aiding the formation of granulomas, in which, owing to the anaerobic environment, the bacilli eventually the. Therefore, IL-8, IL-6 and TNF-α participate in the protection of the patient's tissues against the disease and lead to the 'containmen:" of the disease. 3.4 Deiaytd-Type Hypersensitivity (DTH) The cell-mediated immune reaction occurring in M. tuberculosis infection, in which the main effector cells are activated macrophages, is caiied deiayed-type hypersensitivity (DTH) or type TV hypersensitivity. The tuberculin skin reaction (subcutaneous or intradermal contact with the concentrated derivatives of M. tuberculosis) is a classic example of the DTH reaction mediated through T cells (and cytokines produced by them) via activated macrophages (Ehjert, 1996a) The conversion from tuberculin-negative to tuberculin-positive skin reaction develops within six to eight weeks of infection (Boom, 19J6) and frequently constitutes the first sign of the infection with hi. tuberculosis. The second example of the DTH is the systemic granuloma formation resulting from the inflammatory reaction in tuberculosis The DTH reaction constitutes a part of the immune response to many intracellular infectious microorganisms, particularly those causing chronic diseases such is tuberculosis or leprosy. Although the development of DTH in tuberculosis involves sensitization rather than immunization of the infected person, nevertheless it results in a certain degree of protection. With effective cellular immunity, the infection should be arrested permanently at this stage, with healed granulomas leaving small fibrous and ca.cified lesions and memory CD4 cells. If the cellular immune response is insufficient, macrophages containing viable cells of M. tuberculosis may escape via the intrapulmonary lymphatic vessels which may lead to the rapid spread of infection (Elgert, 1996a; Fenton and Vermeulen, 1996). Although the precise mechanism for the development of the initiated DTH response remains not fully understood, the following has been established: i) the interaction between the macrophage-processed .ind macrophage-presented antigen and the specific receptors present: on CD4 T cells leads to the formation of sensitize** inflammatory T helper cells, referred to as Thl cells; ii) a portion of Thl cells become memory cells; iii) on reactivarion/restimulation Thl cells produce a number of cytokines, the most important being 1 IL-2. which causes the proliferation of antigen-primed Thl cells; 2. GM-CSF. which induces localized haematopoiesis of monocytes and neutrophils; 3. TNF-α and -8 which participate in endothelial cell binding and activation of leucocytes; 4. IFN-7 which is responsible for the enhanced expression of MHC proteins; class II molecules on macrophages and endothelial cells as well as for the activation of macrophages (Elgert. 1996a; Fenton and Vermeulen, 19%). In addition, chemotaxic factors (chemokines) such as interleudns of the IL>8 group may attract monocytes to the sites of antigen deposition and the TNF-6 together with the macrophage-secreted TNF-α acd IL-1 as well as IFN-7 induce and control the movement of leucocytes within the area of inflammation. 3.5 Activation of T cells and their functions Lymphocytes which have left the primary lymphoid organs, e.g. the thymus, and have never encountered their specific antigen and therefore have not responded to it, are referred to as naive T cells. The critical point in their development and in the development of the adaptive immune response is their activation. The activation is brought about by their contact with "professional antigen pressing ceils (APC: macrophages, dendritic cells and B cells) and requires the concomitant presence of two independent signals (Janeway and Travers 1994a and 1994 b). Figure 1 presents a graphic illustration of this process. The first signal is delivered by the binding of MHC-antigen complex to the T-cell antigen specific receptor and its co-receptor (either CD4 or CD8). This signal is transmitted by the T-cell co-receptor, indicating that the antigen has been recognised. The second co-stimulatory signal is delivered to the T cell by the same antigen-presenting cell, APC. The best characterise* co-stimulatory molecules on APC are molecules called B7 and B7.2. The molecule on the surface of the T cell acting as a receptor for B7 is CD28 and the ligation of these two molecules will stimulate the proliferation of the particular done of T cells. Subsequently, an additional receptor called CTLA-4 is expressed on the surface of the activated T cell and binds the B7 molecule with a higher affinity. Naive T cells will respond to a particular antigen only when one APC cell presents both stimulatory signals: antigen specific to the T-cell receptor and a co-stimulating signal (B7, £7.2). Only APC possess the ability to express both classes of MHC molecules as well as to deliver a co-stimulatory signal and therefore to perform the so called "priming" of the naive T cells. It is important to know thai the T cells recognising the specific antigen in the absence of co-stimulatory signal, fail to produce IL-2, do not multiply and become anergic, i.e., unable to respond to a given antigen This dual requirement for the proliferation of T cells is a preventative measure aimed at inhibiting the response of T cells to self tissues, which would b: detrimental to the host. The phenomenon of anergy is frequently encountered in tuberculosis. The interaction of T cells with APCs is influenced to a varying degree by a range of adhesion molecules such as seleccins, integrins, some mucin-like molecules and CD44 molecule The activation of T ceils by APCs results in their clonal proliferation leading to the production of large numbers of antigen-specific lymphocytes and the differentiation of their progeny into armed effector cells. The production of IL-2 is a decisive factor which determines whether a T cell will proliferate and differentiate into effector cells. The antigen presentation takes place with die help of two classes of major histocompatibility molecules (MHC). MHC class I molecules present ro CD8 T-cells antigens originating from the pathogens multiplying in thi cytosol of the macrophages, thus initiating cellular or cell-mediated immune response. MHC molecules class II present antigens derived from ingested extracellular bacteria and toxins. These antigens are presented to CD4 inflammatory cells referred to as Thl cells and to CD4 helper cells, referred to as Th2 cells. The Th2 cells activate the specific B cells, thus initiating the humoral immune response Once the T cells are activated and start the clonal expansion, they can act on any target cell which displays the specific antigen on its surface. Effisctor T cells can perform a number of functions such as: i) killing of infected cells by CD8 cytotoxic T cells; ii) activation of macrophages and peripheral mononuclear cells by CD4 inflammatory cells (an essential activity in tuberculosis leading to the destruction of die phagocyiosed mycobacterial cells); m) activation of B cells to produce antibodies. The first two activities constitute elements of cell-mediated immunity, whereas the third one represents the humoral immunity fJaneway and Travers, 1994a, 1994b and 1994c; Elgert, 1996c). 3.6 Cytokine circuits in tuberculosis Experimental evidence accumulated so far indicates that the protective immune response against M. tuberculosis in man is mediated primarily by CD4 Till cells and mononuclear phagocytes (Kaufmann, 1995a and 1995 b, Fenton and Vermeulen, 1996, Boom, 1996). Among the cytokines secreted it die process of mounting host anti-mycobacterial responses, EL-12, VL-2 and IFM-y appear to be playing the most prominent parts. The treatment of the MtuberculosisAxxkatA. Balb/c mice with IL-12 effectively increased their survival and reduced 10 to 50-fold the numbsr of viable bacilli in their organs (Flynn et al.. 1995). As there exists a distinct possibility of T-ccll hyper-responsiveness at sites of active infection, the production of other cytokines which can inhibit the anti-mycobacterial immune responses such as transforming growth factor B (TGF-fl) also takes place. The intricate interplay between various cytokine circuits which are activated by M. tuberculosis and its constituents may be amplified and contribute to pathology at the site of the infection (Toossi, 1996 K The overall outcome of the host immune response/host defence and the course of the disease will be determined by the balance between macrophagi-activating and -deactivating cytokines. Sortie cytokines e.g. tumour necrosis factor a (ITNF-α) and transforming growth factor B (TGF-ß) may contribute to symptoms of tuberculosis such as tissue destruction, fibrosis formation, fever ind weight loss in addition, in the HIV-infected tuberculosis patients, cytokines may promote viral replication and in this way contribute to the progression of the disease. The well documented roic of IFN-7 in the infection with M, tuberculosis, becomes closci to be understood. The administration of IFN-7 .0 mice deficient in Lhis cytokine prolongs their survival after the challenge with M. tuberculosis (Cooper ttal, 1993; Flynn eral., 1993). The protective effects of IFN-7 appear to be due to ihe enhancement of macrophage activity towards M. tuberculosis, namely due to the upreguiation of TNF-α and 1,25-hydroxy vitamin D (Bermudez and Youn?, 1988; Rook et at., 1987) Both, IFN-7 and TNF-ot increase anti-mycobacterial activity of human (Denis, 1991; Hirsch et ai, 1994) and murine macrophages (Berxmicez and Young, 1988) Both, IFN-7 and TNF-α counteract the effects of TGF-f.. These effects interfere with the production of anti-mycobacterial nitrogen intermediaries within the infected macrophage and down-regulate IFN-7 and TNF-c. The balance between these three macrophage-activating and -deactivating cytolcines, i.e., IFN-7, TNF-α and TGF-0 influences the final outcome of the infecion (.Chantry et al.t 1989; Ding, Nathan and Srimal, 1990; Espevik etal., 1987; Tsunawki, Spom and Nathan, 1988; Toossi, 1996). 3.7 Induction of cytokines by M. tuberculosis' antigens or components The currently available information concerning the involvement of M. tuberculosis' antigens or components on iei. were reported anc .ire thought :o be caused by a functional suppression of T-cell production of IL-2 and expression of IL-2 receptors. The relative numbers of the two main popiilatons/groups of T cells, namely CD4 and CD8, remain unchanged (Toossi Kleinhenz and Ellner. 1936; Kleinhenz and Ellner, 1987: Vanham etal, 3 996), 4. A dominant role played by monocytes in suppression of T-cell responses resulting in me lowering of the synthesis r>f IL-2 is well established (Ellner. 1978; Kleinbenz and Ellner, 1987, Toossi etal., 1989;- and high numbers of these cells are frequently encounirred in cases of active tuberculosis (Toossi 1996). The molecular mechan.sms by which monocytes suppress T-c-tl! rL---p^nse< in patients with ruhercik^is have been to a !ira derree elucidated and were discussed in a recent review by Toossi (1996); 5 Cytokir.es produced by CD4 cells belonging to Th2 subpopulations such as 11^4 and IL 10 are reported by some groups of researchers to be higher in patients with tuberculosis than in a ccntro) group (Sucrel et al, 1994: Toossi, 1996) However, there exists a certain degree of controversy on this subject (Hirsch ex a\, 1996, T OOSSK 1996; AS r>oui of these cytokines have been associated with the ability to de-activate macrophages, their presenc: could adversely affect the course of the disease; 6. Transforming growth factor ß (TGF-ß is a potent immunc.suppret.sor that inhibits the clonal expansion of 1 celh by mrerferim: witi ihe prul ferative signal cf IL 2 and suppresses the production of INF- anc IL-2 ;even at femtomolar coneentrat oris. TGF-ß is known for its auto-induction, through which it can significantly increase its levels at sites of active tuberculosis infection Through the action TGF-ß can have a direct influence on several cytokines and seriously interfere with tie host immune defense mechanisms. 7. Granulocyte-macrophage colony stimulating factor (.GM-CSF) is secreted by macrophages and some T cells. GM-CSF, by acting directly an bone marrow cells, stimulates the expansion of granulocytes ami macrophages. It influences, therefore, both humoral and cell-mediated immune responses (Boom, 1996). 3.9 Involvement of other T cells in tuberculosis Apart from the immune processes dependem on CD4 cells, CD8 T cells are also involved in the immune respo-tse to the tnfecnor. with M. tuberculosis. As CDS cells can be directly syutoxie and have abulatiy to kill the macropnages harbouring mycobacteria, they play a pan in ht destruction of M. xuberculosis, either by lysis of the macrophages or by releasing of M. xuberculosn to the extracellular environment where fliey can be phagocytosed by other activated nucrophaeeRecently carried out Investigations indicate that additional subsets of T cells are imolved in the immune reaction to tuberculosis. These T cells produced large amounts of IFN-7 and varying amounts of IL-2, IL 4, IL-5 and IL-10, and cannot be assigned to the clearly defined Thl or T li2 groups They are classified as ThO subset of CD4 ceils (Boom. 1996) Another subset of T culls which ma> have an important role in the cellular response to the infection with M. tuberculosis are the T cells expressing 76 T-cell receptor (Haanen el ci. l9lH; Boom, 1996, Kaufmann, 1995a>. They have been reported to recognise phosphate-containing non-proteimc ecus components ol ;Lyconaae:,d and, • w .slum, la t.or, win thest: component!, have been s.iown to display a Th: cytokine paitt-m CKauftnarm, 1995a). It has been postulated (Kmftnann. I'?95a) that the rapid activa:ion ol y/8 T cells, preceding that of a/6 T cells could attribute to then" a function of a link between iruate immunity by NK cells and ;he spectre adaptive immunity effected by a/B T cells. The 7/6 T cells appear tc control the locU tissue response at the site of bacterial replication and the TCR<5 (T-cell receptor 5) gene deletion mice mutants were found to be more susceptible to deadi when challenged by M. tuberculosa inocula tolerated by immunocompetent mice (Kaufmann, 1995s.>. The CDlb-restricted a!A T cells produce IFN-γ and express cytolytic activity, in which they resemble Thl cell: (Kaufmann, 1995a). 4. Proposed approach to the prevention of infection with M- tuberculosis and proposed approach to the prevention of rheum stoid arthritis associated with tuberculosis Accepting thai mycohc acids possess immune regulatory properties in spite of their simple, long ;ham fatty acid structure, it is proposed that the prevention of infection with M. tuberculosis could be achieved by a successful induction of humoral and/or cellular memory against niyi-olic acids leading lu a lung-Lerrn protection against the disease. Another approach is based on the assumption that by using appropriate tolerogenic doses of purified, biologically active mycolic acids used alone, in a supportive medium or pharmaceutical currier or excipienr or on appropriate carriers, with or without the simultaneous introduction of anappropriate cytokines (Heath and plyfair 1992 ) it should be possible to successfully modulate die immune responses) m the human body. Such a treatment could potentially help to preveirt or decrease mortality due to tuberculosis a;id be used as a potential treatment of the disease. On the other hand, vaccination with mycolic acids used with or without the appropriate csxriers, may regulate the immune system upon the infection with M tuberculosis. by moculatin;.: the induced response of the u::p:ent. Prevention of rheumatoid arthritis associated with tuberculosis coald be achieved by preventing the generation or auto-immune antibodies directed against collagen. Recently published results of the immunomodulatory properties of a synthetic 10-kE heat shock protein (hsplO) from M. tuberculosis, in relation to adjuvant-induced arthritis in rats, indicate that the administration of this compound could indeed lead to the delayed onset of the disease and the development of less severe symptoms (Ragno et al., 1996) Accepting once again mat mycolic acids possess immunoregulatory properties, the proposed approach is based on the assumption that by using appropriate tolerogenic descs of mycolic tcids, possibly on suitable carrse-s, either nn their own or, simultaneously with appropriate uiterleuJan(s) (Heath and Playrair, 1992). it should be possible to successfully manipulate or regulate the immune response(s) in the human body Such a treatment could potentially help to prevent or delay the onset of rheumatoid arthritis associated with tuberculosis, or decrease the severity of this disease In order to investigate tins approach, adjuvant arthritis was induced in rats, which were treated with mycolic acids either prior to or after the induction of the disease. The induction of Lhis form of arthritis, using a suspension of heat-killed and *rceze-dried cells ot an avirulen; strain of M. Tuberculosis H37 Ra v/as achieved following the method of Wauben. Wagensar-Hilber and Van Eden (1994) Investigations into this approach are set out below in Ex.imple 2 5, Immunogemcity of mycolic acids Immunogenic!^ of a molecule, < •_ us ability tv sidicc an immune reaction, depends on the chenucal struct are and properties of the molecule and on the abiliry of a particular imrn.ne system to recognise it. Many compounds such as proteins, peptides, nucleic acids and polysaccharides are naturally highly immunogenic anc capable of eliciting strong jnmune reactions when recognised as "foreign" by die immune system. On the other hand, the majority of lipid compounds, with the exception of some glycohpids, has until recently not been considered to be immunogenic Mycolic: acids are the major lipids of the cell wall of Mycobacteria and constitute approximately 40% of tie dry weight of these bacteria Mycohc acids are high molecular weight f.-hydroxy fatty acids (C60 to C90). which have moderativel on phate in use poser and are characterised by a highly restricted solubility. Their aliphatic struc.ure and absence of aromaticity suggest dial, sumlarly to other lipid compounds, they should have very weak/limited immunogenic properties (Savelkoul, Claassen and Benncr, 1997) In addition, the mechanism by which lipids could elicit immune responses in a hos: and the manner in which they could be presented to uie immune system, have, until recently, been unknown. However, the evidence for immunogenic ity of mycolic acids, i.e., for their ability to induce an immune reaction, has been accumulated over the last three/fcur years on the basis of the results reported by various :*esearch centers It was observed by die present inventors in 1994 :hat mycolic acids adsorbed to proteins and administered to mice elicit an antibody response (.South African Patent Application No. 95/3077 and International Patent Application No. 95/008:56 relating to the induction of actibodies to mycolic acids upon immunization of mice with mycolic acids adsorbed to proteins). The resxmse appears to be specific for mvcalic acids en two account? It was elicited by bovine serum albemin canjugates but could be detected on ELISA wells coated with mycolic acids-gelatin conjugate in the immunoassay and the response measured oo mycolic acids-gelatin conjugate as antigen could be partly inhibited by co-incubating the antisera with bovine serum albumin-mycolic acids conjugate. The immunogenicity of mycohc acids and their immunoresnalatory properties have been supported by evidence which has recently became available from other sources: i) The discovery of Beckman et c/., (1994) that mycotic acids a:tivate DN T-ce!ls upon presen ation on antigen presenting cells While investigating presentation of non-peptide microbial antigens, Beckman et al , (1994) discovered that mycolic acids originating from M tuberculosis stimu.ated tie proliferation of a rare subset of haman T cell* The group oi stimulated human T cells was identified as double negative, i e., neither CD4* (helper function) nor CD8+ (cytotoxic function). 1-ccLi clones. Similarly, Rosat zt al., (1995) reported stimulation and subsequent expansion of a human 1-ceiJ Jme i.UliDli upon exposure to unique cill-wall lipids isolated by organic extraction from M. tuberculosis and presented on CD lb molecules Additional observations concerning the presentation of lipo-arabinomannan originating from the cell wall fractions of M. leprae by CDl molecules were reported by Sieling e: al , (.1995). who isolated two I well unes responding to the sumulatior wiuh lipoarabinomannan On the basis of these reports and results it appears Liat: i) mycolic acids are in fact jumunogenic at least in terms of eliciting some kind of cellular and humoral immune response, ii) mycolic acids are presented by one of a group of five CDl glycoproteins in humins, of which CDlb plays the major role in presentation of mycolic icids. :i) The role of CDl presented molecules in the presentation of mycotic acids and other mycobacterial lipids CDl molecules constitute a group of glycoprotein;; occurring on antigen presenun|' cells. They appear to perform a novel and unique function, by presenting mycobacterial antigens onguiating from the lhid fraction of bacterial lysaces to the immune system. CDl moiecuies appear to be homologous in their function to pepude-presenting MHC (Major H is tc compatibility Complex) proteins (Beckman et aL. 1994; 1995) t»u. specialized in presenting antigens The compounds presented by CD1 molecules are recognised by human T cells displaying on thai surface receptor chains a and B*' CBeckman et al., 1994, 1995; as well as y and I ch-iins"' (Rosat et a!., 1995V The subset of -y<5 T cells was found lo express only receptors coded b\ Vol" gent and to proliferate on exposure to unique cell-wall lipid? isolated by organic extraction from M. 'tuberculosis, presented on CD lb molecules. Both subsets of .hese T cells were activated en exposure- to the antigens of mycobacterial origin and showed enhanced proliferation GJeckman et al , 1994, 1995; Rosat et al., 1995) Experiments described by Tangri ei al.t (1995), carried out with the mouse CD1 molecules, established peptide sequences binding to this type of aniigen-presenring Ttnlt:i-ule<; and fnimrl cii.ch peptides- to be highly hydrophobic This observation confirms die distiact role which CDt molecules appear :o be playing in the presentation of lipid or other hydrophobic compounds m a nori MHC restricted manner iii) The anticipated role of double negative (DN) T cells in autoimmunity and immunoreguiaticn E'ouble negative (DN) T cells form a small, highly heterogenous group of cells residing in the thymus, comprise several early stages in T-cell development. A small percentage of these cells expresses genes coding for yfr while the remaining DN express gisnes for a. B receptor chains. They precede the arjpeararic-s of the functional receptor chains and the expression of CD4 and CD8 rrarke.rs (Janeway and Travers, i^94d). DN T cells constitute less than 2 % of human lymphocytes prssent in peripheral blood (Niehues et al., 1994). They were found to differ from single positive (SP) subsets of T cells in: i) proliferating in response to the presence of mterlcukin 3 OL-3); n. becoming activated on exposure to non-peptide antigens presented by CD1 molecules; iii) not responding to the stimulation with antigens presented by MHC class I and II molecules (Niehues ei al., 1994). Although the main, function of this small and poorly defined T eel! sibset group remains unknovvt., there is some evidence indicating their involvement in auto-unmunt: reactions and ir. the regulation of aJto4mmuniry The studies of von Boehmer, Kltrberg and Rocha • 1991) and Ki$ielow er al (1985) demonstrated chut in transgenic mice, the receptors for sell antigens were predominantly expressed on DN cells,. In humans, levels of DN T-cell populations were found tc be elevated ir. pa:u:ru.- with auk'-irnmunr d:s>„idtrs sucr as systemic lupus erythematosus (.Shivakumar, Tsokos aid Dana, 1989) and systemic sclerosis (Sakamoto er al.. 1992). The involvement of DN T cells in irnmunoregulation was demonstrated by Niehues er al, (1995a), who reported that the stimulated DN T cells sccreced intsrleukin 10 (IL-10). As IL-10 can downregulate the ixpracsion of MHC proteins cla3i IJ' by the antigen presenting cells .ind, at certain concentrations, can suppress the expression of CDl molecules (Thomssen, Kahan and Londei, 1995) and inhibit the functions or" inflammatory Thl cells (Janeway and Travers, i994a), an important role played by DN T cells in regulating ;tnd probably suppressing immune fun;tio*is and n auto-mununit) L- anticipated. If it is accepted that mycolic acids are immunogenic and, when presented on CDl molecules can activate human DN T cells, it can he postulated that two immune logical phenomena observed m tuberculosa namely the occurrence of anergy ard die induction of post-tuberculosis rheumatoid arthritis could be associated with niycolic acids Anergy, / t , the inability of r.he infected person to mount an itnmune response despite the presence of antigen, is commonly observed during the initial stafe of infection with M tuberculosis and M kprae. it is, believed that the specific immunocompetent iymphocy:es are suppressed as a consequence of either the way in which the molecules are presented to them (Schwartz, 199.3) or as the result of me absence of co-stimulatory signals (Janeway and 1 ravers, 1994a) It the presentation of lipid antigens on CD1 molecules creates the conditions necessary for the nutation of anergy in tuberculosis, it can be hypothesised that mycoJic acids could play a direct role in this phenomenon, probably by die activated DN T cells secreting IL-10 (Niehucs,*/ al . 1995a). Auto-immunity or auto-reactivity is a pathological condition caused by the adaptive inmune response directed at self antigens Such responses can be generally produced by: i - x saceer exposure of normally hidder. seff ant gens (as in the case of sympathetic ophthalmia), n self antigens becoming immunogenic due to chemical, physical or biological changes (as in die case of contact dermatitis). LI) coincidental similariry between a foreign antigen (pathogen) and the seif tissue antigen, referred to as molecular mimicry (as in the case of streptococcal protein M and hum;in heart muscle; (Merck Manual. 1987, Janeway anc 1ravers. 1994eV Plieumatoid arthritis, is an auto-mmune disease associated with the presence of auto-antibodies ane auto-reactive T cells damaging the joints' cartilage (Laycock et al. 1995). The mycobacterial ;tnugens could be implicated in this pa±o ogical state in two ways Either haat-shock proteins of M tuberculosis (HSP 60. HSP 65) elicit the production of aut>antibodies due to the genuine molecular mimicry between them and the aggrecan i.e., the core protein of host ciirtilage (Roitt, 1994; Tizard, 1995b; Voet and Voct, 1995) or the production of sach auto-immunc antibodies is caused by a conramiuauwi uf heat-shock proteins secreted by mycobacteria with itycolic acids. The laner possibility finds some support in the observation reported by Buzas et al., (1995) who could not detect any cross-reactivity between aggrecan and the mycobacterial HSP 65 produced by genetically manipulated E. coli, therefore not contaminated by rnycolic acids. Additional evidence that the HSP 65 may not be directly involved in the induction of adjuvant arthritis, came from Moudgil et al.. 11995) These authors worked with two groups of rats resistant and septible to advar etve and found that they shared identical MHC. As only peptides can be presented by MHC. this finding implies that compounds other than proteins (e.g. lipids) are probably involved in inducing diis form of arthritis Further evidence of the lipid nature of die molecules potentially involved in the 'induction of rheumatoid arthritis comes from the studies of Beech et al., (1995< and Lemonidis el al, (1995) who studied pnstane" -induced ;irthrit s in mice. In both instances the response was characterizec b\ the presence of I-lymphocytes and antibodies, strongly cross-re*ctog with mycobacterial HSP 55. The evidence presented above concerning the role which lipids'oils could play in inducing rheuma(o:d arthritis supports the hypothesis that in the case of tuberolosis this role could be performed by mycolic acids forming natural corjugates; with proteins prese.it in the r footed host. iv) Our observations concerning cross-reactivity of mice Jintisera against ruycolic acids-BSA with gelatin E'unng out experimental work substantiating Patent No 94/2575. it was observed that the antibodies produced during immunization with nwcolic acids-protein con;ugaies cross-reacted with gelatin, the denatured form of collagen. Widtiout wishing to be bound by theory, we propose a hypothesis to sxplin this observation, :iamely that the irunne immune system probably does not recognize mycoLc acids as. such, but rather as modified epitopes on protein molecules. *) A —.i.icul oi. of defined structure, mc.udng itrveral metbyl branches. Similarly during infection with M. tuberculosis, mycolic acids originating from the pathogen may become attached to some proteins present in the host's bod>. sucii as heat-shock proteins known to be expressed on the surface of infected macrophages (Grange, Stanford and Rook, 1995) or host prote.n to create "foreign" epitopes. The presence of such epitopes can lead to the production of autoantibodies and auto-reactive T cells against collagen (gelatin), which could attack host collagen leading to the development of in auto-Lnmune reaction. The auto-antibodies and auto-reacvive T cells thus generated, may significant!}, influence the degree of severirv of the disease and may play a crucial role in TB patient survival. If it is remembered that: i, collagen is the native form of gelatin; li) the mice antibodies generated against mycolic acids-protein conjugates were observed »o recognise gelatin; iii) arthritis can be produced in experimental animals by injection of collagen, collagen reactive T cells or anti-collagen antybodies Brand et al 1995. and iv) collagen is me most abundant proteui in higher vertebrates (Sakai, 1995) and that lungs, where the original contact with M tuberculosa usually tikes place, comprise large cumbers of collagen fibrils (Lccson and Leeson, 1981) the potential of rnycolic acids' attachment to the host's major class of fibrous protein and their involvement in the generation of autoimmune antibodies directed agairst collagen and probably leading to the development 3f rheumatoid arthritis associated with tuberculosis, becomes evident Furthermore, the presence of antibodies recognising collagen /gelatin h a tuberculosis patient could be responsible for impaired resistance against bacterial infection (Bras and Aguas, 1995). Such antibodies could react with the collagen-like region of human serum (segment Clq) thm impairing this serum piotein's u~ucial role in the cytotoxic reaction towards bacteria and infected host ceils The pre:ience of anti-Clq antibodies in patients with systemic lupus erythematosus v-as associated witli persistent bypo-eomplemenritem..a and defective ability to opsonize bacteria, which led to the patients inability to dispose of life-threatening infections (Davies, Norsworfhy and Walport, 1995). EVIMUNOREGULATORY AND IMMUNOGENIC PROPERTIES OF COUNTERCURRENT-PUHIFIED MYCOLIC ACIDS The substantiation/evidence for the immunogenic and lmrnunoregulatory propert.es of countercurrent-purified mycolic acids is illustrated by the three examples described below EXAMPLE I: Protection against tuberculosis in mice provided by the administration of purified mycotic acids 1.1 MATERIALS 1.1.1 Cultures Mycobacterium tuberculosis H37Rv A1CC 27294 - a virulent strain, originally isolated from ;in infected human lung. Type strain of the species. Mycobacteriw.% vaccae ATCC 15483 - a siram originally isolated from cow : milk. Type strain of die species. The cultures were purchased tn lyophilb'.ed form from the American Type Culture Coiled- n (ATCC) Maryland. USA 1.1.2 Media 1.1.2.1 Growth media The following media were used fcr the cultivation ol M tuberculosis: Lowenstein-Jensen (U) medium i slams) and Middlebrook. 7H-10 agar medium (plates). A detailed composition of the ingredients necessary for the preparation of these media as well as the conditions recommended for their sterilization, are given in the Laboratory Manual of Tuberculosis Mediocs, Tuberculosis Research Institute of the SA Medical Research Council 11980. Chapter 6, pp 83-105; Second Eduion. revised by E E Nel. H H Kkeberg and E M S Gamer) The media were prepared by the National Tuberculous Institute of die Medical Research Council of Soudi Africa, in Pretoria 1.1.2.2 Media used for washing and diluting of Mycobacteria The harvested bacteria were washed in sterile 0.9% m/v StCl (Saaxchem, Chemically Pure, RSA). Medium used for die preparation of serial dilutions, preceding the determination of viable counts of M tuberculosis, was prepared by dissolviig Tween 80 (Merck, Chemically Pure) in 0,9% m/\ NaCl (Saarchem, Chemically Pure) to a concentration of 0,01% v/v and distributing it in 9.0 ml aliquots into :e;r-rubes The outciaved media were stored at 4°C 1.1.2.3 Media used for the amplification of the competitive plasmid pGEM-3Z Dulbeccc 's Modified Eagle's Medium (DMEM) manufactured by Life Technologies Inc., Country LB agar comprising: 10% m/v Tryptxoe (Biolat Diagnostics, RSA) 5% m/v Yeast extract (Difco Laboratories. Michigan, USAi 10% m/v NaG (Saarchem, Chemically Pure, RSA) and 1,5% m/v agar (Saarchem, Chemically Pure, RSA) LB broth comprising: 10% m/v Tryptore (Bioiah Diagnostics. RSA) f % m/v Yeast extract (Difco Laboratories, Michigan. USA) and 10% m/v NaC3 (Saarchem. Chemically Pure, RSA). Ampicillin and streptomycin (Lite Technologies Inc., Scotland) Sodium pyruvate (Life Technologies Inc) Agarose (Promega Corporation, Madison, USA) 1.1.3 Reagents 1.1.3.1 For the preparation of the -eagents used for the extraction, derivating and High Performanece Iaquic Chronatoairapiiy fHPLC) analysis of mycolic acids, HPLC Grade memanol (BDH) and double-distilled deionized water were used. Reagent A 25% potassmm hydroxide (Saarchem, Analytical Grade) dissolved in methanol warer (1 1). i.e.. 62.5 g potassium hydroxide v.as dissolved in 125 ml water and 125 ml methanol (BDH, HPLC Grade) was added. Reagent B: Concentrated hydrochloric acid (Saarchem, Analytical Grade) diUted 1:1 with water. Reagent C: 2% potassi am bicarbonate (BDH, Analytical Grade) dissolved in methanol-water (.1:1). 10 g potassium bicarbonate was dissolved in 250 ml water and 250 ml methanol was added. Reagent: D: para-bromophenacylbromide dissolved in a^etonitrile and crown ether (Pierce Chemical Co, Cat. No 48891) was dispensed in 500 ^1 quantities into small amber-coloured screw cap vials with Teflon-coated septa. The caps were tightened aud the vial; were wrapped with Parafilm Reagent D was stored at 4"C. Reagent E. Reagent E was prepared by mixing reagent B 1:1 with methanol. HPLC Standard High Molecular Weight Internal Standard (C-10G) from Ribi InununoChem Research Company, Cat No R-50. The standard, 1 mg, was dissolved m 20 ml chloioform fBDH HPLC r,rade> .n 4'C and aliquots of 100 pl were dispensed into 4 ml amber WISP vials, dried, capper, with Teflon-coated septa and siureQ at 4°C. Chloroform (Saarchem, Analytical Grade. RSA) Methylene chloride (BDH, UK, HPLC-Gradcj Reagents A, B, C and E were prepared fresh prior to experiment;, taking all the .necessary safety precautions. 1 1.3.2 The following reagent:, were used for the preliminary purification of crude bacterial extracts ^funnel extraction") and for the coumercurrent purification of the extracted mycolic acids: Chloroform (Saarchem, Chemically Pure; Methanol (Saarchem.. Chemically Pure) Acetone (Saarchem, Chemically Pure; Sodium chloride (Saarchem, Analytical Grade i Double-distilled deionized water was used for ihe preparation of the required reagent concentrations, i.e.,: 39% v/v methanol 42% v/v chloroform 0,2 M NaCl 1.1.3.3 Reagents used in the 3emi-Quantitative Competitive Reverse Transcriptase Polymerase Chain Reaction (QC-RT-PCR): The following reagents were used' Etbidium bromide (Boehrins^r Mannheim, Germany) Formamide and formaldehyde (BDH. UK) Tns (Hydrox>-methyD-aminonethane (Merck, Germany) I-DTA (Ethylencdiaminete'xa-acetic acid) (Merck.;, Sodium acetate (Merck) TRI-reagtnt (Mo ecular Research Centre Inc. USA) Forma2ol (Molecalar Research Centre Inc) MOPS (3-(A<-morpholin.o) propanssulfonic acid) (Sigma Chemicals, USA) Diethyl pyrocarbonate (DEPC, i Sigma) Oiigo dT primers (Life Technologies Inc., Scotlar.d) Superscript RNase H Reverse Trar scriptase (Life Technologies Inc.) Recombinant Taq Polymerase Dyrtazyme (Finnzyrnes OY) Arnplitaq Gold (Roche Molecular Systems. USA) Qiigen mini preparatory column K.it (Qiagen) I'ni fc DTA hurfet Ins ba^e io ruM disodium ethylene diamine tetraacefatc.2H30, pH adjusted to pH 8,3. 1 1 3.4 Reagents used in 'he purification of a BTCR' CD4\ aBTCR* CDS' single positive (S?) and anTCR*. CD4' and CDS double negative ON) T ce'.ls from the human peripheral blood The reagents used in this part of the experimental work were described by Niehue;; et al, (1994, 1995b) 1.1,4 Experimental animals Eight to twelve weeks old female Balb/c (a tuberculosis-susceptible strain) and C57/blJ6 mice (a tuberculosis-resistant strain) were used in the "immunoregulatory" experiments. The mice were inbred for 11 and 9 generations, respectively, by the Animal Centre at ths South African Institute for Medical Research in Johannesburg. Male mice of corresponding age were used for tne collection of serum necessary for the preparation of mycolic acids/mouse serum conjugates. Seventeen weeks old. Sprague-Dawley female rats \*e:e used for the induction of anti-rnycolic acids antibodies. Feed and water Mice culbes, manufactured by EPOL and tap? autoclaved water were provided ad libitum. Sanitation: Bronocide, manufactured by Essential Medicines (Pty) Ltd, was used for sanitation purposes. I. .5 Plasticware The following plasticw. xc was used Disposable Petri'.-J dishes Promex. RSA ELISA piates (Sterihii. /Ki Sterie traposbi 50 m confnfage tube conning (SA) Disposable tips (Elkav. Denmark) 96-well round bottom m.cruplates (Nunc, Denmark; 1.2 METHODS The folic wing methods v. ere used m the evperimentai u-ork 1-2.1 Cultivation of the bacterial strain* The baciena were cultivated at ITC using LOwenstein-Jensen (LJ) medium slants and Middlebrook 7H-10 agar medium plates. The sterility of all the media was confirmed, before the> were used m the experiments by incubating them at "7°C for 24 h For routine extraction or mycolic acids approximately 4-wcck o d Ki. tuberculosis and 2-week old cultures of M vaccae. grown on LJ slants. were used. When Middiebrook 7H-1D agar medium plates were used, 2-weck old cultures of M. luberculosis were harvested for the extraction of mycolic acids. For the preparation of bactenal suspensions used for the experimental induction »f tuberculosis, approximately 2-week old :ultures of M. tuberculosis, grown on LJ slant.* were used. 1.2.2 Viable and total bacterial counts For the viable count determination, serial suspension? of the harvested bactena were prepared in the diluent medium (as specified under LI.2.2) to a dcisiry corresponding to a McFarland standard 4 (approximately OD of 1,0, using a Beckmai: DU 65 spectrophotometer, at 486 run). Tenfold serial dilutions were prepared usir.g 9 rr.l aliquots of the diluent rr.edium. From the last three dilutions corresponding to 10'\ l0"4 and 105 of the original suspension, aliquots of 0,1 ml (130 µl) were withdrawn and spread over the surface of Middiebrook 7H-1C plates. The plates were incubated at 37°C and the developed colonies counted after two to three weeks for M. tuberculosis and after ore week for the plates seeded with M. vaccae. The dtre:t total count was performed using a Neubauer counting chamber and the lutoclaved cultures of M. tuberculosis and M. vaccae, originally adjusted to a density corresponding to a McFarland standard 4 and suitably ,:. U'CM '.• ith the ciluent medium Statistical analysis ai the bactenal counts included the mean values of bacterial counts and standard deviations. 1.2.3 Preparation of mycotic acids from bacterial samples The preparation of bac:enal samples xrnpnsed three steps.: harvesting of the Mycobacteria cells: saponification ar,d extraction of mycotic acids. Glassware used for the harvesting, extraction, denvaazaiion and HPLC analyses of mycotic acids was washed m 2% (vvi Contrad (Merck), rinsed m water, followed oy rinsing ;n cobrotorm, water. Tschnkai. Grade methanol, water and finally rinsed in ao able distilled deionized water. The washed glassware was dried in a warm air oven. Harvesting was done by scraping the bacteria] growth from the surface of media slants or agar plates (using sterile plastic loops) and by suspending them in Reagent A. Initial bacterial suspensions were prepared in Reagent A, by vortexing the harvested cells with sterile glass beads. Homogenous bacterial suspensions were prepared using sterile tissue homegenizers. Prior to the saponification, the density of the bacterial suspensions was adjusted to a dersity corresponding to a McFarland standard 4. The sap omfication, extraction and den validation of mycotic acids, were carried out as described by Butler, Josi and Kilburn (1991). with minor modifications and are described under the relevant heading*. Saponification of the Mycobacteria in Reagent A was carried out in an autoclave at 121 °C, for 10 mm. 1.2.4 Extraction of mycolic acids The saponified sampler were allowed to cool after autoclavmg. Into 2 mi sampler containing cruce extract, 1,5 ml Reagent B was in'reduced After vortexmg, the pH of each sample was checked and if" necessary, ad uslted to pH 1 with Reagent B Subsequently. 2.0 ml ciloroform was added to each sample and vortexed for 30 seconds. The layers were allowed to separate. The bottom layers were removed with Pasteur pipettes, transferred to amber WISP vials and evaporated to dryness a: 85:C in a heat block-evaporator ur.der a stream of nitrogen. To neutralize -traces of acid carried over, 100 pi of reager.t C was added to each sample ard the fluid evaporated to dryness at 85°C in a heat block-evaporator under a stream of nitrogen. 1.2.5 Storage of the crude extracted mycolic Acids The material obtained trom the large-scale extraction of mycolic acids originating from M. tuberculosis and M vaccae. i e , die crude bacterial extracts, was stored und;i acetone, at 4DC in 4 ml amber WISP vials. To prevent evaporation/drying and the exposure to light, the caps of the WISP vials were covered with Parafilm. 1.2.6 Determination of mycolic acids contents in crude extract) Extracted mycolic acids were dcrivatized as follows To a cooled sample ol crude extract (approximately 10 fig in 2,0 ml Reagent A), an aliquot of 1.0 ml chloroform was introduced, followed by the addition of 100 µl of Reagent D (darivatization reagent). The capped sample:; were vortexed for 30 seconds and heated for 20 minutes at 85°C in a heat block evaporator Subsequent!}. :hc samples were cooled and 1,0 ml of Reagent E added The samples were vortexed for 30 seconds and the layers allowed to separate. The bottom layers were removed with Pasteur pipettes and transferrec to VHSP-vials. The vials were placed in a heat block-evaporator and their contents evaporated to dryness at 85°C using a stream of nitrogen. The residues were res.-pen.ied n. 0,21''. £ (which corresponds to l60 µl) methylene chloride, capped and vortexed. Each reconstituted sample was introduced into a WISP vial conta.nmg 5 ug of the HPLC internal standard (prepared as described under 1.1.3.1). filtered through a C,22 um Millex GV4 filter with a polyethylene housing into another amber-colourec; WISP-vial. The recapped vials were stored at 4°C until ready for HPLC analysis. 1.2.7 HPLC analysis and quantification of mycotic acids Repeatability and accuracy of the pipette used for the distribution of the HPLC standard was determined. The precision was established to be +/- 1% and was confirmed prior to each aiiquoting of the internal standard. For die HPLC analysis 10 ul frorr each sample (maintained on ice during handling), was analyzed. Control samples, i.e., 10 ul of filtered methylene chloride, were run prior lo each set of samples analyzed. If a large number of samples was analyzed, in order to validate the reliability* of the HPLC apparatus, control samples were run after every three or four test samples. The reverse-phase HPLC. analyses wen. carried out using a Waters 600 E System Controller High Perforrrana: Liquid Chromatogiaphy apparatus consisting of: Microsep M741 Data Module Waters 712 WISP Autosampler: Detector (Waters 486 Tunable Absorbance Detector): Column: Now-Pak CIS -i Lira "vQ x 15(: rr.m and an end connector set for steel cartridge coiumns RKC Rex-C 4 Column Temperatixc regulator. Running conditions were: Mobile phase: Solvent A. HPLC Grade methanol Solvent B HPLC Grade m«th>ler.e chloride Flow Rate. 2,5 minim Column temperature 30"C The detector was set at 260 nm Prior to use. the solvent:; were sparged with Instrument Grade helium. High Punty Nitrogen was used to control hydraulics of the WTSP vials auxosampler The HPLC gradient initially comprised 98% (v'v) methanol Solvent A) and 2% (v'v) meth>lene chloride (Solvent B). The gradient was increased linearly to 80% A and 20% B at one minute; 35% A and 65% B at ten minutes, held for 30 seconds and then decreased over 10 seconds back to 98% A and 2% B. This ratio was maintained for 4 minutes to a.low for stab liz£.tion of the system prior to inj?ctio:i of the next sample. Mathematical quantification of mycotic; acids w-as carried out by comparing the combined peak areas of 'Jie tested samples to the peak area of the introduced quantity of the High Molecuhir Weight Interna: HPLC Standard. 1.2.8 Preliminary purification of crude mycobacterial extract:. In order to shorten the time required for the countercurrent purification of the crud; mycobacteria! extracts, an additional preliminary extraction step was introduced. This step had a dual purpose: i't to remove unnecessary cellular n'mponent? from :he crude extract prior to the countercurrent purification and ii) to reduce soap fraction in the crude bacterial extracts A portion of the crude extracted mater al (approximately 3-4 g) was suspended in a minimum volume of the lower phase solvent (usually 100 ml), transferred into a separation funnel and mixed with an equal volume of the upper phase solvent. The phases were allowed to separate and the upper phase was removed and stored at 4°C Into the rsmainirg lower phase in equal volume of the upper orase solvent was again introduced and the process of the phase separation was repeated. The second upper phase was removed and stored at <~°C and the second lower phase was dried in a Buchi Rotocvaporator RE 120, at 75°C and its mass recorded. 1.2.9 Countercurrent purification of mj colic acids originating from M, tuberculosis and M. vaccae Counter-current apparatus A countercurrent apparatus produced by H O POST. Instrument Company Inc., Middle Village, New York was ussd during the investigations. The "trains'" in this model consisted of 2 X 250 inter-connected tubes. Solvent system used in the countercurrent apparatus The solvent system used for the countercurrent separation consisted of: V.% v/v chlorofoi-m (Saarchem, Chemically Pure P.eagent) 39% v/v methanol (Saarchem, Chemically Pure) 19% v/\ 0 2 M NaCl (Saarchem, Chemically Pure). Double-distilled deionh'ed water was used for the preparation of the solvent system. The components were nixed, equilibrated and the upper and lower phases were collected using a -.eparation funnel. The composition of the upper phase was established to be: 15% v/v chloroform. 52% v/v metharo. and 33% v/v 0.2 M NaC'l. The composition of the lower phase was established to be: 68% v '• chloroform. 27% v,v methanol and 3% . v 0,2 Ivl NaCi. The countercurrent purification process was carried out under the following conditions: A countercurrent distribution train comprising 55 tubes, numbered 0-54, was used in the experiments. The upper phase solvent, a volume of 600 :nl, was introduced into a buffer reservoir. A sample of 125 mg of mycol.c acids after the preliminary pur:fication was dissolved in 50 ml of the lower phase solvent, divided into five aliquots and introduced into first five tubes, numbered 0-4. Subset)uently, 10 mi of the upper phase solvent was introduced into each of the firs: five countercurrent rubes. Into the remaining 50 tubes aliquois of 10 mi of the lower phase were introduced Upper phase, in volumes of 10 ml per cycle, was automatically dispensed into tube number 0, repeatedly over 55 cycles resuidng in approximately 5 hour operation Thus, fifty five countercurrent cycles were performed, with each cycle consisting of 10 mixing pcndula and 3 minutes phase separation time Initial load of crude extract after the funnel extraction: 125 mg Number of cycles: 55 Equilibration time: 3 min 1.2.11 Removal of malachite green from the counitercurrent- purified my colic acids To remove traces of malachite green derived from hart-ma I growth media (when M tuberculosis was grown on L J slants), the countereurrent-ourifTed material was selectively precipitated in the following manner. Counter ;urTent-purified mycohc acids (52 mg) were placed in a WISP vial into which 1,0 ml chloroform was in.trodi.iced. The dissolved mycolic acids were trensferred into a pre-weighed round-bottom flask. The vial was rinsed twice with 1,0 ml chloroform .ind the two aliquots of chloroform were added to that already present in the lound-bottom flask. Subsequently, acetone was introduced drop-wise in 500 ul aliquots. In lotal, 26 ml of acetone was introduced and the white flakes of the precipiiated-out rnycolic acids were washed twice with 20 ml acetcne. The acetone supernatant, with the dissolved malachite green was removed and the myco.ic acids dried by evaporation The procedure was carried out at room temperature. L2.12 Determination of my colic acids after counter current purification In order to increase the accuracy of ±; HPLC determiria.ion of mycolic acids, the High Molecuhu Weight lnternal Standard (C-100) was introduced into the countercurrent-puified mycolic acids before the saponification . A sample of 0,5 mg of the countercurrent-purified mycolic acids was introduced into a WISP vial containing 5 us of the High Molecular Weight Internal Standard tC-lO'.M. Saporuficaucn of rnycoliu Jtcids \*di carried out with 2 ml of Reagent A at room temperature. The WISP vial -was vortexed for 30 seconds. The extraction was carried out with 1,5 ml of Reagent B. After vortcxing, the pH of the sample was checked anc if necessary, adjusted to pH 1 with Reagent B. Subsequently, 2,0 ml chloroform was added to each sample and vortexed for 30 seconds. The layers were allowed to separate. The bottom layers were removed with Pasteur pipettes, transferred to amber WISP vials and evaporated to dryness at 85°C in a heat block-evaporator under a stream of nitrogen. To neutralize traces of acid earned over. 100 ul of reagem C was added to each sample and the fluid evaporated to dryness a: 85UC in the heat block cooperator under a that of manger . Therefor;, the main difference between the delcrmination of mycoli: acids after coumercunent purification and in the crude extract was the time of introduction of the Internal Standard. 1.2.13 Determination of yield ol the countercurrent separation in crde- to calculate the approximate yield of purification/separatioEL the amcunt of the rnycoli; acids present in the samples obtained lifter the countercurrent separatio ^'purification was compared to the amount of these compounds present in the crude cellular extract introduced into the couiitercurrent apparatus. The calculations were based on the results obtained by the HPLC ;-Jialysis It "should be stressed that it is essential for the calculation, of the yield of the countercurrent separation, that the myeolic acids detenr..ined by HPLC should be within the tested linear range of the HPLC UV cetector. 1.2.14 Infra-red spectroscopy Samples of mycoLic acids to be analy zed by infra-red spectroscopy were prepared in the following manner. Couritftrcurrent-purified myeolic acids, 1 mg, wtire dissolved in I ml chloroform, introduced into 2C0 mg KBr and thoroughly mixed. After the evaporation of chloroform, a pellet of myeolic acids in KBr was prepared bv using a bhirnacbtu tablet die and applying a force of approximately 1 DO kilonewtons on the sample for 10 minutes. A control pellet was. prepaied using only ciluroform without mvcolic acids added to the preparation. The contro pellet was used to determine the background infra-red spectrum The spectra were analyzed on a Perkin Elmer 1600 series FT-IR system and plotted on a Roland Digital Group X-Y Plotter DXY-1200 1.2.15 Determination of the stability of the countereurrent- purified mycotic acids A pooled sample of the eouiitercurrent-purified mycolic acids was prepared b> introducing live batches of counter current-purified mycolic acids into a container, dissolving diem in chloroform and mixing the contents vt:ry well. The chloroform was evaporated using a Buchi Rotocvaporatoi RE 120. at 75"C and the sample dried under a stream of nitrogen. Trie pooled sample was divided into two pms which constiruted two stock samples. The first stock sample was re-saponified and the second was left as a non-saponified stock sample. From both stock samples individual aliquot* were wiihdrawn and placed at -20LC, 4°C and 25UC. Tliree samples were prepared per each time point and HPLC analyses were caned out after 6 weeks, 3. 6. 9 and 12 months of storage. 1.2.16 Methods used in handling experimental animals in the immunoregulatory experiments 1.2.16.1 Environmental conditions under which the experimental animal were maintained Experimental animals Eight to twelve weeks old lemale Balb/c (a tuberculosis-susceptible strain) and C5"/bLi6 mice (a tuberculosis-resistant strain) were used in the "immuncregulatory" experiments. Experimental animals were accommodated in cages with a floor area of 450 cm", with 8 mice pc: cage. Environmental conditions: lempeiature and humidity in the animal facility were set at 20°C (-/- 1°C) and 40% (+/-- 10%), respecthely. Lighting was provided by mean;; of fluorescent tubes. A light-darkness cycie of alternating 12 hour periods was set up. Cages Mice were housed in transparent polypropylene cages with tight fitting stainless steel lids. Wooden shavings, after autoclaving, were provided as nestling materia' Sanitation Animal rooms, mice cages and glass bottles were cleantd and decontaminated once a week using Bronocide. Water bottles after washing were autoclaved once a week. Glove isolator Mice infected with M tuberculosis HJ7RV were maintained in a glove isolator manufactured by Labotec. South Africa. The isolator was inflated by a positive pressure of 4 atm. If was equipped with an air inlet pre-filter (with the pore size of 0.6 µg through which the incoming air was filtered and an outlet HEPA (High Efficiency Particulate Air) filter (witn a pore size of 0.22 µm) through which the outgoing air was filtered before leaving the isolator. The air-flow rate was regulated at 7 exchanges per hour. Sanitation Animal rooms, mouse cages, the glove isolator and water bottles were cleaned and decontaminated once a wsek using Bronocide. Water bottles. after washing, were autoclaved once a week. 1.2.16.2 Identification of the experimental animals Indhiuii.d identification ot mice ^a< accomplished hy rmiine ear marks. 1.2.16.3 Collection of blood samples and preparation of mouse serum Mice were bled from the tail vein and the blood collected into sterile Eppendorf s tubes The collected blood was incubated a: 373C for one hour and then left at 4°C overnight for die clot to retract. The serum was recovered by cenfifugauon (in a Eieclcman J-6 centrifuge at 1000 g for 15 min), aliquoted in \olumes of 1.0 ml and stored frozen at -70X. 1.2.16.4 Preparation of mycotic acids-mouse serum conjugates The required mass of :n>cohc acids (2,5 mg) was dissolved in 200 fxl chloroform and added to 10,0 ml of mouse serum (see 1.2.19.3), previously filtered through a 0,22 p.m filter. Thus, the volume of dissolved rnycolic acids constituted ?% of ihe volume of mouse serum. The sample was sonicated using a Branson Sonifier B 30 Cell Disruptor, (at 20% duty cycle, output control cf 2, for 50 pulses, at room temperature). The simple was. main used for i hour at room temperature, to allow air bubhit.- formed during sonication to escape. In order to remove chloroform, nitrogen was bubbled through the conjugate until the chloroform odour was removed. The conjugate was prepared immediately before administration to the experimental animals. 1,2.16.5 Preparation of bacterial suspensions for the induction of tuberculosis in mice The ce.is of M tuberculosis H37 Rv, harvested from LJ slants were suspended in the diluting buffer (0,01% v/v Twcen 80 in 0,9% m/v NaCl) and homogenized. After centrifugatiori in a Beckman J-6 centrifuge for 20 rain at i 580 g. the cells were washed with a sterile solution of 0,9% m/v ot NaC and adjusted IO a concentration corresponding to a McFarland standard No.4. After the confirmation of the total direct bacterial count, carried out on an autoclaved suspension in a Neubauer counting chamber, the suspension was further diluted in ihe sterile solution of 0,9% NaCl to obtain concentrations of A/ tuberculosis corresponding to 103, 104 and 105 cells ml. The viable counts of the m>cobacteria in the suspensions were confirmed by piatirg 100 pi aliquotes of die relevant dilutions ontc Middlebrook 7H-10 agar medium, incubating the plates at 37°C for two weeks and counting The number of colony forming units (CTU). The suspensions were introduced into the experimental animals in aliquots of 100 pi per animal. 1.2.16.6 Introduction of the M. tuberculosis H37 Rv suspensions, my colic acids-mouse sen m conjugate and mouse serum The introduction of the bacteria! suspensions and of the mycolic acids corvugaes was earned out via the intravenous route. Prior to injections, mice w;re heated for 5 rain in a heating box until vasodilation of the tail veins could be obscrvec. The respective bactenaJ suspensions were introduced in aiiquots of 100µ1 per mouse. The mycotic acids-mouse serum conjugate wis administered by introducing 25 ng mycolic acids/100 µul mouse serum per mouse. Control animals received 100 µl of mouse serum introduced in the same mamei 1.2.16.7 Experimental set-up [he experimental set-up is presented m Tables 2a - 2d. Table 2a Experimental sef-up for the imnwnoregulatory experiment IR-I1I Table 2b Experimental sit-uji for the munorcguJatorv experiment IK-IV (Balb/c mice) Tabic 2dExperimental set-up for the imuiunoregulatory experiment No V Table 2c Experimental set-up for the immunorcgulatory experiment FR-IV u mortem analyses were performed on control and infected mice. Dh.section of the diseased mice and histological examination of the appropriate organs were carried out by Dr ( H Vorster of the Section of Pathology of the Veterinary Research Institute. Onderstepoort, 0110. Methods used in histopathologica! assessments After dissecting of various organs, i.e . the lungs, spleens and livers, from rnicr cadavers, they were individually weighed and photographed Macroscopic assessment Macroscopic assessment ot the degree of infection in various organs was carried out by comparing individual orgars or .g mating from various groups of experimental mice to the comrol organ'; The evaluators were not aware or the treatment io which individual animals were subjected. Microscopic assessment Fixanon ol the organs was carried out by submerging them in 10% v/v formaldehyde sclution in PBS buffer. The organs/tissues were subsequently embedded in pisraffin-waA and sections of 5 ^im thickness prepared by cutting with microtome. For granuloma counts, organ sections were stained in haematoxylin/eosin solution according to Luna (1968). Lesions observed in the tissues were graded by counting the number of granulomas per field, uiiing 10 fold magnification for the liver and lung tissues and 40 fold magnification for the spleen tissue. A qualitative grading system for the assessment of the severity of lung lesions was devised as follows: 1 - small, well defined granulomas in the lungs; 2 - larger, more diffused granulomas which sometimes fomed extended focal areas of granulomatous pneumonia, occupying less than a third of the lung tissue. Interstitial pneumonia was slightly more pronounced; 3 - mostly fused granulomas which were extensive and affected more than one third of the lung tissue. For counts of acid-fast microorganisms, the sections were stained by Ziehl-Neelsen technique (Heifets and Good, 1994). (Using ttds technique, acid-fast bacteria stain red, nuclei stain dark blue and other tissue constituents are pale blue). Ziehl-Neelsen staining was used for qualitative assessment, which was made by comparing the number of organisms and their density within the stained tissue. The level of infection in the lungs, livers and spleew originating from various groups of mice was compared for each specific organ, but could not be compared among different organs, due to the difference in appearance and size of lesions characteristic for individual organ types. Biochemical assessment Five to seven weeks after the infection with AT tuberculosis, be mice were sacrificed for cytokine profiling, the required organs removed aseptically and soap-frozen in liquid nitrogen The frozen organs were maintained at -70"C and analyzed for die expression of various cytokines. 1.1.17 Methods used in the Semi-Quantitative Competitive Reverse Transcriptase Polymerase Chain Reaction (SQC-RT-PCR) determination Background information: RT-PCR - Principle The Polymerase Chain Reaction (PCR) is a technique used for the amplification of DNA and the complementary DNA (cDNA) or' specific mFNAs, which was invented by Mullis in the late 1980's (Muilts aid Fal^ona 1987; Saiki et al., 1988) for the amplification of DNA sequences in vitro PCR is based on a series o; incubation steps carried out at different temperatures. The template DNA (or cDNA) n; denatured at a temperature abo.e 90°C ^denaturing step). The oligonucleotide primers are then annualed to the single stranded DNA (ssDNA) at a temperature varying between 50°C and 60°C, depending on the type of primers used (annealing step-. This process is followed by an extension of the primers by incorporating ctiviPs. using a heat-res.stant DNA polymerase and an incubation temperature of 70 - 72°C (extension step). The extension products of one primer provide templates for the other primers iti subsequent cycles so that each succsssive cucle essentially doubles the: amoint of DNA synthesised in the previous cycle. The result is the exponential amplification of the target DNA co approximately 2" (n = number of cycles) (Zubay, 1993; Tamarin, 1996). Taking into cDnsideration a number of variables*' which may interfere with the quantification of the exact amount of mRNA originally present in *Jie analyzed sample, an improved technique, that of Semi-Qjautitative Cempeftfive Reverse TnHrLs.:ripta»e Polymerase Chain Rcac:ion, was employed. Sc inning of gels was done with a densitometer (Apple Mac). The density of the unknown cDNA band was compared to the density of that of 6-actin and the concentration of the unknown cDNA could thus be estimated. The relative densitometric measurements were done using a Macintosh NIH Image Program This technique can be used to accurately quantify less than 1 fg (femtogram, 10 s g) of target cDNA obtained frum total RNA after the RT reaction. The accuracy of this method can be improved by using the same master mix for all the samples. The master mix should contain the appropriate primer.;, PCI buffer. dNTPs, Ms;CI; ai:d the polymerase enzyme. The mix should be divided equally between all the tubes used tor the PCR. The competitive plasmid for murine IL-12 p4(> (obtained as a gift from K.JL Bos:. University of Tulane, New Orleans, Louisiana, USA) was a pGEM-3Z derivative with a IL-12 fragment (334 base pairs) cloned into the Xba I site from the muliiclorung sire of the vector (Bost and Clements, 1995). The constructioa of the plasmid was described by Bost and Clements (1995) and Chong, Bost and Clements (1996). *) The variables include: differences k the stability and purity of polymerase enzyme, dNTT and in buffer preparation in various batches; Mg** concentratioa, DNA (.'template) concentration, primer concentration, annealing, extension and denaturing temperature*; length and number of cycles; rate of pnmer-dimer formation; presence of contaminating 1.2.17.1 Preparation of the organs used for RNA extraction The organ* originating from both infected and uninfected mice, used for Che RNA extraction -xperunents were lungs, spleeas and kidney. Msec were sacrificed by rapid cervical dislocation. The organs were removed from each mouse asepucally. and kepi at -70°C after snap-freezing in liquid nitrogen. A single-cell suspension of the spleen was made by cutting the spleen into small pieces on a nylon sieve (70 µm mesh) in the presence of ice :old median (DMEM containing streptomycin and sodium pyruvate). The spleen cells were concentrated by centrifugation and the excess of ;nedaim was removed. The erythrocytes present in the preparation were lysed by hypotonic shock, i.e., by treating the cells with a 1/10 dilution of DMEM tn sterile distilled water for 15 seconds. The lysis of the wells was stopped by adding excess medium. After centrifugation. the excess medium was removed and the cells were snap-frozen in liquid nitrogen (M;Carron, et al, 1984). The cells were maintained as a dry pellet at -70'C. 1.2.17.2 RNA Extraction from control and infected organs RNA was is Dialed from all -the organs using the TRI-Reagent protocol bisea on an acid guanidium thiocynate-phenol-chloroform extraction, a method first developed by Chomczynski and Sacchi (1987). The isolated RNA was quantified by a Shimadzu UV-Visible Recording S v^rrophotometer model UV-160, at ware-lengths of 260 nm and 280 tun. Pure RNA (absorption ratio at 260/280 nm >/= 2,0) was used for PCR. Integrity of the isolated RNA was determined using a denaturing formaldehyde gel (Maniatis, 1982). These denaturing conditions prevent degradation of the RNA by RNascs. The water used in these experiments was diethyl pyro-carbonate (DEPC)-treated. Ethidinm hromide was added to the RNA sample, before it was loaded en the jc'.. at a concentration of 0.5 ng'mi LO enable visuaiiiation of the DNA witt UV light. Pure, undegraded RNA giving die three rRNA bands (the 2SS rRNA, l&S rRNA and tlic 5S rRNA) on agarose gel electrophoresis, was used for the reverse transcriptase reaction (Maniatis. 1982) 1.2.17,3 The optimization of the different cytokine PCRs and the 6-actin PCR Cytokine PCRs were optimised by using different plasmids containing DNA sequence fragments of the various c> tokines to be evaluated/to be tested. These fragments of DNA are deletion mutations of fragments of die wild type cDNA for the individual cytokine. Both the mutated and the wild type cDNA can bs amplified by using the same, primers. Three different plasmids were used for dui purpose: i) a plasmid used for the determination of IL-12 was obtained frcm K Bost (University of Tulane. USA); n) a plasmid used for the determinatiuu of TNF-α was obtained from RL Tarleton (University of Georgia, USA); ii» a plismid used for me determination ot LL-4, 1L-10, IFN-7 and TGF-ß was obtained from R M Locksley (University of California, San Franciso USA). 1.2.17.4 The amplification of the competitor plasmids The competitive plasmids for the determina:ion of IL-12, TNT-α, TGF-ß were amplified after trarns formation in the SURE E. coli strain and isolated with the Qiagen mini preparatory column kit. The recovered plasmids were resuspendeti in TE buffer (10 mM Tris pH 8, 1 mM EDTA) arid stored at -20°C. A list of the sequence of sense and ami-sense primers, their annealing temperatures and the wild type fragment size is given below in Table 3. Tabl e 3 A list of Che sequences of the sense and antiscnse primers, their annealing temperature and the wild type fragment stee (Table Removed) 2.17.5 Semi-Quantitative Competitive Reverse Transcriptase Polymerase Chain Reaction (SQC-RT-PCR) determination of IL-12 The primers were individually diluted in TE to a concentration equal to 132-133 p:nol) PCR reactions, carried out using the MJ Rcseaich Peltier Thermal Cycler (PTC-200), were performed based on the protocol developed oy Boct and Clements (1995) The protocol was adjusted due to a different type of PCR apparatus and :o a different enzyme used (see Table 3) The enzymes used in the SQC-RT-PCR curing the exploratory phase were Dynazyine (Recombinant Taq Polymerase). Axnplitaq Gold (Tiq polymerase) was used in the final PCR experiments Amplitaq Gold is a more sensitive and heat-stable enzyme-antibody complex, which is Activated at temperatures above 90 °C. The PCR conditions for using Jhese two enzymes differ. -or Dynazvme, a 3 ruin Hut Stan (3 min at 96UC followed by 1 min at 30'C,) was required before ihe enzyme was introduced into the reaction mixture After the enzyme was added, tbxee cycles consisting of: 45 sec at 94 DC followed by 7 5 sec at 58°C and 105 ;ecat72=C were run to intitate the syndiesis of che secend CDMA strands. The subsequent amplification cycle consisted of the following shorter steps: 35 sec at 94°C, 45 sec at 58*0 and 75 sec at 72'C This cycle was repeated 29 times. Because Amplitaq Gold enzyme is heat stable, the enzyme was added before the PCR cycling was initiated by an incubation seep for 10 mis at 94°C At this temperature, the enzyme became activated. The rest of the cycling profile remained the same as for the Dynazyme enzyme. The optimum concentration of MgCU and primer was determined for each enzyme in a series of experiments. Each PCR reaction mix consisted of 2 mM MgCl2 (for Dynazyme) or 1,5 raM MgCi: (.for Amplitaq Gold), 0.2 mM dNTP. PCR buffer supplied with the enzyme and 2U polymerase enzyme. 500 ng of each primer and plasmid DNA was added to the mixture. The final reaction mixture volume was. made up to 50nl with sterile deionised water A first QC-RT-PCR approach protocol was described by Bost and Clements U995) for mterleukin 12. For the QC-RT-PCR both the RT mix and the IL-12 p40 plasr.ud DNA were added to the reaction mixture. The RT-mix (for the first stiand cDNA; had a total volume of 23/il. This volume was divided into different percentages eg. 60% , 20% ; 6% ; 3% etc, according to the protocol of Bost and Clements (1995). A constant concentration of plasmid DNA was added to the PCR reaction mix. .'V different approach desciibcd by Chung, BDSC and Clements (1996)was subsequently applied for optimization of PCR conditions. According to tus protocol, the RT-mix was constant at 20% (4 pi of RT-mix were used), and different concentrations of the plasmid DNA were added. Plasmid was. added in the following dilution range: 0.5, 0.25, 0.125, 0.062, 0 032, 0.016 and 0.008 pg. The final PCR product was visualised on a 2% agarose gel containing 0.5 Mg/ml ethidium bromide. It was found that the digested plasmid gave better results than the undigested plasmid due to better denaturaLon of the plasmid material The digestion was performed with the enzyme Xba I at 37°C for ? hours 1.2.17.6 B-actiu PCR: The fl-actn. PCR was performed as an indication of the amount of intact mRNA <\mplitaq Gold was used as DNA polymerase. The muic PCR cycling protocol as that of HL 12 p40 was used, except for the annealing temperature for the 8-actin primers which was 59°C instead of 58°C. The elongation and amplification cycles were the same as thai of IL-12 PCR. The sequences of the primers used are given in Table 3. 1.3 RESULTS and DISCUSSION 1.3.1 The influence of the modified method of purification on yidd and purity of mycolic acids By applying modificatioas to the previously patented purification procedure (SA Patent Applications No 05/1464 and 96/1412). i.e., by using NaCl as described under 1.2.8 acd 12 9. Larger amounts of the extracted myeclic acids could be pur fied in a single run of couni ercurrent separation, without impairing the degree of their purity. This is illuaiaed by the results summarized in Table 4 is well as in Figures 2a and 2b. for mycotic acids originating from hi. tuberculosis and M. vaccae, respectively. Although a yield of approximately 10% m/m of the purified rnycolic acids was previously reported, it was subsequently established that yields of pure rnycolic acids using either mediod, varied between 3 and 10% m/m depending on the particular batch of bacteria used for extraction. Table 4. Yield and purity of the mycotic adds originating from M. tuberculosis, purified using the improved method (Table Removed) 3.2 Structural analysis of mycolic acids originating from M. tuberculosis, using infra-red spectroscopy In order to evaluate the influence of the process of saponification, freezing and ;torage on the conformation of purified mycolic acids, the infra-red spectra of i number o"' samples were analyzed The infra-rtd spectrum of t..ountercurrent-pjrified mycolic acids, originating *:om M. tuberculosis, prior tc saponificaLoi is presented in Fig. 3. "he spectrum in Fig. 3 provides evidence that mycolic acids after eountercurrent purification exist in the methylester form The absent of a broad absorption baud spanning the 3000-^0 JO cm" frequency range indicates that there an; no free carboxylic acids. The intense narrow band at 2800-2950 err. indicates aliphatic nature of the compound. la addirion, the narrow band of absorption at 1750 cm"1 indicates the presence of an ester form. The infra-red spectrum in Fig. 4 shows the pattern observed after resaponific ation of the countercurrent-punfted rnycoiic acids. The anticipated presence of free carbcxylic acid groups cou.d not be confirmed, which suggests that interior or intra jnoleciilai interaction of the atboxyhc acids with other functional croups took place Figure 4 a bo shows that this inter- or ultra-molecular rearrangement of the carboxyiic acids restricts the degree of aliphatic breaching (a decreased absorption in the 1720-1500 cm" range) in comparison to chat of the methyl ester form Tig 3), indicating a conformationally rigid structure. This would imply a significant structural alteration caused by the process of ^saponification The carboxylic acids icarriuigement over time did not manifest as changes observed in the infra-red spectra. In addition, the influence of freezing at • 70LC ar.ci storage a! 10'C on the configuration of niycolic acids was also "ivestigated The respective infra-red spec ra are presented in Figures 5 and o Prior to freezing at 70°C and storage at 10-C, the samples of niycolic acids were freshly resaponiiled freezing at -70"C appears to loosen up the structure of mycolic acids anc leads o the increase nf aliphatic breathing and "out of plate ' bend ng within the molecules This is probably due to the decrease in the strength of van der Waals' forces caused by the withdrawal of water as ice crystals. 1.3.3 Stability of mycolic acids Stability of mycolic acids was investigated by maintaining equal aiiquots of sjunples of countercurrent-purified mycolic acids in the methylester form as well as saponified mycoiic acids, originating from the same Datch, at -20^0, 4CC and 2i°C for 1? mouths in eiihcr :hc iry form, previaptaed under acetone, or dissolved in chloroform. The results obtained after 12 months are summarized in Fig 7 for mycotic acids stored in the dry state. Similar results were obtained for mycoiic acids stored in the acetone-precipitatea and chloroform-dissolved state, but with higher variance On the basis of the HPLC analysis of the various samples ot mycoiic acids stored at -20°C, 4°C and 25°C, it was concluded that mycoiic acids ia either -nethylester or saponified form were stable for at least 12 month*;. The ipparem gradual increase in absorptivity could be an artifact of calibrating the new internal standard with old samples of mycoiic acids but is not due to a chemical process, which would have manifested itself by different values for different temperatures of storage 1.3.4 Imrrjunoregulatory properties of countercurrent-purified mycoiic acids Investigations of the lmmunoregulatory prot>erties of countercurrent-purified l.iycoiic acics were centred on two main aspects, i.e.. I) their ability to extend the survival of the M. tuberculosis- ii> their cytokine profile in various organs over short and long term, with or without concomitant infection with M. tuberculosis. These investigations were based on the following experiments: i. The investigation r.ito the influence of pre- and post-treatment of the experimental mice with resaponified mycolic acids on their survival after the infection with M. tuberculosis; it) The immunoregulatory effect of mycoiic acids on the expression of imerleukin 4 (IL-4), interleukin 10 (IL-10), interkukin 12 (IL-12), jnte-fcron > (IFN-γ). tumour necrosis factor a (TNF-α) and transforming growth factor B tTGF-3) in the lungs of M tuberculosis' infected and non-iniected experimental animals. 3efore the results obtained »n the course of this mvestigation arc presorted and discussed, a number of technical aspect.-; having a direct influence on the -utcon:tr .'i the expenmeius as well as on the repeatability of various irnxnunoreguiatory experiments is listed ar.d briefly discussed below: ! The nfection with .Vf tuberculosis was introduced not by inhalation but by intravenous injections. Although ihe inhalation is a more natural method of infection, it is ver>' difficult to control and consequently to quan.ify the number of mycobacterial cells introduced. The intravenous injections permitted .. rriore dciiua^ introduction of the intended dose of M tuberculosis 2 Tne introduction of the mvcolic acids-mouse col«c acids originating from M. tuberculosis on cytokine profiles in the lungs Lungs usee in the cytokine determinations were removed from mice five woeks after the infection with Si tuberculosis. 1.3.4.4.1 Pre-treatment with mycolic acids and its effect on IL-12 in the lungs Interleukin 12. was determined in the lungs because: it IL-12 i-i mainly expiessed by macrophages, which are abundart in die lungs: ii) i: is 'icncwn to play a ro e as a pro-inflammatory cytokine in protection agair.st tub;rculosis. i: i) its expression in mice ha.- been found to be enhanced by the inrjoducaon of mycolic acids (see section 1.3.4.1). Three doses of mycolic acids, i.e , 12.5 µg, 25 µg and 50 µg were used in IR IV for pre-treatment. The results presented in Fig. 14 indicate thai mycolic acids enhanced IL-12 expression in the lungs up to an optimum dose (25 µg), after which experession was suppressed. This correlated with the protection that was induced by mycolic acids pre-treatment (Fig. 15) Protection by mycolic acids as well as concomitantly enhanced expression of IL-12 was confirmed in IR V (Table 6) 1.3.4.4.2 Pre-treatmeat with mycolic acids and its effect on IFN-γ in the lungs Interleukin 12 is known to exert some of its irnmunoreguiatory properties through th= stimulation of IFN-7, which then provides protection against tuberculosis infection. In order to determine whether this correlation held true for the protection provided by mycolic acid:;, the degree of expression oflFN--, was determined m the lungs, five weeks after the infection with M. 'Mbercu'osi: The results presented in Fig 16 do not clearly support a model .u which IPN-7 j the cytokine stimulated by IL-12 to exert a protective effect in animals .igainst tuberculosis. This also applies to the measurements of TNF-α (results not shown*. In a repeat experiment TJR V as shown in Fig. 17) the results presented in Fig. 16 could not be confirmed for both IFN-7 and TNF-cr. J: was concluded that the semi-quantitative ?CR is not sufficiently reliable in providing qualitative data on subtle differences between IFN-γ and TFN-o; expressions of mycolic acid.} in treated and untreated mice. However, it is adequate to show qualitatively that IFN-γ and TFN-α are expressed upon uifection wiih M. tuberculosis. The data do not exclude the possibility that L^N'-'y and possibly TNF-α may play effector roles in response to the increased IL-12 cXyic:«Mon induced by mycolic acids. 1.3.4.4.3 Pre-trtatment with lnycalic acids and hs effect an TGF- 0 in the lungs The correlation tha: was found between ihe protective effect of mycclic acids and us influence on IL-12 expression indicated that protection was brought about by a pro-inflaairnatory mechanism- The expression in response to pre--reatmeiit with raycoHc acids of two other pro-inflammatory cytokines, IFN-7 and TNF-Q (results not shown), did not yield satisfactory quantitative results, but ar least did not argue against a pre-inflammatory effect induced by mycolic ac:d>. TGF-jj is an anti-infljinrnatory cytokine expressed in macrophages which might respond to mycolic acids. The results in Fig 18 indicate that the levels of expression of the anti-jiflamrraco-y cytokine TGF-3 were already high in the non-infected control .Bice This is the only one of the four cytokines investigated that gave this result. Ir would not be unusual for an ami-inflammatory cytokine in the lungs to maintain a high level of expression under normal conditions when the inflammatory response couh: do harm to normal, uninfected lung tissue After the infection with M. tuberculosis, the levels of TGF-/S expression were not significantly altered, although a broad spread of measured values was 1-beamed indicating the possibility of unstable mJRNA structure. Introduction ; t homologous serum into the arurnals significantly lowered the expression of TGF-0, while there was a tendency to restore this level by addition of mycolic a:ids adsorbed onto the serum Due to the spread of the measurements for e-ery point of data, a dcfaiKe conclusion sased on a fine resolution between experimental groups of animals could not be made TLese measurements of expression of TGF-3 illustrate the complicating roie that homologous serum played in defining the irnmunoregiiUtory role of mycolic acids. Ignoring the effect of the serum carrier, the data might have suggested a reduced expression of TGF 3 under the infuence of mycolic acids, as fig 19 illustrates. 1 J.4.4.4 Post-infection treatment with mycolic acids and its effect on IL-12, IFN-7 and TGF-ß in the lungs Balb/c mice infected with M. tuberculosis and treated with mycolic acids three weeks after the infection, showed protection against tuberculosis (Fig. 12). This was also observed in C57B1/6 mice 'Fig. 13). The measured values for expression of IL-12 in the lungs of Balb/c mice post-treated wita mycolic acids Fig. 20) do not support a model in which this cytokine mediates the protection, as was found in pre-treatment Although the IFN-7 expression levels correlated positively with IL-12 expression levels (Fig. 2.'.). they did not i.:orrelatr with increased survival. Figure 22 indicates a decreased TGF-fi expression 11 the lungs of Balb/c mice chat were treated with 48 µg of mycolic acids after the infection with M. tuberculosis. This decreased TGF-ß expression correlated with a.i increased survival. These obseivauons indicate' a different model of protection provided by m>colic aid; as a potential therapeutic agent against infection with M. wbercuicsis. i.e., the down-regulation of the anu-mflarrunatory process in the kings. 1.3.4.5 The influence of pre-treatment and post-treatment of the experimental mice with resaponifkd mycotic acids originating from M. tuberculosis on cytokine profiles in the spleens Spleens used in the cytokine determinations were removed from mice five weeks aftex the infection with M. tuberculosis. The Thl response has been previously indicated as the protective mode of Th selli to pre vide protection against tuberculosis Because infection with M. -uberculosis was, in our experiments, routed systemically through the toil vein, 'he spleen was regarded as a prime lymphoid organ from which the Thl/Th2 bias could te determined, using IFN-> as the indicator for a Thl and IL-4 as ihe indicator for a Th2 response. These cytokines would have acted antagonistically, if the spleen became biased in one of the two modes upon immune challenge by M. tuberculosis. However, from me results presented 11 Fig. 23 it is evident thai: the infection with M. tuberculosis induced the expression of both cytokines and neitfcer the expression of EFN-7 nor of IL-4 was significantly aliered by the pre-treatment or post-treatment with mycolic a:idi. These results suggest that: ii the spleen aoes not appear to be the organ where the role of mycolic acids as a protector against tuberculosis is decided; ii" me T cells do not appear to play the decisive role in providing protection against tuberculosis upon the administration of mycolic acids. 1.3.4.6 The influence of pre-treatment and post-treatment of the experimental mi a with resaponified mycoiic acids originating from M. vaccae after the infection with M. tuberculosis The immunorcgulaiury eflects of myco.ic acids were found to be present irrespective of the source species of Mycobacterium from which they were obtained. Mycoiic acids extracted from M. vaccae, after the countercurrent purification, stimulated the expression of mterleukin 12 in Baib/c mice (Fig. 24 > and induced protection against tuberculosis in the lungs of the experimental .uiimals (Table 6). These results confirm the effects observed in the mice pre-and post-ire ited with my cole acids originating from M. tuberculosis, described in sections .' 3 4.3 and 1.3.4.5. 1 1.5 ASSESSMENT OF TIIE SIGNIFICANCE Ol THE RESULTS The results obtained in the above experiments indicate that mycohc acids can protect mince againit infection with M tuberculosis, particularly when aiministered before the infection/onset of the disease. The kidneys, liver and iungs in ou:e were found to respond to mycoiic acids even without the itifection with M tuberculoses 'Fig. i). It should be stressed that the lungs from the mycoiic acids-pre-rxeated aiaimals were the only organ m which a significantly reduced tubercle formation could be. observed upun macroscopic post-mortem assessment (Table 6) and were also the nly organ that responded differently to the infection with M. tuberculosis in tcspect of 1L-12 expression. It was, therefore concluded, that die protection provided by mycoiic acids manifested itself mainly in die lungs. Prelinrintry evidence from IR V indicated that mycoiic acids from other species of Mycobacterium (e.g. M vaccae) could also induce immune effects in the luugs of mice infected with tubeiculosis (Tig. 14) The cytokine profiling of the lungs and spleen five weeks after the infection with M. tuberculosis, with or without pre- or post-treatment with mycotic acids, confirmed the observation that the lungs were the responsive organs to the antigenic challenge with mycolic acids. AlUiough cytokine expression in the spleen was induced upon the infection with M. tuberculosis, it was not significantly altered by the pre- or post-treatment with mycolic acids. No bias towards either Thl cr Th2 mode of immune response was evident from the measurements of the IFN-γ and IL-4 expression levels in mice infected with tuberculosis, treated or untreated with mycolic acids (Fig. 23). The results of IL-12. IFN-> and TGF-6 measurements in the lungs of the c xperimental animals support the view that a pro-inflammatory mechanism of protection against tuberculosis was elicited by pre-treatment with mycolic acids. Inter, eukin 12 expression correlated positively with the survival of the M. tuberculosis-infectsd mice, while IFN-γ (although induced) and TNF-α, c;>uld not be determined with sufficient accuracy to clearly demonstrate its role ui the protection. The TGF-8 levels in the organs of the experimental animals remained largely unaltered. During the post-infection treatment with mycolic acids, the levels of expression of IL-12 positively correlated with those of IFN-->. No correlation with the pi erection against Luueicuiotis by mycolic acids was observed. Rather, a down-regulat on of TGF-fl appeared to be a possible mechanism of protective inflammation in the lungs. This mechanism might explain the lower efficiency of the post-infection therapy with mycolic acids. Overall, die mechanistic model of mycolic acids-induced protection against tuberculoses, supported by the experimental evidence provided above, allows tor *iie prediction if a much enhanced protection offered by mycolic acids dguinst the disease, when the infection with M. tuberculosis occurs by (he normal route, i.e., by inhalation into the lungs. It appears possible that the administration of mycolic acids directly into the lungs of tuberculosis-infected individuals via inhaled vapours generated by a nebuiiser could greatly reduce the incidence of infection with M. tuberculosis and also aid towards recovery from the disease. T"he above findings can be extrapolated .o human beings, and it can be expected that the response in humans towards the treatment with mycolic acids ..iiouid be better. This assumption is based on die fact that humans possess mycolic acids presenting molecules, CD lb, expressed on their antigen presenting c?lls, die homologue of which has not yet been identified in mice. Positive evidence was found for short erm prevention or therapy of tuberculosis by mycolic icics in rats ind mice The results obtained would s jggest that Th cells did not participate in die immune response upon challenge with mycolic. acids either before or alter the infection with M. tuberculosis. however, as humans possess CD lb molecules mycolic acids may have a role a human vaccines, i e , for using them to provide long term immune memory against un/ecuon with M. tuberculosis. Tie experimental evidenr,- supports a role of sella of uuiaLc immunity, such as macrophage!; and nsri- killer cells, in responding towards mycolic actds administration in a manner that decreases the pathogenic effects induced in the lu lgs by M. tuberculosis. EXAMPLE 2: Protection against tul>erculosi*-»nduced arthritis in rats 2 MATERIALS 2.1 Culture Mycobacterium tuberculosis R^Rv ATCC 27294 - a violent siraio, originally isolated from an mfected human lung, was used in the experiments. The culture was purchased in lyophilized form from the American Type Culture Collection (ATCC), Maryland, USA. M tuberculosis H37Ra ATCC 27194 - an a virulent strain, was purchased in ivophilized form frorr.. Difco (Cat No: 3114-33-8). 2.1.2 Media 2.1.2 1 Growth media The following media were used for the cultivation of M. twertuiosis Lowenstein-Jensen (IS) medium (slants) and Middlebrcok 71110 *%tu opiates >. A detailed compositjon of the ingredients necessary for the preparation of diese media as well as the conditions recommended for their sterilization, are given in the Laboratory Manual of Tuberculosis Methods. Tuberculosis Research Institute of the SA Medical Research Council (1980, Chapter 6, pp 83-105; Second Edition, revised by E E Ncl, H H Kleeberg and E M S Gatncr). The media were prepared by the National Tuberculosis Institute of the Medical Research Council of South Africa, in Pretoria. 2.1-2.2 Media used for washing and diluting of Mycobacteria The harvested bacteria were washed in sterile 0,9% m/v NaCl (Saarchcm, Chemically Pure, RSA). Medium used for the preparation of serial dilutions preceding the determination of viable counts of M. tuberculosis was prepared by dissolving Tween 80 (Merck, Chemically Pure) in0,9% m,v NaCl (Saarchem. Chemically Pure.) to a concentration of 0,01% v/v and distributing it in 9,0 oil aliquocs into test-tubes. The autoclaved media were stored at *QC 2.1.3 Reagents 2.1.3.1 For the preparation n-' the reagents usee', for ihe CAuactiun, aenvatizanon and High-Performance Liquid Chromatography (HPLC) analysis of mycolic acids, HPLC Grade methanol (BDH) and double-distilled deionized water were used Reagent A: 25% potassiton hydroxide (Saarchem, Analytical Grade) dissolved in methaaol-v/ater (1:1), i.e., 62,5 g potassium hydroxide was dissolved in 12*) mj waicr and 125 ml methanol (BDH, HPLC Grade) was added. Reagent B:Concentrated hydrochloric acid (Saarchem Analytical Grade) diluted 1:1 with water Reagent C:l% potassium bicarbonate (BDH, Analytical Grade) dissolved in methane l-water (1:1), 10 g potassium bicarbonate was dissolved in 250 nil water and 250 nil methanol was added. Reagent D:pa/a-bromophenacyibromide dissolved in acetoninile and crown ether Pierce Chemical Co. Cat. No 48891) was dispensed in 500 ul cuantities into small amber-colouied screw cap vials with Tetlon-coated septa. The caps were tightened and the Mils -vere wrapped u tini; from A/ tuberculosis H37Rv, at a final concentration of 0,067 fig/jil. To prepare the coating untigen, 1 mg mycolic acids was dissolved in 100 ji\ chloroform and the solution introduced into 15,0 ml PBS buffer adjusted to pH 7.4. The solution was autoclaved at 121'C lor one hour. Conjugates: Goat inti-rat antibody conjugated to peroxidase (H J- L chains), Cappel (Cat No 55770V Rabbit anti-human gamma chain specific peroxidase conjugate (Sigma; A 8419). Substrate: O-Phenylenediamine (Sigma; Cat No P-1526) and •rJ:cgeri perox:de BDH). Substrate buffer C, 1 \1 citrate btfler (0,1 M citric acids and C, 1 M Tri sodium citra<:e), adjusted to pH 4,5. 2.1.4 Experimental animals Si* weeks old, female Lewis rats were purchased from Shaw's farm. Blackthorn. Bicester, Oxen, England. This strain of rats is susceptible to the induction of arthrius. The animals were maintained at the Animal Facilities of the Medical Research Council in Pretoria. Feed and water Mice cubes, manufactured by Epol, South Africa and tap, amoclavcd water were provided ad libitum Sanitation: Bronocide - manufactured by Essential Medicines (Pty) Ltd, was used for sanitation purposes. 2.1.5 Plasticware The following plasticware was used: Disposable Petri's dishes (Proraex. RSA) ELISA plates (Steriiin, UK; Sterile, disposable 50 ml centrifuge tubes (Corning, ISA) Disposable tips (Elkay. Denmark) 96-well round bottom mic"opiates (Nunc, Denmark) 2..1 METHODS The following methods were used in the experimental work: 2.2.1 Cultivation of the bacterial strains The mycobacteria for the production of mycolic acids, i.e., M. tuberculosis H37Rv, were cultivated at 37°C using Lowenstein-Jensen iLT) medium «!?.nt^ and Middlebrook 7H-10 agar medium places. The >teniu> of .ill oe media was co:ifirmed. before they were used ,n die experiments by incubating them it 37°C for 24 h. For routine extraction of mycolic acids, approximately 4-week old cultures of M. tuberculosis grown on O slants or 2-week old cultures of M. tuberculosis grown 3n Middlebrook 7H-10 agar medium plates were used. 2.2.2 Viable and total bacterial counts For the viable count determination, serial dilutions of the harvested bacteria w;re suspended m the diluent medium (prepared as specified under 2 1 2.2) to a density corresponding to a McFarland standard 4 t approximately OD oi 1,0. using a Beckraan DU 65 spectrophotometer, at 486 ran,. Tenfold sena, dilutions were prepared using 9 ml aliquots of the diluent medium. From the last three dilutions corresponding to 10 . .0^ and 10s of the original suspension, aliquots of 0,1 ml (100 f.d) were withdrawn and spread over die surface of Middlebrook 7H-10 plates. The plates were incubated at 37°C and the developed colonies counted af:er two to three weeks for M. tuberculosis and after one week for the plates seeded with M. mccae. The direc: total count v,as performed using a Neubauer counting chamber and the autoclave d cultures of hi. tuberculosis and M. vaccae, originally adjusted to a density corresponding to a McFarland standard 4 and suitably diluted with the diluent medium. Statistical ;inalysis of the bacterial counts ncludrd the mean values of bacterial counts and standard deviations 2.2.3 Preparation of mycotic acids from bacterial samples The preparation of bacterial .samples comprised three steps harvesting of the Mycobacterial cells; saponification and extraction of mycolic acids. Glassware used for the harvesting, extraction, derivatization aid HPLC analyse? of mycolic acido was washed in 2% (v.'v) Centred (Merck). raised in water, followed by rinsing m chloroform, water, technical Grade metiancl, water ami finally rinsed JI double distilled de ionized water. Th; washed glassware was dried in a warm air oven. Harvesting was done by M.raping the bacterial growth from the surface of media slants or agar medium plates (using sterile plastic loops) and by suspending them in Reagent A. Initial bacterial suspensions were prepared in Reageni A. b/ vortexmg the harvested cells with sterile glass beads. Homogenous bacterial suspensions were prepared using sterile :issue homogenizes. Prior to saponification, the density of the bacterial suspensions was adjusted to a density corresponding to a McFarland standard 4. This density Df bacteria corresponds to approximai-jsly 10-12 x i08 colony forming units/ml. The saponification, extraction and derivatkanon of mycolic acids were carried out as described by Butler, Jest and Kilburn (1991), with minor -nodif car'.< VJ? and ar. described under the -eievant heading. Saponification of the Myc >bucteria n Reagent A was carried out in an autoclave al 121CC, for 30 min. 2.2.4 Extraction of mycolic acids The saponified samples were allowed to cool after autoclavinir Into 2 ml samples containing crude extract, 1,5 ml Reagent B was introduced. After vortexing, the pH of each sample was checked and if necessary, adjusted to pH 1 with Reagent B. Subsequently. 2.0 ml ch'oroform was added to each sample and vonexed for 30 seconds. The layers were allowed to separate. The bottom layers were removed with Pasteur pipettes, transferred to amber WISP vials and evaporated to dryness at 8>5°C in a heat bloc<-.vaporator under a stream of nitrogen. To neutralize traces of acid carried over, 100 /il of reagent C was added to each sample and the fluid evaporated to dryness at 85°C in the heat block-evaporator under a stream o:~ nitrogen. 2.2.5 Storage of the crude extracted mycolic adds The material obtained from the large-scale extraction of mycolic actds originating from M. tuberculosis H37Rv. the crude bacterial extracts, was stored under acetone, at 4"C in 4 ml amber WISP vials. To prevent evaporation/drying and the exposure to light, the caps of the WISP vials were covered with Parafilri. 2.2.6 Determination of mycolic acids contents in crude extracts Extracted mycolic acids were derivatized as follows: To a cookd sample of crv.de extract (approximately 10 fig in 2,0 ml Reagent A), an aliquot of 1.0 ml chloroform was introduced, followed oy the addition of 100 ul of Reagent D (derivanzation reagent). The capped samples were vortcxed for 30 seconds and heated for 20 minutes at 85°C in a heat block-evaporator. Subsequently, the samples were cooled and 1.0 ml of Reagent E added. The samples were vortexed for 30 seconds and the layers allowed to separate. The bottom layers were removed with Pasteur pipettes and transferred to WTSP-vials. The vials .".ere placed in the neat block-evaporator and their contents evaporated :o dryness at 85"C using a stream of nitrogen. The residues were resuspended in 0.212 g (which corresponds to 160 ,al) methylene chloride, capped and vortexed. Each reconstituted sample was introduced into a WISP vial containing 5 p.g of the HPLC internal standard (prepared as described under 2.1.3-1), filtered through a 0,22 (.mi ivlmcA. VJ * T IULCI v.viuj a puiytuayicuG uuUsfflg uJtO anotfitsi amuci- coloured WISP-vial. The recapped vials were stored at 4°C until ready for HPLC analysis. 2.2.7 HPLC analysis and quantification of mycolic acids Repeatability and accuracy of the pipette used for the diitfd^Mfitm of the HPLC standard was determined. The precision was cstabtHdfJltf to be +/- l°o and it was cenilrrnei prior tc earh aliquoting of the internal standard. for the M'LC analysis I1.) ul from each sample (maintaintd on ice d'^ring handling), wa;> anai:7.ed. Control samples, i.e. 10 ^il of filtered methylene chloride, were ran prior to each set of samples analyzed. If a large number 01 samples was .analyzed, in ordei to -validate die reliability of the HPLC apparatus, control samples were run after every 'Jiree or fo u test samples The reverse-phase HPLC analyses v^re earned out using a Waters 600 E System Controlkr High Performance Liquid Chromatography apparatus consisting of. \1;CIOM-C M741 Data Mod.ik; Waters 711 WISP Autosanipler; Detector (Waters 486 Tunable Absorbance Detector); Column. Nova-Pak CIS 4 jam 3.9 x 150 mm and an end connector set for steel cartridge columns. RKC Rex-C 4 Column Temperature regulator. Running conditions were: Mobile phase: Solvent A HPLC G'adc methanol Solvent B- HP( C Grade methylene -hJ.inde Flov; Rate '2.5 ml -ivn Cokmn temperature 30"C The detector was sei at 260 am. Prior :c> use. the solvents were sparged wiJi Instrument Grade heliuia. High Purity Nitrogen was used to contra hydraulics of the WISP vtals autosampler The HPLC gradient initially comprised 98% (v/v) methanol -'Solvent A) inu 2% r.-'x) methylene chloride (Solvent B). The gracient w.is increased 1 nearly to 80% A and 20% B at one minute: 35% A and 65% B at ten minutes, held for 30 seconds and then decreased over 10 seconds back to 98% A and 2% 6. This ratio was maintained for 4 minutes to allow for stabilization of the system prior to injection of the next sample. Mathematical quantification of mycotic acids was carried out by comparing the combined peak areas of the tested samples to the peak area of the introduced quantity of the High Molecular Weight Internal HPLC Standard 2.2.8 Preliminary [ urification of crude mycobacterial extracts in --,rder u> shorten the t:nv required lor lh." countercurrent purificatkin of the cmd? m>cobacterial extracts, an additional preliminary extraction step %vs> ir^roduced This step had a dual purpose to remove unnecessary cellular components from me crude extract prioi to the countercurrent purification and .11 u- reducu soap friction in the crude bacterial extracts -\ poni.'n of the crude extracted material (approximate!) 3 to 4 g) was suspenceu n a rrunknum volume of the lower phase solvent (usually 100 nil t, transit r:ed into a separ ation funnel arid mixed with an equal volume ox the upper phase solvent The phases were allowed to separate and the apper phas; was removed ind stored at 4°C. Into the remaining lower phase an equal volume of the upper phase solvent was agan introduced and the process of the phase separation was repeated. The second upper phase was removed and stored at 4aC and the second lower phas* was dried in a Buchi Rctoevaporator RJE 120, at 75°C ar.d its mass recorded. 2.2.9 Countercurrent purification of mycotic acids originating from M. tuberculosis Countercurrent apparatus A countercurrent apparatus produced by H O POST, Instrument Conunriv Inc.. Middle Village, New York was used during thc invfitigaucins. The "trams" in this mode! consisted of 2 X 250 mter- Solvent vyitem us«d in the countcrcurrent apparatus The solvent system used UT the eountercuirert reparation consisted of: 42°o v/v chloroform f'Saarchem. C lemically Pure Reagent) ?"->% \ v methanol (S^archem, Chemically Pure) N% w 0.2 M NaC! (Saarchein, Chemically Pure) Double distilled de.omzed \wter w^ U^'LI for die preparation of the solvent bvS'em. The components were rm^ed, equilibrated and the upper aad lower phases wero collected using a separation funnel The composition of the upper phase was established to be: 15% VA chloroform. 52% v/v methanol and 33% v/v 0,2 M Man The composition of the lower phase was established to be: 68% v/v chloroform, 27% v/v methanol and 5% v/v 0.2 M NaCl. The countercurrent purification process was carried out under the following conditions- A countercurrent diitributi< n tra.u composing 55 rubes, numbered 0-54, wa;, itstd ui the exp;rirncruv The upper phase solvent, a volurce of 600: -f mycolic acids after the preliminary purification was dissolved in 50 r.il of the lower phase solvent, jivided into tUv dhquots and introduced in:o first five tubes, numbered ''-4. Subsequently. 10 mi ot the upper pha;e -ohem was introduced int. each of the "";r:>t five countercurrent rubes. Into the remaining 50 tube.s aliquots of 10 ml of the lower phase were .ntioduced. Upper phase, in volume:, of 10 ml per cycle, was automarici ly dispensed iir.o tube number 0, repeatedly over 55 cycles "esuitina in approximate y 5 hoir operation. Thus, fifty five counter current cycles were performer with each cycle consisting of 10 mixing perdula and 3 minutes phase separation time. Initial load of crude e*trict after the funnel extraction 125 mg Number of cycles: S5 Equilibration time: 3 mill 2.2.11 Removal of malachite green from the counter! urrent- purified mycotic acids To remove traces of malachite green derived from bacterial growth media (when M. tuberculosis was grown on LJ slants), the countercunent-purified material was selectively precipitated in trie following manner. Countercurrent-purified mycolic acids (92 mg) were placed in a WISP vial into which 1,0 ml chloroform was introduced. The dissolved mycolic acids were transferred into a pre-weighed round- bottom fla.sk. The vial wa:, rinsed twice wr.li 1,0 ml chloroform and the two aiiquots oi chicroform were added K that already present in the rouMd-bor.r.m rlask Subsequently, acetime was introduced crop-wise in SOU u. aiiquots. In totai. 2!) ml of acetone -vai introduced and the white (lakes of the precipiiated-out mycolic aciis were washed twice with 20 ml acetone The acetone supernatant, with the dissolved malachite green was removed and the myci'hc at,ids dncc b) evaporation. The procecure was carried out at room temperature. 2.2.12 Determination of mycolic ncids after countercurrent purification In order to increase the accuracy of the HPLC determination o:r mycolic acids. ±c High Molecular Weight Internal Standard ('CM 00) was ntrodueec into the couniercurrent-purif.ed mycolic acids before the ::.apomf:C3t'on A sample of 0,5 mg of the countercurrent-purified mycolic acids was introduced into a WISP vial containing 5 p.g of the High Molecular Weight Internal Standard (C-100). Saponification of mycolic acids was carried out with 2 ml of Reagent A at room temperature. The WISP vial was vortexed for 30 seconds. The extraction was carried out with 1,5 nil of Reagent B. After vortexmg, the pH of the sample was checked and if necessaiy, adjusted to pH 1 with Reagent B. Subsequently, 2,0 ml chloroform was added to each sample and vortexed for 30 seconds. The layers were allowed to separate. The bottom layers were removed with Pasteur pipettes, transferred to amber WISP vials and ivaporined to dry lie:., at 8' C 1:1 a heat block-evaporator uiidci a sucam of nitrogen. To naiuahite races of :.cid earned over 100 ^l of reagent ( was added and and pleasend the operated to dr.driness .at 85'( in a heat unlock evap-or for under a stream at muogeu. 2.2.13 Dt-terraioation of yield of the councercurreot separation In order to calculate he aprroximate yield of 'purifieationseparation. the amount of the mytoiic acids presen: in the samples obtained after the countercunem separatioa'p urificatior was compared to the amount of These coarjnundb present ir: thu crude cellar extract introduced into tlie countereiinent apparatus. The calculations were based on the results obtained by the HPLC analysis :t should be stressed that is essential for the calculation of the yled of the cellure under separation, that the riycolic acids determined t»y HPLC shciJd be within the tested linear ransie of the HPLC UV detector. 2.2.14 Methods used in monitoring levels of anti-mycolic acids antibodies Bleeding: the animals were bled from the sublingual vein 12 days after the induction of arthnhs. The blood was collected into sterile centrifuge tubes and allowed to clot for 16 hours at 4CC. The collected serum was Xcentrifuged at 700-750 g for 20 minutes, difuted 1:1 v/v in glycerol and stored at -20"C. ELISA protocol: Coating of ELISA plates: The autoclavcd coating antigen (in PBS buffer. pH 7 4) still hot, was introduced into ELISA wells in aliquots of 50 u,l/well, with ihe solution being continuously stirred. Approximately 3 ug mycotic acids per well were introduced. The coated ELISA plates A'ere incubated at room temperature for 16 hours. Subsequently the antigen solution was removed, the ELISA plates dried and the dry plates were stored at 4"C Blocking cf ELISA plates.: The blocking buffer (0,5% i.m/v) casein in PBS pH 7,4) was introduced in aliquotes of 200 pJ/well. The ELISA plates uere incubated at room temperature for 2 hours. Binding of animal antibodies. Rat sera (mixed with glycerol 1:1 v/v) were diluted further in the diluting buffer 1:10 v/v. The final dilution was therefore 1:20 v/v. Aliquotes of 50 \xl were introduced into wells n duplicate The plates were incubated at room temperature for one hour. The sera were removed and the plates washed three times with the washing buffer using an Anthos Automatic Washer. Quantification of the bound antibodies: Peroxidase anti-rat antibody conjugate ciluted 1:1000 was introduced in aliquotes of 50 µl per well and incubaled at room temperature for 30 minutes. After the removal of the conjugate, the ELiSA plates were washed three times with the washing buffer. The substrate solution comprising 10.0 flag O-phenylenediamme and 8.0 my hydrogen peroxide in 10 mi of 0,1 M citrate buffer pH 4,5, was prepared immediately before use anc introduced in 50 u.1 aliquotes per well The Slates were placed in a dark glace and the colour development was monitored at 15. 30 and 60 minutes intervals using a SLT 340 ATC photometer at a wavelength of 450 run. 2.2.15 Methods used in handling experimental animals in the adjuvant arthritis experiments 2.2.15.1 Environmental conditions under which the experimental animal were maintained Experimental animals: Six weeks clc, female Lewis rats were accommodated in cages with a floor area of 864 cm" and a height of 12.5 cm Four rats were maintained in each cage, except for th2 animals uf group b which were maintained tiree psr cage. The animals were maintained at the Anmral Facilities of the Medical Research Council in Pretoria. Environmental conditions: Temperature and humidity in the animal facility were set at 20°C <+/- 1UC) and 40% (+/- 10%), respectively. Lighting was provided by means of fluorescent tubes. A light-darkness cycle of alternating 12 hour periods was set up. Rats were fed en nutritionally controlled pellets manufactured by Epcl, South Africa-Cages; Rats were housed in transparent polyprupylene cagus with tightly fining stainless stee; lids 'Wooden shavings after autoclaving, were provided a; nestling mater al Sanitation: Aruma: rooms rat cages arid glass bonks were cleaned and decontaminated once a week using Bronocide. Water bottles aft:r washing were autoclaved once a week Identification of the experimental animals: Individual identification was accomplished h makiig ear marks 2.2.15.2 Preparation of the reagent used for the induction of adjuvant arteritis The method used for the induction of adjuvant arthritis was based on the method described -/ Wan-'IT Wager.ur Hilber and Van Eden (1994). In order to obtain a. coarse surface, the xttom of a mortar bowl w;is ground witi coarse grinding paper (100 grain). After the dust particles were removed, freeze-driei cells of M tuberculosis H37 Ra, 100 mg, were transferred into the howl. After die introduction of 3 drops of Freund's Incomplete Adjuvant (FIA) the bacteria were mixed very well with FIA for 2 minutes using a pestle Once a thick paste was abtained, a few additional drops of FIA were introduced into the mortar and mixed with continuous grinding for a further half a minute. The thick paste was transferred into a 50 m. test tube using a glass Pasteur p\p«tte. The mortal bowl was "lius-cd' icvcrai Limes with the remaining FIA using a few drops it a time, until the entire volume of 10 ml was used. The final suspension of the freeze-dried cells of M. tuberculosis H37 Ra in FIA contained 100 mg of cells per 10 ml FIA. 2.2.15.3 Preparation \>f the reagent used for the prevention of adjuvant .I'll ruts An accurately weighed-off sample {lu mgt of freshly saponified mycohc acids, originating from SI tubercuio-'s H37 Rv. wat introduced into 1 nil of FIA in a glass viai and heated on a ruat-block evaporator at 80aC until cempieteb dissolved. After vcrtexing. the dissolve sample was removed from the heat block-evaporator and left ai room temperature :o cool down From this stock solution Lie required concentrations of mycolic ac ds were prepared b> introducinc additional aiiquotes of FLA. 2.2.15.4 Experimental set-up The experimental set-up s presented in Table 7. A day before the start of the -ixpenment, the rats were weighed and trie thickness of the joints of their front and hind limbs was measured with a micrometer. Individual identification of rats was done by making ear marks. Heat-killed and freeze-dried cells of M tuberculosis H37Ra, 100 mg, were suspended in 10 ml of FIA by emulsifying the bacterial cells m FIA using a mortar and pestle. (For details, sec section 2.2.15 2.). The administration of killed ce.ls of M tuberculosis H37Ra suspended in XFIA FIA a one and various doese of myclic acids in FIA us using aliquots of 100 µl. was carried out in the form of ntradermal injections at the base of the rats' tail. The rats were divided into groups and reared as illustrated in Table The raats were laen ald for the or appearance of the symptioms .arthritis such as Sollen limbs necrosis of the tail and nose bleeding. Two week.; into the perment, the diameter of the joints in the front and hind limbs was measured ever- second day Rats' MAss was likewise monitored t-every second day. To determine the level of unti-mycoli: acids antibodies in the sera of the experimental animal, the rats were bled from the sublinguil v-m m the ;ongue on .he 12-th day after the induction of arthritis. Table 7. Experiment^ set-up for the induction of adjuvant arthritis (Table Removed) 2 2.15.5 Methods used in the radiological assessment of arteritis Radiographs of the cadaver limbs vngiiuting from the control, amrui.s and mycotic acids-treated rats were made using a Siemens Pciyinat 50 c lagnostic X-ray machine. Fuji HP.F film and Tm rax T2 detail screens were used at a source to image distance to 09 exposure tactor were 42 KVp and 4mAs to optimise soft tissue visibility and bony detail. Radiological examinations were carried out by Prof. R M Kirbcrger the Section of Pathology of the Veterinary Research Institute, Onderstepoon, Pretoria 2.3 RESULTS and DISCUSSION The results obtained concerning: i) the influence of the modified method of purification on yield ar d purity of mycolic acids. ii> the structural analysis of mycotic acids originating from M. tuberculosis using infra-red spectroscopy; and in) the liability of mycolic acids were presented and discussed in sections 1 3.1. 1 3.2 and 1.3 3 2.3.1 Monitoring of the symptoms of adjuvant arthritis Successful nduction of adjuvant arthritis (experimental details given 2.2.15 2, 2.2.15.3 and 2.2.15.4) was first observed four days after the administration of an artruitis-tnducmg dose of the suspension of M. tuberculosis H37Ra freeze dried cells inFreuiid's Incomplete Adjuvant (FLA.). The first symptoms were these of a necrosis developing a, the site of the injection. Other symptoms were observed at about 11 days after the administration of the cells of M. tuberculous H37Ra in FIA and included swelling of the knuckles and joints as well as nose bleeding (Fig. 25a, ?.5b and Fig. 26b). These symptoms peaked alter approximately 16 to 21 days and subsequently subsided, except for the necrosis. Complete recover)' was observed within the next two weeks. The rats which were prc-treated with 0,1 mg and 0,3 mg mycotic acids developed less severe symptoms than most treated with FlA alone. Three rau pre-treated with I rng MA did not develop any symptoms indicative of the presence of the disease. The fourth rat in the same group (rat Dumber 3 in Fig. 26c j did not receive :he full dose of raycolic acids during pre-trcatment, as part ci the dose leaked out after tie injection. This rat showed only moderate .symptoms of arthritis in the hind limbs. The rats which received injections of FIA only (Fig. 26a), did not show any symptoms No toxic effects were observed among the control rats which were treated w;.th mycolic acids suspended in FlA but without challenge with an arthritis-inducing dose of M. tuberculosis H37Ra in FlA. The result;; obtained in the experiment are summarized in Tables 8a and 8b and illustrated by photographs presented in Figures 25 to 27. Figure 25a and 26b show the typical swelling of the joints and knuckles in the front paws of the experimental rats. Figure 25a illustrates the bleeding from the nose caused by thrombocytopenia induced by the preaence of a high concentration of immune complexes in the blood of the experice als. 1 '> 1 Table 8a Results obtained in rat^ irt ited with raycolic acitb prior to the induction i»r adjuvant Arthntb Table tfb Results obtained in rats en .tted with mycotic acids admiaistu-«d alter the induction of adjutant arthritis (Table Removed) gure 25b ,is wed Fig 26b and 2 show companion between the rat pre treated with lmg MA in FIA arnl the arthritic rat. The pronounced swelling and inflammtion of hind leg oints of the rat in which adjuvant arthritis was successful!} induced can be clearly distinguished from liiose rats that were protectee with mycohc acids pi e-treatmen* (Fig. 25b and 26c). Figure 25c shows the typical deformation of the joints in the hind legs, known as the "swimming position" The emacta.tion caused by adjuvant arthritis is svAcL' from Figure 2". The results obtained in ihe experiment indicate a protective influence of mycolic acids at a dose of 1 mg administered before priming with M, tuberculous H37Ra m FLv EXAMPLE 3; Immunoyenic properties of couutercurrent-purified mycotic acids 3.1 MATERIALS 3.1.1 Culture Mycobacterium tuberculosis H37Rv ATCC 27294 - a virulent strain, originally isolated from an infected huniaa lung Type strain of the species. The culture was purchased in lyophiliztd form from the American Type Culture Collection (ATCC). Maryland, USA 3.1.2 Media 3.1.2.1 Growth media The following media were used for the cultivation of M. tuberculosis. Lowenstein-Jensen ilj) medium (skjitf) and Middlebrook 7H-10 agar medium (plates). A detailed composition of the ingredients necessary for the preparation of these media as well as the conditions recommended for their sterilization, are given in the Laboratory Manual cf Tjberculosis Methods, Tuberculosis Research Institute of the SA Medical Research Council (1980, Chapter 6, pp 83-105; Second Edition, revised by E E Nel, H H Kleebeii and E M S Gainer). The media were prepared by the National Tuberculosis Instituie of the Medical Research foursc ! -if South Af'a.a. in Preiona 3.1.2.2 Media used for washing aod diluting of Mycobacteria The harvested harrena usra washed in sterile 0,9% ui/v NaCl (Saarchem, Chemically Pure, RSA). Medium used for the preparation of serial dilutions, preceding the determina:ion of viable counts of M. tuberculosis, was prepared by dissolving Tween 80 (Merck, Chemically Pure; in 0,9% m/v Ns.Cl (Saarchem, Chemically Pure) to a concentration of 0,01% v/v and distributing it in 9,0 ml ahquots into test-tubev. The auioclaved media were stored at 4°C. 3.1.3 Reagents 3.1.3.1 For the preparation of the reagents used for the extraction, denvatization and High-Performance Liquid Chromatography (HPLC) analysis of mycolic acids, HPLC Grade methanol (BDH) and double-distilled deionized waier were used Reagent A: 25% potassium hydroxide (Saarchem, Analytical Grade) dissolved in methanol-water (1:1), i.e., 62,5 g potassium hydroxide was dissolved in 125 ml water and 125 ml methanol (BDH, KFLC Grade) was added. Reagent B: Concentrated hydrochloric acid (Saarchem, Analytical Grade) diluted 1:1 with water. Reagent C: 2% potassium bicarbonate (BDH. Analytical Grade) dissolved in methanol-water (1:1), 10 g potassium bicarbonate was dissolved in 250 ml water and 250 ml methanol was added. Reagent D: para-bromophenacylbromide dissolved in acetonitrile and crown Mher (Pierse Chemical Co, Cat. Nu 48891) was dispensed in 500 pel quantities into small amber-coloured screw cap vials with Teflon-coated septa. The caps were tightened ard the vials were wrapped witi Parafilm. Reagent D was stored at 4°C. Reagent K: Reagent E was prepared by mixing reagent B 1:1 with methanol. HPLC Standard: High Molecular Weight Internal Standard (O100) from Ribi IrnmunoChem Research Company, Cat No R-50. The standard, 1 mg. was dissolved in 20 ml chloroform (BDH. HPLC Giade) at 43C and aliquots of 100 µl were dispensed into 4 ml amber WISP vials, dried, capped with Teflon-coated septa and stored at 4CC. Chloroform (Saarchem, Analytical Grade, RSA) Methylene chloride \BDH, UK. HPLC-Grade) Reagents A, B, C and E were prepared fresh prior to experiments, talcing all the necessary safety precautions. 5.1.3.2 The foDowing reagents were used for the preliminary purification of crude bacterial extracts ("funnel extraction") and for the couniercurrent purification of the extracted mycolic acids: Chloroform (Saarchem. Chemically Pure; Methanol (Saarchem. Chemically Pure) Acetone tSaarchem. Chemically Pure.) Sodium c'lkride (Saan.hem Analytical Gr.-iJci Double-distUed deioruzed *v,irer was used for the preparation of the required reagent concentrations, i.c ,; 39?; v v methanol 42% v. v chloroform 0.2 M NaCl i 1.3.3 Reagents used m the induction of anti-mycolic acids antibodies Unsaponified mycolic acids originating from M. tuberculosis; Marcol 52 immunization oil manufactured by Esso, RSA. 3.1 3.4 Reagents used in monitoring die production of anri-mycolic a: ;ds antibodies. Glycerol (Merck. Analy:ical Grade) was used for diluting sera of the experiment animals. ELISA reagents: Bask buffer - PBS buffer: 8,0 g NaCl, 0.2 g KC1, 0,2 g KHaP04 and 1.05 g Na2HPO4 per 1/ distilled water, adjusted to pH 7,4. Diluting buffer: 0,5% (rn/v) casein in PBS butter adjusted to pH 7,4 was used for diluting of the experimental animals' sera (mixed with giyccroi i.i) and for trie preparation ut suitable dilutions of immunorcagents Blocking buffer" 0,5^ irn.:v* casein in PBS biffer adjusted to pH "7.4 was used for blocking of ELISA plates Washing buffer: 0,5% (m casein in PBS bur er adjusted to pH n A was used for washing of ELISA plates Coating antigen: unsaporufied m>colic acids originating from M tuberculosis, at a final concentration of 0 067 µg/ µl. To prepare: the coating antigen, 1 mg mycolic acids was dissolved in 100 µl chloroform and the solution introduced into 15,0 ml PBS buffer adjusted to pH 7,4. The solution was autoclaved at 12TC for one hour Conjugates: Goat anti-rat antibody conjugated to peroxidase (H -*- L chains). Cappel (Cat No 55770). Rabbit anti human gamma chain specific peroxidase conjugate (Sigma: A 8419). Substrate: O-Phenylenediaminc (Sigma: Cat No P-1526) and hydrogen peroxide (BDH). Substrate buffer. 0.1 M citrate butter (D.l M citric acids and 0.1 M Tri sodium citrate), adjusted to pH 4.5 EUSA plates were manufactured by Sterilin UK 3.1.3.4 Reagents used in the preparation of SDS-JAGE gels Laenimli buffer: 0,5 M Tris-HCl pH 6,8. 10% v, v glycerol, 10% ra/v SDS and 0,05% m/'v bromc phenol blue CAPS buffer pH 9,0: 3-[Cyclohex>'lamino]-l propane Aphonic acid, tSigma) butter pH y.O IBS buffer pH 7,4: 20 mM Tris and 55 raM NaCl, containing 1 % mv fat-free milk powder and 0,05% v v Tueen 20 SDS-PAGE gels: Sodium dodecyl sulphate pohacrylamide slab electrophoresis gel: a 4% stacking gel and a6S separating gel comprising 30 mM Tris pH 8,0, 200 mM glycine and H mM SDS Substrate: 0,03 mM 4-chloronaphtol 3% v.v hydrogen peroxide in 20 ml methanol, made up to 100 ml with TBS buffer pH 7,4 Immobilon-P Transfer membranes Coomassie blue Human sera Human str& used in the ELISA experiments originated from the Serum Bank of the Medical Research Council Tuberculosis Institute, Pretoria Seventeen weeks old Spraguc-Dawley female rats were lsed for the induction of anti-niycolic acids antibodies. The animals were purchased from the Animal Centre at the South African Institute for Medical Research in Johannesburg Feed and water Mice cubes, manufactured by EPOL and tap, autoclavec water were provided ad libimm Sanitation: Bronocide. manufactured by Essential Medicines (Pryt Ltd. was used for sanitation purposes 3.1.6 Plasticware The following plasticware was used Disposable Petri's dishes (Proinex, RSA) EL1SA plates (Stenhr I'K- Sterile, disposable 50 ml centrifuge rubes (Coming, I" A> Disposable tips (Elkay. Denmark! 96-well round bottom microplates (Nunc, Denmark; 3.2 METHODS The following methods were used in the experimental work' 3.2.1 Cultivation of the bacterial strains The bacteria were cultivated at 37aC using Lowenstein-Jensen (LJ) medium slants and Middiebrook 7H-10 agar medium plates The sterility of all the media was confirmed, before tie> were used m the experiments. b> incubating them at 37°C for 24 h. For routine extraction of mycolic acids approximaiely 4-week aid M. tuberculosis and 2-week old cultures of M. vaccae, gro'vn on LJ slants, were used. When Middlebrook 7H-10 agar medium plates uere used. 2-week old cultures of M. tuberculosis were harvested for the extrac:ion of mycolic acids. For the preparation of bacterial suspensions used lor me experimental induction of tuberculosis, approximately 2-week old cultures of M. tuberculosis, grown on LJ slants were used. 3.2.2 Viable and total bacterial counts For the viable count determination. ser:a- suspensions o+" he harvested bncreria were prepared in the diluent medium (as specified under I 1.2.2) to a density corresponding to a McFarland srajidard 4 (approximately OD of 1.0: using a Bcckman DU 65 spectrophotometer, at 486 nm). Tenfold serial dilutions were prepared using 9 ml aliquots of the diluent medium. From the last three dilutions corresponding to 10-. lCr4 and 10"5 of the original suspension aliquots of 0,1 ml (100 ix\) were withdrawn and spread over the surface of Middlebrook 7H-10 plates The platen were incub;.ted at 37°C and the developed colonies counted after two to three weeks for M. tuberculosis and after one week for the plates seeded with M. vaccae. The direct total count was performed using a Neubauer counting chamber and the autoclaved cultures of M. tuberculosis and M. vaccce. originally adjusted to a density corresponding to a McFarland standard 4 and suitably diluted with the diluent medium Statistical analysis of the bacterial counts included the mean values of bacterial counts and standard deviations. 3.2.3 Preparation of mycolic acids from bacterial samples The preparation of bacterial sample? comprised three steps: harvesting of the Mycobacteria rells: saponification and extraction ot mycohc acids Glassware used for the harvesting, extraction, derivatization and HPLC analyses of mycikluc acid was washed in 2% (v v Courad (Merck) rinsed in water, followed by rinsing in chloroform, water. Technical Grade metuanoi, water and finally rinsed in double distilled deionized water. The washed glassware was dried in a warm air oven. Harvesting was done by scraping the bacterialgrowth from the surface of media siants or agar piates (using steriie piasiic loops) ard by suspending them in Reagent A. Initial bacterial suspensions were prepared in Reagent A, by vortexing the harvested cells with sterile glass beads. Homogenous bacterial suspensions were prepared using sterile tissue homogimizer* Prior TO rb<* saponification, the density of the bacterial suspensions was adjusted to a density corresponding to a McFarland standard 4 The saponification, extraction and denvarization of mycolic acids were carried out as described by Butler, Jost and Kilburn (1991}. with minor modifications and are described under the relevant headings. Saponification of the Mycobacteria in Reagent A was carried out in an autoclave at 12l°C. for 30 mm. 3.2.4. Extraction of mycolic acids The saponified samples were allowed to cool after amoclaving. Into 2 ml samples containing crude extract, 1,5 mi Reagent B wis introduced. After vortexing, the pH of each sample was checked and if necessary, adjusted to pH 1 with Reagent B. Subsequently, 2.0 ml chloroform was added to each sample and vortexed for 30 seconds. The layers were allowed to separate The bottom layers were removed with. Pasteur pipettes, transferred to ambrr WISP vials and evaporated to dryness at 85°C in a heat block-evaporatDi under a stream of nitrogen. To neutralize traces of acid carried over. IOC n) of reagent C was added to each sample and the fluid evaporated to drynsss at 85°C in a heat block-evaporator under a stream of nitrogen 3.2.5 Storage of the crude extracted mycolie acids The material obtained from the large-scale extraction of mycolie acids originating from M. tuberculous and M. \accae. i e . the crude bacterial extracts, was stored under acetone, at 4"C in 4 ml arrber WISP vials. To prevent evaporation drying and the exposure to light, the caps of the WTSP vials were covered with Paraiilm 3.2.6 Determination of mycolie acids contents in en de extracts Extracted mycolie acids were denvanzed a« follows To a cooled sample of crude extract (approximated 10 ag in 2,0 mi Reagent A), an aliquot of LO mi chloroform was introduced, fol"owed bv die addition of 100 ul of Reagent D (derivatizaticn reagent). The :apped samples were vortexed for 30 seconds and heated for 20 minutes at :55°C in a heat block-evaporator. Subsequently, the samples were cooled and 1.0 ml of Reagent E added. The samples were vortexed for 30 seconds and the layers allowed to separate. The bottom layers were removed with Pasteur pipettes and transferred to WISP-vials. The vials were placed in a heat block-evaporator and their contents evaporated to dryness at 85CC using a stream of nitrogen. The residues were resuspended in 0.212 g (which corresponds to 160 ul) methylene chloride, capped and vortexed. Each reco tstituted sample was introcuccd into a WISP Mai contains,.. µg of the HPLC interna, standard (prepared as described under i.i.i.l), filtered through a 0,22 µm Millex GV4 filter with a polyethylene housing into another amber-coloured WISP-vial The recapped vials were stored at 4"C until ready for HPLC analysis 3.2.7 HPi.f! analvsk and nimnttfiratinn nf mvcnlic ieiri<; Repeatability and accuracy of the pipette used for tht: distribution of the HPLC standard was determined. The precision was established TO be -- 1% and was confirmed prior to each aliquoting ot the mien a I .standard. For the HPLC analysis 10 ul from each sample fmaintained on ice during handling) was analyzed. Control samples. ; c 10 µl of filtered methylene chlor.de. were run prior to each set of samples analyzed. If a large number of samples was analyzed, in order to validate the reliability of the HPLC apparatus, control samples were run after every three o> four te^t samples The reverse-phase HPLC analyses were carried out unrig a Waters 600 E System Controller High Performance Liquid Chromatography apparatus consisting of. Microsep M741 Data Module; Waters 712 WISP Autosampler; Detector (Waters 486 Tunable Absorbance Detectoi): Column Nova-Pak C18 4 um 3,9 x 150 mm and an end :onncctor set for steel cartridge RKC Rex-C 4 Column Temperature regulator Running conditions were Mobile phase. Solvent A: HPLC Grade methanol Solvent B: HPLC Crradc methylene chloride Flow Rate 2.5 ml/mm Column temperature 30°C The detector was set at 260 nm. Prior to use, the solvents were sparged with Instrument Grade helium. High Purity Nitrogen was used to control hydraulics of the WISP vials autosampler. The HPLC gradient initially comprised 98% (v/\) methanol (Solvent A) and 2% (v/v) methylene chloride (Solvent B). The gradient was increased linearly to 80% A and 20% B at one minute; 35% A and 65% B at ten minutes, held for 30 seconds and then decreased over 10 seconds back to 98% A and 2% B. This ratio was maintained for 4 minutes to allow for stab ligation of the system prior to injection of the next sample. Mathematical quantification of mycolic acids was carried out by comparing the combined peak areas of the tested samples to the peak rrea of the introduced quantity of the High Molecular Weight Internal HPLC Standard. 3.2.8 Preliminary purification of crude mycobactetial extracts in order to shorten the time required for the countercuxT ?nt purification of the crude mycobacterial extracts, an additional preliminar- extraction step was introduced. This step had a dual purpose 1) to remove unnecessary cellular components ixom he crude extract prior to the countercurrent purification and h> to reduce soap fraction m rfe crude bacteria1 extiacts. A portion of the crude extracted material .approximately was suspended in a minimum volume of the lower phase sol ent (usually H)0 ml). transferred into a separation funnel and mixed with an equal volume of the upper phase solvent. The phases were allowed to separate and the upper phase was removed and stored at 4'C into the remaining lower phase an equal volume of the upper phase solvent was again introduced and the process of the phase separation was repeated The second upper phase was removed and stored at 4"C and the second lower phase was dried in a Buchi Roto evaporator RE 120. at 75°C and its mass recorded. 3.2.9 Countercurrent purification of mvcolie acids < nginarinp from M. tuberculosis and M. vaccae Countercurreot apparatus A countercurrent apparatus produced bv H O POST, instrument Company Inc.. Middle Village. New York was used during the invest) nations The 'trains" in this model consisted of 2 X 250 inter-connected rubes. Solvent system used in the eountereurrcnt apparatus The solvent system used for the countercurrent separati on consisted of: 42% v v chloroform (Saarchem. Chemically Pure Reagent) 39% v'v methanol (Saarchem. Chemically Pure) 19°/o vv 0.2 M NaCl (Saarchem. Chemically Puiei Double-distilled deionized water was used for the preparation of the solvent V, Stt'!U I he components were mixed, equilibrated and the upper J jid lower phases were collected using a separation funnel. The composition of the upper phase was established to be: 15% v/v chloroform. 52% v/v methanol and 33% v/v 0,2 M NaC! The composition oi the lower phase was established to be: 68% v/v chloroform. 27% vv methanol and 5% v/v 0.? VI XaCl. The countercurrent purification process was carried ou under the following conditions A countercurrent distribution train comprising 55 tubes, numbered 0-54, was used tn the experiments. The upper phase solvent, a vciume of 600 ml, was introduced into a buffer reservoir. A sample of 125 mg >f mycolic acids after the preliminary purification was dissolved in 50 ml of the lower phase solvent, divided into five ahquots and introduced into first five rubes, numbered 0-4 Subsequently, 10 ml of the upper phase soivenr was introduced into each of the first five countercurrent tubes. Into the remaining 50 tubes aliquots of 10 ml of the lower phase were introduced. Upper phase, in volumes of 10 ml per cycle, was automatically dispensed into tube number 0. repeatedly o\er 55 cycles resulting ra approximately 5 hour operatior. Thus, fifty tive. countercurrent cycles were performed, with each cycle consisting of 10 mixing pendula and 3 minutes phase separation rime Initial load of crude extract alter the funnel extraction 125 mg Number of cycles: 55 Equilibration time: 3 mm 3JZ.11 Removal of malachite green from the countercurrent-purified mycolic acids To remove traces of malachite green derived from bacterial growth media (when M. tuberculosis was grown oil LJ slants). the countercurrent-purified material was selectively precipitated in the following maimer. Countercurrent-purified mycolic acids (92 mg) were placed in a WISP nal into which 1,0 ml chloroform was introduced. The dissolved mycolic acid: were transferred into a pre-weighed round-bottom flask. The via! was rinsed twice with l 0 ml chloroform and the two aliquots of chloroform were added in that already present in the round-bottom flask.. Subsequently acetone was introduced drop-wise in 500 nl aliquots. In total, 26 ml of acetone was introduced and the white flakes of the precipitated-ouT mycolic acids were cashed twice with 20 ml acetone. The acetone supernatant, with the dissolved malachite green was removed and the mycolic acids dried by evaporation. The procedure was carried out at room temperature 3.2.12 Determination of mycolic acids after countei current purification In order lo increase the accuracy of the HPLC detemunaion of mycotic acids, the High Molecular Weight Internal Standard (C-100) was introduced into the countcrcurrent-purified mycolic acids before the saponification A sample ot 0,5 mg of the countercurrent-punfied mycolic acids was introduced into a WTSP vial containing 5 ug of the Huh Molecular Weight Internal Standard (C-100). Saponification of mycolic .icids was carried out with 2 ml of Rcaeent .A at room temperature. The WTSP vial was vortexed for 30 seconds. The extraction was carried nut with 1 5 ml of Reagent B After vortexing. the pH of the sample was checked and if necessary, adjusted to pH 1 with Reagent B. Subsequently. 2.0 ml chloroform was added to each sample and vortexed for 30 seconds. The layers were allowed to separate The bottom layers were removed with Pasteur pipettes, transferred to amber WTSP vials and evaporated to dryness at 85CC in a heat block-evaporator under a stream of nitrogen To neutralize traces of acid earned over, 100 µ1 of reagent C was added to each sample and the fluid evaporated to dryne ;s at 85aC in the heat block-evaporator tinder a stream of nitrogen Therefore, the main difference between the determination of mycolic acids after countercurren! purification and in the crude extract was the time of lntroduction of the Internal Standard3.2.13 Determination of yield of the countercurrent separation In order to calculate the approximate yield of purification/separation, the amount of the mycolic acids present in the samples obtained after the countercurrent separation/purification was coruparrd to the amount of these compounds present m the crude cellular extract introduced into the countercurreni apparatus. The calculations were based en the results obtained by the HPLC analysis. It should be stressed, that it is essential for the calculation of the yield of the countercurrent separation, that the mycolic acids detenu ned by HPLC should be within the tested linear range of the HPLC UV dete :tor 3.2.14 Infra-red spectroscopy Samples of mycolic acids to be analyzed by infra-red spectroscopy were prepared in the following manner. Couniercurrcnt-purified mycolic acids. 1 mg, were dissolved in 1 ml chloroform, introduced into 200 mg KBr and thoroughly mixed. After the evaporation of chloroform, a pellet of mycolic acids in KBr was prepared by using a Slumadzu tablet die and applying a force of approximately 100 kilonewtons on die sample for 10 minutes. A control pellet was prepared using only chloroform, without mycolic acids added to the preparation. The control pellet was used to detennine the background infra-red spectrum. The spectra were analysed on a Perkin Elmer 1600 scries FT-IR system and plotted on a Roland Digital Group X-Y Plotter DXY-1200. 3.2.15 Determination of the stability of the couatercurrent-purified mycotic acids by introducing five barches of countercurrent-punfieci mycolic acids into a container, dissolving them in chloroform and mixing tl e contents very well The chlorofonn was evaporated using a Buchi Rotoe\ aporator RE 120, at 75°C and the sample dried under a stream of nitrogen The pooled sample was divided into two parts which constituted two stoct samples. The first stock sample was re-saponified and the second was left as a non-saponified stock sample, From both stock samples individual aliquots were withdrawn and placed at -20°C. 4C and 25°C. Three samples were prepared per each time point and HPLC analyses were carried out after 6 -veeks, 3,6.9 and 12 months of storage. 3.2.16 Methods used in the experimental production of anti-mycolic adds antibodies Experimental animals: Spraguc-Dawley female rats 17 weeks old were used. Three animals were used per each antigen dose The animals were maintained at the Animal Facilities of the Medical Research Council in Pretoria Environmental conditions Temperature and humidity in the animal facility were set at 20°C (+ /- 1°C) and 40% t+- 10%). respectively. Lighting was provided by means of fluorescent tubes A light-darkne is cycle of alternating 12 hour periods was set up Cages: Rats were housed in transparent polypropylene cages with tight fitting stainless steel lids. Wooden shavings, after autoclaving, were provided as nestling material. Sanitation: Animal rooms, rat cages and glass bottles were cleaned and decontaminated once a week using Bronocide. Water bottles after washing were autoclaved once a week. Identification of the experimental animals: Individual identification was accomplished by making ear marks. Antigen: Unsaponified mycolic acids originating from M. tuberculosis, suspended in Marcol 52 oil. Dose: Three doses of the antigen were used: 1,0, 0.1 and 0.01 mg mycolic acids in 100 µl of Marcol 52 oil per rat per immunization procedure. A control group received 100 µl Marcol 52 oil. only Route of antigen introduction: The antigen was injected subcuianeously at the underneath site of the rat's tail base. Frequency of immunization: the animals were intmunized at 14 days intervals. needing* the animals were bled from the tongue vein at 14 days intervals. The blood was collected into sterile centrifuge tubes and allowed to clot for 16 hours at 4°C. The collected serum was centrifuged at 700-750 g for 20 minutes, diluted 1:1 v/v in glycerol and stored at -20°C. 3.2.17 Methods used in monitoring levels of znti-mycolic acids antibodies ELISA protocol: Coating of ELISA plates The authoclaved coating antigen (in PBS buffer pH 7.4), still hot. was introduced into ELISA wells in aliquots or 50 µl, well, with the solution being continuously stirred. Approximately 3 µg mycolic acids per well were introduced. The coated ELISA plates were incubated at room temperature for 16 hours. Subsequently, the antigen solution was removed, the ELISA plates dried and die dry plates were stored m 4°C. Blocking of ELISA plates: The blocking buffer (0,5% (mv.i casein in PBS pH 7,4) was introduced in aliquotes of 200 µl/well. The ELISA plates were incubated at room temperamre for 2 hours Binding of animal and human antibodies. Rat or human sera (mixed with glycerol 1:1 v/v) were diluted further in the diluting buffer 1:10 v/v The final dilution was therefore 1:20 v,/v. Aliquotes of 50 µl were introduced into wells in duplicate. The plates were incubated at room tempirature for one hour. The sera were removed and the plates washed three times with the washing buffer using an Anthos Automatic Washer Quantification of the bound antibodies: Peroxidase anti-rat antibody conjugate tor peroxidase ami-human conjugate, diluted 1 -1000 was introduced in aliquotes of 50 M1 per well and incubated at room temperature tor 30 minutes. After the removal of the conjugate, the ELISA plates were washed three tunes with the washing buffer The substrate solution composing 10,0 rag O-phenyleiiediamine and 8,0 mg hydrogen peroxide in 10 ml of 0.1 M citrate buffer pH 4,5, was prepared immediately before use and introduced in iO M1 aliquotes per well. The plates were placed in a dark place and the colour developmcni was monitored at 15, 30 and 60 minutes intervals using a SLT 340 ATC photc meter at a wavelength of 450 nm. 3.2.18 Methods used In evaluating specificity of human anti-mycolic acids antibodies For the determination of the specificity of antibodies recognising mycolic acids, the inhibition ELISA was used. Coating of tie ELISA plates with mycotic acids and blocking of the plates were carried cut as described under 3 2.17. Competition step: Human patient's (patient No 38) serim, 75 (A, was mixed with an equal volume of mycolic acids/mouse serum conjugate (prepared as described in section 1.2.16.4). Human control serurr was likewise mixed with 75 fx\ of the conjugate. Two additional controls we:-e prepared by mixing 75 fi\ of control mouse serum with 75 fil of the human patient's and control human sera. The samples were incubated for 1 hour a< room temperature. Subsequently, 625 /*! of diluting buffer (section 3.1.3.4) was introduced into the each mixture, resulting in a final volume 775 iA (final dilution of the human sera was therefore 1:10). The diluted samples vere mixed and 50 ,ul aiiquots were loaded in triplicate onto ELISA plates, coated with mycolic acids The plates were incubated on an ELISA shake • for 1 hour at room temperature. After washing (three times with the wastiing buffer using an Anthos Automatic Washer) the welh> were aspirated and the anti-buman gamma-chain specific peroxidase conjugate diluted 1:10C0 was introduced into each well. The plates were incubated for 30 minutes at room temperature and again washed three times. The preparation of the substate and quantification of ire bound antibodies was carried out in the same manner as described under 3.2.17. 3.2.22 Preparation of gel electrophoresis Preparation of human sera Patients' sera were centrifuged at 3 000 g, for 10 min at 4°C, using a BHG Hermle centrifuge model 2320. After centrifugation and heat inactivation at 56°C for 30 min, the sera were maintained at -70°C. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) The mouse senim and mycohc acids-mouse serum conjugates (prepared as described under 1.2.16.3 and 1.2.16.4. respectively) were diluted with Laemmli buffer (Laemmli, 1970) comprising 0.5 M Tis-HCl pH 6.8. )07c v/v glycerol, 10% m/v SDS and 0.05 % m'v hromo phenol blue, and separated on a vertical sodium dodecyl sulphate polyacrylamide slab electrophoresis gel system (SDS-PAGE) (Owl system 1.5 mm x 160 mm K 140 mm) The gel consisted of a 4% stacking gei and a 6% separating gel In an electrode buffer comprising 30 mM Tns pH 8,0. 200 mM glycine and 17 mM SDS The SDS-PAGE gels were initially run at a voltage of 6) V/sec for one hour, after wlucii the voltage was turned up to 100 V sec lor additional rwo to three hours The gels were run in an electric field created by the Electrophoretic Constant Power Supply (.ECPS 2000/300) prodtced by Pharmacia Biotechnology Western Blot After the separation of the mouse serum proteins, the £:el was equilibrated in CAPS buffer pH 9,0 (as specified under 3 1.3 4). An inmobilon-P Transfer membrane was equilibrated in methanol for one minute and then washed with CAPS (3-fCyclohexylaminoJ-l propane >ulphonic acid Sigma) buffer. The separated mouse proteins were transferred from the SDS-PAGE gel to the Immobilon membrane with a Biorad Transbloi-SP semi-dry transfer cell (pov^er supply: ECPS 2000/300 from Pharmacia Biotechnology) The strips present on the Immobilon membrane were c jt out and blocked by incubation in TBS buffer pH 7.4 (20 mM Tris. 55 mM NaCl) containing I % m/v fat-free milk powder and 0,059 \;\ Tween 20 Each strip of the Immobilon membrane contained one la.ie of the mouse serum and one lane of the mouse serum-mycolic acids conmgite. The control strip comprised one lane of the standard Low Molecular Weight Markers one lane of me mouse serum and of the mouse serum-mycolic icids conjugate The control strip was stained with Coomassie blue. The remaining strips were individually incubated in either patient or control sera at 4°C for 16 hours Ilie sera of both types were diluted 1:6 v v in the bloc king buffer CTBS pH 7,4, 1% m/v fat free milk powder and 0.05% v.'v Tw«=en 20). The membrane strips were subsequently incubated with a mixture of anti-uuinui igG +- lgM peroxidase conjugate diluted 1:5)0 with the blocking buffer, at room temperature for three hours and excess antibody was removed by three rinses in TBS buffer pH 7,4 containing 1% m/v fat free milk powcer. The blots were developed by adding the subsnate. i.e 0,03 mM 4-chioronaphtoi, 3% v/v hydrogen peroxide in 20 mi methanol, made up to 100 ml with TBS buffer pH 7,4 3.2.23 Methods used in the purification of CD4* single positive (SP), CD8single positive.,CD4-and CD8-double negative (DN) α ß TCR positive cells from the human peripheral blood Purification of these cells was performed according to the procedure described by Niehues et ai, (1994 with small modifications; Peripheral blood mononuclear cells (PBMC) were isolated from 100 to 200 ml of blood from healthy individuals, usmg density gradient centrifugation over Ficoll-Hypaque. Red blood cells v.ere lysed with 0.(15 M NH,C1 and the remaining cells were resuspended in PBS buffer containing l°'r> v v B90% viable. The assessment of the cell viability was carried out using propidium iodide staining and Fluorescence Activated Cell Sorter (FACS). Cells that did not bind to WT31 in the first selection step were irradiated (30Oy) and used as autologous total APCs (Antigen Presenting Cells). Induction of CD1 on autologous APC was carried out as described by Poreelli, Morita and Brenner (1992). Human monocytes were isolated from leucocyte concentrates originating from blood of normal donors by plastic adherence and detached by incubation at 37°C in PBS with 0,53 mM EDTA. Adherent c«Us (»rnprised typically more than 90% C^ \3* and MHC class II+ but tested negative for CDla, -b and -c by surface staining. To induce COl expression, monocytes were cultured in RPM1 1640 lissue culture medium containing 10% fetal calf serum with 100 units/ml of GM-CSF and IL-4 for 60 hours. The cells were collected by desorption using PBS with 0,53 mM EDTA. Proliferation assay: The cells were suspended in RPM1 1640 tissue culture medium exploraineted with 2 mM glutamine, 0,25% m/v refobacine and 5% v/v heat. pooled human AB serum. SP and DN cells were plated in triplicated round-bottpmed 96-well tissue culture inicroplatet, stimulated with ravcnlte acids (at concentrations of 5. 25, and 50 µg/ml and a mutagen, phyliiliwmsnliiHiilii (PHA), at a concentration of 3.3 µg/ml). The cells were incubated m72 hours at 37^C in a humidified CO, incubator (5% CO,). During the final 16 hours of incubation: the cells in the nncroplates were pulsed with 3H-thyrnidine (0.5 uCi'well). Proliferation of SP and DN cells was determined by incorporation of ^H-thymidine as measured by standard liquid scintillation counting. Dose response curves were generated. Autologous, irradiated (30 Gy) APC or CDT APC cells were added at a ratio of 4:1 in all experiments. The results were expressed at nean cpm +/- SbM, from which background values (medium alone) were subtracted. 3.3 RESULTS and DISCUSSION The results obtained concerning: i) the influence of the modified method of purification on yield and purity of mycolic acids; ii) the structural analysis of mycolic acids originating from M. tuberculosis using infra-red spectroscops. and iii) the stability of mycolic acids were presented and discussed in sections 1.3.1, 1.3.2 and 1.3.3. 3-"1 Investigations of the immunogenic properties of cot ntercurrent-purified mycolic acids These investigations were based on the following experiments: 3.3.1 The indurtion of antibodies against mycotic acids in the experimental rals; 3.3.2 The detection of anti- mycolic acids antibodies in human tuberculosis patients; 3.3.3 Response of human CD4 T cells to the in vi.ro stimulation with mycolic acids. The fcllowing results were obtained. 3.3.1 The induction of antibodies against my:olic acids in the experimental animals In order to determine the lmmunogeniciry of mycolic acids, suspensions of a methylester form of mycolic acids in oil were used for the immunization of Sprague-Dawley rats, as described under 3.2.16. The antibody response was monitored and the ELISA results obtained after a treatment period of 3 months, are presented in Fig. 28. A dose related response was observed for the induction of antibodies specific for mycolic acids, immobilized on the ELISA plates as described under 3.2.17. The results presented in Fig. 28 support the hypothesis ihat mycolic acids are immunogenic in respect of being able to induce anti-mycolic acids antibodies. 3.3.2 The detection of anti- mycolic acids antibodies in human tuberculosis patients Two out of 58 human tuberculosis patients sera screened, revealed the presence of antibodies recognising methyl-ester form of mycohc acids (Jig. 29;. The specificity of these antibodies was confirmed by an inhibition ELISA reaction carried out as described under 3.2.18. The results obtained are presented in Fig 30. The results imply that mycolic acids shed from the ceil walls of M. tuberculosis infecting the human host may induce the tbrmution of anti-mycoiic acids antibodies in patients. However, the antibodies ar: produced at a low frequency, or there is a stage of the infection during which the antibodies are not detected. The antibodies are specific (Fig. 30), but their affinity is low due to the high serurn concentration required to give a detecable signal, Anti-mycohc acids antibodies from human tuberculosis patients recognise a preferred serum protein of -'- 80 kDa on control mouse serum exposed to purified mycolic acids in methyl-ester form. An illustration of this observation is given in Fig. 31. The result presented in Fig. 31 could not be easily reprocuced with the sera of other human tuberculosis patients, probably due to the fact that these antibodies occur at low frequencies. The -'- 80 kDa mouse protein appears to enhance the antigenicity of mycolic acids and its human homologue may therefore also increase the immunogemcity of mycolic acids in blood of human tuberculosis patients. 3.3.3 Response of human CD4 T cells to the in vitro stimulation with mycolic acids In order to establish the extent of the human T-cell response to mycolic acids stimulation, besides the known stimulation of double legative (US) T cells (Porzelli. Morita and Brenner. 1992. Beckman et a!., 1394), CD4 T cells and CDS T cells were exposed to CD1 -expressing antigen presenting cells and the cell proliferation was measured. The results arc presentee in Figures 32a and 32b. The results in Fig. 32a indicate stronger stimulation of CD4 T cells by mycolic acids at a frequency higher than that of DN T ceils. This effect was not previusly reported by Beckman et al (1995) or other researchers. On the other hand, upon exposure to non-CDl expressing APCs, CD4 T cells also showed a significant but weak cell proliferation alter mycolic acids treatment (Fig. 32b). No stimulauon of DN T cells was observed. This result may, therefore, suggest a non-CDl dependent way of mycolic acids presentation by APC cells in order to activate CD4 T cells On the basis of these results, immunoregulatory proper.ies of mycolic acids appear to be mediated by both DN and CD4 T cells, bu: not by CD8 T cells. CONCLUSIONS 1. On the basis of the results reported above it can be concluded that mycoiic acids originating from M. tuberculosis H37Rv purified using counter-current purification, possess irnmunoregulatory and immunogenic properties ?nd can be used in the prophylaxis and'or therapy of diseases, particularly those induced by or associated with Mycobacteria 2 Mycoiic acids can protect mice against infection with At tuberculosis, especially when administered before the infection/onset :>! the disease. 3. The protection provided by mycoiic acidi manifested itself mainly in the lungs The lungs from the mycoiic acids-pre-treated animals were the only organ in which a significantly reduced tubercle rormation could be observed upon macroscopic post-mortem assessment. 4. On the basis of cytokine profiling of IL-12, IFN-T, TNF-α and TGF-ß inhe organs of the experimental animals, mycoiic acids ippear to induce upon pretreatment a pro-inflammatory mechanism of protection against 5. Freenanaary from IR V indicates that mycoiic acids from other spects, of Mycobacterium (e.g. M. vaccaej could also induce the expteert inferance effects in the lungs. v Mycoiic acids ampair the development of arthritic symptoms when introduced before the administration of an arthritis-indu<:ing dose of heat-killed and freeze-dried cells of M. tuberculosis H37Ra. This reveals an innuncsepressive regulatory property of mycoiic acids, which might be applied in the prevention of auto-immune side-effects of bacterial infections, particularly in the case of M tuberculosis or mycobacterial infections. 7. The results do not exclude a protective effect of mycohc acids treatment nfter the administration of arthritis-inducing doses of M tuberculosis H37Ra. 8. The immunogenic properties of mycohc acids were confirmed in the experiments in which they induced the formation of artibodies in the experimental animals upon immunization with these compounds Anti-mycolic acids antibodies occurring spontaneously and detected in human serum were found to be specific but ot low affinity 9. Countercurrent-purified mycolic acids were also found to stimulate human DN and CD4 T cells, but did not appear to have an effect on CD8 T cells. 10. No toxic effects of mycolic acids were detected in control rats within the tested doses, i.e between 8 and 50 ug for mice and 0:1 and t mg for rats. REFERENCES Agrewala, J N and G C Mishra. 1995. A 38-kDa antigen of Mycobacterium tuberculosis predominantly induces the secretion of interleukin-2, ititerferon-gamma and IgG2a antibodies. Microbiol Immunol, 39, 801-808. Beagly, K W, K Fujihasni, C A Black et al. 1993. 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A composition according to claim 1 or claim 2, wherein the purified lipid cell-wall component is a cell-wall component from a bacterium which produces mycolic acids 4 A composition according to claim 3, wherein the bacterium is selected from the genus Mycobacterium, the genus Corynebaaenum, the genus Nocardia and the genus Rhodococcus. 5 A composition according to claim 4. wherein the bacterium is selected from the genus Mycobacterium. 6. A composition according to claim 5, wherein the. bacterium is Mycobacterium tuberculosis. A composition according to any one of claims 1 to 6 wherein the purified lipid cell-wall component is a purified mycolic acid or mixture of purified mycolic acids. 8 A composition according to any one of claims I to 7, wherein the pharmaceutically acceptable carrier is an oil 9. A composition according 10 claim S. wherein the oil is Marcol 52. 10 A composition according to any one of claims 1 to 7, wherein the pharmaceutically acceptable carrier is a vapourised liquid. 11. A composition according to claim 10. wherein the vapourised liquid is a saline solution. 12. A composition according to any one of claims 1 to 11. which comprises a therapeutic, prophylactic or tolerogenic amount of the ourified lipid cell-wall component. 13. A composition according to claim 12, which comprises about 5 µg or less of the purified lipid cell-wall component per ml of composition. 14. A composition according to claim 13. which comprises about 1 µg of the purified lipid cell-wall component per ml of composition. 15. A composition according to any one of claims 1 to 14. wherein a unit dose for administration to a human subject comprises from about 5 to 10 mg of the purified lipid cell-wall component 16 A vaccine for a microbial infection or an immune disorder comprising a composition according to any one of claims 1 to 15 17. A vaccine according to claim 16. wherein the microbial infection is a mycobacterial infection 18 A vaccine according to claim 17 wherein the mycobacterial infection is tuberculosis. 19. A vaccine according to claim lb, wherein me immune disorder is an autoimmune disorder. 20. A vaccine according to claim 10 wherein the autoinimune disorder is arthritis. 21. A method of treatment of a microbial infection in a subject comprising the step of administering to the subject a purified bacterial lipid cell-wall component or analog or derivative thereof or a composition according to any one of claims 1 to 15 or a vaccine according to any one of claims 16 to 18. 22. A method according to claim 21, which is a prophylactic method or a therapeutic method. 23. A method according to claim 22, which is an immunorrgulatory method. 24. A method according to claim 22 or claim 23, which is a prophylactic method which enhances resistance or reduces susceptibility to a microbial infection in a subject 25. A method according to claim 24. wherein the prophylactic method promotes an inflammatory response in an infected organ of the subject. 26. A method according to claim 25, wherein the infected organ is the lungs 27. A method according to claim 22 or claim 23, wherein the prophylactic method or the therapeutic method modulates or manipilates the humoral immune system or cellular immune system or both in a subject. 28. A method according to any one of claims 21 to 27. wherein the purified lipid cell-wall component is a purified mycolic acid or a mixture of purified mycotic acids. 29. A method according to any one of claims 21 to 28 whtrein the microbial infection is a mycobacterial infection. 30. A method according to claim. 29. wherein the mycoba;terial infection is tuberculosis. 31. A purified bacterial lipid cell-wall component or analog or derivative thereof or a composition according to any one of claims 1 to 15 or a vaccine according to any one of claims 16 to 18 for use in a method of treatment of a microbial infection in a subject. 32. Use of a purified bacterial lipid cell-wall component or analog or derivative thereof or a composition according to any one of claims 1 to 15 or a vaccine according to any one of claims 16 to 18 in a method of making a medicament for use in a method of treatment of a microbial infection in a subject 33. A purified lipid cell-wall component according to claim 31 or claim 32, wherein the method of treatment is a prophylactic method or a therapeutic method 34 A purified lipid cell-wall component according to claim 33, wherein the method of treatment is an immunoregulatory method 35. A purified lipid cell-wall component according to claim 33 or claim 34, wherein the method of treatment is a prophylactic method which enhances resistance or reduces susceptibility to a microbial infection in a subject. 36 A purified lipid cell-wall component according to claim 35, wherein the prophylactic method promotes an inflammatory response in an infected organ of the subject. 37. A purified lipid cell-wall component according to claim 36, wherein the infected organ is the lungs 38. A purified lipid cell-wall component according to claim 33 or claim 34, wherein the prophylactic method or the therapeutic method modulates or manipulates the humoral immune system or cellular immine system or both in a subject. 30. A purified lipid cell-wall component according to any one of claims 31 to 38, which is a purified mycolic acid or a mixture of purified mycolic acids. 40. A purified lipid cell-wall component according to any one of claims 31 to 39, wherein the microbial infection is a mycobacterial infection. 41. A purified lipid cell-wall component according to claim 40, wherein the mycobacterial infection is tuberculosis. 42. A method of diagnosing an immune disorder in a subject comprising the steps of: contacting a sample from the subject with a purified lipid cell-wall component or analog or derivative thereof or with a composition according to any one of claims 1 to 15; and detecting any reaction between the purified lipid cell-wall component and the sample 43. A method according to claim 42, wherein the step of detecting any reaction between the purified lipid cell-wall component and the sample comprises detecting the binding of an antibody present in the sample to the purified lipid cell-wall component. 44. A method of treatment of an immune disorder in a subject comprising the step of administering to the subject a purified bacterial lipid cell-wall component or analog or derivative thereof or a composition according to any one of claims 1 to 15 or a vaccine according to am one of claims 16, 19 and 20. 45. A method according to claim 44, which is a prophylactic method or a therapeutic method 46. A method according to claim 45. which is an lmmunoregulatory method. 47 A method according to claim 45 or claim 46. which is a prophylactic method which enhances resistance or reduces susceptibility to an immune disorder in a subject. 48. A method according to claim 47, wherein the prophylactic method suppresses inflammation of the joints in the subject. 49. A method according to claim 45 or claim 46, wherein the prophylactic method or the therapeutic method modulates or manipulates the humoral immune system or cellular immune system or both in a subject. 50. A method according to any one of claims 44 to 49, wherein the lipid cell-wall component is a purified mycolic acid or a mixture of purified maycolic acids. 51. A method according to any one of claims 44 to 50, wherein the immune disorder is an inflammatory condition or allergy. 5? A method according to claim 51. wherein the inflammatory condition is an autoimmune disease. 53. A method according to claim 52, wherein the autoimmune disease is arthritis. 54. A purified bacterial lipid cell-wall component or analog or derivative thereof or a composition according to any one of claims i to 15 or a vaccine according to any one of claims 16. 19 and 20 for use in a method of treatment of an immune disorder in a subject. 55. Use of a purified bacterial lipid cell-wall component or analog or derivative thereof or a composition according to any one of claims. 1 to 15 or a vaccine according to any one of claims 16, 19 and 20 in a method of making a medicament for use in a method of treatment of an immune disorder in a subject. 56. A purified lipid cell-wall component according to clam 54 or claim 55, wherein the method of treatment is a prophylactic method or a therapeutic method. 57 A purified lipid cell-wall component according to claim 56, wherein the method of treatment is an immunoregulatary method. 58. A purified lipid cell-wall component according to claim 56 or claim 57, wherein the method of treatment is a prophylactic method which enhances resistance or reduces susceptibility to an immune disorder in a subject. 59. A purified lipid cell-wall component according to claim 58, wherein the prophylactic method suppresses inflammation of the joints in the subject. 60. A purified lipid cell-wall component according to claim 56 or claim 57, wherein the prophylactic method or the therapeutic method modulates or manipulates the humoral immune system or cellular immune system or both in a subject. 61. A purified lipid cell-wall component according to any one of claims 54 to 60, which is a purified mycolic acid or a mixture of purified mycolic acids. 62. A purified lipid cell-wall component according to any one of claims 54 to 61, wherein the immune disorder is an inflammatory condition or allergy. 61 A purified lipid cell-wall component according to claim 62. wherein the inflammatory condition is an autoimmune disease. 64 A purified lipid cell-wall component according to claim 63, wherein the autoimmune disease is arthritis 65 A method of separating and purifying a specific microbial cell-wall component of a lipid or carbohydrate nature or a de:ivative or analog thereof from an extracted mixture of the cell-wall component or derivative or analog thereof and contaminants or from a synthetic mixture of the cell-wall component or derivative or analog thereof and contaminants comprising the steps of: dissolving the extracted mixture or synthetic mixture in a bi-phasic solvent containing sodium chloride to form a solution; allowing the solution to separate to form an upper phase and a lower phase; subjecting the phases to countercurrent distribution/separation comprising a required number of cycles to separate the microbial cell-wall component or analog or derivative thereof in the upper phase or the lower phase; and removing the separated microbial cell-wall component or derivative or analog thereof from the upper or lower phase 66 A method according to claim 65, which also comprises the additional pre-purification steps of: dissolving the extracted mixture of cell-wall component or derivative or analog thereof and contaminants or the synthetic mixture of the cell-wall component or derivative or analog thereof and contaminants in a first solvent without sodium chloride; adding thereto a second solvent without sodium chloride; mixing and allowing the solution to separate to form a first upper phase (second solvent) and a first lower phase (first solvent); and removing the first upper phase and/or the first lower phase for further processing. 67. A method according to claim 65 or claim 66, wherein the lower phase containing the extracted mixture is removed and subjected to countercurrent distribution/separation comprising a required number of cycles to separate the microbial cell-wall component or analog or derivative thereof in a second upper phase or lower phase and the separated microbial cell-wall component or derivative or analog thereof is removed from the second upper or lower phase. 68. A method according to any one of claims 65 10 67, which also comprises the additional post-purification steps of; dissolving the extracted microbial cell-wall component or derivative or analog thereof in a suitable solvent; and adding a precipitant to the solution to precipitate out the dissolved farther purified microbial cell-wall component or derivative or analog thereof. 69 A method according to claim 68 wherein the solvent is chloroform. 70. A method according to claim 68 or claim 69, wherein the precipitant is acetone. 71. A method according to any one of claims 65 to 70, when also comprises the steps of: saponifying a microbial culture prior to preparing therefrom an extracted mixture of a cell-wall component or drivative or analog thereof on which to perform the method: and resaponifying the separated and purified microbial cell-wall component or derivative or analog thereof. 72. Detection means for detecting the presence of antibodies to a purified mycolic acid or mixture of purified mycolic acids comprising a solid phase and a purified mycolic acid or a mixture of purified mycolic acids in a methylester form or in a freshly resaponified form associated therewith. 73. Detection means according to claim 72. wherein the solid phase is an ELISA plate. 74. A composition substantially as hereinbefore described with reference to the accompanying drawings. 75. A vaccine substantially as herein described with reference to the accompanying drawings. 76. A purified bacterial lipid cell-wall component substantially as herein described with reference to the accompanying drawings.