Antibody reactivity to Mycobacterium tuberculosis-specific regions of differences 1 and regions of differences 9 proteins and peptides in rabbits, mice, and humans.

Background: The major antigens encoded by Mycobacterium tuberculosis-specific genomic regions of differences (RDs) could be useful in the development of new vaccines and/or diagnostic reagents using T-cell and/or antibody assays. In particular, RD1 proteins PE35, PPE68, ESXA, ESXB, and RD9 protein ESXV and their peptides have been identified as major T-cell antigens. However, little is known about their antibody reactivities in different mammalian species. This study aims to determine the antigen-specific antibody reactivities to the above antigens and their peptides in three different mammalian species, i.e., rabbits, mice, and humans. Methods: Sera were obtained from (i) rabbits immunized with purified recombinant proteins PE35, PPE68, ESXA, ESXB, and ESXV; (ii) mice immunized with recombinant DNA vaccine constructs of pUMVC6 and pUMVC7 containing RD1 and RD9 genes; and (iii) tuberculosis (TB) patients and healthy humans. Enzyme-linked immunosorbent assays (ELISAs) were performed with the sera to determine the antibody reactivity to purified recombinant proteins, peptide pools, and individual peptides of RD1 and RD9 proteins. Results: The ELISA results with sera from rabbits immunized with pure recombinant proteins showed positive antibody reactivity with all of the immunizing proteins and their synthetic peptide pools. Testing of the sera with individual peptides showed positive antibody reactivity with PE35 peptides P1 (aa 1-25), P2 (aa 16-40), P5 (aa 61-85), and P6 (aa 76-99); PPE68 peptides P9 (aa 121-145), P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371); all peptides (P1 to P6) of ESXA and ESXB; and ESXV peptides P1 (aa 1-25), P2 (aa 16-40), P3 (aa 31-55), P5 (aa 61-85), and P6 (aa 76-94). The sera from mice immunized with DNA vaccine constructs showed antibody reactivity to all proteins and the peptide P6 (aa 76-99) of PE35 and peptides P19 (aa 271-295) and P24 (aa 346-371) of PPE68. In humans, the peptides P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371) of PPE68 and the peptides P4 (aa 46-70), P5 (aa 61-85), and P6 (aa 76-94) of ESXV showed positive reactivity with sera from TB patients and healthy controls. Conclusion: The results demonstrate the presence of several antibody epitopes in each protein, but variations in the epitopes recognized were observed among mice, rabbits, and humans, which could be due to mammalian species differences and/or mode of antigen delivery.

Synergistic induction of apoptosis and chemosensitization of human colorectal cancer cells by histone deacetylase inhibitor, scriptaid, and proteasome inhibitors: potential mechanisms of action.

Histone deacetylase inhibitors (HDACIs) exhibit modest results as single agents in preclinical and clinical studies against solid tumors; they often fall short and activate nuclear factor kappa-B (NFκB). Co-administration of HDACI with proteasome inhibitors (PIs), which interrupt NFκB pathways, may enhance HDACI-lethality. The goal of this study was to determine whether PIs could potentiate HDACI, scriptaid (SCP)-mediated lethality, to unravel the associated mechanisms and to assess the effects of the combined inhibition of HDAC and proteasome on chemotherapy response in human colorectal cancer cells. Cancer cells were exposed to agents alone or in combination; cell growth inhibition was determined by MTT and colony formation assays. HDAC-, proteasome-, NFκB-activities, and reactive oxygen species (ROS) were quantified. Induction of apoptosis and cell cycle alterations were monitored by flow cytometry. Expression of cell cycle/apoptosis and cytoprotective/stress-related genes was determined by real-time qRT-PCR and EIA, respectively. Potentiation of cancer cell sensitivity to chemotherapies by SCP/PIs was also evaluated. SCP and PIs: MG132, PI-1, or epoxomicin interact synergistically to potently inhibit cancer cell growth, alter cell cycle, induce apoptosis, reduce NFκB activity, and increase ROS generation. These events are associated with multiple perturbations in the expression of cell cycle, apoptosis, cytoprotective, and stress-related genes. Co-administration of SCP and PIs strikingly increases the chemosensitivity of cancer cells (122-2 × 10(5)-fold) in a drug and SCP/PIs-dependent manner. This combination regimen markedly reduced the doses of chemotherapies with potent anticancer effects and less toxicity. A strategy combining HDAC/proteasome inhibition with chemotherapies warrants further investigation in colorectal cancer.

Mycobacterial antigen-induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non-diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette-Guérin (BCG)-vaccinated healthy subjects.

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non-diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell-mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non-diabetic TB patients and Mycobacterium bovis bacilli Calmette-Guérin (BCG)-vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen-induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)-gamma, interleukin (IL)-2, tumour necrosis factor (TNF)-beta], and Th2 (IL-4, IL-5, IL-10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1(pool) stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1(pool) in healthy subjects. In response to complex mycobacterial antigens, both IFN-gamma and TNF-beta were secreted by PBMC of all groups whereas diabetic TB patients secreted IL-10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN-gamma and IL-10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.

Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG.

Comparative genomics has identified several regions of differences (RDs) between the infectious Mycobacterium tuberculosis and the vaccine strains of Mycobacterium bovis BCG. We aimed to evaluate the cellular immune responses induced by antigens encoded by genes predicted in 11 RDs. Synthetic peptides covering the sequences of RD1, RD4 to RD7, RD9 to RD13, and RD15 were tested for antigen-induced proliferation and secretion of Th1 cytokine, gamma interferon (IFN-gamma), by peripheral blood mononuclear cells (PBMC) obtained from culture-proven pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects. Among the peptide pools, RD1 induced the best responses in both donor groups and in both assays. In addition, testing of TB patients' PBMC for secretion of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, and IL-1beta), Th1 cytokines (IFN-gamma, IL-2, and TNF-beta), and Th2 cytokines (IL-4, IL-5, and IL-10) showed differential effects of RD peptides in the secretion of IFN-gamma and IL-10, with high IFN-gamma/IL-10 ratios (32 to 5.0) in response to RD1, RD5, RD7, RD9, and RD10 and low IFN-gamma/IL-10 ratios (<1.0) in response to RD12, RD13, and RD15. Peptide-mixing experiments with PBMC from healthy subjects showed that secretion of large quantities of IL-10 in response to RD12 and RD13 correlated with inhibition of Th1 responses induced by RD1 peptides. In conclusion, our results suggest that M. tuberculosis RDs can be divided into two major groups--one group that activates PBMC to preferentially secrete IFN-gamma and another group that activates preferential secretion of IL-10--and that these two groups of RDs may have roles in protection against and pathogenesis of TB, respectively.

Assessment of in vitro immunity to Mycobacterium tuberculosis in a human peripheral blood infection model using a luciferase reporter construct of M. tuberculosis H37Rv.

Protective immune responses to tuberculosis in man are primarily cell-mediated and require the interaction of specific T cells, cytokines and activated macrophages. In the present study, Mycobacterium tuberculosis H37Rv labelled with luciferase reporter enzyme was used to analyse the anti-mycobacterial immunity in man using an in vitro whole blood infection model. Peripheral blood samples obtained from M. bovis bacille Calmette-Guérin (BCG)-vaccinated tuberculin-positive healthy volunteers (n = 23) were cultured with M. tuberculosis H37Rv reporter strain. The growth of bacteria in the whole blood cultures was monitored after 48 and 96 h of infection. The results showed that the growth of M. tuberculosis was significantly inhibited after 96 h (P < 0.029) of culture. Among the cytokines studied, interleukin (IL)-10 and IL-12 were not detected at all, whereas low levels of interferon (IFN)-gamma after 96 h (0.4 IU/ml) and tumour necrosis factor (TNF)-alpha after 48 (135 pg/ml) and 96 h (47 pg/ml) of culture were detected in the supernatants of whole blood infected with M. tuberculosis. The magnitude of bacterial growth correlated directly with the concentration of TNF-alpha detected after 48 h (r = 0.722) and 96 h (r = 0.747) of culture (P

Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle.

The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.

The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobacterium tuberculosis.

The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. The mammalian cell entry (mce)3 operon is one of four homologous mce operons that encodes six putative invasin-like exported proteins (Mce3A-F), possibly involved in entry and survival of M. tuberculosis inside macrophages. We have recently shown that Mce3A, Mce3D and Mce3E are expressed and elicit antibody responses in a majority of human subjects during natural infection with M. tuberculosis. In this study, we demonstrate the expression of Mce3A-F proteins and their mRNA during in vitro growth of M. tuberculosis. To demonstrate the expression of mce3A-F proteins, the antibodies were raised in rabbits against three pure proteins (Mce3A, Mce3D and Mce3E), and their specificity was checked by immunoblotting with recombinant Mce1A-F proteins encoded by mce1 operon. The antibodies were also generated against all the six Mce3 proteins, which were expressed and purified as fusion proteins with glutathione S-transferase (GST) as the fusion partner (GST-Mce3A-F). The antibodies reacted, in each case, with a protein of expected molecular mass (Mr) for the corresponding Mce3 protein in the cell wall fraction but not in the soluble fraction of in vitro-grown M. tuberculosis cells. The presence of mRNA for mce3A-F genes was also shown by using mce3A-F gene-specific primers, and total RNA isolated from in vitro-grown M. tuberculosis cells by reverse transcription-polymerase chain reaction (RT-PCR). Pretreatment of the RNA preparation with RNase A abolished amplification in RT-PCR confirming that mce3A-F mRNA rather than genomic DNA was being amplified. The data show that Mce3A-F encoded by the mce3 operon are expressed during in vitro growth of M. tuberculosis.

In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis.

The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell-mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette-Guérin (BCG)-vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen-induced proliferation and interferon (IFN)-gamma secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8.4, Mtb9.8, Mtb9.9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen-induced proliferation and IFN-gamma secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9.8 and Mtb39A in proliferation assays (median SI = 6.2 and 6.4, respectively) and of Mtb9.8, Mtb39A, Mtb40 and Ag85B in IFN-gamma assays (median delta IFN-gamma= 15.5, 10.8, 7.8 and 8.1 U/ml, respectively). BCG-vaccinated healthy donors showed weak (<30% responders) to moderate (31-50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12] and the immunosuppressive cytokine IL-10, the complex CF and CW antigens as well as the recombinant Mtb9.8, Mtb9.9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL-10, while Mtb8.4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL-10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.

Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis.

The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

Human Th1 cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class II molecules.

We have used a synthetic-peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT-6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT-6-specific CD4+ T-cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA-DR-typed tuberculosis patients with complete antigen in vitro. The established T-cell lines were then screened for proliferation and interferon-gamma (IFN-gamma) secretion in response to eight overlapping 20-mer peptides covering the ESAT-6 sequence. The response of the T-cell lines to ESAT-6 and peptides from a human leucocyte antigen (HLA)-heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti-HLA class I and class II antibodies suggested that the T-cell lines recognized ESAT-6 in association with HLA-DR and -DQ molecules. Furthermore, testing of selected T-cell lines with ESAT-6 and the peptides in the presence of autologous and allogeneic HLA-DR- and -DQ-typed antigen-presenting cells identified HLA-DR2, -DR52 and -DQ2 amongst the HLA molecules involved in the presentation of ESAT-6 and its peptides to human Th1 cells. In addition, the T-cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT-6 and peptides. In conclusion, the recognition of ESAT-6 by IFN-gamma-secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.

The immunomodulatory effects of prolonged intravenous infusion of propofol versus midazolam in critically ill surgical patients.

Both propofol and midazolam are known to inhibit immune function. The aim of this study was to investigate cytokine production in critically ill surgical patients as early markers of immune response to prolonged infusion of propofol and midazolam. The study enrolled 40 elective patients who were to receive long-term sedation for more than 2 days. Patients were randomly allocated to one of two equally sized groups. Central venous blood samples for measurement of interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were drawn prior to the start and after 48 h of infusion. After 48 h, propofol caused significant increases in IL-1beta (24%), IL-6 (23%) and TNF-alpha (4.8 times) levels, while midazolam caused significant decreases in IL-1beta (21%), IL-6 (21%) and TNF-alpha (19%). Both agents caused significant decreases in IL-8 levels (propofol: 30%, midazolam: 48%, p < 0.05). Propofol caused significant decreases in IL-2 levels (68%, p < 0.001) but increases in IFN-gamma (30%, p < 0.05), whereas there was no significant change with midazolam compared with the pre-infusion level. In conclusion, during 48 h of continuous infusion, propofol stimulated, while midazolam suppressed, the production of the pro-inflammatory cytokines IL-1beta, IL-6 and TNF-alpha, and both caused suppression of IL-8 production. Propofol inhibited IL-2 production and stimulated IFN-gamma production, whereas midazolam failed to do so. Therefore, sedative agents may have clinical implications in high-risk and immunocompromised patients.

The effect of halothane and isoflurane on plasma cytokine levels.

The aim of this study was to investigate the effect of halothane vs. isoflurane on cytokine production during minor elective surgery. Forty adult patients, ASA I-II were randomly allocated to receive halothane or isoflurane. Venous samples for interleukin (IL)-1beta, IL-2, IL-6, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were taken before anaesthesia, before incision, at the end of anaesthesia and 24 h postoperatively. In both groups, IL-6 and TNF-alpha levels remained low throughout the study period. Before incision, in both groups IL-1beta and IFN-gamma showed a decrease (p<0.01 for IL-1beta in isoflurane group and p<0.05 for the others) compared with pre-induction. By the end of anaesthesia and surgery, IL-1beta had increased significantly (p<0.05) and IFN-gamma had decreased significantly (p<0.05) in both groups compared with pre-incisional levels. By 24 h postoperatively in both groups, IL-1beta had decreased significantly (p<0.05), whereas IFN-gamma had increased significantly (p<0.05) compared with the end of anaesthesia and surgery level. Pre-incisionally, IL-2 increased in the halothane group (p<0.01), whereas it decreased significantly in the isoflurane group (p<0.001) compared with the pre-induction level. By the end of anaesthesia and surgery and by 24 h postoperatively, IL-2 had decreased significantly in the halothane group (p<0.001), whereas it increased significantly in the isoflurane group (p<0.001) compared with pre-incision and end of anaesthesia and surgery levels, respectively.

TNF alpha-mediated tissue damage in mouse footpads primed with mycobacterial preparations.

Tissue sites involved in certain types of inflammation become sensitive to the destructive effect of a subsequent injection of tumour necrosis factor alpha (TNF alpha). To try to further delineate the cascade of effector and regulatory events controlling this activity, a new model is described and its main properties characterized. C57BL/GrFa mice received mycobacterial products subcutaneously in the footpads. Recombinant TNF alpha was injected 24 h later into the same sites. To assess the tissue-destructive effect of TNF alpha in these "primed" footpads, swelling and haemoglobin content of injected footpads were measured, 16 h and 20 h respectively after the injection of TNF alpha. When loaded with either Escherichia coli LPS (10 micrograms) or Mycobacterium vaccae soluble sonicate (17 micrograms), footpads were reactive to the subsequent injection of 1 microgram recombinant TNF alpha, as assessed by both swelling and haemoglobin content. When C57BL/GrFa mice received 10(9) autoclaved M. vaccae subcutaneously in the back 10 days before the footpad was "primed" with soluble M. vaccae sonicate, the destructive effect of TNF alpha was significantly enhanced, becoming 5-10-fold greater than that seen in sites "primed" with an optimal dose of LPS. This higher reactivity was abrogated by a single dose of anti-CD4 given just before the injection of TNF alpha. This local reactivity to TNF alpha of skin sites loaded with mycobacterial products is compared to the local LPS-dependent Shwartzman reaction, and the relevance of this assay as a model with which to delineate the mechanisms of tissue damage in tuberculosis is discussed.

A model for the investigation of factors influencing haemorrhagic necrosis mediated by tumour necrosis factor in tissue sites primed with mycobacterial antigen preparations.

Mycobacterial lesions and skin sites challenged with soluble mycobacterial antigen are very sensitive to the necrotizing effect of tumour necrosis factor (TNF). We have used a model that permits separate quantitative assessment of swelling and haemorrhage to show that when these reactions are elicited in mice that have not been deliberately immunized, pretreatment of the mice with lipopolysaccharide (LPS), or with a MoAb to CR3 which blocks emigration of myeloid cells into the tissues, will block both the swelling and the haemorrhage. On the other hand, treatment with an inhibitor of platelet-activating factor (PAF), or with misoprostol (a synthetic prostaglandin E1 analogue), or with cobra venom factor (CVF) which depletes complement, preferentially blocks the haemorrhagic component, while leaving the swelling relatively unaltered. As swelling occurs before the haemorrhage is seen, it is possible that these factors act at a late stage in the cascade of events leading to the tissue damage. However, LPS and CVF were able to inhibit swelling and haemorrhage in the massive reactions elicited in pre-immunized animals, whereas the PAF inhibitor had no detectable effect.

Cytokines and the Koch phenomenon.

We outline the mechanisms contributing to the human form of the Koch phenomenon, which we define as necrosis occurring within 24-48 h of injection of mycobacterial antigen into the skin of past or present tuberculosis patients. It is probable that tissue damage mediated in the same way occurs in the lesions themselves. We suggest that the necrosis is mediated in part by cytokines, particularly Tumour Necrosis Factor (TNF), and that this occurs for three reasons. First, Mycobacterium tuberculosis evokes an immunoregulatory abnormality characterised by raised agalactosyl IgG. This abnormality, also found in rheumatoid arthritis, Crohn's disease, and Erythema Nodosum Leprosum, seems to be associated with dysregulation of cytokine release. Secondly, M. tuberculosis itself triggers further cytokine release. Thirdly, the normally protective role of TNF is distorted by several interacting properties of components of M. tuberculosis, which render the cytokine toxic to the host tissues. The immunoregulatory abnormality may be susceptible to correction by immunotherapy.

New insights into the immunopathology of tuberculosis.

Tuberculosis is characterised by fever, weight loss and necrosis in both lesions and tuberculin skin test sites (Koch phenomenon), although the antigens of Mycobacterium tuberculosis are not directly toxic to the tissues. The tissue damage appears to be due to several interacting factors. First, M. tuberculosis induces an immunoregulatory disorder of which a raised percentage of agalactosyl IgG is a marker. This is seen also in rheumatoid arthritis and Crohn's disease and is associated with tissue-damaging inflammation. Subsequently, several properties of M. tuberculosis exacerbate this disorder by triggering cytokine release, and rendering tissues sensitive to the toxicity of tumor necrosis factor (TNF). Moreover, M. tuberculosis, but not bacillus Calmette-Guérin or several Mycobacterium avium strains, produces a factor which increases the toxicity of TNF for individual cells. Thus, M. tuberculosis may distort the normal protective role of TNF so that this cytokine becomes toxic to the host. The immunoregulatory disorder associated with agalactosyl IgG appears to be susceptible to immunotherapy, so novel types of treatment for the immunopathological component of tuberculosis are being explored.