Patterns of T and B cell responses to Mycobacterium tuberculosis membrane-associated antigens and their relationship with disease activity in rheumatoid arthritis patients with latent tuberculosis infection.

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3-6, 9-12 and 15-18 months after baseline) were performed in 41 patients who were classified empirically as 'high' (HR-T/HR-B) or 'low' (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA.

Mucosal Therapy of Multi-Drug Resistant Tuberculosis With IgA and Interferon-γ.

New evidence has been emerging that antibodies can be protective in various experimental models of tuberculosis. Here, we report on protection against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) infection using a combination of the human monoclonal IgA 2E9 antibody against the alpha-crystallin (Acr, HspX) antigen and mouse interferon-gamma in mice transgenic for the human IgA receptor, CD89. The effect of the combined mucosal IgA and IFN-γ; treatment was strongest (50-fold reduction) when therapy was applied at the time of infection, but a statistically significant reduction of lung bacterial load was observed even when the therapy was initiated once the infection had already been established. The protection involving enhanced phagocytosis and then neutrophil mediated killing of infected cells was IgA isotype mediated, because treatment with an IgG version of 2E9 antibody was not effective in human IgG receptor CD64 transgenic mice. The Acr antigen specificity of IgA antibodies for protection in humans has been indicated by their elevated serum levels in latent tuberculosis unlike the lack of IgA antibodies against the virulence-associated MPT64 antigen. Our results represent the first evidence for potential translation of mucosal immunotherapy for the management of MDR-TB.

Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease.

Changes in expression of membrane antigens may accompany the transition of Mycobacterium tuberculosis (Mtb) from 'dormant' to 'active' states. We have determined whether antibody and T cell responses to Mtb membrane (MtM)-associated antigens, especially the latency-induced protein alpha crystallin (Acr), can discriminate between latent tuberculosis infection (LTBI) and active TB (ATB) disease. Study subjects comprised a previously described cohort of healthcare workers (HCWs, n = 43) and smear-positive ATB patients (n = 10). HCWs were further categorized as occupational contacts (OC, n = 30), household contacts of TB (HC, n = 8) and cured TB (CTB, n = 5). Levels (ΔOD) of serum antibody isotypes (IgG, IgA and IgM) were determined by ELISA and blood T cell proliferative responses were determined by flow cytometry using Ki67 protein as marker for DNA synthesis. Antibodies to MtM and Acr were predominantly IgG and their levels in HCWs and ATB did not differ significantly. However, HCWs showed a significantly higher level of anti-MtM IgM and a significantly lower level of anti-Acr IgA antibodies than the ATB patients. Also, a larger proportion of HCWs showed a high (>1) ΔODAcr/ΔODMtM ratio for IgG. HCWs also showed a higher, though not significantly different from ATB, avidity of anti-MtM (IgG) antibodies. A higher proportion of HCWs (35% of OC, 62.5% of HC and 20% of CTB), compared with ATB (10%) showed a positive T cell response to Acr along with significant difference (P <0.05) between HC and ATB. A significant correlation (r = 0.60, P <0.0001) was noted between T cell responses of HCWs towards Acr and MtM (reported earlier by us) and both responses tended to decline with rising exposure to the infection. Even so, positive responses to Acr (38.5%) were significantly lower than to MtM (92%). Neither antibody nor T cell responses to either antigen appeared affected by BCG vaccination or reactivity to tuberculin. Results of the study suggest that the levels of IgM antibodies to MtM, IgA antibodies to Acr and proliferative T cell responses to both the antigens can potentially discriminate between LTBI and active TB disease. They also underscore the necessity of SOPs for antibody assays.

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection.

One-third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single-center prospective observational study, we analyzed IgG-antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA-binding protein 1 (MDP1) and alpha-crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18-month follow-up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.

[On-line analysis of T and B cell epitopes of α-crystallin (Rv2031c) derived from Mycobacterium tuberculosis].

Objective To predict the epitopes of Mycobacterium tuberculosis dormancy-related protein α-crystallin (Rv2031c). Methods The homology between Rv2031c and human proteins was analyzed online by BLAST alignment. B- and T-cell epitopes of Rv2031c were predicted by Protean of DNAStar software. RANKPEP and SYPEPITHI were used to predict the epitopes of T helper (Th) cells; while SYFPEPI, BIMAS, and NetCTL were used to predict the epitopes of cytotoxic T lymphocytes (CTLs). Results Rv2031c had low homology with human proteins. Eight potential epitopes of B-cells, 7 epitopes of Th cells and 3 epitopes of CTLs were predicted in Rv2031c. Conclusion Rv2031c, which has many potential epitopes of Th cells, CTLs and B-cells, is expected to be a promising candidate for the development of tuberculosis vaccines.

Boosting with recombinant MVA expressing M. tuberculosis α-crystallin antigen augments the protection imparted by BCG against tuberculosis in guinea pigs.

Tuberculosis (TB) is one of the major causes of mortality all over the globe. BCG, the only vaccine available against this disease has been successful in preventing the severe forms of childhood TB. However, the unsatisfactory performance of BCG in controlling the adult pulmonary tuberculosis has made the development of an effective vaccine against M. tuberculosis a prime objective of the TB research. In this study, a genetically stable, marker-free recombinant MVA expressing α-crystallin of M. tuberculosis (rMVA.acr) was generated which was further evaluated for its ability to impart protection as a booster vaccine against tuberculosis in a heterologous prime boost approach. Our results demonstrated that intradermal delivery of rMVA.acr was able to efficiently boost the BCG induced protection against M. tuberculosis infection in guinea pigs by significantly reducing the pulmonary bacillary load (1.27 log10 fewer bacilli) in comparison to BCG vaccination alone. In addition, boosting BCG vaccinated animals with intramuscular delivery of rMVA.acr resulted in significantly superior protective efficacy in both lungs and spleen with 0.83 log10 and 0.74 log10 CFU fewer bacilli, respectively, when compared to animals vaccinated with BCG only. These findings establish the promise of this prime-boost strategy involving rMVA.acr in enhancing the efficacy of BCG.

Detection of anti-HspX antibodies and HspX protein in patient sera for the identification of recent latent infection by Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a pathogen causing tuberculosis (TB) a spectrum of disease including acute and asymptomatic latent stages. Identifying and treating latently-infected patients constitutes one of the most important impediments to TB control efforts. Those individuals can remain undiagnosed for decades serving as potential reservoirs for disease reactivation. Tests for the accurate diagnosis of latent infection currently are unavailable. HspX protein (α-crystallin), encoded by Rv2031c gene, is produced in vitro by M. tuberculosis during stationary growth phase and hypoxic or acidic culture conditions. In this study, using standard, and Luminex xMAP® bead capture ELISA, respectively, we report on detection of anti-HspX IgG and IgM antibodies and HspX protein in sera from acute and latent TB patients. For the antibody screen, levels of IgG and IgM antibodies were similar between non-infected and active TB patients; however, individuals classified into the group with latent TB showed higher values of anti-HspX IgM (p = 0.003) compared to active TB patients. Using the bead capture antigen detection assay, HspX protein was detected in sera from 56.5% of putative latent cases (p< 0.050) compared to the background median with an average of 9,900 pg/ml and a range of 1,000 to 36,000 pg/ml. Thus, presence of anti-HspX IgM antibodies and HspX protein in sera may be markers of latent TB.

Difference in Antibody Responses to Mycobacterium tuberculosis Antigens in Japanese Tuberculosis Patients Infected with the Beijing/Non-Beijing Genotype.

The Beijing genotype Mycobacterium tuberculosis (MTB), notorious for its virulence and predisposition to relapse, could be identified by spoligotyping based on genetic heterogeneity. The plasma samples from 20 cases of Beijing and 16 cases of non-Beijing MTB infected individuals and 24 healthy controls (HCs) were collected, and antibodies against 11 antigens (Rv0679c142Asn, Rv0679c142Lys, Ag85B, Ag85A, ARC, TDM-M, TDM-K, HBHA, MDP-1, LAM, and TBGL) were measured by ELISA. Compared to the HCs, the MTB infected subjects showed higher titers of anti-Ag85B IgG (positivity 58.2%) and anti-ACR IgG (positivity 48.2%). Of note, anti-ACR IgG showed higher titer in Beijing MTB infected tuberculosis (TB) patients than in HC (Kruskal-Wallis test, p < 0.05), while the levels of anti-Ag85B, anti-TBGL, anti-TDM-K, and anti-TDM-M IgG were higher in non-Beijing TB patients than in HC. Moreover, anti-Ag85B IgG showed higher response in non-Beijing TB patients than in Beijing TB patients (p < 0.05; sensitivity, 76.9% versus 44.4%). The sensitivity and specificity analysis showed that 78.8% Beijing infected individuals were negative in anti-TBGL-IgG or/and anti-Ag85B-IgG, while 75.0% of those were positive in anti-TBGL-IgA or/and anti-ACR-IgG tests. These results indicate the possibility of developing antibody-based test to identify Beijing MTB.

Active tuberculosis patients have high levels of IgA anti-alpha-crystallin and isocitrate lyase proteins.

SETTING: Mexico City, Mexico. OBJECTIVE: To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). DESIGN: Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. RESULTS: We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. CONCLUSION: ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.

DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection.

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.

Prime-boost vaccination strategy with bacillus Calmette-Guérin (BCG) and liposomized alpha-crystalline protein 1 reinvigorates BCG potency.

Bacillus Calmette-Guérin (BCG) remains the only available and most widely administered vaccine against Mycobacterium tuberculosis (Mtb), yet it fails to protect vaccinated individuals either from primary infection or reactivation of latent tuberculosis (TB). Despite BCG's variable efficacy against TB, the fact remains that BCG imparts protection in children against the disease, indicating that BCG possesses a wide protective antigenic repertoire. However, its failure to impart protection in adulthood can be linked to its failure to generate long-lived memory response and elicitation of an inadequate immune response against latency-associated antigens. Therefore, to improve the protective efficacy of BCG, a novel vaccination strategy is required. Consequently, in the present study, we have exploited the vaccination potential of liposomized α-crystalline 1 (Acr1L), a latency-associated antigen to induce enduring protective immunity against Mtb in BCG-primed animals. It is noteworthy that an increase in the multi-functional [interferon (IFN)-γ(hi) /tumour necrosis factor (TNF)-α(hi) ] CD4 and CD8 T cells were observed in BCG-primed and Acr1L-boosted (BCG-Acr1L) animals, compared to BCG alone. Further, substantial expansion of both central memory (CD44(hi) /CD62L(hi) ) and effector memory (CD44(hi) /CD62L(lo) ) populations of CD4 and CD8 T cells was noted. Importantly, BCG-Acr1L exhibited significantly better protection than BCG, as evidenced by a reduction in the bacterial burden and histopathological data of the lungs. In essence, BCG-Acr1L could be a potent future vaccination strategy to reinvigorate BCG potency.

T cell response to alpha crystallin and Mycobacterium tuberculosis specific antigens using ex-vivo elispot assay for detecting latent tuberculosis infection in Addis Ababa, Ethiopia.

BACKGROUND: Mycobacterium tuberculosis (Mtb) persists in a state of non-replication or stationary phase, but resulting in active tuberculosis (TB) when the immune system is suppressed. Alpha-crystallin (ACR) is one of the bacterial antigens characterized known to be related to shifting of the bacilli from growth to a non-replicating persistent state. OBJECTIVE: To compare the ex-vivo responsiveness of active TB patients, close household contacts and healthy controls to specific Mtb antigens. METHODOLOGY: Antigen-specific interferon-gamma (IFN-g) responses were measured to a 16kDa-alpha crystallin (ACR) antigen along with its peptides and other Mtb antigens (ESAT-6, CFP-10, PPD, TB10.3 and Ag85A) in 39 active TB patients, 23 close household contacts and 25 community controls, using ex-vivo ELISPOT RESULT: The proportion of responders to ACR was 36% in active TB patients (76 +/- 14 spot forming cells), 48% in close household contacts (123 +/- 31 spot forming cells) and 76% in community controls (165 +/- 29 spot forming cells) indicating the presence of latency more in the community controls compared to the other groups. Sixty percent of community controls (131 +/- 27 spot forming cells), 61% of healthy household contacts (138 +/- 3 spot forming cells) and 54% of TB patients (198 +/- 37 spot forming cells) showed ESAT-6-specific T cell responses. CONCLUSION: Antigen specific T cell response based on ex-vivo ELISPOT assay using combined ACR and ESAT-6/ CFP-10 antigens can be used as indicator of underlying latent TB infection in tropical setting where tuberculosis is endemic.

Latency-associated protein Acr1 impairs dendritic cell maturation and functionality: a possible mechanism of immune evasion by Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) in latently infected individuals survives and thwarts the attempts of eradication by the immune system. During latency, Acr1 is predominantly expressed by the bacterium. However, whether M. tuberculosis exploits its Acr1 in impairing the host immunity remains widely unexplored. Hence, currently we have investigated the role of Acr1 in influencing the differentiation and function of dendritic cells (DCs), which play a cardinal role in innate and adaptive immunity. Therefore, for the first time, we have revealed a novel mechanism of mycobacterial Acr1 in inhibiting the maturation and differentiation of DCs by inducing tolerogenic phenotype by modulating the expression of PD-L1; Tim-3; indoleamine 2, 3-dioxygenase (IDO); and interleukin 10. Furthermore, Acr1 interferes in the differentiation of DCs by targeting STAT-6 and STAT-3 pathways. Continuous activation of STAT-3 inhibited the translocation of NF-κB in Acr1-treated DCs. Furthermore, Acr1 also augmented the induction of regulatory T cells. These DCs displayed decline in their antigen uptake capacity and reduced ability to help T cells. Interestingly, M. tuberculosis exhibited better survival in Acr1-treated DCs. Thus, this study provides a crucial insight into a strategy adopted by M. tuberculosis to survive in the host by impairing the function of DCs.

Adjunctive immunotherapy with α-crystallin based DNA vaccination reduces Tuberculosis chemotherapy period in chronically infected mice.

By employing modified Cornell model, we have evaluated the potential of adjunctive immunotherapy with DNA vaccines to shorten the tuberculosis chemotherapy period and reduce disease reactivation. We demonstrate that α-crystallin based DNA vaccine (DNAacr) significantly reduced the chemotherapy period from 12 weeks to 8 weeks when compared with the chemotherapy alone. Immunotherapy with SodA based DNA vaccine (DNAsod) reduced the pulmonary bacilli only as much as DNAvec. Both DNAacr and DNAsod, although significantly delayed the reactivation in comparison to the chemotherapy alone, this delay was associated with the immunostimulatory sequences present in the vector backbone and was not antigen specific. Both DNA vaccines resulted in the production of significantly higher number of TEM cells than the chemotherapy alone, however, only in the case of DNAsod, this enhancement was significant over the DNAvec treatment. Overall, our findings emphasize the immunotherapeutic potential of DNAacr in shortening the duration of TB chemotherapy.

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis.

BACKGROUND: In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB. METHODS/PRINCIPAL FINDINGS: In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin--a key latency antigen of M. tuberculosis to boost the BCG induced immunity. 'BCG prime-DNA boost' regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log(10) and 1.96 log(10) fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3(+)) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3(+) Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.

A novel human IgA monoclonal antibody protects against tuberculosis.

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.

Clinical usefulness of alpha-crystallin antibodies in non-small cell lung cancer patients.

The non-invasive approach of finding biomarkers in peripheral blood of cancer patients makes it useful for clinical application and cancer screening. The aim of the study was to explore the clinical utility of alpha-crystallin antibodies as markers for diagnosis of non-small cell lung cancer (NSCLC) and screening among high-risk groups. Alpha-crystallin antibodies were detected with enzyme-linked immunosorbent assay (ELISA) in 51 NSCLC patients, 38 high-risk chronic obstructive pulmonary disease (COPD) patients and 52 age and sex matched healthy volunteers. Alpha-crystallin IgG antibodies differed significantly between the groups of cancer patients and the healthy volunteers (P<0.001). A cut-off value of 0.317 discerned NSCLC patients with sensitivity 62%, and specificity 72% among the control group. The assay was effective in distinguishing the patients with and without lymphogenic metastatic spread of the disease (P=0.045): sensitivity 60%, and specificity 70%. The clinical significance of this marker has a modest implication in lung cancer diagnosis and screening in high-risk groups. Its importance as a prognostic marker or a marker of disease recurrence and lymph node micrometastasis should be further explored.

Prevention of the post-chemotherapy relapse of tuberculous infection by combined immunotherapy.

We report that a recently developed combined immunotherapy (CIT) has the capacity to prevent a spontaneous relapse of replicating Mycobacterium tuberculosis bacilli in the lungs of BALB/c, C57Bl/6 or C3H/HeJ strains of mice, following 4weeks of non-sterilising treatment with isoniazid and rifampicin. The CIT regimen, represented by recombinant IFNgamma, anti-alpha crystalline monoclonal IgA antibody and IL-4 neutralizing polyclonal antibody, reduced the 8-week relapse of viable bacterial counts in the lungs most significantly, when CIT was inoculated during the 5th week post infection, i.e. during the 3rd week of chemotherapy. Although CIT enhanced lung granuloma area, nitric oxide, cytokine and chemokine levels in lung washings significantly, these could not be directly associated with the beneficial effect of CIT on the degree of relapse in the lungs. These results represent a proof-of-principle, that the described CIT, when combined with chemotherapy, could have potential for future development of a shorter regimen of tuberculosis treatment.

High incidence of antibodies to lens proteins in sera from patients with uveitis.

BACKGROUND: Uveitis is an intraocular inflammatory disease in which autoimmune reactions have been discussed in playing an important role. Many data in this respect derived from the animal model of "experimental autoimmune uveitis", where several organ-specific autoantigens have been described such as the retinal S-antigen and the interphotoreceptor retinoid-binding protein. However, their diagnostic and pathogenic role in humans has been controversially discussed. In recent studies, the possible relevance of betaB1-crystallin, present in lens and ciliary body, has been outlined. We, therefore, wanted to analyse whether sera from patients with uveitis might contain antibodies to lens proteins METHODS: Human lenses from cadaveric eyes were shock frozen, homogenized, and resuspended. The resulting suspension (human lens protein fraction--HLPF) was analyzed for antigenicity by ELISA and Western blotting with patients' sera. A total of 165 patients with uveitis, 54 patients with scleritis and episcleritis, 56 patients with other eye diseases, and 112 healthy blood donors were studied. RESULTS: Twenty-six (49%) of the 53 patients with anterior uveitis, 17 (32%) of the 53 patients with intermediate uveitis and 7 (22%) of 32 patients with posterior uveitis reacted in the ELISA with the HLPF. Antibodies to lens antigens were detected in one-third of patients with panuveitis and retinal vasculitis. In contrast, only 12% of the healthy blood donors were positive in the ELISA. The number of patients with an autoimmune response to alpha-crystallins in Western blot predominated in all investigated groups. CONCLUSIONS: These data indicate that antibodies to lens proteins occur in a high incidence in sera from patients with uveitis. Forty-nine percent of the patients with anterior uveitis and only 12% of healthy controls were positive in the ELISA. In our groups of patients and controls the autoantibodies reacted in the Western blot predominantly with alpha-crystallin. Further studies are required to analyze in more detail the clinical and etiopathogenetic relevance of the antilens antibodies in uveitis.

Analysis of IgG antibody patterns against retinal antigens and antibodies to alpha-crystallin, GFAP, and alpha-enolase in sera of patients with "wet" age-related macular degeneration.

BACKGROUND: The aim of this study was to compare the IgG antibody patterns against retinal antigens in sera of patients with age-related macular degeneration (AMD) and healthy subjects to learn more about possible immunological aspects of this disease and to identify some of the most important antigens. METHODS: Sera of 140 patients were analyzed: healthy volunteers (CO, n=101) and patients with "wet" age-related macular degeneration (AMD, n=39). The sera were tested against western blots of bovine retinal antigens. The IgG antibody patterns were analyzed by multivariate statistical techniques and some antigens were identified via LC-MS/MS. RESULTS: All patients showed complex patterns of IgG antibodies against retinal antigens. The discriminant analysis revealed a statistical significant difference between the antibody profiles of the AMD and the CO group (P=0.000023). Not only up-regulations of antigen-antibody-reactivities in the AMD group at some molecular weight ranges, e.g. at 46 and 52 kDa, could be seen, but also down-regulations, e.g. at 18 and 36 kDa. The 18 kDa antigen band was identified as alphaB-crystallin, the band at 46 kDa as alpha-enolase, and one at 52 kDa as glial fibrillary acidic protein. CONCLUSIONS: We could demonstrate that both groups (wet AMD and CO) show complex IgG antibody patterns against retinal antigens, which are highly specific for each group. This provides further hints for the immunological basis of the disease. These changes in the antibody profiles in "wet" AMD could represent a secondary response to retinal damage or can play a causative role in the disease.