[ROLE OF VARIOUS ANTIGENIC PREPARATIONS OF FRANCISELLA TULARENSIS IN FORMATION OF ALLERGY REACTION IN HUMANS AND ANIMALS].

AIM: Study the role of LPS in induction of anti-tularemia immunity in humans and animals. MATERIALS AND METHODS: Activity of various antigenic preparations of tularemia microbe, including highly purified from protein and S- and R-LPS, was studied using leukocytolysis reaction with blood of vaccinated humans and guinea pigs and skin allergy test (guinea pigs). RESULTS: Only the whole cells of Francisella tularensis, killed in protein non-denaturating conditions and conserving full S-LPS structure (tularin⁺) were shown to be inductors of delayed-type hypersensitivity reaction. Alterations in LPS structure (tularin⁻) results in a significant decrease, and denaturation of bacterial proteins (during boiling) results in a complete loss of immune stimulating properties of the preparations. Purified LPS preparations and O-polysaccharide fraction of S-LPS are not able to activate cell-mediated immunity. CONCLUSION: The presence of LPS with the full structure affects the ability of antigenic preparations of F. tularensis to cause allergic reactions, and thus, form cell-mediated antitularemia immunity. LPS of F. tularensis can not be excluded as an adjuvant and provides the most effective presentation of epitopes of protein molecules for interaction with receptors of T-lymphocytes.

[Dual role of photosensibilizator merocyanine 540 as a bactericidal agent and immune reaction regulator].

AIM: Develop conditions for inactivation of staphylococcus by using photosensibilizator merocyanine 540 (MC540) for the production of antigenic preparation (AP). Study some of immune reactions to AP and the possibility of regulation of DTH reaction to AP under the effect of MC540. MATERIALS AND METHODS: Merocyanine 540 (MC540, Sigma-Aldrich, Switzerland) is used in the study. MC540 and Staphylococcus aureus, strain 78 (Sa78) were irradiated by light of a mercury-quartz lamp DRSH-250 (Zelenograd). C56BL/6 line mice were immunized once by subcutaneous administration of AP. DTH reaction was tested 7 days after the immunization. Functional activity of peritoneal exudate macrophages was determined 1 and 9 days after the immunization. Immune modulating effect of MC540 in DTH was determined after its per os administration to mice 1 hour after AP sensibilization. RESULTS: In order to obtain AP, S. aureus suspension at the concentration of 2.5 x 10(7) CFU/ml in 25 microM MC540 solution and 0.25 M NaCl solution were exposed to irradiation for 5 minutes. During DTH reaction induction its intensity dependence on AP dose was revealed. A persistent increase of a lysosomatic enzyme cathepsin D in macrophages of peritoneal exudate after a single administration of AP was noted. During MC540 irradiation an accumulation of photoproducts that have a pronounced immune suppression effect in DTH reaction had a dose-dependent character. CONCLUSION: Use of saline allows to increase bactericidal potential of a photosensibilizator (PS). However during therapy of localized forms of infection a possible immune modulating effect of PS on macro organism should be considered. By varying PS dose and irradiation time not only maximum bactericidal effect can be achieved but also regulation of inflammatory reactions in the area of PS effect can be ensured.

Refractory Trichophyton rubrum infection in lamellar ichthyosis.

A 10-month-old boy with congenital lamellar ichthyosis presented with a chronic Trichophyton rubrum infection. There was no history of atopy or immunosuppression, and examination revealed high total immunoglobulin E (IgE) with a positive specific IgE for T. rubrum. Multiple treatments with fluconazole were necessary to control the infection. T. rubrum is present worldwide and is responsible for the vast majority of chronic dermatophytosis. Lamellar ichthyosis is a risk factor for chronic dermatophytosis because of excessive keratin and the barrier defect. A delayed-type hypersensitivity reaction to T. rubrum is associated with cure, whereas immediate hypersensitivity and IgE are not protective and may lead to chronic infection. Atopy and the Th2 profile therefore seem to be associated with chronic dermatophytosis. The association between ichthyosis and atopy is well documented. T. rubrum also has an interesting ability to evade immunity, which helps explain the chronic infection. Finally, in ichthyosis, it is likely that fluconazole has difficulty penetrating the acanthotic stratum corneum, which explains treatment failure. We report this case to alert clinicians to the possible association between lamellar ichthyosis and chronic dermatophytosis and to report the difficulties of management.

Suppression of experimental autoimmune encephalomyelitis by extracellular adherence protein of Staphylococcus aureus.

Multiple sclerosis (MS) is a devastating inflammatory disorder of the central nervous system (CNS). A major hallmark of MS is the infiltration of T cells reactive against myelin components. T cell infiltration is mediated by the interaction of integrins of the beta1 and beta2 family expressed by lymphocytes with their endothelial counter-receptors, vascular cell adhesion molecule 1 and intercellular adhesion molecule (ICAM)-1, respectively. We have reported previously that extracellular adherence protein (Eap) of Staphylococcus aureus exerts antiinflammatory activities by interacting with ICAM-1 and blocking beta2-integrin-dependent neutrophil recruitment. Here, we report that Eap inhibits experimental autoimmune encephalomyelitis (EAE) in mice. In vitro, Eap reduced adhesion of peripheral blood T cells to immobilized ICAM-1 as well as their adhesion and transmigration of TNF-activated human endothelium under static and shear flow conditions. These inhibitory effects were corroborated in two mouse models of inflammation. In a delayed-type hypersensitivity model, both T cell infiltration and the corresponding tissue edema were significantly reduced by Eap. In addition, Eap administration prevented the development of EAE and markedly decreased infiltration of inflammatory cells into the CNS. Strikingly, intervention with Eap after the onset of EAE suppressed the disease. Collectively, our findings indicate that Eap represents an attractive treatment for autoimmune neuroinflammatory disorders such as MS.

Immunization by a bacterial aerosol.

By manufacturing a single-particle system in two particulate forms (i.e., micrometer size and nanometer size), we have designed a bacterial vaccine form that exhibits improved efficacy of immunization. Microstructural properties are adapted to alter dispersive and aerosol properties independently. Dried "nanomicroparticle" vaccines possess two axes of nanoscale dimensions and a third axis of micrometer dimension; the last one permits effective micrometer-like physical dispersion, and the former provides alignment of the principal nanodimension particle axes with the direction of airflow. Particles formed with this combination of nano- and micrometer-scale dimensions possess a greater ability to aerosolize than particles of standard spherical isotropic shape and of similar geometric diameter. Here, we demonstrate effective application of this biomaterial by using the live attenuated tuberculosis vaccine bacille Calmette-Guérin (BCG). Prepared as a spray-dried nanomicroparticle aerosol, BCG vaccine exhibited high-efficiency delivery and peripheral lung targeting capacity from a low-cost and technically simple delivery system. Aerosol delivery of the BCG nanomicroparticle to normal guinea pigs subsequently challenged with virulent Mycobacterium tuberculosis significantly reduced bacterial burden and lung pathology both relative to untreated animals and to control animals immunized with the standard parenteral BCG.

Biological properties of an influenza A virus-specific killer T cell clone. Inhibition of virus replication in vivo and induction of delayed-type hypersensitivity reactions.

We tested two biological properties of a continuously growing mouse cytotoxic T cell line, L4, which is specific for influenza A virus and has been cloned and recloned many times. We previously reported that L4 cells are H-2 restricted and cross-reactive for all type A influenza viruses, whereas they do not recognize type B influenza viruses. They bear Thy-1 and Lyt-2 markers. In the present study, we show that L4 cytotoxic T cells protect mice against a lethal influenza infection on transfer to syngeneic recipients, and reduce virus titers in the lungs of mice challenged with a heterologous type A influenza virus. This provides further support for the active role of cytotoxic T cells in limiting virus replication in influenza infection. We could also demonstrate that the cloned cytotoxic T cells induce a delayed-type hypersensitivity skin reaction in the footpads of mice challenged with live or inactivated influenza virus. This reaction can be observed at 24 h, but has declined by 48 h. A clone of cells derived from L4 that has lost its cytotoxic potential and its ability to recognize infected cells did not induce a delayed-type hypersensitivity reaction in the presence of virus. Thus, cytotoxic T cells actively killing influenza virus-infected cells are able to induce a delayed-type hypersensitivity skin reaction to homologous and heterologous type A influenza viruses.

Trehalose dimycolate elicits eosinophilic skin hypersensitivity in mycobacteria-infected guinea pigs.

Delayed-type hypersensitivity represents high levels of protein Ag-specific adaptive immunity induced by mycobacterial infection, and can be monitored in the Ag-challenged skin. Besides protein Ags, recent evidence has suggested that a substantial immunity directed against glycolipid Ags is also elicited in response to mycobacterial infection, but skin hypersensitivity to this class of Ags has not been fully assessed. To address this issue directly, glycolipid-specific skin reactions were evaluated in guinea pigs infected with Mycobacterium avium complex (MAC). Significant skin induration was observed in MAC-infected, but not mock-infected, guinea pigs, following intradermal administration of a mixture of MAC-derived glycolipids. Surprisingly, this glycolipid-specific skin response involved up-regulated expression of IL-5 mRNA in situ and marked local infiltration of eosinophils. Challenge experiments with individual glycolipid components detected an outstanding capability for trehalose dimycolate (TDM), but not a structurally related glycolipid, glucose monomycolate, to elicit the skin response. T lymphocytes derived from the spleen of MAC-infected, but not uninfected, guinea pigs specifically responded to TDM in vitro by up-regulating IL-5 transcription, and this response was not blocked by Abs that reacted to the known guinea pig group 1 CD1 proteins. Finally, the eosinophilic skin hypersensitivity to TDM was also elicited in guinea pigs vaccinated with bacillus Calmette-Guerin, which contrasted sharply with the classical delayed-type hypersensitivity response to the purified protein derivative. Therefore, the TDM-elicited eosinophilic response defines a new form of hypersensitivity in mycobacterial infection, which may account for local infiltration of eosinophils often observed at the site of infection.

Group A streptococci: localization in rabbits and guinea pigs following tissue injury.

Rabbits injected intravenously with extracellular products ("toxins") of group A streptococci develop myocardial, muscular, and hepatic lesions. When such animals are then challenged with fluorochrome-labeled group A streptococci or with titanium oxide particles the labeled bacteria or particles localize within phagocytic cells in the tissue lesions caused by the toxins. Similarly, labeled streptococci or titanium oxide particles will also localize within phagocytic cells in skin lesions of guinea pigs that develop delayed hypersensitivity to tuberculin or to bovine gamma globulin. It is proposed that a combined mechanism of injury and localization of bacteria in damaged tissues may be responsible for poststreptococcal sequelae or other chronic inflammatory diseases.

Induction of delayed-type hypersensitivity and interferon-gamma to Candida albicans and anti-hen-egg white lysozyme antibody as phenotypic markers of enhanced bovine immune response.

Delayed-type hypersensitivity (DTH) is a protective localized cell-mediated immune response (CMIR), primarily against intracellular pathogens. DTH is widely used in research to assess immune responsiveness and has been a valuable diagnostic test in commercial settings. In pigs and other species both antibody (AMIR) and CMIR have been considered as reliable phenotypic markers of selection programs for disease resistance. Therefore in cattle, it was also considered important to find antigen/adjuvant combinations capable of inducing AMIR and CMIR without interfering with diagnostic tests. The objectives of the present study were to evaluate the combined use of hen-egg white lysozyme (HEWL) and Candida albicans adjuvanted with Quil A in lactacting Holstein cows for the induction of anti-HEWL antibody, as well as DTH and IFN-gamma to C. albicans as phenotypic markers of enhanced immune responsiveness. Thirty one lactating Holstein cows were immunized with HEWL to induce antibody responses and C. albicans to sensitize for DTH. Two test antigens, candin and C. albicans whole cell (CaWC), were used to induce the effector phase of DTH. PBS was used as the negative control. In addition, two different skin sites (neck versus tail) were tested to evaluate differences in skin site responsiveness. C. albicans-induced IFN-gamma production, as an indicator of a type 1 response, was evaluated by ELISA. Microscopic evaluation of skin samples at DTH sites was performed in five randomly selected cows and these skin biopsies were scored based on inflammation and cell infiltration. Results demonstrated the presence of classical DTH response to C. albicans, in that DTH responses peaked at 24 h post-intradermal injections and cell infiltration was composed largely of mononuclear leukocytes, typical of DTH skin reactions in cattle. The only difference in test antigens was that DTH to candin showed a higher early response (6 h) than CaWC and a rapid decrease in inflammation from 24 to 48 h. The neck was significantly more sensitive than the tail skin-fold as a DTH test site. IFN-gamma was detected on days 14 and 21 post-immunization in plasma from blood incubated with candin. Significant primary and secondary anti-HEWL antibodies were also detected, indicating that this combination of test antigens could be used as phenotypic markers of immune responsiveness in cattle.

Sex Differences in Mouse Popliteal Lymph Nodes.

Females have more robust immune responses than males, well-illustrated by the degree of inflammation elicited during delayed-type hypersensitivity (DTH) responses. Here, we have investigated underlying sex differences that may contribute to differential footpad DTH responses using wildtype and four core genotypes (FCG) mice and popliteal lymphnode cellularity and gene expression. DTH responses in XX and XY FCG females showed no role for almost all genes expressed on sex chromosomes. After then filtering-out genes differentially expressed between XX and XY females, only one gene was sexually differentially expressed in wildtype mice, glycosylation-dependent cell adhesion molecule 1 (Glycam1), expressed 7-fold higher in females. Glycam1 facilitates leukocyte entry through high endothelial venules. Consistent with greater Glycam1 expression, female nodes contained twice as many cells. While females had more memory T cells in their nodes, males had a higher percentage of T regulatory cells. This sexual dimorphism in wildtype animals manifested pre-pubertally, was enhanced post-pubertally, and was eliminated by castration. The formation of male gonads is determined by the expression of Sry. Sry overexpression, which does not affect testosterone levels, produced an exaggerated male phenotype. We conclude that Sry expression through formation of the male gonad indirectly negatively impacts the potential for local inflammation.

The relative importance of CD4+ and CD8+T cells in immunity to pulmonary paracoccidioidomycosis.

Protective immunity in paracoccidioidomycosis (PCM) is believed to be mediated by cellular immunity, but the role of T cell subsets has never been investigated. The aim of this study was to characterize the function of CD4+ and CD8+ T cells in the immunity developed by susceptible, intermediate and resistant mice after P. brasiliensis infection. In susceptible mice, depletion of CD4+ T cells did not alter disease severity and anergy of cellular immunity but diminished antibody production. Anti-CD8 treatment led to increased fungal loads, but restored DTH reactivity. In resistant mice, both CD4+ and CD8+ T cells control fungal burdens and cytokines although only the former regulate DTH reactions and antibody production. In the intermediate strain, deficiency of whole T and CD8+ T cells but not of CD4+ T or B cells led to increased mortality rates. Thus, in pulmonary PCM: (a) irrespective of the host susceptibility pattern, fungal loads are mainly controlled by CD8+ T cells, whereas antibody production and DTH reactions are regulated by CD4+ T cells; (c) CD4+ T cells play a protective role in the resistant and intermediate mouse strains, whereas in susceptible mice they are deleted or anergic; (d) genetic resistance to PCM is associated with concomitant CD4+ and CD8+ T cell immunity secreting type 1 and type 2 cytokines.

Investigations and implications of associations between mycobacterial purified protein derivative hypersensitivity and MAP-antibody ELISA in Irish dairy cows.

Intradermal testing, involving administration of purified protein derivative (PPD), to elicit a delayed hypersensitivity (DTH) response, is used as a diagnostic tool for bovine tuberculosis (bTB) and to aid in the identification of exposure to Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD). Further research is required to increase the diagnostic value of skin testing for MAP. The aim of this study was to investigate if animals showing DTH reactions to PPD had an associated increase in MAP ELISA response, thereby identifying potential cases of sub-clinical JD. A 139-cow dairy herd was recruited to the study. During the mandatory annual bTB test, skin thickness measurements (mm) were recorded at the site of avian and bovine PPD administration. Cows were categorised based on recording no DTH, DTH at both PPD administration sites and DTH at one PPD site only. Blood samples were collected pre and post bTB testing, and ELISA tested. Generalised estimating equations were performed to identify associations between DTH responses and MAP ELISA results. Significant associations were identified between PPD DTH responses and MAP ELISA readings. Animals with DTH at both avian and bovine PPD sites were most likely to test ELISA positive in the post-PPD period relative to other categories. Further research is required to identify whether skin thickness increases post-PPD and associated increase in ELISA response, identifies animals previously exposed to MAP, or if results are due to cross reactivity.

The effect of tuberculin testing on the development of cell-mediated immune responses during Mycobacterium bovis infection.

Protection against tuberculosis (TB) is associated with Th1-type cell-mediated immunity (CMI). Whilst the intradermal injection of partially purified derivatives of tuberculin (PPD) represents the classic test assessing the delayed type hypersensitivity (DTH) response used in both humans and cattle for diagnosing TB, it has been suggested that the test may modulate host CMI responses. To investigate the kinetics of the development of the DTH response and its subsequent effect on CMI responses, groups of 6-month old calves were inoculated intranasally with 8 x 10(4) cfu of Mycobacterium bovis, subjected to the comparative intradermal tuberculin test (TT) using bovine and avian PPD (PPD-B, PPD-A) at various time intervals post-infection, and immune responses compared. These included DTH, lymphocyte proliferation, IgG production, and synthesis of the cytokines: IFNgamma, IL-10, IL-4, IL-6, and IL-13. All animals were subjected to post-mortem examination. The kinetics of the development of the DTH response assessed in the TT was such that infected cattle could be identified as early as 3 weeks post-infection, which correlated with the detection of an antigen-specific IFNgamma response. Transient increases in plasma-derived IFNgamma as a result of TT during an established TB infection were more pronounced when blood was stimulated with PPD-A compared with PPD-B stimulation. This has the potential to mask diagnosis of infection as a result of the stronger avian-bias if the IFNgamma test is used the week following TT. Disease pathology was not affected by TT. A transient failure to a second TT was observed in 1 of 30 animals and the time (post-infection) at which the TT is administered may be of significance. In serum, IgG responses to PPD-B, which were undetectable prior to TT, were elevated after TT and were most pronounced in cattle that were TT at 6 weeks post-infection. Other cytokines were also affected by the TT; IL-4 mRNA levels increased and IL-6 mRNA levels decreased, whilst PPD-B specific IL-10 protein synthesis was enhanced. These observations may offer the potential for further diagnostic assays that could complement the TT and IFNgamma test.

Delayed-type hypersensitivity to a recombinant mycobacterial antigen, MPB64, in guinea pigs sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG.

Recombinant MPB64 (rMPB64), a mycobacterial antigen, was obtained from an Escherichia coli clone transformed with a recombinant expression vector, pMAL64c. The rMPB64 was examined for the activity to elicit delayed-type hypersensitivity (DTH) in guinea pigs injected with liver Mycobacterium tuberculosis H37Rv or live M. bovis BCG Tokyo. It was found that rMPB64 has the same reactivity as native MPB64 (nMPB64) or MPT64 (nMPT64) and the potency to elicit DTH was 13.4 times higher than that of PPD. Because MPB64 is secreted only by living M. tuberculosis and some strains of BCG, it is possible to use this antigen for the diagnosis of tuberculosis.

Identification of a novel immunodominant antigen Rv2645 from RD13 with potential as a cell-mediated immunity-based TB diagnostic agent.

Search for novel specific antigens are urgently needed for the detection of tuberculosis (TB). In this study, we evaluated the diagnostic potential of a novel Mycobacterium tuberculosis (M.tb)-specific candidate antigen (Rv2645) from DNA segment region of differentiation (RD) 13 of M.tb and investigated T-cell recognition during natural infection in humans and experimental mice. Rv2645-specific IFN-γ levels were much higher in the peripheral blood mononuclear cells (PBMCs) of TB patients than that in healthy donors (HDs) (including Bacille Calmette-Guerin (BCG)-vaccinated donors). The enzyme-linked immunospot (ELISPOT) assay with Rv2645 had a high overall agreement (98.0%) with the results from the clinical T-SPOT.TB with 10-kD culture filtrate protein (CFP10) and 6-kD early secreted antigenic target (ESAT6) peptides. The combination of Rv2645 and CFP10-ESAT6 was better than the individual protein, with increased sensitivity and a similar specificity of 96.0% and 98.2%, respectively. Rv2654 also induced M.tb-specific skin-test responses in heat-inactivated M.tb H37Rv immunized mice. Epitope mapping revealed that Rv264530-44 and Rv2645136-143 may be the dominant T-cell and B-cell epitopes, respectively, of Rv2645. This is the first report demonstrating the Rv2654 is a strongly recognized T-cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.

Establishment of protective immunity against cerebral cryptococcosis by means of an avirulent, non melanogenic Cryptococcus neoformans strain.

The opportunistic fungal pathogen, Cryptococcus neoformans, shows a marked predilection for the central nervous system (CNS). This can be partially explained by its ability to synthesize melanin starting from the catecholamines, highly concentrated at the CNS level. Two cryptococcal strains, the avirulent non-melanogenic strain Sb26 and the virulent melanogenic revertant strain Sb26Rev, were used in a murine model of intracerebral (i.c.) infection, in order to evaluate their virulence and immunomodulating properties at the cerebral level. We found that, unlike Sb26Rev, Sb26 i.c. infection was never lethal regardless of the challenging dose. Sb26Rev infection resulted in massive CNS tissue damage, associated with little or no cytokine response, as established by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Differently, Sb26 infection failed to alter CNS structure, while inducing IL-12 p40, TNF-alpha, IL-1beta, IFN-gamma and iNOS specific-gene expression as well as IL-12, TNF-alpha and IL-1beta cytokine production. Interestingly, all Sb26 infected mice survived a subsequent lethal challenge with Sb26Rev. The phenomenon was associated with enhanced IL-12, TNF-alpha and IL-1beta production and was strictly specific, as shown by heterologous challenges and delayed type of hypersensitivity assay. Overall, we provide evidence that protective immunity against cerebral cryptococcosis is established by means of an avirulent strain of C. neoformans.

Role of T-cell subsets in protection, delayed type of hypersensitivity and granuloma formation during Yersinia enterocolitica infection in mice.

Studies were undertaken with (C57BL/6 x DBA/2) B6D2 F1 mice as a prototype of a strain resistant to Y. enterocolitica. The growth of Y. enterocolitica in liver and spleen following intravenous infection was determined. Restriction of growth of Y. enterocolitica in the spleen and liver started, when a delayed type of hypersensitivity (DTH) became elicitable. Mice were treated with monoclonal antibodies (mAb) specific to T-cell surface markers; injection of these antibodies leads to marked depletion of the specific T-cell subset. After selective in vivo depletion the three characteristic T-cell mediated phenomena, DTH, anti-bacterial protection and granuloma formation were investigated. DTH to Y. enterocolitica soluble antigen was abolished in mice treated with anti-Thy1.2 or anti-CD4 mAbs, while anti-CD8 mAbs had no effect. The elimination of bacteria from the spleens of infected animals was inhibited by the application of either anti-Thy1.2 or anti-CD8 mAbs, while anti-CD4 mAbs had a marginal effect on anti-bacterial protection. The accelerated development of mononuclear cell foci in the liver of immune mice was also inhibited by the application of anti-CD4 and anti-CD8 mAbs. Thus, it appears that specific T-lymphocytes play an important role in murine Yersiniosis. The present model is valuable for the investigation of the cellular immune response to this important enteric pathogen.

Delayed-type hypersensitivity to Mycobacterium leprae soluble antigens as a test for infection with the leprosy bacillus.

BACKGROUND: Mycobacterium leprae (M. leprae) soluble antigen (MLSA) reagents have been developed with the aim of finding a reagent, comparable to tuberculin, which could identify individuals infected with the leprosy bacillus. They have yet to be evaluated fully in human populations. METHODS: More than 15000 individuals living in a leprosy endemic area of northern Malawi were skin tested with one of five batches of MLSA prepared using two different protocols. The main difference in preparation was the introduction of a high G centrifugation step in the preparation of the last three ('second-generation') batches. RESULTS: The prevalence of skin-test positivity (delayed-type hypersensitivity (DTH)) and association with the presence of a BCG scar were greater for first (batches A6, A22) than second (batches AB53, CD5, CD19) generation reagents. The association of positivity with M. leprae infection was investigated by comparing results among known (household) contacts of leprosy cases, and among newly diagnosed leprosy patients with those in the general population. While positivity to 'first-generation' antigens appeared to be associated with M. leprae infection, positivity to later antigens was unrelated either to exposure to leprosy cases or presence of leprosy disease. There were geographical differences in the prevalence of DTH to the various batches, probably reflecting exposure to various mycobacteria in the environment. CONCLUSIONS: Our results suggest that the 'second-generation' batches have lost antigens that can detect M. leprae infections, but that they retain one or more antigens which are shared between M. leprae and environmental mycobacteria. Natural exposure to these both sensitizes individuals and provides natural protection against M. leprae infection or disease. Identification of antigens present in these groups of skin test reagents may assist in production of improved skin test reagents.

The detection of Mycobacterium leprae protein and carbohydrate antigens in skin and nerve from leprosy patients with type 1 (reversal) reactions.

Type 1 (reversal) reactions are the most common immunological complications of leprosy. These episodes of delayed hypersensitivity produce severe local immunopathology and ultimately nerve damage. To date, the Mycobacterium leprae antigens associated with type 1 reactions have not been identified. Using monoclonal antibodies to defined protein and carbohydrate M. leprae epitopes (65, 35 and 28 kd and lipoarabinomannan [LAM]) in a two-step immunoperoxidase staining technique, M. leprae antigens were demonstrated in skin and nerve biopsies from patients in reversal reaction. Antigen presence and staining patterns were similar in skin and nerve lesions, implying that the pathological processes are similar in the two sites. Antigens were present both in macrophages and Schwann cells but also as a diffuse extracellular infiltrate associated with the inflammatory infiltrate. The 28-kd antigen was present most strongly and may be a potential candidate antigen for initiating type 1 reactions. LAM also stained strongly and persisted after treatment. The possible roles of LAM and 65 kd in the cellular events of type 1 reactions are discussed.

The effect of UV irradiation on infection of mice with Borrelia burgdorferi.

These studies addressed the hypothesis that UV radiation (UVR) could affect immune responses in mice infected with Borrelia burgdorferi. Immunity against the Lyme spirochete B. burgdorferi was studied in a murine model of UV-induced immune suppression. Borrelia-specific cellular and humoral responses were examined following immunosuppressive doses of UVR. Low-passage Borrelia were injected intradermally at the base of the tail following irradiation. At various time points after infection the blood was cultured for the presence of Borrelia and the serum analyzed for Borrelia-specific antibodies. Two weeks after infection one hind-limb joint was cultured for the presence of spirochetes and the contralateral joint was examined histologically for arthritis formation. The results demonstrated that UV irradiation, administered at the site of infection or at a distant site, suppressed Borrelia-specific cellular and humoral responses in infected mice. Suppression of delayed-type hypersensitivity and antibody responses to UV was abrogated by administration of anti-interleukin (IL)-10 after UV irradiation. In addition, UV irradiation altered the dissemination pattern of the bacteria from the skin into the blood and exacerbated arthritis when compared with unirradiated controls. From these studies we concluded that UV irradiation can modulate the immune response to Borrelia and exacerbate the subsequent arthritic component of Lyme disease in mice. Furthermore, our studies suggest that IL-10 is in part responsible for the suppression of both cellular and humoral responses in addition to playing a role in the development of Lyme arthritis.