[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.

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.

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.

Humoral and delayed-type hypersensitive responses against listeria monocytogenes phosphatidylinositol-specific phospholipase C in experimentally infected buffaloes.

The kinetics of antibody production against phosphatidylinositol-specific phospholipase C (PI-PLC) and the isolation pattern of Listeria monocytogenes from bacteriological samples were studied following oral infection of buffalo calves with 3 x 10(9) cells each of pathogenic L. monocytogenes. Antibodies to PI-PLC appeared by 4-8 days post infection (PI), with a peak between days 7 and 16 PI, when tested by indirect plate-ELISA. Subsequently, antibody titres in all the animals declined and became undetectable on days 26-35 PI onwards until the study concluded on day 211 PI. Dot-ELISA could detect the antibodies to PI-PLC 1-2 days earlier and at higher titres as compared to plate-ELISA. L. monocytogenes could be recovered from faeces, nasal swabs and haemocultures from days 2 to 33, days 2 to 21 and days 11 to 17 PI, respectively. Antibodies to PI-PLC were detected during the course of active infection but their titres declined sharply once animals became culturally negative. Sonicated antigen elicited the highest delayed-type hypersensitivity response, followed by PI-PLC and listeriolysin O.

Significant accumulations of cathepsin B and prolylendopeptidase in inflammatory focus of delayed-type hypersensitivity induced by Mycobacterium tuberculosis in mice.

To clarify what kinds of proteinases are secreted into the foci of allergic-inflammation involving delayed-type hypersensitivity reaction, we examined the characteristic releases of various proteinases into the foci of Mycobacterium tuberculosis (M. tuber.)-induced delayed-type allergic-inflammation in mice. The significant activities of cathepsin B and prolylendopeptidase were observed in the washing-fluids of subcutaneous inflammatory foci of M. tuber.-induced delayed-type allergic-inflammation, but not M. tuber.-induced acute-inflammation. The SDS-resistant complex of cathepsin B and a protein substrate with apparent molecular mass of 74 kDa was observed by Western blot analysis. On the other hand, no significant accumulations of other proteinases, such as matrix metalloproteinases, cathepsin D, and serine proteinases, were determined. CA-074, a specific inhibitor of cathepsin B, suppressed both swelling and cathepsin B activity in the footpad having M. tuber.-induced delayed-type allergic-inflammation in vivo. These results suggest that cathepsin B may play an important role in the formation of M. tuber.-induced delayed-type allergic-inflammation.

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis.

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.

Ocular surface inflammation induced by Propionibacterium acnes.

PURPOSE: In the eye, is commonly isolated in the lid, conjunctiva, and meibomian gland secretion. Well known as a causative bacterium of granulomatous endophthalmitis and a potent inflammatory stimulus, reportedly induces a delayed-type hypersensitivity (DTH) response and forms granulomas in the liver and lung in animal models. In this study, we examined whether can induce a DTH response in the cornea. METHODS: Six- to 8-week-old female Lewis rats were immunized with heat-killed suspension of and assessed as to DTH response via ear challenge at 2 weeks after immunization. At 3 weeks after immunization, suspension was injected in the rat corneal stroma, which was then observed biomicroscopically at 6, 24, and 48 hours after injection. Phenol-killed suspension was also used for the comparison. Histological examination was also performed on the corneal tissues, using hematoxylin and eosin staining as well as immunohistochemical staining against CD4 and CD8 T cells. RESULTS: Rats immunized with suspension showed significantly higher ear swelling values at both the 24- and 48-hour measurements than did the naïve controls (p < 0.005). Massive cellular infiltration with stromal edema was observed biomicroscopically at 48 hours after injection of suspension in the corneal stroma. Histological study showed that the cell infiltration pattern was similar to that of DTH in the skin, i.e., neutrophils infiltrated at 6 hours, followed by mononuclear cells that, including macrophages and lymphocytes, increased and mixed with neutrophils, accompanied by stromal edema at 48 hours. Immunohistochemical study revealed that CD4 T-cell infiltration in the corneal stroma appeared to predominate over CD8 T-cell infiltration. CONCLUSIONS: These results indicate that can induce a DTH response in the cornea and may be a causative bacterium of ocular surface inflammation.

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.

Recombinant HIV-1 glycoprotein 120 induces distinct types of delayed hypersensitivity in persons with or without pre-existing immunologic memory.

Induction of T cell help is critical in HIV-1 control and potentially in prevention by immunization. A practical approach is needed to identify HIV-1-specific helper activities in vivo. We explored the feasibility of measuring delayed-type hypersensitivity (DTH) following intradermal injection of recombinant soluble HIV-1(MN) glycoprotein 120 in HIV-1-infected, vaccinated, and exposed individuals. DTH reactions were elicited within 48 h in 16 of 29 untreated, infected patients and in 24 of 30 uninfected vaccinees. Concomitant envelope-specific lymphoproliferation in vitro was undetectable among 9 infected patients tested with positive envelope-specific DTH. By contrast, no 48-h DTH reactions occurred among 25 high risk and 32 low risk, uninfected volunteers. However, 7--12 days after injection, 10 (40%) high risk and 11 (34%) low risk individuals developed induration resembling DTH, and the cellular infiltrates contained monocytes and T cells. Five of 18 examined also developed anti-gp120 Abs. The very delayed time course and lack of correlation with previous Ag exposure clearly distinguish this reaction from DTH. Thus, HIV-1 skin testing can identify persons with HIV-specific recall responses resulting from infection, in the absence of in vitro lymphoproliferation, and from vaccination. In contrast, very late reactivities may signify chemotactic properties of the envelope protein and/or herald the induction of primary HIV-specific Th1-type immunity.

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.

Tumor necrosis factor receptor 2 plays a minor role for mycobacterial immunity.

Tumor necrosis factor (TNF) signalling via the TNF receptor 1 (TNF-R1) is required for host resistance to mycobacterial infection. The role of TNF-R2 in anti-mycobacterial immunity is not known. Therefore, we compared TNF-R1 and TNF-R2 knockout (KO) mice infected with Mycobacterium bovis BCG (10(7) CFU, i.v.). While the bacterial burden of TNF-R1-deficient mice was significantly increased and the mice succumbed to infection between 4 and 5 weeks, TNF-R2 KO mice were less sensitive, and only 3 of 10 mice died within 12 weeks. Wild-type (WT) mice were resistant to BCG infection. The inability to clear the infection of TNF-R1 KO mice was associated with a reduced delayed-type hypersensitivity (DTH) response to purified protein derivative and severe impairment in forming granulomas with reduced macrophage recruitment and activation, and diminished expression of adhesion molecules. By contrast, TNF-R2 KO mice developed normal DTH response and mature mycobactericidal granulomas as the WT mice. Therefore, anti-mycobacterial immunity is largely dependent on TNF signalling via the TNF-R1, while activation of TNF-R2 plays a minor role.

Transcription-coupled and global genome repair differentially influence UV-B-induced acute skin effects and systemic immunosuppression.

Exposure to UV-B radiation impairs immune responses in mammals by inhibiting especially Th1-mediated contact hypersensitivity and delayed-type hypersensitivity. Immunomodulation is not restricted to the exposed skin, but is also observed at distant sites, indicating the existence of mediating factors such as products from exposed skin cells or photoactivated factors present in the superficial layers. DNA damage appears to play a key role, because enhanced nucleotide excision repair (NER) strongly counteracts immunosuppression. To determine the effects of the type and genomic location of UV-induced DNA damage on immunosuppression and acute skin reactions (edema and erythema) four congenic mouse strains carrying different defects in NER were compared: CSB and XPC mice lacking transcription-coupled or global genome NER, respectively, as well as XPA and TTD/XPD mice carrying complete or partial defects in both NER subpathways, respectively. The major conclusions are that 1) transcription-coupled DNA repair is the dominant determinant in protection against acute skin effects; 2) systemic immunomodulation is only affected when both NER subpathways are compromised; and 3) sunburn is not related to UV-B-induced immunosuppression.

Fatal Mycobacterium bovis BCG infection in TNF-LT-alpha-deficient mice.

Neutralization of TNF or disruption of TNF-R1 leads to fatal Mycobacterium bovis BCG infection. Here we used TNF-LT-alpha-deficient mice to test whether a complete disruption of TNF and LT-alpha reduces further host resistance to BCG infection. The bacterial burden especially in the lungs of TNF-LT-alpha-deficient mice was significantly increased and the mice succumbed to infection between 8 and 10 weeks. In the absence of TNF-LT-alpha the granulomatous response was severely impaired and delayed. The cells in the granulomas of TNF-LT-alpha-deficient mice expressed low levels of MHC class II and ICAM-1. They contained a few T cells and F4/80-positive macrophages expressing little iNOS and acid phosphatase activity. By contrast, the lethal action of endotoxin was dramatically reduced in BCG-infected TNF-LT-alpha-deficient mice. In summary, in the absence of TNF-LT-alpha the recruitment and activation of mononuclear cells in response to BCG infection were significantly delayed and reduced resulting in immature granulomas allowing uncontrolled fatal infection.

Effects of laparotomy, and carbon dioxide and air pneumoperitoneum, on cellular immunity and peritoneal host defences in rats.

OBJECTIVE: To assess the effects of laparotomy, and insufflation of carbon dioxide and air, on the immune system in rats. DESIGN: Randomised laboratory study. SETTING: Teaching hospital, Turkey. ANIMALS: 77 Wistar rats randomly allocated to 2 groups one of which was sensitised with dinitrofluorobenzene (DNFB, n = 43) and one of which was not (n = 34). INTERVENTIONS: The DNFB group was sensitised and subdivided into control (n = 8), laparotomy alone (n = 7), and insufflation with carbon dioxide (CO2) for 30 and 60 mins (n = 7 in each) or room air for 30 and 60 mins (n = 7 in each). A week later DNFB was reapplied to the ears. In the group not sensitised with DNFB the animals were subdivided similarly, the corresponding numbers in each group being, 6, 6, 6, 6, 5, and 5. MAIN OUTCOME MEASURES: Delayed type hypersensitivity (DTH) measured by ear swelling in the DNFB group, and peritoneal bactericidal activity, total free peritoneal cell counts (TPC), and cell types in the non-sensitised group. RESULTS: There were significant differences in the degree of ear swelling in the DNFB group between control and laparotomy groups (p = 0.0001) and between control and both insufflations of air (p = 0.002 and p = 0.0003, respectively). In the non-sensitised group peritoneal bactericidal activity was significantly increased after 7 hours in the 60 mins air insufflation group (p = 0.04). At 24 hours there were no differences among the groups. TPC were not affected. The number of peritoneal polymorphonuclear leucocytes (PMN) was significantly higher in the laparotomy alone group than in the control or any of the insufflation groups (p < 0.05). CONCLUSIONS: Laparotomy and air insufflation depressed cell-mediated immunity. Peritoneal bactericidal activity was affected only after 60 minutes of air insufflation.

IL-10 gene knockout mice show enhanced Th1-like protective immunity and absent granuloma formation following Chlamydia trachomatis lung infection.

We previously reported that higher IL-10 production is correlated with lower IFN-gamma production, weaker delayed hypersensitivity (DTH), and slower organism clearance following chlamydial infection in mice. To assess more directly the role of IL-10, we examined protective immunity and pathological reaction in C57BL/6 IL-10 gene knockout (KO) and wild-type mice. The results showed that in the absence of endogenous IL-10, mice had significantly accelerated chlamydial clearance and developed significantly stronger DTH responses, which could be inhibited by local delivery of rIL-10. Consistent with the enhancement of DTH responses, IL-10 KO mice showed stronger and more persistent CD4 T cell-dependent IFN-gamma production and significant elevation of IL-12 and TNF-alpha production. Additionally, wild-type, but not IL-10 KO, mice showed granuloma formation that was correlated with higher levels of Th2 cytokine (IL-5) production at the later stages of infection. Moreover, chlamydial infection, unlike parasitic protozoan infection, did not induce significant acute toxicity in IL-10 KO mice, which may be due to the low (undetectable) levels of systemic release of proinflammatory cytokines. These results suggest that IL-10 inhibits the priming and expansion of Th1-like T cell responses and that IL-10 plays a role in the fibrotic reaction seen with chlamydial infection.

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.

T-cell immune responses in Mycobacterium avium-infected mice.

Mycobacterium avium infection was substantially more severe in C57BL/6 (Bcgs) than in (C57BL/6 x DBA/2)F1 hybrid (Bcgr) mice both in terms of bacterial growth in the spleens and lungs and in host survival. Prior Mycobacterium bovis BCG vaccination resulted in increased resistance as well as enhanced tuberculin hypersensitivity to both PPD-S (Mycobacterium tuberculosis) and PPD-A (M. avium). Mice heavily infected with M. avium were used as T-cell donors in an adoptive transfer system. Substantial resistance was observed for both recipient hosts regardless of the genotype of the donor strain. Transfer of resistance was ablated by treatment of the immune spleen cells with anti-Thy 1.2 monoclonal antibody and complement or by cyclophosphamide treatment. Spleen cells which were monodepleted of L3T4+ or Lyt-2+ T cells did not lose their ability to transfer resistance against a subsequent challenge. However, when these cells were doubly deleted, all resistance was ablated in both the BCG-susceptible and -resistant mice. The recipient host expressed a detectable adoptive immune response although the donor had been unable to reduce the growth of the primary M. avium infection in vivo.

Virus-induced delayed-type hypersensitivity reaction is sequentially mediated by CD8+ and CD4+ T lymphocytes.

After subcutaneous inoculation into the hind foot of a mouse, the lymphocytic choriomeningitis (LCM) virus multiplies locally, attaining 10(7)-10(8) mouse infectious units per g of tissue; elimination commences around day 7. About 1 day earlier, the foot begins to swell, which is regarded as a delayed-type hypersensitivity (DTH) reaction. To answer the question of whether the local inflammatory response is involved in virus clearance, we needed to known what cells mediate both these phenomena. With three different procedures--namely, depletion in vivo of defined cells by treatment of mice with monoclonal antibodies ("serologic surgery"), adoptive immunization with negatively selected cells, and adoptive immunization with cells from mice differing at the major histocompatibility gene complex--it is shown that the LCM virus-induced local DTH reaction consists of two phases that are sequentially mediated by (first) class I-restricted cytotoxic/suppressive CD8+ and (second) class II-restricted helper/inducer CD4+ T lymphocytes. In contrast, for virus elimination only the former subset of T lymphocytes was found to be needed. Thus, an association may exist between the CD8+ cell-mediated component of the local DTH response and control of the infection, but the CD4+ cell-mediated part appears to be of doubtful antiviral relevance.

Alterations in the antigenic structure of two major HSV-1 glycoproteins, gC and gB, influence immune regulation and susceptibility to murine herpes keratitis.

We previously demonstrated that anterior chamber (AC) injection of HSV-1 before or simultaneous with topical corneal HSV-1 infection resulted in cellular immune tolerance of HSV-1 Ag and a reduced frequency of corneal stromal lesions. In the present study, we have investigated the role of the HSV-1 cell-surface glycoproteins gC and gB in the induction of tolerance, and the resulting reduced susceptibility to HSV-1 corneal stromal disease. These studies utilized mutant strains of HSV-1 with deletion or point mutations in the gene coding for gC or gB. Groups of mice received topical corneal infections with wild-type HSV-1, followed by AC injection of the same eye with wild-type HSV-1 or a mutant strain. Varying the antigenic composition of the virus injected into the AC resulted in three distinct patterns of immune responsiveness. In agreement with our previous findings, AC injection of wild-type HSV-1 induced a state of HSV-1 specific tolerance that extended to both the delayed type hypersensitivity (DTH) and CTL responses. A mutant strain lacking gC (gC-) induced partial tolerance characterized by undetectable CTL activity but a normal DTH response. A mutant strain lacking gB (gB-) caused partial suppression of the CTL response and no reduction of the DTH response. Thus, whereas gB may be involved in CTL tolerance induction in this model, gC clearly is not involved. In contrast, both gC and gB must be present in the AC to induce detectable DTH tolerance. The latter interpretation was strengthened by the observation that AC injection of a mixture of gC- (expressing normal gB) and gB- (expressing normal gC) effectively suppressed the DTH response to wild-type HSV-1. A panel of mar mutants with individual point mutations affecting gC and gB was used to identify the epitopes responsible for induction of DTH tolerance. Two of the gC mutants failed to induce DTH tolerance to wild-type HSV-1 when injected into the AC, suggesting that the sites on the gC molecule that are altered by these mutations are important for the induction of DTH tolerance. Similarly, one of the mar mutants for gB uniformly failed to suppress the DTH response, while another had a variable effect. The unique pattern of cellular immune reactivity exhibited by the mice receiving simultaneous topical corneal infection with wild-type HSV-1 and AC injection of gC- (no CTL but normal DTH) was associated with significantly reduced susceptibility to HSV-1 corneal stromal lesions.(ABSTRACT TRUNCATED AT 400 WORDS)