Effect of a single exposure to ultraviolet radiation on Mycobacterium bovis bacillus Calmette-Guerin infection in mice

BALB/c and C3H mice were exposed on the dorsal skin to 45 kJ/m2 of UVB radiation from FS-40 sunlamps 3 days before infection with 1 x 10(6) live units of Mycobacterium bovis bacillus Calmette-Guérin (BCG) (Tice strain) in the footpad. At regular intervals, groups of mice were tested for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli, and the course of infection was monitored by measuring the size of the infected footpad, enlargement of the draining lymph node, and the number of bacteria in the spleen and lymph node. In both strains the DTH response to PPD was significantly delayed in UV-treated mice compared to unirradiated mice, when tested 21 and 42 days after BCG infection. By day 50, no significant difference was detected in the DTH response between irradiated and unirradiated mice. UV treatment reduced the size of the lymph node draining the site of BCG infection in both strains of mice and the size of the infected footpad in C3H mice but not in BALB/c mice. In both strains of mice the total number of bacteria in the spleen and the draining lymph node increased after UV irradiation. When irradiated 3, 5, 18, or 21 days after BCG infection, BALB/c mice also showed a significant decrease in their DTH response to PPD, indicating that the UV-induced suppression of BCG occurs both at the induction and the elicitation stages of the immune response. Thus, mice exposed to a single dose of UV radiation either before or after BCG infection showed an impaired DTH response to mycobacteria, which was accompanied by an increase in the multiplication of bacteria in the tissues, even though the organisms were introduced at an unirradiated site. These studies demonstrate that a systemic effect of UV irradiation can interfere with the development and expression of immunity to pathogenic bacteria in mice.     

Regulation of antitubercular immunity in mice by genes of the H-2 complex

Development of DTH reaction and survival time after M. tuberculosis H37Rv infection have been studied in H-2 congenic and recombinant mice pretreated with high doses of BCG vaccine. In addition, in vitro proliferation of lymphocytes from infected CBA, B6 and 4R mice to PPD was studied in the presence of anti-I-A and anti-I-E mAbs. High doses of BCG vaccination (1 mg/mouse) have led to a significant inhibition of DTH and diminution of survival time in B10.M (H-2f) mice only, and to opposite effects in all other strains tested (H-2a, b, d, k, h4). In I-A+, I-E- 4R mice anti-I-Ak mAbs abrogated lymphocyte proliferation to PPD completely, while in I-A+, I-E- CBA mice only the mixture of anti-I-Ak and anti-I-Ek mAbs was effective.     

B-cell-mediated depression of the granulomatous response to BCG in mice

The depression of the granulomatous response to Mycobacterium bovis strain BCG in mice infected intravenously with 2 x 10(7) CFU of the microorganism turned out to be mediated by various types of cells arising at different times after infection. Anti-PPD B lymphocytes were found to play a major role at Day 1 after infection and to be no longer effective 4 days later. At this time the depression was mediated by anti-idiotype B lymphocytes, whereas T lymphocytes proved to be involved in later phases of the infectious process. These results show that B lymphocytes may be of critical importance in the regulation of cell-mediated immune reactions to this facultative intracellular parasite.     

Prostaglandin-mediated suppression of delayed-type hypersensitivity to infected erythrocytes during Babesia microti infection in mice

The mechanism of suppression of delayed-type hypersensitivity (DTH) to intraerythrocytic Babesia microti which occurs during infection in mice was examined. The suppression was not specific for anti-parasite DTH; infected mice immunized and challenged with sheep red blood cells had a similar depression of anti-sheep red blood cell DTH. Sublethal or lethal irradiation did not significantly alter the suppression of the DTH response, and cyclophosphamide pretreatment of infected mice also had no effect on suppression. Multiple passive transfer experiments using serum or regional lymph node cells from immunized or infected and immunized (suppressed) donor animals failed to demonstrate any ability to transfer suppression of DTH. Adherent cells from the spleens or peritoneal exudates of suppressed mice, however, did significantly depress the ability of immunized mice to express a DTH response. The cells responsible for this suppression were Thy 1- and nonspecific esterase+. Treatment of suppressive cell populations with 10 micrograms/ml indomethacin for 24 hr in vitro abrogated their suppressive ability, and in vivo administration of indomethacin to suppressed mice also restored DTH to normal levels. By examining levels of prostaglandin E2 (PGE2) in supernates of cultured peritoneal exudate cells from immune or suppressed mice, it was shown that infected mice had peritoneal exudate cells which produced significantly more PGE2 than similar cells from immune mice. These data suggest that B. microti infection elicits synthesis of PGE2 by macrophage-like cells which results in suppression of DTH to parasite as well as heterologous antigens.     

Alteration of the immune response to Mycobacterium bovis BCG in mice exposed chronically to low doses of UV radiation

BALB/c mice were exposed on shaved dorsal skin to 1 minimal erythemal dose (MED) of UVB radiation (2.25 kJ/m2) from a bank of six FS-40 sunlamps three times per week. The total number of irradiations ranged from 1 to 27. At regular intervals, groups of mice were injected in the left hind foot pad with 1 x 10(6) live mycobacteria (Mycobacterium bovis BCG) 3 days after the last UVB exposure. The mice were tested 21 and 42 days after infection for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli by injecting PPD into the right hind foot pad and measuring the foot pad swelling 24 hr later. The course of infection was followed by assessing the number of bacterial colony forming units in the lymph node draining the site of BCG infection and the spleen. Mice exposed from 1 to 15 times to 1 MED of UV radiation showed a significant suppression in their DTH response to PPD compared with the unirradiated mice. At the same time, the number of bacterial colony-forming units in the lymph node and spleen of the UV-irradiated mice was greater than in control mice. With continued exposure to UVB, however, the DTH response recovered to a normal level, and there was no longer an increase in the number of viable bacteria in the lymphoid organs. These results indicate that early in the course of chronic UV irradiation, mice were impaired in their ability to mount a DTH response to BCG and to clear these bacteria from their lymphoid organs; later the mice recovered from these effects of UV, with continued treatment. A dose-response study using single doses of UV radiation indicated that a dose of 2.7 kJ/m2 suppressed the DTH response by 50%. Thus, exposure of mice to a single or multiple low doses of UV radiation prior to infection can interfere with systemic immunity to mycobacteria.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

The genetic regulation of host response to inoculation with Mycobacterium bovis (BCG) and Mycobacterium tuberculosis H37RV

Vaccination with M. bovis (BCG) essentially prolonged survival time (ST) of several strain mice, with the exception, of CBA/N, infected with M. tuberculosis H37Rv. ST of CBA/N, differing from CBA by xid mutation, was not prolonged by vaccination. Mouse strains with alternative alleles of BCG gene (s and r) and fzy gene as a genetic marker for Bcg5 were used for segregation analysis. It was shown that ST, the level of DTH reaction of mice infected with M. tuberculosis H37Rv, and protective effect of BCG vaccination did not depend on Bcg gene. However, Bcg gene, apparently, regulate the DTH response to PPD in mice only vaccinated with M. bovis (BCG).     

Delayed-Type Hypersensitivity Skin Reaction to HBsAg and Immunohistopathologic Study of Liver in Patients with Acute Type B Viral Hepatitis

In an attempt to clarify the role of cellular immunity in the pathogenesis of acute type B viral hepatitis (AHB), we studied delayed-type hypersensitivity (DTH) skin reaction to hepatitis B surface antigen (HBsAg) and immunohistopathology of livers in patients with AHB. DTH skin reaction to HBsAg developed early in the convalescent phase in all 14 patients with AHB. In contrast, the production of anti-HBs was significantly delayed in these patients, compared with healthy controls immunized with HB vaccine intradermally (P< 0.001). These observations suggest that DTH to HBsAg but not anti-HBs may be associated with recovery from AHB. In the biopsied livers obtained from patients with AHB, the proliferation of Kupffer cells was extensive and immunohistopathologic studies revealed an accumulation of CD-4 positive lymphocytes in the portal area, a finding which suggested a DTH reaction in the liver with AHB. CD-8 positive cells had infiltrated the lobule and made contact with affected hepatocytes, thereby indicating that a cytotoxic T cell response is involved in damaging of the infected cells. All these observations taken together, we propose that not only a cytotoxic T cell response but also a DTH reaction may be involved in the pathogenesis of AHB.     

Antigen specific lymphocyte transformation, delayed hypersensitivity and protective immunity. I. Kinetics of the response

The association between protective immunity, delayed hypersensitivity (DTH) and in vitro antigen specific lymphocyte transformation (ASLT) has been studied during the course of BCG infection in mice. The peak ASLT response was obtained 2 weeks after a subcutaneous footpad infection using lymph node cells draining the immunization site. This response decreased to a low but statistically significant level by week 4 and persisted indefinitely thereafter. A similar course of events was observed in splenic cell cultures, but the size of the response was smaller. The reactive cells were shown to be T-lymphocytes. The ability to express DTH to tuberculin purified protein derivative coincided with the emergence of cells capable of responding in ASLT assays. In contrast, there was a poor association between ASLT and protective immunity: lymphocytes from lymph nodes that did not drain the immunization site conferred substantial protective immunity but nonetheless failed to respond significantly in ASLT assays. The latter failure could not be attributed to insufficiency of sensitized lymphocytes and was independent of the antigen concentration and the culture incubation time. It was concluded that the ASLT reactive cells were either immunoblasts or recent progeny of these cells.     

Induction of delayed-type hypersensitivity and antitumor immunity by systemic BCG

Both delayed-type hypersensitivity (DTH) and antitumor resistance induced in mice by intravenous (i.v.) and local injection of highly immunogenic irradiated Meth A cells were potentiated by prior systemic BCG infection. DTH and antitumor immunity were not elicited by i.v. injection of poorly immunogenic irradiated mastocytoma cells, P 815 (MA), but were induced by the local injection of these cells when animals were systemically infected with BCG. The level of the potentiated response corresponded with the dose of immunogen up to an optimum, beyond which additional immunogen was suppressive. At all dose levels the subcutaneous (s.c.) route of immunogen inoculation was more effective than the i.v. route. Significant DTH was first detected 7 days after the local administration of immunogen and was correlated with antitumor immunity. Systemically administered BCG grew mainly in the liver and spleen until the development of maximal tuberculin sensitivity when the number of organisms decreased. However, the small number of mycobacteria that reached the peripheral lymph nodes remained constant after maximal tuberculin sensitivity but failed to augment the cell proliferation that occurred in these lymph nodes as a result of the local inoculation of irradiated tumor cells. Autoradiographs of such nodes revealed proliferation in the thymus-dependent areas whereas nodes from mice immunized with immunogen alone manifested B- as well as T-cell activity. Local immunization in both BCG-infected and uninfected hosts was also associated with a proliferative response in the red pulp of the spleen but the BCG-infected hosts differed conspicuously by virtue of the presence of tubercles and depletion of lymphoid cells from the periarteriolar sheath. Immunity generated by the local administration of immunogen in systemically infected mice was tumor specific and could be adoptively transferred with spleen cells.     

Cutaneous leishmaniasis in anti-IgM-treated mice: enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway

The contribution of B cells and antibodies to either the resistance or susceptibility to cutaneous leishmaniasis has been investigated in mouse strains rendered B cell-deficient by treatment with anti-mouse IgM antisera from birth (mu-suppressed). These studies confirm that immunity to cutaneous disease in a normally resistant mouse strain (C3H/HeJ) is independent of antibody, but that B cells and/or antibodies are required for the evolution of suppressed DTH and the consequent disease susceptibility of BALB/c mice. Anti-IgM-treated BALB/c mice, which lacked detectable anti-leishmanial antibodies during the course of infection, displayed a sustained DTH response to leishmanial antigen and were able to control their cutaneous lesions. The enhanced resistance of mu-suppressed mice could be completely abrogated by transfer of suppressor T cells from infected control animals into mu-suppressed mice before their infection. Thus the suppressor T cells, which are generated during leishmanial infection in BALB/c mice, can effect suppression in the absence of antibody. Evidence that B cells or antibodies are required for the generation of suppressor T cells was demonstrated by using BALB/c mice in which suppressor T cells fail to be generated during infection as a result of prior sublethal irradiation. Splenic T cells from normal mice could overcome the resistance conferred by sublethal irradiation, whereas splenic T cells from mu-suppressed mice could not. Thus the enhanced resistance of mu-suppressed BALB/c mice appears to be a consequence of their lack of functional expression of a B cell-dependent T cell critical to the suppressor pathway.     

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.Abbreviations DTH delayed-type hypersensitivity - DNFB dinitrofluorobenzene - FN18 Fava Netto's antigen obtained from isolate Pb18 - FN265 Fava Netto's antigen obtained from isolate Pb265 - SRBC sheep red blood cells     

Virus-specific delayed-type hypersensitivity (DTH). Cells mediating lymphocytic choriomeningitis virus-specific DTH reaction in mice

We had previously shown that the local lymphocytic choriomeningitis virus-induced delayed-type hypersensitivity (DTH) reaction in mice consists of two well delineated phases that are mediated by CD8+ and CD4+ T lymphocytes, respectively. These findings have been confirmed and extended by showing that the first CD8+ cell-dependent part of the response was enhanced by either the presence of CD4+ cells or systemic treatment with IL-2 and that it developed in the absence of detectable numbers of mononuclear phagocytes, whereas the later CD4+ cell part required monocytes or related elements. Furthermore, in the DTH reaction that was elicited with noninfectious viral Ag in mice previously immunized by infection, only the CD4+ cells participated. Thus, the two phases of the lymphocytic choriomeningitis-viral DTH reaction are principally different, which has to be taken into account when trying to assess the relevance of DTH during this virus infection.     

Mycoplasma pulmonis depresses humoral and cell-mediated responses in mice

Humoral and cell-mediated immune responses to sheep red blood cells (SRBC) were studied in mice infected experimentally with Mycoplasma pulmonis. The hemagglutinating (HA) antibody against SRBC was evaluated at 0, 3, 5, 7, 14, 21 and 28 days postinfection (PI). Antibody tiers during all days PI were depressed significantly (p less than 0.05) in infected mice as compared to noninfected controls. The HA antibody, which is of the IgM class, peaks at day 5 PI. There is no shift in the kinetics of the humoral response in M. pulmonis infected mice. Cellular immune responses were evaluated by a delayed-type hypersensitivity (DTH) reaction and the lymphocyte transformation technique. Mice were sensitized at 0,3,5,7,14, 21 and 28 days PI with SRBC and challenged by footpad injection of SRBC 7 days later. The DTH reaction measured at 24 hours after challenge was depressed significantly (p less than 0.05) in all infected animals. After a transient enhancement on day 3 PI, the DTH responses remained depressed through day 28 PI. The lymphocyte transformation test showed a significantly (p less than 0.05) depressed response except on days 5 and 7 PI. These results indicate that M. pulmonis infection in mice suppresses the humoral antibody and cell-mediated immune responses.     

Infection with Bacillus Calmette-Guérin activates murine thymus-independent (B) lymphocytes.

Thymus-independent (B) lymphocytes from BCG-infected mice respond with enhanced or depressed levels of proliferation relative to the nonspecific levels of stimulation induced by tuberculin. The type of response obtained depends on how BCG was administered and the source of the lymphocytes. The proliferation of splenic B lymphocytes was depressed when BCG was given i.v. and unchanged when BCG was given subcutaneously, whereas the lymph node B cell response was enhanced regardless of the route of injecting BCG. BCG was found also to be a mitogen for normal B cells in vitro and to stimulate polyclonal activation of B cells in vivo. Finally, evidence is presented which indicates that the depressed splenic B cell response to tuberculin may be due to the activation of suppressor T cells.     

BCG—PPD的制备及应用

用三氯醋酸(TCA)和硫酸铵(AS)综合法,由卡介菌苗(BCG)培养滤液中提纯制得卡介菌素纯蛋白衍生物(BCG—PPD)。BCG—PPD的纯度和结核菌素纯蛋白衍生物国际标准(PPD-S)及中国标准(PPD—C)相近,高于加拿大标准(PPD—CT68)和丹麦标准(PPD—RT23)。在BCG免疫豚鼠中,BCG—PPD的皮肤迟发型变态反应(DTH)大于结核菌素纯蛋白衍生物(PPD)的DTH反应。在结核菌感染豚鼠组中,BCG—PPD的DTH反应小于PPD的DTH反应.在检查333名新生儿接种BCG12周后的免疫     

Murine model of BCG lung infection: Dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes

C57B1/6 female mice were infected with an intrapulmonary dose of 2.5 × 10⁴ BCG(Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1,2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4–6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3⁺ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1⁺) per lung increased by about 50%, 5–6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNγ producing non-T cells outnumbered T cells producing IFNγ. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNγ producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNγ response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNγ, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine     

IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis bacille Calmette-Guerin infection

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-gamma production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that gammadelta T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-gamma in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.     

Delayed-type hypersensitivity to Babesia microti-infected erythrocytes in mice

Strong delayed-type hypersensitivity (DTH) to Babesia microti was elicited when intraerythrocytic parasites (IEP) were inoculated subcutaneously into the flank of normal mice 6 to 14 days before challenge in the ipsilateral footpad with 10(8) IEP. Intraperitoneal or intravenous administration of antigen did not sensitize mice for DTH. When challenge was given 21 days after immunization, the response was approximately half of the maximum and then rose again slowly over the next 3 weeks to levels that were not significantly different from those maximal values. The response was similar in seven strains of mice, regardless of sex. The response was classified as a true DTH reaction on the basis of kinetics, histology, and the transfer of responsiveness with immune T lymphocytes of the Ly 1+ phenotype, but not with serum. The reaction was specific for IEP since control groups given two injections of red blood cells from uninfected syngeneic mice (NRBC) or one injection of NRBC or sheep red blood cells (SRBC) and one of IEP never developed significant footpad swelling. Freed parasites obtained by osmotic rupture, density gradient sedimentation, and lethally irradiated IEP were also effective for elicitation of DTH. Anti-IEP DTH was expressed in a dose-dependent fashion with 10(6), 10(7), or 10(8) parasites sufficing for immunizing inoculum as long as 10(8) parasites were used as the challenge dose. Mice immunized and challenged with 10(8) lethally irradiated IEP (60 krad, 60Co), were protected against subsequent intraperitoneal challenge with 10(8) viable IEP. If mice were infected intraperitoneally with 10(8) IEP at any time between 21 days before immunization to 2 hr after challenge, their ability to respond to immunization and challenge was profoundly depressed. These data suggest that development of a strong anti-parasite DTH response can occur in parallel with resistance to infection, but is not a rapid sequela of bloodborne infection.