Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants

In this study, we report on the safety and skin delayed-type hypersensitivity (DTH), responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG), Montanide ISA 720 (MISA) or Monophosphoryl lipid A (MPLA) in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) levels were also measured and these were compared with DTH. Only those animals immunized with alum-BCG reacted adversely to the inoculum by producing ulcerative erythematous skin indurations. Non-parametric analysis of variance followed by a post-test showed significantly higher DTH responses in the MISA+Ag group compared with other immunized groups (p < 0.001). The MPLA+Ag group indicated significantly lower DTH responses to the sonicate antigen compared with the AlBCG+Ag group. There was a significant correlation between the DTH and cytokine responses (p < 0.0001). Based on this study we conclude that Leishmania donovani sonicate antigen containing MISA 720 is safe and is associated with a strong DTH reaction following immunization.

Effect of treatment with cyclophosphamide in low doses upon the onset of delayed type hypersensitivity in mice chronically infected with Trypanosoma cruzi: involvement of heart interstitial dendritic cells

Acute infection with Trypanosoma cruzi results in intense myocarditis, which progresses to a chronic, asymptomatic indeterminate form. The evolution toward this chronic cardiac form occurs in approximately 30% of all cases of T. cruzi infection. Suppression of delayed type hypersensitivity (DTH) has been proposed as a potential explanation of the indeterminate form. We investigated the effect of cyclophosphamide (CYCL) treatment on the regulatory mechanism of DTH and the participation of heart interstitial dendritic cells (IDCs) in this process using BALB/c mice chronically infected with T. cruzi. One group was treated with CYCL (20 mg/kg body weight) for one month. A DTH skin test was performed by intradermal injection of T. cruzi antigen (3 mg/mL) in the hind-footpad and measured the skin thickness after 24 h, 48 h and 72 h. The skin test revealed increased thickness in antigen-injected footpads, which was more evident in the mice treated with CYCL than in those mice that did not receive treatment. The thickened regions were characterised by perivascular infiltrates and areas of necrosis. Intense lesions of the myocardium were present in three/16 cases and included large areas of necrosis. Morphometric evaluation of lymphocytes showed a predominance of TCD8 cells. Heart IDCs were immunolabelled with specific antibodies (CD11b and CD11c) and T. cruzi antigens were detected using a specific anti-T. cruzi antibody. Identification of T. cruzi antigens, sequestered in these cells using specific anti-T. cruzi antibodies was done, showing a significant increase in the number of these cells in treated mice. These results indicate that IDCs participate in the regulatory mechanisms of DTH response to T. cruzi infection.

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human Schistosomiasis: I. Granuloma formation and modulation around polyacrylamide antigen-conjugated beads

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.

Cyclophosphamide effect on paracoccidioidomycosis in the rat

Paracoccidioidomycosis is an endemic fungal disease widely distributed throughout Latin America. The potent immunosuppressor cyclophosphamide (CY) has been used to modulate host immune response to Paracoccidioides brasiliensis in an experimental model. Inbred male Buffalo/Sim rats weighing 250-300 g were inoculated with 5 x 10(6) P. brasiliensis cells of the yeast phase form by intracardiac route. One group of animals was treated with 20 mg/kg body weight at days +4, +5, +6, +7, +11 and +12 post-infection (pi.), while a control group was infected alone. No mortality was recorded in either group. Treated rats presented: a) a decrease in granuloma size, which contained less fungal cells; b) a lack of specific antibodies up to 35 days pi., and c) a significant increase in the footpad swelling test (DTH) against paracoccidioidin. Splenic cell transfer from CY-treated P. brasiliensis-infected donors to recipients infected alone led to a significant increase in DTH response in the latter versus untreated infected controls. Likewise, in treated infected recipients transferred with untreated infected donor spleen cells, footpad swelling proved greater than in controls. Thus, it would seem that each successive suppressor T lymphocyte subset belonging to the respective cascade may be sensitive to repeated CY doses administered up to 12 days pi.. Alternatively, such CY schedule may induce the appearance of a T cell population capable of amplifying DTH response.

American tegumentary leishmaniasis: langerhans cells in montenegro skin test

This work analyzed the histopathology and epidermal Langerhans cells (LC) of Montenegro skin test (MST) in patients with American tegumentary leishmaniasis (ATL) in order to in situ characterize and compare the immunological reaction of the two major clinical forms of ATL, localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). MST histopathology of both LCL and MCL showed superficial and deep perivascular inflammatory infiltrate composed mainly of lymphocytes and histiocytes. Epidermal LC population was higher in MST biopsies taken from LCL patients when compared to MCL group, at 48 and 72 hours after antigen inoculation. Increased number of epidermal LC displayed in MST biopsies of LCL patients represents specific cellular immunity against parasites. The decrease of LC in MST biopsies of MCL patients does not necessarily indicate a worse specific cellular immunity in this clinical form of leishmaniasis.

Cytokine profile in Montenegro skin test of patients with localized cutaneous and mucocutaneous leishmaniasis

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL.

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection

In Amazonian Brazil, the Cebus apella monkey (Primates: Cebidae) has been associated with the enzootic cycle of Leishmania (V.) shawi, a dermotropic parasite causing American cutaneous leishmaniasis (ACL). It has also been successfully used as animal model for studying cutaneous leishmaniasis. In this work, there has been investigated its susceptibility to experimental Leishmania (L.) infantum chagasi-infection, the etiologic agent of American visceral leishmaniasis (AVL). There were used ten C. apella specimens, eight adult and two young, four males and six females, all born and raised in captivity. Two experimental infection protocols were performed: i) six monkeys were inoculated, intra-dermal via (ID), into the base of the tail with 2 x 10(6) promastigotes forms from the stationary phase culture medium; ii) other four monkeys were inoculated with 3 x 10(7) amastigotes forms from the visceral infection of infected hamsters by two different via: a) two by intravenous via (IV) and, b) other two by intra-peritoneal via (IP). The parameters of infection evaluation included: a) clinical: physical exam of abdomen, weigh and body temperature; b) parasitological: needle aspiration of the bone-marrow for searching of amastigotes (Giemsa-stained smears) and promastigotes forms (culture medium); c) immunological: Indirect fluorescence antibody test (IFAT) and, Delayed-type hypersensitivity (DTH). In the six monkeys ID inoculated (promastigotes forms) all parameters of infection evaluation were negative during the 12 months period of follow-up. Among the four monkeys inoculated with amastigotes forms, two IV inoculated showed the parasite in the bone-marrow from the first toward to the sixth month p.i. and following that they cleared the infection, whereas the other two IP inoculated were totally negative. These four monkeys showed specific IgG-antibody response since the third month p.i. (IP: 1/80 and IV: 1/320 IgG) toward to the 12th month (IP: 1/160 and IV: 1/5120). The DTH-conversion occurred in only one IV inoculated monkey with a strong (30 mm) skin reaction. Considering these results, we do not encourage the use of C. apella monkey as animal model for studying the AVL.

PREVALENCE OF PARACOCCIDIOIDOMYCOSIS INFECTION BY INTRADERMAL REACTION IN RURAL AREAS IN ALFENAS, MINAS GERAIS, BRAZIL

This study aimed to estimate the prevalence of paracoccidioidal infection by intradermal reaction (Delayed-Type Hypersensitivity, DTH) to Paracoccidioides brasiliensis in rural areas in Alfenas, Southern Minas Gerais (MG) State, Brazil, and to assess risk factors (gender, occupation, age, alcohol intake and smoking) associated with infection. We conducted a population-based cross-sectional study using intradermal tests with gp 43 paracoccidioidin in 542 participants, who were previously contacted by local health agents and so spontaneously attended the test. Participants underwent an interview by filling out a registration form with epidemiological data and were tested with an intradermal administration of 0.1 mL of paracoccidioidin in the left forearm. The test was read 48 hours after injection and was considered positive if induration was greater than or equal to 5 mm. Out of 542 participants, 46.67% were positive to the skin test. Prevalence increased in accordance with an increase of age. There was statistical significance only for males. Occupation, alcohol intake and smoking habits were not significantly associated with the risk of paracoccidioidomycosis infection. There is relevance of paracoccidioidomycosis infection in such rural areas, which suggests that further epidemiological and clinical studies on this mycosis should be done in the southern part of Minas Gerais State.

Boosting the suppressive effects of Ascaris suum components in IFN-γ-deficient mice

High molecular weight components from Ascaris suum extract suppress ovalbumin-specific immunity in mice. In IFN-γ-deficient mice, ovalbumin-specific delayed-type hypersensitivity reactions are more strongly downregulated by these suppressive components. Here, the cellularity of the delayed-type hypersensitivity reaction in IFN-γ-deficient mice and the increased downregulation induced by Ascaris suum components were analyzed. IL-12p40-dependent neutrophilic influx was predominant. Suboptimal doses of the suppressive fraction from this nematode completely inhibited the hypersensitivity reaction, thus indicating intensification of the immunosuppression under conditions of intense recruitment of IFN-γ-independent neutrophils.

Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Complete immunization against Trypanosoma cruzi verified in individual mice by complement-mediated lysis

Experimental systems to assay immunity against Trypanosoma cruzi usually demonstrate partial resistance without excluding the establishment of sub-patent infections in protected animals. To test whether Swiss mice immunized with attenuated parasites might develop complete resistance against virulent T. cruzi, experiments were performed involving challenge with low numbers of parasites, enhancement of local inflammation and the combination of natural and acquired resistance. Absence of infection was established after repeated negative parasitological tests (including xenodiagnosis and hemoculture), and lack of lytic antibody was tested by complement mediated lysis. Immunization with 10(7) attenuated epimastigotes conferred protection against the development of high levels of parasitemia after challenge with Tulahuen strain, but was unable to reduce the number of infected animals. However, when a strong, delayed-type hypersensitivity reaction was triggered at the site of infection by injecting a mixture of virulent and attenuated T. cruzi, a significant proportion of immunized animals remained totally free of virulent infection. The same result was obtained when the immunization experiment was performed in four month old Swiss mice, displaying a relatively high natural resistance and challenged with wild, vector-borne parasites. These experiments demonstrate that complete resistance against T. cruzi can be obtained in a significant proportion of animals, under conditions which replicate natural, vector delivered infection by the parasite.

Resolution of an Infection with Leishmania braziliensis Confers Complete Protection to a Subsequent Challenge with Leishmania major in BALB/c Mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-g (IFN-g ) but low amounts of interleukin-4. The IFN-g produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-g abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.

Skin reactions to thimerosal and Leishmania in dogs from a leishmaniasis endemic area: it is better to keep them apart

Positive Montenegro's skin test is a delayed type hypersensitivity reaction widely used as indicative of previous infection with Leishmania in both humans and dogs. Montenegro's antigen consists of a crude Leishmania antigen solution, usually containing thimerosal as preserving agent. In this work it is shown that a large proportion of dogs (11 out of 56) examined in an endemic area of leishmaniasis presented induration at the site of injection of a diluent containing thimerosal alone. This clearly demonstrates that thimerosal leads to a high number of false positive skin reactions in dogs and that its use in Montenegro's skin test antigenic preparations should be avoided.

Leishmania (Leishmania) major-infected rhesus macaques (Macaca mulatta) develop varying levels of resistance against homologous re-infections

Seven rhesus macaques were infected intradermally with 10(7) promastigotes of Leishmania (Leishmania) major. All monkeys developed a localized, ulcerative, self-healing nodular skin lesion at the site of inoculation of the parasite. Non-specific chronic inflammation and/or tuberculoid-type granulomatous reaction were the main histopathological manifestations of the disease. Serum Leishmania-specific antibodies (IgG and IgG1) were detected by ELISA in all infected animals; immunoblot analyses indicated that numerous antigens were recognized. A very high degree of variability was observed in the parasite-specific cell-mediated immune responses [as detected by measuring delayed-type hypersensitivity (DTH) reaction, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production] for individuals over time post challenge. From all the recovered monkeys (which showed resolution of the lesions after 11 weeks of infection), 57.2% (4/7) and 28.6% (2/7) animals remained susceptible to secondary and tertiary infections, respectively, but the disease severity was altered (i.e. lesion size was smaller and healed faster than in the primary infection). The remaining monkeys exhibited complete resistance (i.e. no lesion) to each rechallenge. Despite the inability to consistently detect correlates of cell-mediated immunity to Leishmania or correlation between resistance to challenge and DTH, lymphocyte transformation or IFN-gamma production, partial or complete acquired resistance was conferred by experimental infection. This primate model should be useful for measuring vaccine effectiveness against the human disease.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.