Alexandrina Sartori

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat‐shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis‐infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65‐treated mice and infected pCDNA3‐treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon‐γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor‐α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65‐treated mice were able to produce significant levels of interferon‐γ and to restrict the growth of bacilli.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Immune complex glomerulonephritis in experimental kala‐azar

Summary In the present work we demonstrate that hamsters infected with L. donovani eliminate large quantities of immunoglobulins in the urine. This alteration is clearly a consequence of a conspicuous immune complex glomerulonephritis readily detectable 7 days after the beginning of infection. L. donovani antigens and hamsters immunoglobulins (Igs) were revealed as granular deposits in the mesangial areas and contiguous loops of the glomeruli.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA‐HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non‐obese diabetic (NOD) mouse develops insulin‐dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin‐producing β cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA‐HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA‐HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA‐HSP65‐injected mice. The protective effect of DNA‐HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4+ and CD8+ T cells infiltration, appearance of CD25+ cells influx and an increased staining for interleukin (IL)‐10 in the islets. These results show that DNA‐HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

Impact of malnutrition on immunity and infection

Malnutrition may be a consequence of energy deficit or micronutrient deficiency. It is considered the most relevant risk factor for illness and death, particularly in developing countries. In this review we described the magnitude of this problem, as well as its direct effect on the immune system and how it results in higher susceptibility to infections. A special emphasis was given to experimental models used to investigate the relationship between undernutrition and immunity. Malnutrition is obviously a challenge that must be addressed to health authorities and the scientific community.

Immune complex glomerulonephritis in experimental kala‐azar II: Detection and characterization of parasite antigens and antibodies eluted from kidneys of Leishmania donovani‐infected hamsters

In a previous report analysing kidney sections by immunofluorcscence we showed that hamsters infected with L. donovani develop a glomerulonephritis (GN) associated with deposition of hamster immunoglobulins and parasite antigens in the glomeruli. In this study we characterize these immune components eluted from the kidneys. The eluted immunoglobulins showed specificity for L. donovani antigens and hamster immunoglobulins (rheumatoid factor‐like activity). The four isotypes IgG1. IgG2, IgA and IgM were detected. Several L. donovani antigens were detected in the renal eluates by Western blot and immuneprecipitation using 125 I‐labelled eluates. Proteins with mol. wt of 134, 82, 52, 31, and 26 kD were detected by Western blot and proteins with 134, 110, 93, 89 and 48 kD were detected by immunoprecipitation. With the exception of the 134 kD protein which was recognized by both rabbit anti‐promastigote and rabbit anti‐amastigote sera all the others were recognized only by the anti‐amastigote serum. The 134 kD protein was the only one isolated from the kidneys of infected hamster immunocomplexed with IgG and was the only one detected in a promastigote lysate using IgG from L. donovani‐ infected hamsters.

Treatment with vitamin D/MOG association suppresses experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model to study multiple sclerosis (MS). Considering the tolerogenic effects of active vitamin D, we evaluated the therapeutic effect of myelin oligodendrocyte glycoprotein (MOG) associated with active vitamin D in EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG emulsified with Complete Freund’s Adjuvant plus Mycobacterium tuberculosis. Animals also received two intraperitoneal doses of Bordetella pertussis toxin. One day after immunization, mice were treated with 0,1μg of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) every other day during 15 days (on days 1, 3, 5, 7, 9, 11, 13 and 15). MOG (150μg) was co-administered on days 3 and 11. The administration of 1,25(OH) 2D3 or MOG determined significant reduction in EAE incidence and in clinical scores. When MOG was associated with 1,25(OH) 2D3 the animals did not develop EAE. Spleen and central nervous system (CNS) cell cultures from this group produced less IL-6 and IL-17 upon stimulation with MOG in comparison to the EAE control group. In addition, this treatment inhibited dendritic cells maturation in the spleen and reduced inflammatory infiltration in the CNS. The association of MOG with 1,25(OH) 2D3 was able to control EAE development.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

BackgroundOur group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases.MethodsIn this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ.ResultsDNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete.ConclusionThe data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases.

Persistent Inflammation in the CNS during Chronic EAE Despite Local Absence of IL-17 Production

Experimental autoimmune encephalomyelitis (EAE) is an artificially induced demyelination of the central nervous system (CNS) that resembles multiple sclerosis in its clinical, histopathological, and immunological features. Activated Th1 and Th17 cells are thought to be the main immunological players during EAE development. This study was designed to evaluate peripheral and local contribution of IL-17 to acute and chronic EAE stages. C57BL/6 mice were immunized with MOG plus complete Freunds adjuvant followed by pertussis toxin. Mice presented an initial acute phase characterized by accentuated weight loss and high clinical score, followed by a partial recovery when the animals reached normal body weight and smaller clinical scores. Spleen cells stimulated with MOG produced significantly higher levels of IFN-γ during the acute period whereas similar IL-17 levels were produced during both disease stages. CNS-infiltrating cells stimulated with MOG produced similar amounts of IFN-γ but, IL-17 was produced only at the acute phase of EAE. The percentage of Foxp3+ Treg cells, at the spleen and CNS, was elevated during both phases. The degree of inflammation was similar at both disease stages. Partial clinical recovery observed during chronic EAE was associated with no IL-17 production and presence of Foxp3+ Treg cells in the CNS.