A single administration of human adipose tissue-derived mesenchymal stromal cells (MSC) induces durable and sustained long-term regulation of inflammatory response in experimental colitis.

Current therapies for inflammatory bowel diseases (IBD) are aimed at controlling the exacerbated response in the gut, but no treatment is fully effective for many refractory patients. Mesenchymal stromal cells (MSC) are multi-potent cells with regulatory immunosuppressive activity that may control inflammatory diseases. In this study, we investigated the short- and especially the long-term protective effects of MSC on experimental colitis. We show that MSC elicited protection to acute intestinal inflammation with gain of weight, improvement in the clinical disease score and expressive reduction in the mortality rate of treated mice. MSC changed the population of neutrophils, eosinophils and augmented the frequency of CD4 T lymphocytes in the gut-draining lymph nodes, together with reduced accumulation of these cells in the colon intraepithelial compartment. Interestingly, there were increased levels of programmed death 1 (PD-1) and glucocorticoid-induced tumour necrosis factor receptor family-related receptor (GITR) in the spleen regulatory T cells of mice that received MSC treatment, which also presented a reversal in the pattern of immune response in the gut, with diminished inflammatory, T helper type 1 (Th1) and Th17 profile, in contrast to augmented Th2 responses. Most strikingly, this balanced response elicited by a single administration of MSC during the acute colitis persisted long-term, with restored goblet cells, eosinophils and maintenance of elevated gut interleukin (IL)-4, besides increased CD4+ CD25+ PD-1+ cells in the spleen and reduced Th17 response in mesenteric lymph nodes (MLN) of treated mice on day 60. Taken together, our findings provided a significant contribution to translational immunology by pointing human adipose tissue-derived MSC as a novel therapeutic approach with long-term beneficial regulatory effects in experimental colitis.

Validation of Mycobacterium tuberculosis dihydroneopterin aldolase as a molecular target for anti-tuberculosis drug development.

An early step of target validation in antimicrobial drug discovery is to prove that a gene coding for a putative target is essential for pathogen's viability. However, little attention has been paid to demonstrate the causal links between gene essentiality and a particular protein function that will be the focus of a drug discovery effort. This should be considered an important step in target validation since a growing number of proteins are found to exhibit multiple and unrelated tasks. Here, we show that the Mycobacterium tuberculosis (Mtb) folB gene is essential and that this essentiality depends on the dihydroneopterin aldolase/epimerase activities of its protein product, the FolB protein from the folate biosynthesis pathway. The wild-type (WT) MtFolB and point mutants K99A and Y54F were cloned, expressed, purified and monitored for the aldolase, epimerase and oxygenase activities using HPLC. In contrast to the WT MtFolB, both mutants have neither aldolase nor epimerase activities in the conditions assayed. We then performed gene knockout experiments and showed that folB gene is essential for Mtb survival under the conditions tested. Moreover, only the WT folB sequence could be used as a rescue copy in gene complementation studies. When the sequences of mutants K99A or Y54F were used for complementation, no viable colonies were obtained, indicating that aldolase and/or epimerase activities are crucial for Mtb survival. These results provide a solid basis for further work aiming to develop new anti-TB agents acting as inhibitors of the aldolase/epimerase activities of MtFolB.

5-ALA-mediated photodynamic therapy reduces the parasite load in mice infected with Leishmania braziliensis.

Photodynamic therapy (PDT) has proven to be an effective alternative for the treatment of cutaneous leishmaniasis. Skin lesions consist of ulcers with well-defined raised edges, and granular floor. Th1 immune response is the protective profile in patients infected with Leishmania. In this study, the photodynamic therapy with 5-aminolevulinic acid, the parasitic load, and the modulation of the immune response was evaluated in mice infected with Leishmania braziliensis. Balb/c mice were infected with L. braziliensis and subsequently treated with three sections of PDT. The parasite load and mRNA expression of cytokines (IFN-γ, IL-4, IL-17, IL-22, IL-27, IL-10) and transcription factors (GATA-3, Foxp3 and T-bet) were analysed by quantitative PCR. The parasite load in the treated group was significantly lower than in the untreated group (P<.0001); in PDT treated animals, we observed an increase in IFN-γ and T-bet mRNA (P=.012 and P=.0071). There was a significant reduction in mRNA expression of IL-22 associated with an increased expression of IL-27 mRNA in the animals treated with light only (P=.0001). 5-ALA associated with photodynamic therapy promotes a reduction in parasite load and an increased expression of IFN-γ and T-bet mRNA.

Evaluation of DPPD, a single recombinant Mycobacterium tuberculosis protein as an alternative antigen for the Mantoux test.

Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult particularly because sensitization with non-tuberculous mycobacteria leads to false positive tests. Using the guinea pig model of tuberculosis, we have recently described a recombinant antigen (DPPD) that could circumvent this problem. The DPPD gene is unique to the M. tuberculosis complex organisms and is absent in the organisms representative of all other members of the Mycobacterium genus. Moreover, DPPD induced strong DTH in 100% of the guinea pigs infected with M. tuberculosis and in none of the guinea pigs immunized with nine different species of Mycobacterium. Here we present results of a clinical investigation using DPPD. Mantoux test using both PPD and DPPD was initially performed in 26 patients with confirmed pulmonary tuberculosis and in 25 healthy PPD negative individuals. The results indicated that both PPD and DPPD elicited DTH in 24 out of the 26 patients. No DTH was observed in any of the PPD negative individuals. In addition, a small clinical trial was performed in a population of 270 clinically healthy and randomly selected individuals. DPPD produced a bimodal histogram of skin reaction size and PPD produced a skewed histogram. Because the DPPD gene is not present in non-tuberculous bacilli, these results suggest that this molecule can be an additional tool for a more specific diagnosis of tuberculosis.

In vitro granuloma formation, NO production and cytokines profile from human mononuclear cells induced by fractionated antigens of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the main mode of protection against this fungal infection. We investigated in vitro the response of peripheral blood mononuclear cells (PBMC) from paracoccidioidomycosis (PCM) patients presenting different clinical forms to antigenic fractions from P. brasiliensis yeast cell lysate (PbAg). These fractions designated F0 to FV were obtained using anion-exchange chromatography on a FPLC system. Our studies showed variation in the cellular responses induced by different antigenic fractions. The fraction F0 caused significant decrease in cellular proliferation, granuloma formation, accompanied by significant elevation in the production of IL-10. The fractions FII and FIII increased in vitro granuloma formation associated with high production of TNF-alpha. Besides that, FII and FIII evoked decrease in NO production but not F0 that induced very high levels, among patients with PCM from acute form. The findings suggest that P. brasiliensis antigenic components participate in the modulation or activation of PBMC response in PCM, and IL-10 and NO could be important in the regulation of in vitro granuloma formation.

Synergistic signal of an anti-hamster T cell monoclonal antibody on antigen-induced T cell proliferation.

1. The use of monoclonal antibodies (mAb) has permitted the identification of T cell surface antigens and the classification of these antigens based upon phenotype and function. Some of these monoclonal antibodies can identify antigens specifically involved in T lymphocyte activation and are also able to induce, under certain conditions, T cell proliferation. 2. We describe a new mAb raised against hamster T cells which binds to a 45-kDa cell surface antigen expressed on 45% of thymic cells and 90% of mature T lymphocytes. This mAb, designated X2VA, alone does not cause T cell proliferation, but increases in a synergistic manner T cell proliferation when these cells are cultured in the presence of specific antigens, or used in mixed lymphocyte reactions. 3. When the X2VA mAb is used as a single signal for the T cells it induces the production of a T cell growth factor, suggesting that the synergist effect observed during antigen-induced T cell proliferation is mediated by one or more cytokines. 4. Our results indicate that the X2VA mAb recognizes an antigen which is expressed during T cell ontogenesis and which is involved in hamster T cell activation.

[Tests of inflammatory activity in endemic pemphigus foliaceus]. / Provas de atividade inflamatória no pênfigo foliáceo endêmico.

Endemic pemphigus foliaceus (EPF) has its pathogenesis frequently associated to autoimmune phenomena. In this paper, a few routine laboratory tests, usually disturbed in some autoimmune diseases, were taken in 20 patients with EPF, which were screened for antinuclear antibodies (ANA), rheumatoid factor (RF), C-reactive protein (CRP) and changes of erythrocyte sedimentation rate (ESR), serologic proteins electrophoresis and total leucocyte count. The CRP was found in 60% of cases, leukocytosis in 85%, high ESR in all of them and mild alterations in serologic proteins analysis. No ANA or RF was found. Although widely accepted as nonspecific tests, we believe that an association of the laboratory routine tests with clinical findings, can prove to be helpful in the follow up care of these patients.

[Hanseniasis virchowiana in Chagas cardiopathy: an autopsy report]. / Hanseníase virchowiana em cardiopata chagásico: relato de necropsia.

This is a case report of lepromatous infection diagnosed at necropsy, with cardiac alterations directly caused by mycobacteria, in a 34-year-old black male with the cardiac form of Chagas' disease. The possible role of inflammatory mediators on cardiac dysfunction, and the possibility that immune depression may be due to factors associated with heart failure, as congestive splenomegaly and splenic infarctions, are emphasized.

Cytokines and chemokines production by mononuclear cells from parturient women after stimulation with live Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can cause variable clinical symptoms or can even be asymptomatic in immunocompetent individuals. More severe symptoms are observed in immunocompromised patients and congenital transmission of the parasite has been reported. The objective of this study was to evaluate the response of peripheral blood mononuclear cells (PBMC) in parturient and non-pregnant women exposed to live tachyzoites of T. gondii strain RH or ME49. PBMC were isolated from parturient and non-pregnant women with negative or positive serology for toxoplasmosis and cultured with live tachyzoites of the two T. gondii strains for 24 h. Next, the cell culture supernatants were collected and levels of CCL2, CCL5, IL-6, IL-10, IL-12, and TNF-α produced by PBMC after tachyzoite exposure were measured. Live tachyzoite forms of T. gondii significantly inhibited the synthesis of CCL2 in seropositive parturient women, whereas a stimulatory effect on CCL5 was observed in seronegative parturient women. Cells from T. gondii-seronegative non-pregnant women produced significantly higher levels of TNF-α and IL-12, demonstrating the proinflammatory profile induced by the presence of the parasite in culture. The results suggest that the immunomodulation seen during pregnancy contributes to the development of an environment that facilitates escape of the parasite from the immune response.

Selective inability of spleen antigen presenting cells from Leishmania donovani infected hamsters to mediate specific T cell proliferation to parasite antigens.

Golden hamsters (Mesocricetus auratus) infected with Leishmania donovani develop a disease similar to human kala-azar. There is conspicuous hypergammaglobulinaemia and their T cells do not respond to stimulation by parasite antigens. The impairment of the cellular immune response seems to be restricted to parasite antigens since infected animals are able to develop a T cell response to the mitogen Concanavalin A (Con-A) and, after sensitization, to the antigens keyhole limpet haemocyanin (KLH) and human serum albumin (DNP-HSA). In the present investigations we studied the role played by infected macrophages in the development of the cellular unresponsiveness present in visceral leishmaniasis. Adherent spleen cells from infected hamsters were unable to present L. donovani antigens to antigen specific T cells, however they were able to present KLH. Conversely, T cells from infected animals did not respond to parasite antigens even when these antigens were presented by normal syngeneic macrophages. Interestingly, lymphocytes from inguinal lymph nodes of infected animals sensitized in their foot pad with parasite antigens proliferated well when stimulated in vitro with L. donovani antigens. These results suggest that the defect in the cellular immune response of the L. donovani infected hamsters is a consequence of a selective inability of their antigen presenting cells to process and present parasite antigens to T cells.

Aspectos clínicos e imunopatológicos da ceratoplastia com membrana amniótica xenógena fresca e conservada em glicerina: estudo experimental em coelhos / Clinical and immunopatho logical aspects of keratoplasty with fresh and preserved in glycerin xenogenous amniotic membrane: experimental study in rabbits

Avaliaram-se, por meio da análise clínica, histopatologia e imunoistoquímica, os efeitos da aplicação da membrana amniótica xenógena fresca e conservada em glicerina, sobre os mecanismos imunológicos da superfície ocular. Para tal, utilizaram-se 40 coelhos, distribuídos em dois grupos experimentais, os quais foram avaliados por 21 dias. A avaliação clínica revelou que a membrana amniótica xenógena conservada em glicerina estimulou uma resposta inflamatória aguda maior que a membrana aplicada fresca. A análise histopatológica indicou que ambas se comportaram de forma semelhante a partir da primeira semana de pós-operatório, apresentando as alterações clássicas da resposta inflamatória da córnea, com o predomínio de infiltrado do tipo polimorfonuclear. A análise imunoistoquímica indicou que, ainda aos 21 dias, a resposta imune local é inespecífica, permitindo concluir que a resposta imune específica na córnea é tardia e que a córnea é um sítio privilegiado para aplicação de enxertos com características imunológicas diferentes, visto que não houve o estímulo para o desenvolvimento de uma resposta mais específica nos grupos avaliados durante toda a execução do experimento.

The dynamics of the inflammatory response and the mechanism involved in the healing process of cornea treated by keratoplasty, applying fresh and glycerin preserved xenogenous amniotic membranes as method to cover experimental ulcers were studied using 40 rabbits. The animals were allotted into two groups and evaluated during 21 days. The eyes were evaluated by histopathological study and immunohistochemical reaction. The clinical evaluation showed that the xenogenous amniotic membrane preserved in glycerin stimulated a greater acute inflammatory response than the one caused by fresh membrane. The histopathological analysis indicated that both membranes reacted in a very similar way from the first week post-surgery, presenting the classical alterations of the inflammatory response on the cornea. The immunohistochemical technique indicated that in no moment of the observation, the local immunologic response was specific. It was concluded that the specific immunologic response on the cornea took place later and that is a privileged site to use grafts with different immunological characteristics, once there was not a stimulus to the development of a more specific response in none of the evaluated groups throughout the experiment.

Interleukin-5 and interleukin-10 are major cytokines in cerebrospinal fluid from patients with active neurocysticercosis

Neurocysticercosis (NCC) is a common neurological disorder especially in developing countries, caused by infection of the brain with encysted larvae of the tapeworm Taenia solium. Seizures are a common finding associated with this disease. The objective of the present study was to evaluate the correlation between the levels of various cytokines present in the cerebrospinal fluid (CSF) of patients with NCC and the severity of the disease. The levels of the cytokines IL-1î, TNF-alpha, IL-5, IL-10 and IFN-gamma were determined in the CSF of 22 patients with active NCC, 13 patients with inactive NCC and 15 control subjects. CSF from patients with active NCC presented significantly higher IL-5 levels compared to control subjects. IL-5 and IL-10 levels in CSF from NCC patients with inflammatory CSF were significantly higher than those detected in non-inflammatory CSF. These results show a predominant Th2 lymphocyte activation in human NCC and also indicate the possible use of cytokines in the CSF as a marker for the differential diagnosis between inactive disease and the active form of NCC

Synergistic signal of an anti-hamster T cell monoclonal antibody on antigen-induced T cell proliferation

1.The use of monoclonal antibodies (mAb) has permitted the identification of T cell surface antigens and the classification of these antigens based upon phenotype and function. Some of these monoclonal antibodies can identify antigens specifically involved in T lymphocyte activation and are also able to induce, under certain conditions, T cell proliferation. 2. We describe a new mAb raised against hamster T cells which binds to a 45-KDa cell surface antigen expressed on 45% of thymic cells and 90% of mature T lymphocytes. This mAb,d esignated X2VA, alone does not cause T cells proliferation, but increases in a synergistic manner T cell proliferation when these cells are cultured in the presence of specific antigens, or used in mixed lymphocyte reactions. 3. When the X2Va mAb is used as single signal for the T cells it induces the production of a T cell growth factor, suggesting that the synergist effect observed during antigen-induced T cell proliferation is mediated by one more cytokines. 4. Our results indicate that the X2Va mAb recognizes an antigen which is expressed during T cell ontogenesis and which is involved in hamster T cell Activation