Evaluation of systemic immunity in atypical cutaneous leishmaniasis caused by Leishmania (L.) infantum chagasi.

In some central-American countries, Leishmania (L.) infantum chagasi infection can cause non-ulcerated or atypical cutaneous leishmaniasis (NUCL) in addition to the classic clinical form, visceral leishmaniasis (VL). Little is known about the host-parasite relationship that can contribute to the determination of one or another clinical form. The present study had the objective to evaluate the humoral and cellular immunity in the sera of individuals affected by NUCL to improve the comprehension of this atypical host-parasite interaction. Based on clinical and laboratory diagnosis, serum of 80 individuals was collected to evaluate the cytokines and immunoglobulins profile of NUCL (n = 47), VL patients (n = 5), and negative controls (n = 28). Cytokines were detected using Cytokine Bead Array (CBA) Human Th1/Th2/Th17 kit according to the manufacturer's instructions; class (IgG and IgM), and subclass of (IgG1 and IgG2) immunoglobulins was evaluated by ELISA using specific antigens. The concentration of TNF-α, IFN-γ, IL-2 and IL-4 cytokines in NUCL, VL and control was present below the detection threshold of CBA kit. IL-6, IL-10 and IL-17A cytokines was lower in NUCL compared to LV patients. Regarding to immunoglobulins, NUCL patients produced 4.0 times more IgG than the control, while VL patients produced 6.6 times more; and IgM level was 1.6 times higher in NUCL and 2.6 times in VL patients compared to the control. Concerning the immunoglobulins subclass, only VL patients showed positive reaction for IgG1, and IgG2 did not show positive reaction among the groups. The results showed a weak cellular and humoral systemic immune response in NUCL patients.

The expression of FOXP3 in lesions of several forms of leprosy in patients co-infected with HIV.

BACKGROUND: Brazil remains endemic for infection by the human immunodeficiency virus (HIV) and leprosy, having a major impact on public health and the life quality of affected patients. Although the relevance of this co-infection is recognized, several aspects, such as the immune response, are not yet fully understood. The objective of this study was to investigate the expression of FOXP3+ Treg cells in leprosy skin lesions and to correlate their clinical forms, laboratory characteristics (CD4, CD8, and CV), and the immune reconstitution syndrome in HIV-leprosy co-infection. METHODOLOGY/PRINCIPAL FINDINGS: An observational, cross-sectional, and analytical study was carried out comparing four groups of patients: those with concomitant diagnosis of leprosy and HIV infection without a leprosy reaction, those with leprosy and HIV co-infection patients with a reverse reaction (RR), those with leprosy without HIV and without reaction, and those with leprosywithout HIV and with RR. The patients were diagnosed at a dermatology outpatient clinic located in Belém, Pará, Brazil, from 2003 to 2017. In the sample studied, there was a positive correlation between FOXP3+ cell density and viral load, negative correlation with blood CD4+ (not statistically significant), significant positive correlation in CD8 count in patients with leprosy reaction, and positive relationship in patients with IRIS. The density of cells expressing FOXP3 was higher in the BL/LL forms in patients without HIV, although the difference was not statistically significant. However, the cell mean was higher in the TT/BT forms in patients co-infected with leprosy and HIV, showing contradictory results. CONCLUSIONS/SIGNIFICANCE: These findings support that higher activity of the HIV may stimulate or result in a higher expression of FOXP3-Tregs and that they may be involved in active immunosuppression observed at the infection site at the tissue level. This supports the need to expand studies on FOXP3+ Treg cells in co-infected patients.

Peripheral nerve abnormality in HIV leprosy patients.

BACKGROUND: The geographical overlap of HIV (human immunodeficiency virus) and leprosy infection has become increasingly frequent and worrying, bringing many clinical issues. Peripheral neuropathy is very frequent in leprosy because of the predilection of its etiologic agent by Schwann cells of the peripheral nervous system, and it also affects individuals with HIV as one of the most common neurological manifestations. METHODOLOGY/PRINCIPAL FINDINGS: The present study compared a cohort of 63 patients diagnosed with leprosy and coinfected with HIV with a cohort of 64 patients with leprosy alone, who were followed at the outpatient clinic of the Nucleus of Tropical Medicine of the Federal University of Pará, Brazil. We observed that HIV-coinfected leprosy patients presented greater odds of overall peripheral nerve damage (nerve function impairment-NFI) than patients with leprosy alone. More sensitive damage was observed, especially in patients coinfected with multibacillary forms. Leprosy patients coinfected with HIV presented higher chances of motor damage with improvement over time using multidrug therapy (MDT) and highly active antiretroviral therapy (HAART), along with a greater extent of damage and occurrence of neuritis. The data suggest that in addition to patients presenting possible damage caused by leprosy, they also had a greater damage gradient attributable to HIV disease, but not related to HAART because most of these patients had been on the treatment for less than a year. Neuritis was treated with prednisone at doses recommended by the WHO, and coinfected patients had the highest rate of clinical improvement in the first 60 days. CONCLUSIONS/SIGNIFICANCE: The clinical characteristics of the two diseases should be considered in leprosy patients coinfected with HIV for better diagnosis and treatment of peripheral neuropathy. We suggest that new simplified assessment tools that allow the evaluation of the NFI of these patients be developed for use in the service.

Evaluation of Regulatory Immune Response in Skin Lesions of Patients Affected by Nonulcerated or Atypical Cutaneous Leishmaniasis in Honduras, Central America.

In Honduras, Leishmania (L.) infantum chagasi causes both visceral leishmaniasis (LV) and nonulcerated or atypical cutaneous leishmaniasis (NUCL). NUCL is characterized by mononuclear inflammatory infiltration of the dermis, composed mainly of lymphocytes followed by macrophages with discrete parasitism. Considering that little is known about the pathogenesis of NUCL, the aim of this study was to evaluate the regulatory response in situ in skin lesions of patients affected by NUCL. Biopsies (n = 20) from human cutaneous nonulcerative lesions were collected and processed by usual histological techniques. The in situ regulatory immune response was evaluated by immunohistochemistry using antihuman CD4, FoxP3, IL-10, and TGF-ß antibodies. CD4+, FoxP3+, TGF-ß+, and IL-10+ cells were observed in the dermis with inflammatory infiltration in all studied cases and at higher densities compared to the normal skin controls. A positive and strong correlation was observed between CD4+ and FoxP3+ cells, and a positive and moderate correlation was observed between FoxP3+ and TGF-ß+ but not with IL-10+ cells. The data suggest that T regulatory FoxP3+ cells and the regulatory cytokines, especially TGF-ß, play an important role in the immunopathogenesis of NUCL, modulating a cellular immune response in the skin, avoiding tissue damage, and leading to low tissue parasitic persistence.

Immunopathological characterization of human cutaneous leishmaniasis lesions caused by Leishmania (Viannia) spp. in Amazonian Brazil.

American cutaneous leishmaniasis (ACL) is a chronic infectious disease caused by different protozoan species of Leishmania, and it is endemic in both tropical and subtropical countries. Using immunohistochemistry, we investigate the density of CD68+, lysozyme+, CD1a+, factor XIIIa+, CD4+, CD8+, CD56+, interferon (IFN)-γ+, and inducible NO synthase (iNOS+) cells. These cells were analyzed from 22 biopsy samples obtained from the lesions of ACL patients, whose infection was caused by Leishmania (Viannia) spp. Histopathological analysis showed dense mononuclear inflammatory infiltration in the dermis, which was composed of lymphocytes, macrophages, plasma cells, and discrete tissue parasitism. Granulomatous reactions were also present in the majority of cases. The density of the activated macrophages was higher than that of inactivated macrophages in the lesions. The density of Langerhans cells (CD1a+) was lower than that of dermal dendrocytes (factor XIIIa+). The density of CD8+ T lymphocytes was higher than that of CD4+ T lymphocytes. The cellular density of these immunological markers in relation to the species of Leishmania demonstrated that L. (Viannia) sp. lesions had higher IFN-γ expression than that Leishmania (Viania) braziliensis lesions. The evaluation of these markers, according to disease progression, did not reveal any significant differences. L. (Viannia) sp. infection leads to a favorable immune response in the host, as predominantly represented by lysozyme+, factor XIIIa+, CD8+ T cells, and the expression of (IFN)-γ+ at the lesion site.

Preclinical diagnosis of American visceral leishmaniasis during early onset of human Leishmania (L.) infantum chagasi-infection.

American visceral leishmaniasis (AVL) is an infectious disease, often with long-duration evolution, caused by Leishmania (L.) infantum chagasi. However, although the disease is considered the major clinical manifestation of the link between L. (L.) i. chagasi and the human immune response, we have recently identified five clinical-immunological profiles of infection in the Brazilian Amazon: three asymptomatic (Asymptomatic Infection--AI, Sub-clinical Resistant Infection--SRI, and Indeterminate Initial Infection--III), and two symptomatic ones [Symptomatic Infection--SI (=AVL) and Sub-clinical Oligosymptomatic Infection--SOI]. We confirm here the preclinical diagnosis of AVL through the IgM-antibody response in a case of an early infection (profile III) that evolved to the full disease after 6 weeks.

Dynamic of the cellular immune response at the dermal site of Leishmania (L.) amazonensis and Leishmania (V.) braziliensis infection in Sapajus apella primate.

The purpose of this study was to characterize the immunopathological response in the skin of S. apella infected with Leishmania (L.) amazonensis and L. (V.) braziliensis parasites, the main causative agents of localized cutaneous leishmaniasis in South America. In infected animals, amastigote forms of L. (L.) amazonensis could be detected till 120 days postinfection (PI), while, in L. (V.) braziliensis infection, parasites could be detected until 180 days PI in the skin sections. CD20(+) cells were detected throughout the experimental time in both groups as well as in CD3(+) cells, which appeared to be activated because high densities of inducible nitric oxide synthase (iNOS(+)) cells were detected at 60 and 90 days PI in both studied groups. After 60 and 120 days PI, decrease in iNOS(+) cells was observed in L. (L.) amazonensis and L. (V.) braziliensis, respectively, which was associated with parasite clearance. Increase in lysozyme(+) cells was observed during the experimental infections, which also can be associated with parasite killing.

Severe visceral leishmaniasis in children: the relationship between cytokine patterns and clinical features

Introduction The relationship between severe clinical manifestations of visceral leishmaniasis (VL) and immune response profiles has not yet been clarified, despite numerous studies on the subject. This study aimed to investigate the relationship between cytokine profiles and the presence of immunological markers associated with clinical manifestations and, particularly, signs of severity, as defined in a protocol drafted by the Ministry of Health (Brazil). Methods We conducted a prospective, descriptive study between May 2008 and December 2009. This study was based on an assessment of all pediatric patients with VL who were observed in a reference hospital in Maranhão. Results Among 27 children, 55.5% presented with more than one sign of severity or warning sign. Patients without signs of severity or warning signs and patients with only one warning sign had the highest interferon-gamma (IFN-γ) levels, although their interleukin 10 (IL-10) levels were also elevated. In contrast, patients with the features of severe disease had the lowest IFN-γ levels. Three patients who presented with more than two signs of severe disease died; these patients had undetectable interleukin 2 (IL-2) and IFN-γ levels and low IL-10 levels, which varied between 0 and 36.8pg/mL. Conclusions Our results showed that disease severity was associated with low IFN-γ levels and elevated IL-10 levels. However, further studies with larger samples are needed to better characterize the relationship between disease severity and cytokine levels, with the aim of identifying immunological markers of active-disease severity. .

Molecular markers of mucosa harboring gastric adenomas.

CONTEXT: Gastric adenoma is a precursor lesion of the adenocarcinoma. OBJECTIVE: To characterize gastric adenomas according to the mucin immunoexpression and to evaluate the immunoexpression of p53, p16ink4a, BCL-2, cyclin D, Ki-67, in the adenoma and in the gastric mucosa harboring adenoma. METHODS: Forty gastric specimens from 20 patients were classified as intestinal (MUC2-goblet cell mucin) or foveolar (MUC5AC-gastric-foveolar mucin) adenomas. Immunohistochemistry was performed using streptavidin-biotin-complex method. RESULTS: Twelve (60%) patients were men. The mean age was 67.9±12.9 years-old. Intestinal adenomas were detected in 13 (65%) patients and gastric type in 7 (35%). Low-grade dysplasia was present in 13 (65%) of the adenomas, high-grade in 3 (15%), and adenocarcinoma within the polyp in 4 (20%). Six (30%) patients had synchronous adenocarcinoma. p53 immunoexpression was observed in 6/20 (30%) of adenomas, and in 2/6 (33.3%) of synchronous tumors. There was an association between p53 immunoexpression and intestinal type of adenoma/tumor, P=0.04. There was no association between p16ink4a, Bcl-2, cyclin D and Ki-67 and adenoma clinicopathological characteristics. CONCLUSION: Immunohistochemistry may be useful to classify the adenomas subtypes and may define the pathway of adenoma to carcinoma sequence.

Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector.

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n=24) and asymptomatic (n=14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p=0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p=0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p=0.027) and was directly correlated with antibody levels (p=0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas.

Molecular markess of mucosa harboring gastric adenoass

Context Gastric adenoma is a precursor lesion of the adenocarcinoma. Objective To characterize gastric adenomas according to the mucin immunoexpression and to evaluate the immunoexpression of p53, p16ink4a, BCL-2, cyclin D, Ki-67, in the adenoma and in the gastric mucosa harboring adenoma. Methods Forty gastric specimens from 20 patients were classified as intestinal (MUC2 - goblet cell mucin) or foveolar (MUC5AC - gastric-foveolar mucin) adenomas. Immunohistochemistry was performed using streptavidin-biotin-complex method. Results Twelve (60%) patients were men. The mean age was 67.9 ± 12.9 years-old. Intestinal adenomas were detected in 13 (65%) patients and gastric type in 7 (35%). Low-grade dysplasia was present in 13 (65%) of the adenomas, high-grade in 3 (15%), and adenocarcinoma within the polyp in 4 (20%). Six (30%) patients had synchronous adenocarcinoma. p53 immunoexpression was observed in 6/20 (30%) of adenomas, and in 2/6 (33.3%) of synchronous tumors. There was an association between p53 immunoexpression and intestinal type of adenoma/tumor, P = 0.04. There was no association between p16ink4a, Bcl-2, cyclin D and Ki-67 and adenoma clinicopathological characteristics. Conclusion Immunohistochemistry may be useful to classify the adenomas subtypes and may define the pathway of adenoma to carcinoma sequence. .

Contexto Adenoma gástrico é uma lesão precursora do adenocarcinoma. Objetivo Melhor caracterizar os adenomas de acordo com a imunoexpressão de mucinas e avaliar a imunoexpressão de p53, p16ink4a, BCL-2, cyclin D, Ki-67, nos adenomas e na mucosa gástrica adjacente. Métodos Quarenta espécimes gástricos provenientes de 20 pacientes portadores de adenomas foram classificados como do tipo intestinal (MUC2 – mucina presente nas células caliciformes) ou gástrico (MUC5AC – mucinas de padrão foveolar). Realizou-se imunoistoquímica para p53, p16ink4a, BCL-2, cyclin D e Ki-67 pelo método do complexo da estreptavidina-biotina. Resultados Doze (60%) pacientes eram homens e a média de idade foi de 67,9 ± 12,9 anos. Os adenomas foram classificados como do tipo intestinal em 13 (65%) pacientes e do tipo gástrico em 7 (35%). Displasia (neoplasia intraepitelial) de baixo grau estava presente em 13 (65%), displasia de alto grau em 3 (15%), e adenocarcinoma no pólipo adenomatoso em 4 (20%) pacientes. Observou-se immunoexpressão do p53 em 6/20 (30%) adenomas, e em 2/6 (33,3%) dos tumores sincrônicos. Houve associação entre imunoexpressão do p53 e adenoma/tumor tipo intestinal, P = 0.04. Não houve associação entre imunoexpressão do p16ink4a, Bcl-2, ciclina D e Ki-67 e as características clinicopatológicas dos adenomas. Conclusão Imunoistoquímica pode ser utilizada para caracterizar os subtipos de adenoma e talvez indicar o caminho de carcinogênese. .

Severe visceral leishmaniasis in children: the relationship between cytokine patterns and clinical features.

INTRODUCTION: The relationship between severe clinical manifestations of visceral leishmaniasis (VL) and immune response profiles has not yet been clarified, despite numerous studies on the subject. This study aimed to investigate the relationship between cytokine profiles and the presence of immunological markers associated with clinical manifestations and, particularly, signs of severity, as defined in a protocol drafted by the Ministry of Health (Brazil). METHODS: We conducted a prospective, descriptive study between May 2008 and December 2009. This study was based on an assessment of all pediatric patients with VL who were observed in a reference hospital in Maranhão. RESULTS: Among 27 children, 55.5% presented with more than one sign of severity or warning sign. Patients without signs of severity or warning signs and patients with only one warning sign had the highest interferon-gamma (IFN-γ) levels, although their interleukin 10 (IL-10) levels were also elevated. In contrast, patients with the features of severe disease had the lowest IFN-γ levels. Three patients who presented with more than two signs of severe disease died; these patients had undetectable interleukin 2 (IL-2) and IFN-γ levels and low IL-10 levels, which varied between 0 and 36.8pg/mL. CONCLUSIONS: Our results showed that disease severity was associated with low IFN-γ levels and elevated IL-10 levels. However, further studies with larger samples are needed to better characterize the relationship between disease severity and cytokine levels, with the aim of identifying immunological markers of active-disease severity.

Susceptibility of peritoneal macrophage from different species of neotropical primates to ex vivo Leishmania (L.) infantum chagasi-infection.

This study examined the susceptibility of peritoneal macrophage (PM) from the Neotropical primates: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus and Callimico goeldii to ex vivo Leishmania (L.) infantum chagasi-infection, the etiological agent of American visceral leishmaniasis (AVL), as a screening assay for evaluating the potential of these non-human primates as experimental models for studying AVL. The PM-susceptibility to infection was accessed by the PM-infection index (PMI) at 24, 72 h and by the mean of these rates (FPMI), as well as by the TNF-α, IL-12 (Capture ELISA) and Nitric oxide (NO) responses (Griess method). At 24h, the PMI of A. azarae infulatus (128) was higher than those of C. penicillata (83), C. goeldii (78), S. sciureus (77) and C. jacchus (55). At 72h, there was a significant PMI decrease in four monkeys: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) and C. jacchus (55/12), with exception of C. goeldii (78/54). The FPMI of A. azarae infulatus (82.5) and C. goeldii (66) were higher than C. jacchus (33.5), but not higher than those of C. penicillata (60.5) and S. sciureus (57.5). The TNF-a response was more regular in those four primates which decreased their PMI at 24/72 h: C. jacchus (145/122 pg/mL), C. penicillata (154/130 pg/mL), S. sciureus (164/104 pg/mL) and A. azarae infulatus (154/104 pg/mL), with exception of C. goeldii (38/83 pg/mL). The IL-12 response was mainly prominent in A. infulatus and C. goeldii which presented the highest FPMI and, the NO response was higher in C. goeldii, mainly at 72 h. These findings strongly suggest that these New World primates have developed a resistant innate immune response mechanism capable of controlling the macrophage intracellular growth of L. (L.) i. chagasi-infection, which do not encourage their use as animal model for studying AVL.

Susceptibility of peritoneal macrophage from different species of neotropical primates to Ex vivo Leishmania (L.) infantum chagasi-infection / Susceptibilidade do macrófago peritoneal de diferentes espécies de primatas neotropicais para a infecção ex vivo por Leishmania (L.) infantum chagasi

This study examined the susceptibility of peritoneal macrophage (PM) from the Neotropical primates: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus and Callimico goeldii to ex vivo Leishmania (L.) infantum chagasi-infection, the etiological agent of American visceral leishmaniasis (AVL), as a screening assay for evaluating the potential of these non-human primates as experimental models for studying AVL. The PM-susceptibility to infection was accessed by the PM-infection index (PMI) at 24, 72 h and by the mean of these rates (FPMI), as well as by the TNF-α, IL-12 (Capture ELISA) and Nitric oxide (NO) responses (Griess method). At 24h, the PMI of A. azarae infulatus (128) was higher than those of C. penicillata (83), C. goeldii (78), S. sciureus (77) and C. jacchus (55). At 72h, there was a significant PMI decrease in four monkeys: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) and C. jacchus (55/12), with exception of C. goeldii (78/54). The FPMI of A. azarae infulatus (82.5) and C. goeldii (66) were higher than C. jacchus (33.5), but not higher than those of C. penicillata (60.5) and S. sciureus (57.5). The TNF-a response was more regular in those four primates which decreased their PMI at 24/72 h: C. jacchus (145/122 pg/mL), C. penicillata (154/130 pg/mL), S. sciureus (164/104 pg/mL) and A. azarae infulatus (154/104 pg/mL), with exception of C. goeldii (38/83 pg/mL). The IL-12 response was mainly prominent in A. infulatus and C. goeldii which presented the highest FPMI and, the NO response was higher in C. goeldii, mainly at 72 h. These findings strongly suggest that these New World primates have developed a resistant innate immune response mechanism capable of controlling the macrophage intracellular growth of L. (L.) i. chagasi-infection, which do not encourage their use as animal model for studying AVL.

Este estudo examinou a susceptibilidade do macrófago peritoneal (PM) dos primatas neotropicais: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus e Callimico goeldii para a infecção ex vivo por Leishmania (L.) infantum chagasi, o agente etiológico da leishmaniose visceral americana (LVA), como método de triagem para avaliar o potencial desses primatas como modelo de estudo da LVA. A susceptibilidade do PM para a infecção foi investigada através do índice de infecção do PM (PMI) a intervalos de 24, 72 horas e, ainda, pela média dessas taxas (FPMI), assim como, pelas respostas do TNF-α, IL-2 (ELISA de captura) e óxido nítrico (NO) (método de Griess). Às 24hs da infecção experimental, o PMI do primata A. azarae infulatus (128) foi maior que aqueles de C. penicillata (83), C. goeldii (78), S. sciureus (77) e C. jacchus (55). Às 72hs, houve uma redução significativa do PMI de quatro primatas: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) e C. jacchus (55/12), com exceção de C. goeldii (78/54). O FPMI dos primatas A. azarae infulatus (82.5) e C. goeldii (66) foi maior que do primata C. jacchus (33.5), porém, não foi maior que dos primatas C. penicillata (60.5) e S. sciureus (57.5). A resposta do TNF-α foi mais regular nos quatro primatas que reduziram o PMI no intervalo de 24-72hs: C. jacchus (145/122 pg/µL), C. penicillata (154/130 pg/µL), S. sciureus (164/104 pg/µL) e A. azarae infulatus (154/104 pg/µL), com exceção de C. goeldii (38/83 pg/µL). A resposta de IL-12 foi, principalmente, marcante nos primatas A. azarae infulatus e C. goeldii, os quais apresentaram as maiores taxas do FPMI, e a resposta do NO foi maior no primata C. goeldii, em especial no intervalo de 72hs. Estes achados sugerem, fortemente, que estes primatas neotropicais parecem ter desenvolvido mecanismos resistentes de resposta imune inata capaz de controlar o crescimento intracelular da infecção por L. (L.) i. chagasi no macrófago, o que não encoraja o uso destes primatas como modelo de estudo da LVA.

Leishmania (Viannia) shawi purified antigens confer protection against murine cutaneous leishmaniasis.

OBJECTIVE: Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis. MATERIALS AND METHODS: Soluble antigen from L. (V.) shawi promastigotes was submitted to reverse phase HPLC to purify F1, F2, F3, F4 and F5 antigens. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 µg protein. After 1 week, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 8 weeks, those same mice were sacrificed and parasite burden as well as the cellular and humoral immune responses were evaluated. RESULTS: F1 and F5-immunized mice restrained lesion progression and parasite load in the skin. However, only the F1 group was able to control the parasitism in lymph nodes, which was associated with low IL-4 and high IFN-γ production; IgG2a isotype was increased in this group. Immunizations with F2, F3 and F4 antigens did not protect mice. CONCLUSION: The capability of antigens to restrain IL-4 levels and increase IFN-γ was associated with protection, such as in immunization using F1 antigen.

Analysis of the protective potential of antigens released by Leishmania (Viannia) shawi promastigotes.

Leishmania (Viannia) shawi causes cutaneous lesions in humans. Parasite antigens conferring significant protection against American tegumentar leishmaniosis (ATL) might be important for the development of effective vaccine. Therefore, this work evaluates the protective effect of antigenic fractions released by L. shawi. Antigens released by promastigotes to culture medium were concentrated and isolated by SDS-PAGE. The three main fractions LsPass1 (>75 kDa), LsPass2 (75-50 kDa) and LsPass3 (<50 kDa) were electro-eluted according with their molecular mass. Immunized BALB/c mice were challenged with L. shawi promastigotes and the course of infection monitored during 5 weeks. LsPass1-challenged mice showed no protection, however, a strong degree of protection associated to smaller lesions and high expression of IFN-γ and TNF-α by CD4(+) T, CD8(+) T and double negative CD4CD8 cells was achieved in LsPass3-challenged mice. Furthermore, LsPass2-challenged mice showed an intermediated degree of protection associated to high levels of IFN-γ, IL-4 and IL-10 mRNA. In spite of increased expression of IFN-γ and TNF-α, high amounts of IL-4 and IL-10 mRNA were also detected in LsPass3-challenged mice indicating a possible contribution of these cytokines for the persistence of a residual number of parasites that may be important in inducing long-lasting immunity. Therefore, LsPass3 seems to be an interesting alternative that should be considered in the development of an effective vaccine against ATL.

Human cutaneous leishmaniasis: interferon-dependent expression of double-stranded RNA-dependent protein kinase (PKR) via TLR2.

We investigated the type I interferon (IFN-1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW-264.7 macrophages infected with L. (L.) amazonensis or HEK-293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2-knockout (KO) or IFNR-KO mice were infected, and the levels of PKR, IFN-1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN-1 and PKR-positive cells. Leishmania infection increased the expression of PKR and IFN-ß on induction of PKR-promoter activity. The observed effects required the engagement of TLR2. TLR2-KO macrophages expressed low IFN-ß and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania-induced IFN-1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN-1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant-negative PKR-expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN-1-expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN-1/PKR axis in the Leishmania infection.

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms.

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-ß concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-γ, TGF-ß and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.

Evaluation of Helicobacter pylory colonization by serologic test (IgG) and dyspepsia in volunteers from the countryside of Monte Negro, in the Brazilian western Amazon region / Avaliação da colonização por Helicobacter pylori através de teste sorológico (IgG) e de dispepsia em voluntários da população rural de Monte Negro (RO), região da Amazônia ocidental

The present study intended to analyze the seroprevalence of Helicobacter pylori, IgG, and its relation to dyspepsia in a population from the western Amazon region. During the "Projeto Bandeira Científica", a University of São Paulo Medical School program, in Monte Negro's rural areas, state of Rondônia, 266 blood samples were collected from volunteers. The material was tested for IgG antibodies anti-Helicobacter pylori by ELISA method and the participants were also interviewed on dyspepsia, hygiene and social aspects. Participants aged between five and 81 years old (34 years on average), 149 (56 percent) were female and 117 (44 percent) male. We found 210 (78.9 percent) positive, 50 (18.8 percent) negative and six (2.3 percent) undetermined samples. Dyspeptic complaints were found in 226 cases (85.2 percent). There was no statistical association between dyspepsia and positive serology for H. pylori. We concluded that the seroprevalence in all age categories is similar to results found in other studies conducted in developing countries, including those from Brazil. On the other hand, the seroprevalence found in Monte Negro was higher than that reported in developed countries. As expected, there was a progressive increase in the positivity for H. pylori in older age groups.

Este trabalho tem por objetivo analisar a soroprevalência do Helicobacter pylori, IgG, em população rural da Amazônia, e sua correlação com queixa dispéptica. No Projeto Bandeira Científica da FMUSP, em Monte Negro - RO, foram coletadas 266 amostras sangüíneas nos assentamentos rurais do município. Foram pesquisados anticorpos da classe IgG dirigidos contra Helicobacter pylori pelo método ELISA e aplicados questionários sobre dispepsia, aspectos sociais e epidemiológicos. Os pacientes tinham idades entre cinco e 81 anos (média de 34 anos); 149 (56 por cento) do sexo feminino e 117 (44 por cento) do sexo masculino. Foram encontradas 210 (78.9 por cento) amostras positivas, 50 negativas (18.8 por cento) e seis indeterminadas (2.3 por cento). A queixa de dispepsia foi encontrada em 226 casos (85.2 por cento). Não houve associação significativa entre os sintomas dispépticos e a soro positividade para H. pylori. Concluímos que a soro prevalência para todas as faixas etárias é comparável com os resultados de outros estudos realizados em países em desenvolvimento, e maior que aquela encontrada nos países desenvolvidos. Houve aumento progressivo da positividade com a idade, como citado na literatura.

Endemic hepatitis B and C virus infection in a Brazilian Eastern Amazon region.

CONTEXT: Hepatitis B and hepatitis C infection has been an important cause of morbidity and mortality around the world. However there are few investigations regarding the prevalence and possible risk factors for these diseases in Brazil, particularly in Amazon region, where there are some endemic focus. OBJECTIVES: To determine the prevalence of hepatitis B and hepatitis C in the city of Buriticupu, MA, located in the Brazilian Eastern Amazon region, and try to explore the risk factors for these infections in that area. METHODS: Two hundred forty three subjects (46.5% male and 53.5% female) were investigated. RESULTS: The prevalence of past or current infection of hepatitis B and C virus was, respectively, 40.74% and 5.76%. Positivity for HBsAg was found in 2.88% of the subjects. The prevalence of current infection or chronic virus carriers found was 2.88% (HBsAg). There was a statistically significant relationship between the sera-prevalence of anti-HBc and the distance of the residence from the city center which may reflect an indirect association between the infection and precarious conditions of existence. Individuals with age equal or greater than 60 years were also more likely to be anti-HBc positive which could only reflect that older people have a longer history of exposure to hepatitis B infection. The prevalence of hepatitis C is higher than the worldwide estimate. CONCLUSION: Buriticupu may be considered endemic for hepatitis B and C. Hepatitis B infection could be related to precarious living conditions and old age. Hepatitis C was not associated with the variables investigated in the present investigation.