DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes.

BACKGROUND: Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome. OBJECTIVES: In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes. METHODS: DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture. RESULTS: In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations. CONCLUSION: Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

Chagas disease in Virgem da Lapa, Minas Gerais, Brazil: left ventricle aneurysm and the risk of death in the 24-year interval.

BACKGROUND: Left ventricular aneurysm (LVA) is indicator of high morbidity in Chagas' disease. A cross-sectional study performed identified LVA in 18.8% of the chronic chagasic patients (CCP). OBJECTIVE: Determine the risk of death of patients with chronic chagasic cardiopathy (CCC) and LVA in 24-year interval. MATERIAL AND METHODS: In 1995 a cohort of 298 CCP was evaluated by anamnesis, physical examination, EKG and ECHO and classified in groups: G0 = 86 without cardiopathy; G1 = 156 with cardiopathy without LVA and G2 = 56 with cardiopathy and LVA. 38 patients of G0 and G1 used benznidazole. Information about the deaths was obtained in the notary, death certificates, hospital records and family members. FINDINGS: Were registered 113 deaths (37.9%): 107 (35.9%) attributed to cardiopathy and 6 (2.0%) to other causes (p < 0.05). Amongst these 107 deaths, 10 (11.6%) occurred in G0; 49 (31.4%) occurred in G1 and 48 (85.7%) occurred in G2 (p < 0.05). The risk of death was 2.7 and 7.4 times significantly higher in G2, than in G1 and G0, respectively. CONCLUSION: Chronic chagasic patients with LVA and ejection fraction < 45% have a higher risk of death than those without.

Comparative evaluation of lesion development, tissue damage, and cytokine expression in golden hamsters (Mesocricetus auratus) infected by inocula with different Leishmania (Viannia) braziliensis concentrations.

The golden hamster (Mesocricetus auratus) is a susceptible model to Leishmania (Viannia) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L. (V.) braziliensis preparations were standardized to contain 10(4), 10(5), or 10(6) parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti-Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10(4) parasites presented nodular lesions, while those infected with 10(6) parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10(4) and 10(5) inocula or 10(4) and 10(6) inocula. High IFNG expression, anti-Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10(4) parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10(5) and 10(6) parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil.

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

[First South american network of biomedical research. Education and biotechnology for health]. / Primera Red Sudamericana de Biomedicina. Investigación, educación y biotecnología aplicadas a la salud.

It is in our interest, in this brief manuscript, to report the creation of the first program of regional integration of a network of research institutes in Biomedicine belonging to members of the MERCOSUR countries. We discuss some of the foundations that gave sustenance to its creation and its objectives in the medium and long term. In addition, we consider the potential of the results of this program in the fields of applied medical research, education and biotechnology.

Dual Role of Insulin-Like Growth Factor (IGF)-I in American Tegumentary Leishmaniasis.

BACKGROUND: Cytokines and growth factors involved in the tissue inflammatory process influence the outcome of Leishmania infection. Insulin-like growth factor (IGF-I) constitutively present in the skin may participate in the inflammatory process and parasite-host interaction. Previous work has shown that preincubation of Leishmania (Leishmania) amazonensis with recombinant IGF-I induces accelerated lesion development. However, in human cutaneous leishmaniasis (CL) pathogenesis, it is more relevant to the persistent inflammatory process than progressive parasite proliferation. In this context, we aimed to investigate whether IGF-I was present in the CL lesions and if this factor may influence the lesions' development acting on parasite growth and/or on the inflammatory/healing process. Methodology. Fifty-one CL patients' skin lesion samples from endemic area of L. (Viannia) braziliensis infection were submitted to histopathological analysis and searched for Leishmania and IGF-I expression by immunohistochemistry. RESULTS: In human CL lesions, IGF-I was observed preferentially in the late lesion (more than 90 days), and the percentage of positive area for IGF-I was positively correlated with duration of illness (r = 0.42, P < 0.05). IGF-I was highly expressed in the inflammatory infiltrate of CL lesions from patients evolving with good response to therapy (2.8% ± 2.1%; median = 2.1%; n = 18) than poor responders (1.3% ± 1.1%; median: 1.05%; n = 6; P < 0.05). CONCLUSIONS: It is the first time that IGF-I was detected in lesions of infectious cutaneous disease, specifically in American tegumentary leishmaniasis. IGF-I was related to chronicity and good response to treatment. We may relate this finding to the efficient anti-inflammatory response and the known action of IGF-I in wound repair. The present data highlight the importance of searching nonspecific factors besides adaptive immune elements in the study of leishmaniasis' pathogenesis.

Leishmania (V.) braziliensis infection promotes macrophage autophagy by a LC3B-dependent and BECLIN1-independent mechanism.

Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phagocytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimination. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirming BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell.

Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children.

The diagnosis of visceral leishmaniasis (VL) generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes). This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR) is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6%) that is similar to that from the PCR of bone marrow aspirates (91.1%) and higher than that achieved with microscopy (80%) or culture (26.7%) methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

An open toolkit for tracking open science partnership implementation and impact.

Serious concerns about the way research is organized collectively are increasingly being raised. They include the escalating costs of research and lower research productivity, low public trust in researchers to report the truth, lack of diversity, poor community engagement, ethical concerns over research practices, and irreproducibility. Open science (OS) collaborations comprise of a set of practices including open access publication, open data sharing and the absence of restrictive intellectual property rights with which institutions, firms, governments and communities are experimenting in order to overcome these concerns. We gathered two groups of international representatives from a large variety of stakeholders to construct a toolkit to guide and facilitate data collection about OS and non-OS collaborations. Ultimately, the toolkit will be used to assess and study the impact of OS collaborations on research and innovation. The toolkit contains the following four elements: 1) an annual report form of quantitative data to be completed by OS partnership administrators; 2) a series of semi-structured interview guides of stakeholders; 3) a survey form of participants in OS collaborations; and 4) a set of other quantitative measures best collected by other organizations, such as research foundations and governmental or intergovernmental agencies. We opened our toolkit to community comment and input. We present the resulting toolkit for use by government and philanthropic grantors, institutions, researchers and community organizations with the aim of measuring the implementation and impact of OS partnership across these organizations. We invite these and other stakeholders to not only measure, but to share the resulting data so that social scientists and policy makers can analyse the data across projects.

Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA).

BACKGROUND: The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. METHODS: We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. RESULTS: HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. CONCLUSION: Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development.

Suppression of allergic inflammatory response in the skin of alloxan-diabetic rats: relationship with reduced local mast cell numbers.

BACKGROUND: Diabetic patients are refractory to allergic inflammatory diseases. In this study, the influence of alloxan-induced diabetes on allergic skin inflammation was investigated. METHODS: Diabetes was induced by intravenous injection of alloxan into male Wistar rats, and the analyses were performed 21 days later. Animals were actively sensitized with a mixture of aluminium hydroxide plus ovalbumin and challenged intradermally with ovalbumin on day 14. RESULTS: Diabetic sensitized rats exhibited a less pronounced antigen-induced protein extravasation in the dorsal skin when compared with normal animals. Also, fragments of the dorsal subcutaneous tissue from diabetic sensitized rats showed a reduction in histamine release after stimulation with antigen in vitrowhen compared with fragments obtained from nondiabetic sensitized rats. Optical microscopy analysis revealed that the dorsal skin of diabetic rats showed a marked reduction in dermis thickness, as compared with that seen in normal animals. A significant decrease in the number of skin mast cells was also noted, a phenomenon that paralleled with the reduction in the expression of extracellular matrix components laminin, fibronectin and collagen. Administration of insulin into diabetic rats restored basal mast cell numbers as well as the levels of laminin, fibronectin and collagen. CONCLUSIONS: Our findings show that alloxan diabetes induces downregulation of the skin allergic inflammatory response in rats, and this was correlated with reduction in local mast cell numbers and expression of extracellular matrix components. Lastly, these alterations were reversed with insulin treatment.

DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes

BACKGROUND Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome. OBJECTIVES In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes. METHODS DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture. RESULTS In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations. CONCLUSION Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

Cutaneous leishmaniasis.

Cutaneous leishmaniasis is endemic in the tropics and neotropics. It is often referred to as a group of diseases because of the varied spectrum of clinical manifestations, which range from small cutaneous nodules to gross mucosal tissue destruction. Cutaneous leishmaniasis can be caused by several Leishmania spp and is transmitted to human beings and animals by sandflies. Despite its increasing worldwide incidence, but because it is rarely fatal, cutaneous leishmaniasis has become one of the so-called neglected diseases, with little interest by financial donors, public-health authorities, and professionals to implement activities to research, prevent, or control the disease. In endemic countries, diagnosis is often made clinically and, if possible, by microscopic examination of lesion biopsy smears to visually confirm leishmania parasites as the cause. The use of more sophisticated diagnostic techniques that allow for species identification is usually restricted to research or clinical settings in non-endemic countries. The mainstays of cutaneous leishmaniasis treatment are pentavalent antimonials, with new oral and topical treatment alternatives only becoming available within the past few years; a vaccine currently does not exist. Disease prevention and control are difficult because of the complexity of cutaneous leishmaniasis epizoology, and the few options available for effective vector control.

IFNG +874T/A polymorphism is not associated with American tegumentary leishmaniasis susceptibility but can influence Leishmania induced IFN-gamma production.

BACKGROUND: Interferon-gamma is a key cytokine in the protective responses against intracellular pathogens. A single nucleotide polymorphism (SNP) located in the first intron of the human IFN-gamma gene can putatively influence the secretion of cytokine with an impact on infection outcome as demonstrated for tuberculosis and other complex diseases. Our aim was to investigate the putative association of IFNG+874T/A SNP with American tegumentary leishmaniasis (ATL) and also the influence of this SNP in the secretion of IFN-gamma in vitro. METHODS: Brazilian ATL patients (78 cutaneous, CL, and 58 mucosal leishmaniasis, ML) and 609 healthy volunteers were evaluated. The genotype of +874 region in the IFN-gamma gene was carried out by Amplification Refractory Mutational System (ARMS-PCR). Leishmania-induced IFN-gamma production on peripheral blood mononuclear cell (PBMC) culture supernatants was assessed by ELISA. RESULTS: There are no differences between +874T/A SNP frequency in cases and controls or in ML versus CL patients. Cutaneous leishmaniasis cases exhibiting AA genotype produced lower levels of IFN-gamma than TA/TT genotypes. In mucosal cases, high and low IFN-gamma producers were clearly demonstrated but no differences in the cytokine production was observed among the IFNG +874T or A carriers. CONCLUSION: Our results suggest that +874T/A polymorphism was not associated with either susceptibility or severity to leishmaniasis. Despite this, IFNG +874T/A SNP could be involved in the pathogenesis of leishmaniasis by influencing the amount of cytokine released by CL patients, although it could not prevent disease development. On the other hand, it is possible that in ML cases, other potential polymorphic regulatory genes such as TNF-alpha and IL-10 are also involved thus interfering with IFN-gamma secretion.

Is Leishmania (Viannia) braziliensis parasite load associated with disease pathogenesis?

BACKGROUND: Leishmania (Viannia) braziliensis is the main etiological agent of tegumentary leishmaniasis in the Americas. Parasite molecular diversity and host immune status contribute to extensive variations in its clinical presentation within endemic areas of Brazil. Pentavalent antimonials have been used for more than 60 years as the first-line drug for all cases, despite the potential for severe side effects and refractoriness. In Rio de Janeiro, Brazil, most L. (V.) braziliensis infections are benign with a scarcity of parasites, although metastasis and refractory infections can arise. In this scenario, the use of novel molecular tools can be useful for diagnosis and to assess tissue parasitism, and is of benefit to clinical and therapeutic management. METHODS: In this study, parasite load was assessed by real-time PCR based on the leishmanial small subunit ribosomal RNA gene. RESULTS AND CONCLUSION: The data revealed a tendency to higher tissue parasitism in the skin compared to mucous lesion sites and a reduction with disease progression. Parasite load was lower in poor compared to good responders to antimonials, and was also reduced in recurrent lesions compared to primary ones. However, parasite load became higher with sequential relapses, pointing to an immune system inability to control the infection. Therefore the parasite burden does not seem to be a good predictor of disease progression.

Haematogenous dissemination of Leishmania (Viannia) braziliensis in human American tegumentary leishmaniasis.

One of the potential dangers of American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis is the development of mucosal lesions. Haematogenous dissemination of the parasite is the most likely mechanism to explain this occurrence, but most attempts to isolate the parasite from blood have so far been unsuccessful. The presence of Leishmania in peripheral blood was therefore evaluated by PCR using DNA samples isolated from patients presenting active cutaneous or mucosal disease, and from individuals cured by antimonial treatment as well as individuals without a past history of leishmaniasis but with a positive Montenegro skin test, all living in L. (V.) braziliensis-endemic areas. Leishmania DNA was found not only in those patients presenting active cutaneous (24.8%) or mucosal (35%) lesions, but also in samples isolated from healed individuals (27.3%) as well as in asymptomatic skin-test-positive residents of endemic areas (37.5%). Overall, PCR showed the presence of parasite DNA in the blood of 26.2% of the 225 examined samples. These data suggest that persistence of parasites within the host may last for many years and, rather than being a risk factor, might be important in maintaining the protective response in those living in endemic areas.

Comparative immunohistological analysis of the Montenegro skin test reaction in asymptomatic infection and in acute and chronic cutaneous leishmaniasis.

INTRODUCTION: The Montenegro skin test reaction and leishmaniasis lesions share fundamental characteristics of a delayed type hypersensitivity reaction. OBJECTIVES: To determine whether the Montenegro skin test reaction (response to leishmanin) might approximate and thereby provide insight into the early inflammatory and immune response to Leishmania infection. MATERIALS AND METHODS: We compared the inflammatory response in biopsies of acute (evolution time < or = one month), and chronic lesions (evolution time > or = 6 months) with the Montenegro skin test reaction in the corresponding patients, and with the Montenegro skin test of asymptomatically infected volunteers. RESULTS: The proportion of CD4+ and CD8+ T lymphocytes, mononuclear phagocytes and granulocytes were similar in acute lesions and in their corresponding Montenegro skin test reactions. In contrast, CD4+ lymphocytes (32.6%) represented a significantly lower, and B cells (20%) and macrophages (27%) a significantly higher proportion of the cellular infiltrate in chronic lesions as compared to reactions in the corresponding skin test site (CD4+: 43.7%, B cells: 0.9%; macrophages: 17.5%). CD8+ T lymphocytes and macrophages were positively associated (P = 0.038) in the Montenegro skin test of asymptomatically infected individuals whereas CD8+ and CD4+ T cells were positively associated in the Montenegro skin test of chronic patients (P = 0.002). Notably, B cells were markedly more frequent in chronic lesions (20%) than in acute lesions (5.3%) (P = 0.002). CONCLUSION: The Montenegro skin test distinguished the cellular immune response to Leishmania in asymptomatic infection and chronic disease and may provide a surrogate of the early response to infection.