Presence of parasite DNA in clinically unaffected nasal mucosa during cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.

OBJECTIVES: We aimed to detect Leishmania DNA carriage in nasal mucosa of individuals with cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis. METHODS: A cross-sectional study was performed in all individuals with CL without nasal lesions (n = 153) attended within 2 years in an endemic area of L. (Viannia) braziliensis in Bahia (Brazil). An otorhinolaryngologist assessed the clinical status of the nasal mucosa by anterior rhinoscopy and endoscopic examinations. Swab samples were collected for parasite DNA detection by PCR from all individuals before standard treatment for leishmaniasis. A second evaluation 3 months after treatment was performed to assess clinical outcomes. RESULTS: Parasite DNA was detected in 7.8% (12/153) of clinically healthy nasal mucosa of individuals with CL. Interestingly, DNA was more frequently identified in individuals with more skin lesions (median 1.5, interquartile range (IQR) 1-3.5 versus 1.0, IQR 1-1.5; p 0.044), or larger injuries (median 2.7, IQR 2-3.8 versus 1.6, IQR 1-2.5; p 0.013). Additionally, the disease of those individuals with positive PCR evolved more frequently to unusual forms of leishmaniasis (recidiva cutis and disseminated) (45.5% (5/11) versus 11.5% (14/122); p 0.009), and required more cycles of treatment to reach clinical cure (median 2, IQR 1-4 versus 1, IQR 1-2; p 0.05). CONCLUSION: These findings suggest an early parasite tropism to nasal mucosa in L. (Viannia) braziliensis infection and a clinical phenotype of CL cases associated with parasite DNA in nasal mucosa. Future studies should evaluate whether PCR of nasal swab samples could serve as a prognostic tool for individuals at risk of mucocutaneous leishmaniasis.

The Centre for Data and Knowledge Integration for Health (CIDACS): Linking Health and Social Data in Brazil.

The Centre for Data and Knowledge Integration for Health (CIDACS) was created in 2016 in Salvador, Bahia-Brazil with the objective of integrating data and knowledge aiming to answer scientific questions related to the health of the Brazilian population. This article details our experiences in the establishment and operations of CIDACS, as well as efforts made to obtain high-quality linked data while adhering to security, ethical use and privacy issues. Every effort has been made to conduct operations while implementing appropriate structures, procedures, processes and controls over the original and integrated databases in order to provide adequate datasets to answer relevant research questions. Looking forward, CIDACS is expected to be an important resource for researchers and policymakers interested in enhancing the evidence base pertaining to different aspects of health, in particular when investigating, from a nation-wide perspective, the role of social determinants of health and the effects of social and environmental policies on different health outcomes.

Challenges in the research and development of new human vaccines.

The field of vaccinology was born from the observations by the fathers of vaccination, Edward Jenner and Louis Pasteur, that a permanent, positive change in the way our bodies respond to life-threatening infectious diseases can be obtained by specific challenge with the inactivated infectious agent performed in a controlled manner, avoiding the development of clinical disease upon exposure to the virulent pathogen. Many of the vaccines still in use today were developed on an empirical basis, essentially following the paradigm established by Pasteur, "isolate, inactivate, and inject" the disease-causing microorganism, and are capable of eliciting uniform, long-term immune memory responses that constitute the key to their proven efficacy. However, vaccines for pathogens considered as priority targets of public health concern are still lacking. The literature tends to focus more often on vaccine research problems associated with specific pathogens, but it is increasingly clear that there are common bottlenecks in vaccine research, which need to be solved in order to advance the development of the field as a whole. As part of a group of articles, the objective of the present report is to pinpoint these bottlenecks, exploring the literature for common problems and solutions in vaccine research applied to different situations. Our goal is to stimulate brainstorming among specialists of different fields related to vaccine research and development. Here, we briefly summarize the topics we intend to deal with in this discussion.

Challenges in the research and development of new human vaccines

The field of vaccinology was born from the observations by the fathers of vaccination, Edward Jenner and Louis Pasteur, that a permanent, positive change in the way our bodies respond to life-threatening infectious diseases can be obtained by specific challenge with the inactivated infectious agent performed in a controlled manner, avoiding the development of clinical disease upon exposure to the virulent pathogen. Many of the vaccines still in use today were developed on an empirical basis, essentially following the paradigm established by Pasteur, "isolate, inactivate, and inject" the disease-causing microorganism, and are capable of eliciting uniform, long-term immune memory responses that constitute the key to their proven efficacy. However, vaccines for pathogens considered as priority targets of public health concern are still lacking. The literature tends to focus more often on vaccine research problems associated with specific pathogens, but it is increasingly clear that there are common bottlenecks in vaccine research, which need to be solved in order to advance the development of the field as a whole. As part of a group of articles, the objective of the present report is to pinpoint these bottlenecks, exploring the literature for common problems and solutions in vaccine research applied to different situations. Our goal is to stimulate brainstorming among specialists of different fields related to vaccine research and development. Here, we briefly summarize the topics we intend to deal with in this discussion.

Changes in amounts of total salivary gland proteins of Lutzomyia longipallpis (Diptera: Psychodidae) according to age and diet.

Saliva plays important roles in facilitation of a bloodmeal, lubrication of mouthparts, and parasite transmission for some vector insects. Salivary composition changes during the lifetime of an insect, and differences in the salivary profile may influence its functions. In this report, the amount and profile of salivary gland protein of the American visceral leishmaniasis vector Lutzomyia longipalpis (Lutz & Neiva, 1912) were analyzed at different times of insect development and diet. Protein content from unfed female sand flies increased significantly with age, and a significant difference was observed in sugar-fed females during the first 10 d of adult life. Salivary protein content sharply decreased 1 d after blood feeding, with gradual increase in concentration the following days. SDS-polyacrylamide gel electrophoresis analysis revealed that most polypeptides present in the saliva of sugar-fed also were present in the saliva of blood-fed females. Understanding changes in sand fly's saliva contents at distinct days after emergence and the influence of a bloodmeal in this aspect may reveal the role played by saliva during leishmaniasis transmission.

Role of sand fly saliva in human and experimental leishmaniasis: current insights.

Leishmaniases are wide spread diseases transmitted to their vertebrate host by infected sand fly. The saliva from these arthropods contains a vast repertoire of pharmacologically active molecules that hampers the host's haemostatic, inflammatory and immune responses. The early interactions between Leishmania and the host's immune response are closely linked to disease evolution or protection against the protozoan, and the ectoparasite saliva contributes directly to these interactions. Current studies have depicted these features, and these relations are being widely explored. There are concrete indications that the host response against sand fly saliva influences disease outcome in leishmaniasis. Additionally, there are demonstrations that immunization with whole sand fly saliva, or its components, leads to protection against leishmaniasis in different host species. The combination of these evidences opens up optimistic perspectives for improving vaccine development against Leishmania infection.

Leishmanial infection: analysis of its first steps: a review

The first steps in leishmaniasis are critical in determining the evolution of the disease. Major advances have recently been done in understanding this crucial moment. Fundamental research in parasite-vector interaction, parasite biology, insect saliva, and vertebrate host response have shed new light and uncovered a most fascinating and complex moment in leishmaniasis. We review here some of these aspects and we try to connect them in a logical framework.

Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a major aetiological agent of childhood diarrhoea in developing countries. The structural repeating protein A subunit, BfpA, found in the bundle-forming pilus, is one of the virulent factors for EPEC pathogenesis. Recombinant BfpA in laying hens elicited sustained and vigorous antibody production. Immunoglobulin Y (IgY) anti-BfpA antibodies were recovered from egg yolk, purified and characterized. Immunoadsorption with whole extracts of the isogenic E. coli EPEC adherence factor (EAF) strain that lacks BfpA rendered the resulting IgY preparations capable of: (a) recognizing purified or recombinant BfpA proteins in a dose-dependent fashion; (b) blocking the colonization of HeLa cells by EPEC EAF+, in vitro; (c) specifically identifying E. coli bearing EAF+; and (d) inhibiting the growth of E. coli EAF+ but not the EAF strain. IgY anti-BfpA is potentially useful as a specific, low-cost immunobiological reagent to screen human faecal specimens for the presence of EPEC.

Leishmania (Leishmania) chagasi infection alters the expression of cell adhesion and costimulatory molecules on human monocyte and macrophage.

The initial steps of Leishmania infection in humans are largely unknown. There is limited information on the Leishmania infected human monocytes, the first cells that the parasite lives in, particularly related to costimulatory molecules. We show here that Leishmania (L.) chagasi infection avoids inducing proinflammatory molecules and has striking down modulating effects on human monocytes or macrophages. It does not induce CD54, interleukin (IL)-12 or tumour necrosis factor-alpha, potent proinflammatory cytokines and down modulates CD11b expression in monocytes. Lipopolysaccharide stimulated IL-12 (p40) levels, CD54 and HLA-DR expression are diminished in infected monocytes as well as interferon-gamma stimulated HLA-DR and HLA-ABC expression in infected macrophages. There is a negative correlation between CD54 and CD86 expression in both monocytes and macrophages. The depressed expression of class I and II molecules, absence of key proinflammatory cytokines and impaired expression of costimulatory molecules induced by L. chagasi could leave the immune system, at least in its initial phases in anergy or ignorance.

Leishmanial infection: analysis of its first steps. A review.

The first steps in leishmaniasis are critical in determining the evolution of the disease. Major advances have recently been done in understanding this crucial moment. Fundamental research in parasite-vector interaction, parasite biology, insect saliva, and vertebrate host response have shed new light and uncovered a most fascinating and complex moment in leishmaniasis. We review here some of these aspects and we try to connect them in a logical framework.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases.

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-gamma, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ

Differences in gamma interferon production in vitro predict the pace of the in vivo response to Leishmania amazonensis in healthy volunteers.

The initial encounter of Leishmania cells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensis response of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-gamma) (n = 16) (low producers) and those who produced large amounts of this cytokine (n = 16) (high producers). IFN-gamma production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-gamma in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-gamma production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-gamma upon stimulation of their peripheral blood mononuclear cells with Leishmania promastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-gamma producers there was an increased frequency of activated CD8(+) T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-gamma as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmania cell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.

Treatment of multiple sclerosis patients with interferon-beta primes monocyte-derived macrophages for apoptotic cell death.

Although interferon (IFN)-beta has shown a significant clinical benefit in multiple sclerosis (MS), its mechanism of action remains unclear. We found that IFN-beta treatment of patients with MS resulted in a significant increase in apoptotic cell death (measured by annexin V staining and nuclear fragmentation) of monocyte-derived macrophages, as compared with cells derived from patients before treatment. Stimulation of the cells with IFN-beta in vitro resulted in an even further increase of annexin V binding, as well as increased Fas (CD 95, APO-1) expression. However, no increased Fas expression, apoptotic monocytes, or monocytopenia were observed upon in vivo treatment. This indicates that IFN-beta does not deliver a death signal to monocytes but rather primes for subsequent macrophage apoptosis upon activation or differentiation.

Interleukin 8 as a vaso-occlusive marker in Brazilian patients with sickle cell disease.

Sickle cell disease has a worldwide distribution and is a public health problem in Brazil. Although vaso-occlusive crisis (VOC) is one of the most important clinical features of the disease, there are still several steps of its pathogenesis which are unknown. The increase of the chemotactic factor interleukin 8 (IL-8) has been reported to be involved in sickle cell disease crisis, but this has not been demonstrated conclusively. In the present study we analyzed serum IL-8 levels by ELISA and hematological parameters and hemoglobin patterns by standard techniques in 23 (21 SS and 2 SC) Brazilian patients with sickle cell syndromes during VOC caused by different inducing factors, 22 (21 SS and 1 SC) sickle cell patients out of crisis, and 11 healthy controls. Increased IL-8 levels were observed in 19 of 23 VOC patients (79.2%), 3 of them with more than 1,000 pg/ml. Seventeen of 22 (77.3%) non-crisis patients showed low IL-8 levels (less than 15 pg/ml). Healthy controls had low IL-8 levels. A significant difference in serum IL-8 levels was observed between crisis and non-crisis sickle cell patients (P<0.0001). There was no correlation between IL-8 levels and hematological data or hemoglobin patterns. High serum IL-8 levels were observed in VOC patients independently of the crisis-inducing factor. We conclude that in the studied population, IL-8 concentration may be a useful VOC marker, although the mechanism of the pathogenic process of sickle cell VOC syndromes remains unclear.

Interleukin 8 as a vaso-occlusive marker in Brazilian patients with sickle cell disease

Sickle cell disease has a worldwide distribution and is a public health problem in Brazil. Although vaso-occlusive crisis (VOC) is one of the most important clinical features of the disease, there are still several steps of its pathogenesis which are unknown. The increase of the chemotactic factor interleukin 8 (IL-8) has been reported to be involved in sickle cell disease crisis, but this has not been demonstrated conclusively. In the present study we analyzed serum IL-8 levels by ELISA and hematological parameters and hemoglobin patterns by standard techniques in 23 (21 SS and 2 SC) Brazilian patients with sickle cell syndromes during VOC caused by different inducing factors, 22 (21 SS and 1 SC) sickle cell patients out of crisis, and 11 healthy controls. Increased IL-8 levels were observed in 19 of 23 VOC patients (79.2 percent), 3 of them with more than 1,000 pg/ml. Seventeen of 22 (77.3 percent) non-crisis patients showed low IL-8 levels (less than 15 pg/ml). Healthy controls had low IL-8 levels. A significant difference in serum IL-8 levels was observed between crisis and non-crisis sickle cell patients (P<0.0001). There was no correlation between IL-8 levels and hematological data or hemoglobin patterns. High serum IL-8 levels were observed in VOC patients independently of the crisis-inducing factor. We conclude that in the studied population, IL-8 concentration may be a useful VOC marker, although the mechanism of the pathogenic process of sickle cell VOC syndromes remains unclear

In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe.

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects.

Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis.

Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.

Characterizing subpopulations of neoplastic cells in serous effusions. The role of immunocytochemistry.

OBJECTIVE: To analyze the role of immunochemistry in serous effusions. STUDY DESIGN: We analyzed cell blocks of 18 pleural and 18 peritoneal effusions diagnosed as malignant (18), benign (14) and suspicious (4). They were immunostained by the avidin-biotin complex method with a panel of four monoclonal antibodies--CEA, Ber-EP4, LeuM1 (CD15) and p53--and, for lectins (Ulex europaeus) UEA-l, ConA and ConBr. RESULTS: Seventeen of the 18 cases of adenocarcinoma were positive for CEA (95%), 12 (66.6%) for Ber-EP4, 11 (61%) for CD15 and 11 (61%) for p53. Twelve of the 18 (66.6%) were positive for UEA-1, CEA, Ber-EP4 and CD15. UEA-1 did not react with mesothelial cells. p53 Gave a positive reaction in only one case, reactive mesothelial cells. ConA and ConBr reacted indiscriminately with benign and malignant cells; thus, it was not useful in distinguishing between these cells. CONCLUSION: In this context no antibody used alone is reliable for corroborating a diagnosis, but the selective use of a small panel of three markers (CEA, Ber-EP4 and LeuM1) can be very useful in solving diagnostic difficulties in the cytodiagnosis of serous effusions.