Impaired macrophage and memory T-cell responses to Bacillus Calmette-Guerin nonpolar lipid extract.

Introduction: The attenuation of BCG has led to the loss of not only immunogenic proteins but also lipid antigens. Methods: Thus, we compared the macrophage and T-cell responses to nonpolar lipid extracts harvested from BCG and Mycobacterium tuberculosis (Mtb) to better understand the role of BCG lipids in the already known diminished responses of the vaccine strain. Results: Relative to Mtb, nonpolar lipid extract from BCG presented a reduced capacity to trigger the expression of the genes encoding TNF, IL-1b, IL-6 and IL-10 in RAW 264.7 macrophages. Immunophenotyping of PBMCs isolated from healthy individuals revealed that lipids from both BCG and Mtb were able to induce an increased frequency of CD4+ and CD8+ T cells, but only the lipid extract from Mtb enhanced the frequency of CD4-CD8-double-negative, γσ+, CD4+HLA-DR+, and γσ+HLA-DR+ T cells relative to the nonstimulated control. Interestingly, only the Mtb lipid extract was able to increase the frequency of CD4+ memory (CD45RO+) T cells, whereas the BCG lipid extract induced a diminished frequency of CD4+ central memory (CD45RO+CCR7-) T cells after 48 h of culture compared to Mtb. Discussion: These findings show that the nonpolar lipids of the BCG bacilli presented diminished ability to trigger both proinflammatory and memory responses and suggest a potential use of Mtb lipids as adjuvants to increase the BCG vaccine efficacy.

Leishmania braziliensis causing human disease in Northeast Brazil presents loci with genotypes in long-term equilibrium.

BACKGROUND: Leishmaniases are neglected tropical diseases that inflict great burden to poor areas of the globe. Intense research has aimed to identify parasite genetic signatures predictive of infection outcomes. Consistency of diagnostic tools based on these markers would greatly benefit from accurate understanding of Leishmania spp. population genetics. We explored two chromosomal loci to characterize a population of L. braziliensis causing human disease in Northeast Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Two temporally distinct samples of L. braziliensis were obtained from patients attending the leishmaniasis clinic at the village of Corte de Pedra: (2008-2011) primary sample, N = 120; (1999-2001) validation sample, N = 35. Parasites were genotyped by Sanger's sequencing of two 600 base pairs loci starting at nucleotide positions 3,074 and 425,451 of chromosomes 24 and 28, respectively. Genotypes based on haplotypes of biallelic positions in each locus were tested for several population genetic parameters as well as for geographic clustering within the region. Ample geographic overlap of genotypes at the two loci was observed as indicated by non-significant Cusick and Edward's comparisons. No linkage disequilibrium was detected among combinations of haplotypes for both parasite samples. Homozygous and heterozygous genotypes displayed Hardy-Weinberg equilibrium (HWE) at both loci in the two samples when straight observed and expected counts were compared by Chi-square (p>0.5). However, Bayesian statistics using one million Monte-Carlo randomizations disclosed a less robust HWE for chromosome 24 genotypes, particularly in the primary sample (p = 0.04). Fixation indices (Fst) were consistently lower than 0.05 among individuals of the two samples at both tested loci, and no intra-populational structuralization could be detected using STRUCTURE software. CONCLUSIONS/SIGNIFICANCE: These findings suggest that L. braziliensis can maintain stable populations in foci of human leishmaniasis and are capable of robust genetic recombination possibly due to events of sexual reproduction during the parasite's lifecycle.

Rainfall and other meteorological factors as drivers of urban transmission of leptospirosis.

BACKGROUND: Leptospirosis is an important public health problem affecting vulnerable urban slum populations in developing country settings. However, the complex interaction of meteorological factors driving the temporal trends of leptospirosis remain incompletely understood. METHODS AND FINDINGS: From March 1996-March 2010, we investigated the association between the weekly incidence of leptospirosis and meteorological anomalies in the city of Salvador, Brazil by using a dynamic generalized linear model that accounted for time lags, overall trend, and seasonal variation. Our model showed an increase of leptospirosis cases associated with higher than expected rainfall, lower than expected temperature and higher than expected humidity. There was a lag of one-to-two weeks between weekly values for significant meteorological variables and leptospirosis incidence. Independent of the season, a weekly cumulative rainfall anomaly of 20 mm increased the risk of leptospirosis by 12% compared to a week following the expected seasonal pattern. Finally, over the 14-year study period, the annual incidence of leptospirosis decreased significantly by a factor of 2.7 (8.3 versus 3.0 per 100,000 people), independently of variations in climate. CONCLUSIONS: Strategies to control leptospirosis should focus on avoiding contact with contaminated sources of Leptospira as well as on increasing awareness in the population and health professionals within the short time window after low-level or extreme high-level rainfall events. Increased leptospirosis incidence was restricted to one-to-two weeks after those events suggesting that infectious Leptospira survival may be limited to short time intervals.

In silico comparisons of lipid-related genes between Mycobacterium tuberculosis and BCG vaccine strains.

Despite highly variable efficacy, BCG (Bacillus Calmette-Guérin) is the only vaccine available to prevent the tuberculosis (TB). Genomic heterogeneity between attenuated BCG strains and virulent Mycobacterium tuberculosis might help to explain this vaccine's impaired capacity to induce long-term protection. Here, we investigate the lipid-related genes absent in attenuated BCG strains in order to correlate changes in both lipid metabolism and cell-wall lipid content to vaccine impairment. Whole genome sequences of M. tuberculosis H37Rv and the six most used BCG strains worldwide were aligned and the absent regions functionally categorized. Genomes of the BCG strains showed a total of 14 non-homologous lipid-related genes, including those belonging to mce3 operon, as well as the gene echaA1, which encodes an enoyl-CoA hydratase, and the genes encoding phospholipases PlcA, PlcB and PlcC. Taken together, the depletion of these M. tuberculosis H37Rv genomic regions were associated with marked alterations in lipid-related genes of BCG strains. Such alterations may indicate a dormant-like state and can be determining factors to the vaccine's inability to induce long-term protection. These lipids can be further evaluated as an adjuvant to boost the current BCG-based vaccine.

Whole blood mRNA expression-based targets to discriminate active tuberculosis from latent infection and other pulmonary diseases.

Current diagnostic tests for tuberculosis (TB) are not able to predict reactivation disease progression from latent TB infection (LTBI). The main barrier to predicting reactivation disease is the lack of our understanding of host biomarkers associated with progression from latent infection to active disease. Here, we applied an immune-based gene expression profile by NanoString platform to identify whole blood markers that can distinguish active TB from other lung diseases (OPD), and that could be further evaluated as a reactivation TB predictor. Among 23 candidate genes that differentiated patients with active TB from those with OPD, nine genes (CD274, CEACAM1, CR1, FCGR1A/B, IFITM1, IRAK3, LILRA6, MAPK14, PDCD1LG2) demonstrated sensitivity and specificity of 100%. Seven genes (C1QB, C2, CCR2, CCRL2, LILRB4, MAPK14, MSR1) distinguished TB from LTBI with sensitivity and specificity between 82 and 100%. This study identified single gene candidates that distinguished TB from OPD and LTBI with high sensitivity and specificity (both > 82%), which may be further evaluated as diagnostic for disease and as predictive markers for reactivation TB.

Differential Host Pro-Inflammatory Response to Mycobacterial Cell Wall Lipids Regulated by the Mce1 Operon.

The cell wall of wild-type (WT) Mycobacterium tuberculosis (Mtb), an etiologic agent of tuberculosis (TB) and a Mtb strain disrupted in a 13-gene operon mce1 (Δmce1) varies by more than 400 lipid species. Here, we examined Mtb lipid-induced response in murine macrophage, as well as in human T-cell subpopulations in order to gain an insight into how changes in cell wall lipid composition may modulate host immune response. Relative to WT Mtb cell wall lipids, the non-polar lipid extracts from Δmce1 enhanced the mRNA expression of lipid-sense nuclear receptors TR4 and PPAR-γ and dampened the macrophage expression of genes encoding TNF-α, IL-6, and IL-1ß. Relative to untreated control, WT lipid-pre-stimulated macrophages from healthy individuals induced a higher level of CD4-CD8- double negative T-cells (DN T-cells) producing TNF-α. Conversely, compared to WT, stimulation with Δmce1 lipids induced higher mean fluorescence intensity (MFI) in IL-10-producing DN T cells. Mononuclear cells from TB patients stimulated with WT Mtb lipids induced an increased production of TNF-α by CD8+ lymphocytes. Taken together, these observations suggest that changes in mce1 operon expression during a course of infection may serve as a strategy by Mtb to evade the host pro-inflammatory responses.

Tissue Damage in Human Cutaneous Leishmaniasis: Correlations Between Inflammatory Cells and Molecule Expression.

Cutaneous leishmaniasis (CL) is caused by the bite of the infected sand fly, which inoculates parasites of Leishmania spp and triggers an immune response. An exacerbated cutaneous inflammatory response is crucial for controlling parasite burden but can also promote tissue damage. This study aimed to characterize the populations of natural killer (NK), CD57+, CD4+, and CD8+ T cells, CD20+ B cells, as well as CD68+ macrophages, in biopsies of ulcerated CL lesions, and quantify the production of perforin+, grazyme B+, interleukin 1 beta (IL-1ß+) and Tumor Necrosis Factor (TNF-α+ cells). We then correlated these parameters with necrosis, inflammation and the number of amastigotes. CD4+ T cells were positively correlated to the extent of inflammation, B cells and IL-1ß+ were associated with the extent of necrosis, CD68+ macrophages and perforin were correlated with the number of amastigotes, and CD57+ NK cells was correlated to CD68+ macrophages and amastigotes. In sum, the finding suggests that the production of cytotoxic granules and cytokines by inflammatory cells contributes to tissue damage in CL lesions.

Serological biomarkers for monitoring response to treatment of pulmonary and extrapulmonary tuberculosis in children and adolescents.

Key measures to halt the spread of tuberculosis (TB) include early diagnosis, effective treatment, and monitoring disease management. We sought to evaluate the use of serum immunoglobulin levels against antigens present in cell envelope of Mycobacterium tuberculosis to monitor TB treatment response in children and adolescents with pulmonary (PTB) or extrapulmonary TB (EPTB). Blood samples were collected prior to and one, two, and six months following treatment initiation. Serum immunoglobulin levels against cardiolipin, sulfatide, mycolic acid and Mce1A protein were measured by ELISA. Serum from 53 TB patients and 12 healthy participants were analyzed. After six months of successful treatment, there was a significant decrease (p < 0.0001) in IgM levels against cardiolipin, sulfatide, mycolic acid and Mce1A protein and IgG levels against Mce1A protein when compared to baseline immunoglobulin levels. There was no significant variation in antibody levels during follow-up between participants with PTB and EPTB, confirmed and unconfirmed TB diagnosis, and HIV infection status. Antibody levels in control participants without TB did not decrease during follow-up. These results suggest that immunoglobulin responses to mycobacterial cell wall products may be a useful tool to monitor treatment response in children and adolescents with PTB or EPTB.

Lipidomic Analysis Reveals Serum Alteration of Plasmalogens in Patients Infected With ZIKA Virus.

Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) in the Flavivirus genus of the Flaviviridae family. Since the large outbreaks in French Polynesia in 2013-2014 and in Brazil in 2015, ZIKV has been considered a new public health threat. Similar to other related flavivirus, ZIKV is associated with mild and self-limiting symptoms such as rash, pruritus, prostration, headache, arthralgia, myalgia, conjunctivitis, lower back pain and, when present, a short-term low grade fever. In addition, ZIKV has been implicated in neurological complications such as neonatal microcephaly and Guillain-Barré syndrome in adults. Herein, serum lipidomic analysis was used to identify possible alterations in lipid metabolism triggered by ZIKV infection. Patients who presented virus-like symptoms such as fever, arthralgia, headache, exanthema, myalgia and pruritus were selected as the control group. Our study reveals increased levels of several phosphatidylethanolamine (PE) lipid species in the serum of ZIKV patients, the majority of them plasmenyl-phosphatidylethanolamine (pPE) (or plasmalogens) linked to polyunsaturated fatty acids. Constituting up to 20% of total phospholipids in humans, plasmalogens linked to polyunsaturated fatty acids are particularly enriched in neural membranes of the brain. The biosynthesis of plasmalogens requires functional peroxisomes, which are important sites for viral replication, including ZIKV. Thus, increased levels of plasmalogens in serum of ZIKV infected subjects suggest a link between ZIKV life cycle and peroxisomes. Our data provide important insights into specific host cellular lipids that are likely associated with ZIKV replication and may serve as platform for antiviral strategy against ZIKV.

Molecular epidemiology and in vitro evidence suggest that Leishmania braziliensis strain helps determine antimony response among American tegumenary leishmaniasis patients.

Antimony is the first line drug for treating American tegumentary leishmaniasis (ATL) in Brazil. In this country, Leishmania braziliensis causes at least three distinct forms of disease: localized cutaneous (CL), mucosal (ML) and disseminated leishmaniasis (DL). All forms can be found in Corte de Pedra, Northeast Brazil. ML and DL respond poorly to antimony, in contrast to CL. The L. braziliensis population causing ATL in Corte de Pedra is genetically very diverse, with strains of the parasite associating with the clinical form of leishmaniasis. We tested the hypotheses that antimony refractoriness is associated with L. braziliensis genotypes, and that parasites from ML and DL present greater in vitro resistance to antimony than L. braziliensis from CL. Comparison of geographic coordinates of living sites between antimony responders and non-responders by Cusick and Edward́s test showed that refractoriness and responsiveness to the drug were similarly wide spread in the region (p>0.05). Parasites were then genotyped by sequencing a locus starting at position 425,451 on chromosome 28, which is polymorphic among L. braziliensis of Corte de Pedra. Haplotype CC- in CHR28/425,451 was associated with risk of treatment failure among CL patients (Fishers exact test, p=0.03, odds ratio=4.65). This haplotype could not be found among parasites from ML or DL. Finally, sensitivity to antimony was evaluated exposing L. braziliensis promastigotes to increasing concentrations of meglumine antimoniate in vitro. Parasites from ML and DL were more resistant to antimony at doses of 2mg/100µL and beyond than those isolated from CL (Fisher's exact test, p=0.02 and p=0.004, respectively). The intrinsically lower susceptibility of L. brazliensis from ML and DL to antimony parallels what is observed for patients' responsiveness in the field. This finding reinforces that ML and DL patients would benefit from initiating treatment with drugs currently considered as second line, like amphotericin B.

Dynamics of American tegumentary leishmaniasis in a highly endemic region for Leishmania (Viannia) braziliensis infection in northeast Brazil.

BACKGROUND: American Tegumentary Leishmaniasis (ATL) caused by Leishmania braziliensis is endemic in Corte de Pedra, Northeast Brazil. Most L. braziliensis infections manifest as localized cutaneous leishmaniasis (CL). Disseminated manifestations include mucosal leishmaniasis (ML), present at a low constant level for several decades, and newly emerging disseminated leishmaniasis (DL). Surprisingly, DL has recently surpassed ML in its spatial distribution. This led us to hypothesize that distinct forms of ATL might spread in different patterns through affected regions. METHODOLOGY/PRINCIPAL FINDINGS: We explored the incidence and geographic dispersion of the three clinical types of ATL over a span of nearly two decades in Corte de Pedra. We obtained the geographic coordinates of the homes of patients with ATL during 1992-1996, 1999-2003 and 2008-2011. The progressive dispersion of ML or DL in each time period was compared to that of CL in 2008-2011 with the Cusick and Edward's geostatistical test. To evaluate whether ATL occurred as clusters, we compared each new case in 2008-2011 with the frequency of and distance from cases in the previous 3 to 12 months. The study revealed that DL, ML and CL actively spread within that region, but in distinct patterns. Whereas CL and DL propagated in clusters, ML occurred as sporadic cases. DL had a wider distribution than ML until 2003, but by 2011 both forms were distributed equally in Corte de Pedra. The incidence of ML fluctuated over time at a rate that was distinct from those of CL and DL. CONCLUSIONS/SIGNIFICANCE: These findings suggest that CL and DL maintain endemic levels through successive outbreaks of cases. The sporadic pattern of ML cases may reflect the long and variable latency before infected patients develop clinically detectable mucosal involvement. Intimate knowledge of the geographic distribution of leishmaniasis and how it propagates within foci of active transmission may guide approaches to disease control.

Regulação gênica da via ampk pelo exercício físico: revisão sistemática e análise in silico / Ampk pathway gene regulation by physical exercise: systematic review and in silico analysis / Regulación génica de la vía ampk por el ejercicio físico: revisión sistemática y análisis in silico

RESUMO Introdução: Novos estudos de regulação gênica do exercício físico por meio de técnicas pós-genômicas em ensaios de resistência (endurance) e força caracterizam a transcriptômica do exercício físico. Entre os genes afetados, destacamos a via da proteína quinase ativada por AMP (AMPK), cuja ativação ocorre durante o exercício como resultado das alterações dos níveis de fosfato energético da fibra muscular. Objetivo: Avaliar a via de sinalização da AMPK por revisão sistemática da expressão de genes e análise in silico. Método: Foi efetuada uma revisão sistemática para avaliar a regulação gênica da via de sinalização AMPK, caracterizando os genes estudados na literatura, as variações de regulação obtidas, na forma de fold change e tipos de exercício usados. Resultados: A via de sinalização AMPK mostrou 133 genes no repositório KEGG (Kyoto Encyclopedia of Genes and Genomes), os quais foram confrontados com a revisão sistemática da literatura, totalizando 65 genes. Dezessete genes apresentaram UR e 24 mostraram DR com relação ao seu respectivo controle. Além destes, 20 genes estavam presentes nos trabalhos, apresentando tanto UR e DR e quatro genes não apresentaram dados de regulação. Verificou-se regulação específica em função do tipo de exercício efetuado. Discussão: Dos 133 genes da via AMPK, 48,8% foram amostrados nos trabalhos revisados, indicando que uma parte significativa da via é regulada pelo exercício. O estudo apresentou a regulação gênica básica de dois mecanismos para a recuperação energética, a biogênese mitocondrial e o bloqueio da gliconeogênese. Conclusão: Este trabalho mostrou que o exercício atua ativamente na via de sinalização da AMPK, na importância da regulação via PGC-1α e no papel de outros genes, regulando a expressão de mais da metade dos genes amostrados.

ABSTRACT Introduction: New studies of gene regulation by physical exercise through post-genomic techniques in endurance and strength tests characterize the physical exercise transcriptomics. Among the affected genes, we highlight the AMP-activated protein kinase (AMPK) pathway, the activation of which occurs during exercise because of changes in muscle fiber energetic phosphate levels. Objective: To evaluate the AMPK signaling pathway by systematic review of gene expression and in silico analysis. Method: A systematic review was performed in order to assess the gene regulation of AMPK signaling pathway, characterizing the genes studied in the literature, regulation variations obtained in the form of fold change, and types of exercise performed. Results: The AMPK signaling pathway showed 133 genes in the KEGG repository (Kyoto Encyclopedia of Genes and Genomes), which were compared with the systematic review of the literature, totaling 65 genes. Seventeen genes presented UR and 24 showed DR in relation to their respective control. In addition to these, 20 genes were present in the literature, presenting both UR and DR and four genes showed no regulatory data. Specific regulation was verified according to the type of exercises performed. Discussion: Of the 133 genes of the AMPK pathway, 48.8% were sampled in the revised studies indicating that a significant part of the pathway is regulated by exercise. The study presented the basic gene regulation of two mechanisms for energy recovery, mitochondrial biogenesis, and gluconeogenesis blockade. Conclusion: This work showed that the exercise actively works in the AMPK signaling pathway, in the importance of regulation via PGC-1α and in the role of other genes, regulating the expression of more than half of the genes sampled.

RESUMEN Introducción: Nuevos estudios de regulación génica del ejercicio físico por medio de técnicas pos-genómicas en ensayos de resistencia (endurance) y fuerza caracterizan la transcriptómica del ejercicio físico. Entre los genes afectados, destacamos la vía de la proteína quinasa activada por AMP (AMPK), cuya activación ocurre durante el ejercicio como resultado de las alteraciones de los niveles de fosfato energético de la fibra muscular. Objetivo: Evaluar la vía de señalización AMPK por revisión sistemática de la expresión de genes y análisis in silico. Método: Se ha efectuado una revisión para evaluar la regulación génica de la vía de señalización AMPK, caracterizando los genes estudiados en la literatura, las variaciones de regulación obtenidas en forma de fold change y tipos de ejercicios utilizados. Resultados: La vía de señalización AMPK mostró 133 genes en el repositorio KEGG (Kyoto Encyclopedia of Genes and Genomes), los cuales fueran confrontados con la revisión sistemática de la literatura, totalizando 65 genes. Diecisiete genes presentaron UR y 24 mostraron DR con respecto a su respectivo control. Además de estos, 20 genes estaban presentes en los trabajos, presentando tanto UR y DR y cuatro genes no presentaron dados de regulación. Se observó una regulación específica en función del tipo de ejercicio efectuado. Discusión: De los 133 genes de la vía AMPK, 48,8% fueron muestreados en los trabajos revisados, indicando que una parte significativa de la vía es regulada por el ejercicio. El estudio presentó la regulación génica básica de dos mecanismos para la recuperación energética, la biogénesis mitocondrial y el bloqueo de la gluconeogénesis. Conclusión: Este trabajo mostró que el ejercicio actúa activamente en la vía de señalización AMPK, en la importancia de la regulación vía factor PGC-1a y en el papel de otros genes, regulando la expresión de más de la mitad de los genes muestreados.

Bacterial immunostat: Mycobacterium tuberculosis lipids and their role in the host immune response.

The lipid-rich cell wall of Mycobacterium tuberculosis is a dynamic structure that is involved in the regulation of the transport of nutrients, toxic host-cell effector molecules, and anti-tuberculosis drugs. It is therefore postulated to contribute to the long-term bacterial survival in an infected human host. Accumulating evidence suggests that M. tuberculosis remodels the lipid composition of the cell wall as an adaptive mechanism against host-imposed stress. Some of these lipid species (trehalose dimycolate, diacylated sulphoglycolipid, and mannan-based lipoglycans) trigger an immunopathologic response, whereas others (phthiocerol dimycocerosate, mycolic acids, sulpholipid-1, and di-and polyacyltrehalose) appear to dampen the immune responses. These lipids appear to be coordinately expressed in the cell wall of M. tuberculosis during different phases of infection, ultimately determining the clinical fate of the infection. This review summarizes the current state of knowledge on the metabolism, transport, and homeostatic or immunostatic regulation of the cell wall lipids, and their orchestrated interaction with host immune responses that results in bacterial clearance, persistence, or tuberculosis.

Bacterial immunostat: mycobacterium tuberculosis lipids and their role in the host immune response

Abstract: The lipid-rich cell wall of Mycobacterium tuberculosis is a dynamic structure that is involved in the regulation of the transport of nutrients, toxic host-cell effector molecules, and anti-tuberculosis drugs. It is therefore postulated to contribute to the long-term bacterial survival in an infected human host. Accumulating evidence suggests that M. tuberculosis remodels the lipid composition of the cell wall as an adaptive mechanism against host-imposed stress. Some of these lipid species (trehalose dimycolate, diacylated sulphoglycolipid, and mannan-based lipoglycans) trigger an immunopathologic response, whereas others (phthiocerol dimycocerosate, mycolic acids, sulpholipid-1, and di-and polyacyltrehalose) appear to dampen the immune responses. These lipids appear to be coordinately expressed in the cell wall of M. tuberculosis during different phases of infection, ultimately determining the clinical fate of the infection. This review summarizes the current state of knowledge on the metabolism, transport, and homeostatic or immunostatic regulation of the cell wall lipids, and their orchestrated interaction with host immune responses that results in bacterial clearance, persistence, or tuberculosis.

Characterization of the Histopathologic Features in Patients in the Early and Late Phases of Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL), characterized by an ulcerated lesion, is the most common clinical form of human leishmaniasis. Before the ulcer develops, patients infected with Leishmania (Viannia) braziliensis present a small papule at the site of the sandfly bite, referred to as early cutaneous leishmaniasis (E-CL). Two to four weeks later the typical ulcer develops, which is considered here as late CL (L-CL). Although there is a great deal known about T-cell responses in patients with L-CL, there is little information about the in situ inflammatory response in E-CL. Histological sections of skin biopsies from 15 E-CL and 28 L-CL patients were stained by hematoxilin and eosin to measure the area infiltrated by cells, as well as tissue necrosis. Leishmania braziliensis amastigotes, CD4+, CD8+, CD20+, and CD68+ cells were identified and quantified by immunohistochemistry. The number of amastigotes in E-CL was higher than in L-CL, and the inflammation area was larger in classical ulcers than in E-CL. There was no relationship between the number of parasites and magnitude of the inflammation area, or with the lesion size. However, there was a direct correlation between the number of macrophages and the lesion size in E-CL, and between the number of macrophages and necrotic area throughout the course of the disease. These positive correlations suggest that macrophages are directly involved in the pathology of L. braziliensis-induced lesions.

Atypical Manifestations of Cutaneous Leishmaniasis in a Region Endemic for Leishmania braziliensis: Clinical, Immunological and Parasitological Aspects.

BACKGROUND: Atypical cutaneous leishmaniasis (ACL) has become progressively more frequent in Corte de Pedra, Northeast Brazil. Herein we characterize clinical presentation, antimony response, cytokine production and parasite strains prevailing in ACL. METHODOLOGY/PRINCIPAL FINDINGS: Between 2005 and 2012, 51 ACL (cases) and 51 temporally matched cutaneous leishmaniasis (CL) subjects (controls) were enrolled and followed over time in Corte de Pedra. Clinical and therapeutic data were recorded for all subjects. Cytokine secretion by patients' peripheral blood mononuclear cells (PBMC) stimulated with soluble parasite antigen in vitro, and genotypes in a 600 base-pair locus in chromosome 28 (CHR28/425451) of the infecting L. (V.) braziliensis were compared between the two groups. ACL presented significantly more lesions in head and neck, and higher rate of antimony failure than CL. Cytosine-Adenine substitutions at CHR28/425451 positions 254 and 321 were highly associated with ACL (p<0.0001). In vitro stimulated ACL PBMCs produced lower levels of IFN-γ (p = 0.0002) and TNF (p <0.0001), and higher levels of IL-10 (p = 0.0006) and IL-17 (p = 0.0008) than CL PBMCs. CONCLUSIONS/SIGNIFICANCE: ACL found in Northeast Brazil is caused by distinct genotypes of L. (V.) braziliensis and presents a cytokine profile that departs from that in classical CL patients. We think that differences in antigenic contents among parasites may be in part responsible for the variation in cytokine responses and possibly immunopathology between CL and ACL.

The gp63 Gene Cluster Is Highly Polymorphic in Natural Leishmania (Viannia) braziliensis Populations, but Functional Sites Are Conserved.

GP63 or leishmanolysin is the major surface protease of Leishmania spp. involved in parasite virulence and host cell interaction. As such, GP63 is a potential target of eventual vaccines against these protozoa. In the current study we evaluate the polymorphism of gp63 in Leishmania (Viannia) braziliensis isolated from two sets of American tegumentary leishmaniasis (ATL) cases from Corte de Pedra, Brazil, including 35 cases diagnosed between 1994 and 2001 and 6 cases diagnosed between 2008 and 2011. Parasites were obtained from lesions by needle aspiration and cultivation. Genomic DNA was extracted, and 405 bp fragments, including sequences encoding the putative macrophage interacting sites, were amplified from gp63 genes of all isolates. DNA amplicons were cloned into plasmid vectors and ten clones per L. (V.) braziliensis isolate were sequenced. Alignment of cloned sequences showed extensive polymorphism among gp63 genes within, and between parasite isolates. Overall, 45 different polymorphic alleles were detected in all samples, which could be segregated into two clusters. Cluster one included 25, and cluster two included 20 such genotypes. The predicted peptides showed overall conservation below 50%. In marked contrast, the conservation at segments with putative functional domains approached 90% (Fisher's exact test p<0.0001). These findings show that gp63 is very polymorphic even among parasites from a same endemic focus, but the functional domains interacting with the mammalian host environment are conserved.

Spatiotemporal Determinants of Urban Leptospirosis Transmission: Four-Year Prospective Cohort Study of Slum Residents in Brazil.

BACKGROUND: Rat-borne leptospirosis is an emerging zoonotic disease in urban slum settlements for which there are no adequate control measures. The challenge in elucidating risk factors and informing approaches for prevention is the complex and heterogeneous environment within slums, which vary at fine spatial scales and influence transmission of the bacterial agent. METHODOLOGY/PRINCIPAL FINDINGS: We performed a prospective study of 2,003 slum residents in the city of Salvador, Brazil during a four-year period (2003-2007) and used a spatiotemporal modelling approach to delineate the dynamics of leptospiral transmission. Household interviews and Geographical Information System surveys were performed annually to evaluate risk exposures and environmental transmission sources. We completed annual serosurveys to ascertain leptospiral infection based on serological evidence. Among the 1,730 (86%) individuals who completed at least one year of follow-up, the infection rate was 35.4 (95% CI, 30.7-40.6) per 1,000 annual follow-up events. Male gender, illiteracy, and age were independently associated with infection risk. Environmental risk factors included rat infestation (OR 1.46, 95% CI, 1.00-2.16), contact with mud (OR 1.57, 95% CI 1.17-2.17) and lower household elevation (OR 0.92 per 10m increase in elevation, 95% CI 0.82-1.04). The spatial distribution of infection risk was highly heterogeneous and varied across small scales. Fixed effects in the spatiotemporal model accounted for the majority of the spatial variation in risk, but there was a significant residual component that was best explained by the spatial random effect. Although infection risk varied between years, the spatial distribution of risk associated with fixed and random effects did not vary temporally. Specific "hot-spots" consistently had higher transmission risk during study years. CONCLUSIONS/SIGNIFICANCE: The risk for leptospiral infection in urban slums is determined in large part by structural features, both social and environmental. Our findings indicate that topographic factors such as household elevation and inadequate drainage increase risk by promoting contact with mud and suggest that the soil-water interface serves as the environmental reservoir for spillover transmission. The use of a spatiotemporal approach allowed the identification of geographic outliers with unexplained risk patterns. This approach, in addition to guiding targeted community-based interventions and identifying new hypotheses, may have general applicability towards addressing environmentally-transmitted diseases that have emerged in complex urban slum settings.