Fernando Tobias Silveira

Evidence for a cluster of genes on chromosome 17q11–q21 controlling susceptibility to tuberculosis and leprosy in Brazilians

The region of conserved synteny on mouse chromosome 11/human 17q11–q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Zlr score 2.34; P=0.01) and D17S1795 (Zlr 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Zlr 2.04; P=0.02). Combined analysis shows significant linkage (peak Zlr 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A–8.4 Mb–CCL18–32.3 kb–CCL4–6.04 Mb–STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

Genome-wide scans for leprosy and tuberculosis susceptibility genes in Brazilians

Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1, 405 markers (10 cM map) were typed in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9 chromosomal regions respectively with provisional evidence (P<0.05) for linkage. At stage 2, 58 markers from positive regions were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers typed in all families; 42 positive markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3, 20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.85, 1.78; P=0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3 regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P=5.8 × 10−5; D17S1868, 2.38, P=0.0005; D20S889, 1.51, P=0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India. (151 words).

Genome-wide scan for visceral leishmaniasis susceptibility genes in Brazil

A genome-wide scan was conducted for visceral leishmaniasis (VL) in Brazil. Initially, 405 markers were typed in 22 multicase pedigrees (28 nuclear families; 174 individuals; 66 affected). Non-parametric multipoint analysis detected nine chromosomal regions with provisional evidence (logarithm of the odds (LOD) scores 0.95–1.66; 0.003<P<0.018) for linkage. To confirm linkage, 132 individuals (43 affected) from 19 independently ascertained families were genotyped across these regions. Three regions (6q27, 7q11.22 and 17q11.2–q21.3) retained evidence (LOD scores 1.08, 1.34, 1.14; P=0.013, 0.007, 0.011) for linkage. To determine which genes contribute to linkage at 17q11.2–q21.3, 80 single nucleotide polymorphisms were genotyped in 98 nuclear families with 183 affected individuals. Family-based association test analysis indicated associations at two chemokine genes, CCL1 and CCL16, that lie 1.6 Mb apart, show some extended linkage disequilibrium with each other, but each lie within different clusters of candidate CCL genes. Multiple genes may therefore contribute to the linkage peak for VL at 17q12.

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‐β on induction of PKR‐promoter activity. The observed effects required the engagement of TLR2. TLR2‐KO macrophages expressed low IFN‐β 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.—De Carvalho Vivarini, A., Pereira, R. M. S., Dias Teixeira, K. L., Calegari‐Silva, T. C., Bellio, M., Laurenti, M. D., Corbett, C. E. P., de Castro Gomes, C. M., Soares, R. P., Mendes Silva, A., Silveira, F. T., Lopes, U. G. Human cutaneous leishmaniasis: interferon‐dependent expression of double‐stranded RNA‐kinase (PKR) via TLR2. FASEB J. 25, 4162–4173 (2011). www.fasebj.org

Genetic analysis of multicase families of visceral leishmaniasis in northeastern Brazil: no major role for class II or class III regions of HLA.

Familial aggregation, high relative risk to siblings, and segregation analysis, suggest genetic control of visceral leishmaniasis in Brazil. Class II gene effects in mice, and high circulating tumour necrosis factor α in humans, provide reasons to target HLA. Fifteen polymorphic markers across 1.03 Mb (DQB1 to TNFa) were genotyped (87 multicase families; 638 individuals). Model-based parametric analyses using single-point combined segregation and linkage in COMDS, or multi-point linkage in ALLEGRO, failed to detect linkage. Model-free nonparametric affected sibling pair (SPLINK) or NPLall score (ALLEGRO) analyses also failed to detect linkage. Information content mapping confirmed sufficient marker information to detect linkage. Analysis of simulated data sets demonstrated that these families had 100% power to detect NPLall scores of 5 to 6 (>LOD4; P < 0.00001) over the range (7% to 61%) of age-related penetrances for a disease susceptibility gene. The extended transmission disequilibrium test (TDT) showed no consistent allelic associations between disease and the 15 loci. TDT also failed to detect significant associations between extended haplotypes and disease, consistent with failure to detect significant linkage disequilibrium across the region. Linkage disequilibrium between adjacent groups of markers (HLADQ/DR; 82–1/82–3/-238bpTNFA; LTA/62/TNFa) was not accompanied by significant global haplotype TDT associations with disease. The data suggest that class II/III regions of HLA do not contain major disease gene(s) for visceral leishmaniasis in Brazil.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs

Canine visceral leishmaniasis (CVL) is recognizable by characteristic signs of disease and is highly lethal. The infection, however, may be quite inapparent in some seropositive dogs, and this has raised the polemic question as to whether or not such animals can be a source of infection for Lutzomyia longipalpis, the vector of American visceral leishmaniasis (AVL). In this study we have examined 51 dogs with acute CVL from an AVL area in Pará State, northern Brazil, and compared the parasite density, amastigotes of Leishmania (L.) infantum chagasi, in the skin, lymph node and viscera of symptomatic with that of nine asymptomatic but seropositive dogs (IFAT-IgG). Post-mortem biopsy fragments of these tissues were processed by immunohistochemistry, using a polyclonal antibody against Leishmania sp. The X² and Mann Whitney tests were used to evaluate the means of infected macrophage density (p < 0.05). There was no difference (p > 0.05) in the skin (10.7/mm² x 15.5/mm²) and lymph node (6.3/mm² x 8.3/mm²), between asymptomatic and symptomatic dogs, respectively. It was higher (p < 0.05), however, in the viscera of symptomatic (5.3/mm²) than it was in asymptomatic (1.4/mm²) dogs. These results strongly suggest that asymptomatic or symptomatic L. (L.) i. chagasi-infected dogs can serve as a source of infection, principally considering the highest (p < 0.05) parasite density from skin (10.7/mm² x 15.5/mm²), the place where the vetor L. longipalpis takes its blood meal, compared with those from lymph node (6.3/mm² x 8.3/mm²) and viscera (1.4/mm²x 5.3/mm²).

Teste de aglutinação direta no sorodiagnóstico da leishmaniose visceral no estado do Pará

Avaliou-se o teste de aglutinacao direta (TAD) no sorodiagnostico da leishmaniose visceral (LV) humana e de canideos (cao e raposa Cerdocyon thous), sendo os resultados comparados com aqueles obtidos em imunofluorescencia indireta (IFI) e ensaio imunoenzimatico (ELISA). Utilizaram-se soros: humanos (303): individuos com LV confirmada (16), suspeitos de LV (65), em outras condicoes (102), controles negativos (15), individuos em area endemica (105): de caes (82): de area endemica (68), Salvaterra/Marajo/PA. (21 parasitologicamente positivos), e controles negativos (14), de Belem; de raposas (9): especimes capturados na Ilha do Marajo. Antigenos para o TAD foram preparados a partir de promastigotas de Leishmania (Leishmania) donovani e L. (L.) chagasi. Aqueles utilizados em ELISA e IFI, respectivamente, de promastigotas (antigeno soluvel) e amastigotas deL. (L.) chagasi. Em humanos, a especificidade e sensibilidade do TAD, utilizando-se antigeno deL. (L.) donovani, foram altas (98,4% e 100%, respectivamente) e comparaveis as de IFI (97,5% e 100%). ELISA foi menos especifico (84,8%), embora igualmente sensivel. Em caes, o TAD foi mais especifico com antigeno de L. (L.) donovani do que com aquele preparado a partir de L. (L.) chagasi. Entretanto, ELISA e TAD foram menos sensiveis (ambos 71,4%) que IFI (100%). Essa diferenca foi reflelida nos resultados de caes de area endemica, 87% dos quais foram positivos para IFI, mas somente 54% para ELISA e 49% para o TAD. Resultados similares foram observados com raposas, quando todos os 9 soros foram positivos para IFI, 7 de 9 (78% ) positivos para ELISA, enquanto que o TAD nao revelou nenhum resultado positivo. Concluiu-se que o TAD, usando antigeno de L. (L.) donovani e um teste util para LV humana; sua utilizacao em ampla escala e indicada, desde que monitorada por um laboratorio de referencia que garanta qualidade dos antigenos. Contudo, nao e a melhor opcao em inqueritos sorologicos caninos, ja que foi menos especifico que ELISA e, especialmente, IFI, na deteccao de anticorpos em casos infeccao canina.

Sobre a sensibilidade da cultura de leucócitos circulantes na detecção de Leishmania no sangue periférico de pacientes com leishmaniose tegumentar

Foi investigada a presenca de Leishmania, atraves da cultura de leucocitos circulantes, no sangue periferico de 60 pacientes portadores de leishmaniose tegumentar americana, nas suas diferentes formas clinicas, assim como nas principais fases evolutivas da doenca. Biopsias de lesoes cutâneas e/ou de mucosa desses pacientes foram obtidas com a finalidade de isolar e caracterizar os parasitas, atraves da tecnica de anticorpos monoclonais. Dos 60 pacientes examinados, foram isoladas 40 amostras de Leishmania das lesoes biopsiadas, sendo 5 de Leishmania (V.) brasiliensis, 3 de L. (V.) guyanensis, 1 de L. (V.) lainsoni, 13 de L. (L.) amazonensis e 18 nao puderam ser caracterizados a nivel especifico, porem, reagiram com anticorpos monoclonais do grupo braziliensis. Quanto apesquisa atraves das culturas de leucocitos circulantes, esta revelou resultados completamente negativos. Com base nesses achados, os autores concluiram ser pouco consistente atribuir valor a cultura de leucocitos para o diagnostico da leishmaniose tegumentar.

Fauna flebotomínica da Serra dos Carajás, Estado do Pará, Brasil, e sua possível implicação na transmissão da leishmaniose tegumentar americana

Serra dos Carajas, located in the southeast of Para State, Brazil, is a rich tropical forest where species of Leishmania sp. of medical interest are found, such as Leishmania (V.) braziliensis, L. (V.) lainsoni, L. (V.) shawi and L. (L.) amazonensis. They are transmitted by the following phlebotomi: Psychodopygus complexus or Ps. wellcomei, Lutzomyia ubiquitalis, Lu. whitmani and Lu. flaviscutellata. Considering the increase of immigrants in the region of the Carajas project, this study aimed to assess the Phlebotominae fauna and their possible participation in the transmission of American cutaneous leishmaniasis (ACL). The phlebotomi were captured from December 2005 to September 2007 at the following locations: i) Parauapebas Botanical Park; ii) an environmental protection area; and iii) Tapirape-Aquiri National Forest. During the 172 days of collection, 10 CDC (18 h to 6 h) and 2 Shannon (18 h to 20 h) light traps were used. Of the 22,095 phlebotomi captured, 6,789 (31%) were male and 15,306 (69%) were female, and they belonged to 69 species and three genera, including Psychodopygus, Lutzomyia and Brumptomyia. A total of 19 (0.16%) natural infections of the following species were detected: Ps. davisi (4), Ps. h. hirsutus (3), Lu. umbratilis (3), Lu. richardward (2), Lu. brachipyga (2), Lu. ubiquitalis (2), Lu. trinidadensis (1) and Lu. migonei (1). Although no infection was found in Ps. wellcomei/complexus, the main vector of L. (V.) braziliensis in the region, this species was the most prevalent (16%), followed by Ps. davisi (15.4%), Ps. carrerai (4.2%), Lu. shawi (3.9%), Lu. brachipyga (2.5%) and Lu. richardward (1.2%). These results show the importance of these phlebotomi as possible vectors of ACL in Serra dos Carajas.

Phenotypic characterization of Leishmania spp. causing cutaneous leishmaniasis in the lower Amazon region, western Pará state, Brazil, reveals a putative hybrid parasite, Leishmania (Viannia) guyanensis × Leishmania (Viannia) shawi shawi

We phenotypically characterized 43 leishmanial parasites from cutaneous leishmaniasis by isoenzyme electrophoresis and the indirect immunofluorescence antibody test (23 McAbs). Identifications revealed 11 (25.6%) strains of Leishmania (V.) braziliensis, 4 (9.3%) of L. (V.) shawi shawi, 7 (16.3%) of L. (V.) shawi santarensis, 6 (13.9%) of L. (V.) guyanensis and L. (V.) lainsoni, 2 (4.7%) of L. (L.) amazonensis, and 7 (16.3%) of a putative hybrid parasite, L. (V.) guyanensis/L. (V.) shawi shawi. McAbs detected three different serodemes of L. (V.) braziliensis: I-7, II-1, and III-3 strains. Among the strains of L. (V.) shawi we identified two populations: one (7 strains) expressing the B19 epitope that was previously considered to be species-specific for L. (V.) guyanensis. We have given this population sub-specific rank, naming it L. (V.) s. santarensis. The other one (4 strains) did not express the B19 epitope like the L. (V.) shawi reference strain, which we now designate as L. (V.) s. shawi. For the first time in the eastern Brazilian Amazon we register a putative hybrid parasite (7 strains), L. (V.) guyanensis/L. (V.) s. shawi, characterized by a new 6PGDH three-band profile at the level of L. (V.) guyanensis. Its PGM profile, however, was very similar to that of L. (V.) s. shawi. These results suggest that the lower Amazon region – western Pará state, Brazil, represents a biome where L. (V.) guyanensis and L. (V.) s. shawi exchange genetic information.