Use of phylogeny to resolve the taxonomy of the widespread and highly polymorphic African giant shrews (Crocidura olivieri group, Crocidurinae, Mammalia).

The aim of this study is to provide a better understanding of the genetic relationships within the widespread and highly polymorphic group of African giant shrews (Crocidura olivieri group). We sequenced 769 base pairs (bp) of the mitochondrial cytochrome b gene and 472 bp of the mitochondrial control region over the entire geographic range from South Africa to Morocco. The analyses reveal four main clades associated with different biomes. The largest clade occurs over a range covering Northwest and Central Africa and includes samples of C. fulvastra, C. olivieri, and C. viaria. The second clade is composed of C. goliath from Gabon, while South African C. flavescens, and C. hirta form two additional clades. On the basis of these results, the validity of some taxa in the C. olivieri group should be re-evaluated.

The role of triple infection with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus (HIV) type-1 on CD4+ lymphocyte levels in the highly HIV infected population of North-Central Nigeria

We set out to determine the seroprevalence of hepatitis B and C among human immunodeficiency virus type-1 (HIV-1) infected individuals in North-Central Nigeria to define the influence of these infections on CD4+ lymphocytes cells among our patients as access to antiretroviral therapy improves across the Nigerian nation. The CD4+ values of 180 confirmed HIV-1 infected individuals were enumerated using a superior fluorescence-activated cell sorter system. These patients were tested for the presence of hepatitis B surface antigen and anti-hepatitis C virus (HCV) using third generation enzyme-linked immunosorbent assays. Fifty (27.8%) patients had active hepatitis B virus (HBV) infection while 33 (18.3%) tested positive for anti-HCV antibody. Of these infections, 110 (61.1%), 37 (20.6%), and 20 (11.1%) had HIV only, HBV/HIV-only, and HCV/HIV-only respectively. A HBV/HCV/HIV coinfection prevalence of 7.2% (13 patients) was recorded. Patients coinfected with HIV/HBV/HCV appeared to have lower CD4+ counts (mean = 107 cells/µl; AIDS defining) when compared to HBV/HIV-only (mean = 377 cells/µl), HCV/HIV-only (mean = 373 cells/µl) and patients with mono HIV infection (mean = 478 cells/µl). Coinfection with HBV or HCV is relatively common among HIV-infected patients in Nigeria and should be a big consideration in the initiation and choice of therapy.

Comparison of the epidemiology, profile of mutations, and clinical response to antiretrovirals among subtypes B and F of the human immunodeficiency virus type 1

The authors compared demographic aspects and profile of mutations in 80 patients with subtypes B and F of human immunodeficiency type 1 (HIV-1). Genotyping of the pol region of the reverse transcriptase was performed using the ViroSeqTM Genotyping System. A total of 61 (76.2%) patients had subtype B and 19 (23.8%) subtype F of the HIV-1. Subtype F tended to be more frequent in heterosexuals and women with a low educational level, but without statistical significance. The frequency of mutations related to nucleoside reverse transcriptase inhibitors and protease inhibitors (PI) was the same in the two subtypes, but mutations related to PI at the codons 63, 77, and 71 were more frequent in subtype B, while mutations at the codons 36 and 20 predominated in subtype F. Sixty-two of the 80 patients infected with subtypes B and F were submitted to antiretroviral therapy for an average of 18-22 months. Undetectable viral loads at the end of follow-up were similar in the two groups, representing 63.8% of subtype B and 73.3% of subtype F (p = 0.715). CD4 lymphocyte counts before and after treatment were similar in the two groups. This study, despite pointing to possible epidemiological and genetic differences among subtypes B and F of HIV-1, suggests that the use of highly active antiretroviral therapy is equally effective against these subtypes.

Phylogenetic analysis of Biomphalaria tenagophila (Orbigny, 1835) (Mollusca: Gastropoda)

Mitochondrial DNA of Biomphalaria tenagophila, a mollusc intermediate host of Schistosoma mansoni in Brazil, was sequenced and characterised. The genome size found for B. tenagophila was 13,722 bp and contained 13 messenger RNAs, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). In addition to sequencing, the mitochondrial DNA (mtDNA) genome organization of B. tenagophila was analysed based on its content and localization of both coding and non-coding regions, regions of gene overlap and tRNA nucleotide sequences. Sequences of protein, rRNA 12S and rRNA 16S nucleotides as well as gene organization were compared between B. tenagophila and Biomphalaria glabrata, as the latter is the most important S. mansoni intermediate host in Brazil. Differences between such species were observed regarding rRNA composition. The complete sequence of the B. tenagophila mitochondrial genome was deposited in GenBank (accession EF433576). Furthermore, phylogenetic relationships were estimated among 28 mollusc species, which had their complete mitochondrial genome deposited in GenBank, using the neighbour-joining method, maximum parsimony and maximum likelihood bootstrap. B. tenagophila was positioned at a branch close to B. glabrata and Pulmonata molluscs, collectively comprising a paraphyletic group, contrary to Opistobranchia, which was positioned at a single branch and constituted a monophyletic group.

Phylogeny, ultrastructure, histopathology and prevalence of Myxobolus oliveirai sp. nov., a parasite of Brycon hilarii (Characidae) in the Pantanal wetland, Brazil

This paper presents the morphological, histological and ultrastructural characteristics of Myxobolus oliveirai sp. nov., a parasite of the gill filaments in Brycon hilarii from the Brazilian Pantanal. Out of 216 B. hilariispecimens examined (126 wild and 90 cultivated), 38.1% of wild specimens (n = 48) were infected. The parasites form elongated plasmodia primarily in the tip of gill filaments, reaching about 3 mm in length. A thorough comparison with all the Myxobolus species described from South American hosts, as well as nearly all the Myxobolus species described so far is provided. Partial sequencing of the 18S rDNA gene revealed a total of 1,527 bp. The Myxobolus species parasite of B. hilarii did not match any of the Myxozoa available in GenBank. In the phylogenetic analysis, M. oliveirai sp. nov. composed a monophyletic group with eight other species: five species of Myxobolus parasites of mugilid fishes, two parasites of pangasiid and one of centrarchid. Infection prevalence values of the parasite revealed no significant differences between wet and dry seasons or between males and females. The importance of the infection to the farming of the host species is emphasized.

Conditional deletion of ferritin h in mice reduces B and T lymphocyte populations.

The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fth(lox/lox) ; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fth(lox/lox) ;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Prevalence of occult hepatitis B virus infection in kidney transplant recipients

In this cross-sectional study, 207 hepatitis B surface antigen (HBsAg)-negative kidney transplant recipients were evaluated based on demographic and epidemiological data and on the levels of serological markers of hepatitis B virus (HBV) and hepatitis C virus infection and liver enzymes. Patients with HBV or human immunodeficiency virus infection were excluded. Sera were analysed for the presence of HBV-DNA. HBV-DNA was detected in two patients (1%), indicating occult hepatitis B (OHB) infection (the HBV-DNA loads were 3.1 and 3.5 IU/mL in these patients). The results of the liver function tests were normal and no serological markers indicative of HBV infection were detected. The prevalence of OHB infection was low among kidney transplant recipients, most likely due to the low HBsAg endemicity in the general population of the study area.

BAFF mediates survival of peripheral immature B lymphocytes.

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.

Efficacy of entecavir and tenofovir in chronic hepatitis B under treatment in the public health system in southern Brazil

There are about 350 million hepatitis B virus (HBV) carriers worldwide and chronic HBV is considered a major public health problem. The objective of the present study was to assess the effectiveness of the nucleos(t)ide analogues tenofovir (TDF) and entecavir (ETV) in the treatment of chronic HBV. A cross-sectional study was carried out from March-December 2013, including all patients with chronic HBV, over 18 years of age, undergoing therapy through the public health system in southern Brazil. Only the data relating to the first treatments performed with TDF or ETV were considered. Retreatment, co-infection, transplanted or immunosuppressed patients were excluded. Six hundred and forty patients were evaluated, of which 336 (52.5%) received TDF and 165 (25.8%) ETV. The other 139 (21.7%) used various combinations of nucleos(t)ide analogues and were excluded. The negativation of viral load was observed in 87.3% and 78.8% and the negativation of hepatitis B e antigen was achieved in 79% and 72% of those treated with ETV or TDF, respectively. Negativation of hepatitis B surface antigen was not observed. There was no occurrence of adverse effects. This is a real-life study demonstrating that long-term treatment with ETV and TDF is both safe and effective.

Statistical Analysis of Eco-Toxicologic Data of Mammals from a Polluted Area

Several eco-toxicological studies have shown that insectivorous mammals, due to theirfeeding habits, easily accumulate high amounts of pollutants in relation to other mammal species. To assess the bio-accumulation levels of toxic metals and their in°uenceon essential metals, we quantified the concentration of 19 elements (Ca, K, Fe, B, P,S, Na, Al, Zn, Ba, Rb, Sr, Cu, Mn, Hg, Cd, Mo, Cr and Pb) in bones of 105 greaterwhite-toothed shrews (Crocidura russula) from a polluted (Ebro Delta) and a control(Medas Islands) area. Since chemical contents of a bio-indicator are mainly compositional data, conventional statistical analyses currently used in eco-toxicology can givemisleading results. Therefore, to improve the interpretation of the data obtained, weused statistical techniques for compositional data analysis to define groups of metalsand to evaluate the relationships between them, from an inter-population viewpoint.Hypothesis testing on the adequate balance-coordinates allow us to confirm intuitionbased hypothesis and some previous results. The main statistical goal was to test equalmeans of balance-coordinates for the two defined populations. After checking normality,one-way ANOVA or Mann-Whitney tests were carried out for the inter-group balances

Use of Granada medium to detect group B streptococcal colonization in pregnant women.

Direct inoculation onto Granada medium (GM) in plates and tubes was compared to inoculation into a selective Todd-Hewitt broth (with 8 microg of gentamicin per ml and 15 microg of nalidixic acid per ml) for detection of group B streptococci (GBS) in pregnant women with 800 vaginal and 450 vaginoanorectal samples. Comparatively, GM was found to be as sensitive as the selective broth for the detection of GBS in vaginal specimens and more sensitive than selective broth for the detection of GBS in vaginoanorectal samples (96 versus 82%). The use of GM improved the time to reporting of a GBS-positive result by at least 24 h and reduced the direct cost of screening. We have also found that the inconvenience of anaerobic incubation of GM plates can be avoided when a cover slide is placed upon the inoculum, because aerobic incubation in GM plates with cover slides causes GBS to develop the same pigmentation that it develops with incubation under anaerobic conditions. These data support the routine use of GM plates or tubes as a more accurate, easier, and cheaper method of identification of GBS-colonized women compared to the enrichment broth technique.

Molecular phylogenetics of shrews (Mammalia: Soricidae) reveal timing of transcontinental colonizations.

We sequenced 2167 base pairs (bp) of mitochondrial DNA cytochrome b and 16S, and 1390 bp of nuclear genes BRCA1 and ApoB in shrews taxa (Eulipotyphla, family Soricidae). The aim was to study the relationships at higher taxonomic levels within this family, and in particular the position of difficult clades such as Anourosorex and Myosorex. The data confirmed two monophyletic subfamilies, Soricinae and Crocidurinae. In the former, the tribes Anourosoricini, Blarinini, Nectogalini, Notiosoricini, and Soricini were supported. The latter was formed by the tribes Myosoricini and Crocidurini. The genus Suncus appeared to be paraphyletic and included Sylvisorex. We further suggest a biogeographical hypothesis, which shows that North America was colonized by three independent lineages of Soricinae during middle Miocene. Our hypothesis is congruent with the first fossil records for these taxa. Using molecular dating, the first exchanges between Africa and Eurasia occurred during the middle Miocene. The last one took place in the Late Miocene, with the dispersion of the genus Crocidura through the old world.

Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome.

BAFF (BLyS, TALL-1, THANK, zTNF4) is a member of the TNF superfamily that specifically regulates B lymphocyte proliferation and survival. Mice transgenic (Tg) for BAFF develop an autoimmune condition similar to systemic lupus erythematosus. We now demonstrate that BAFF Tg mice, as they age, develop a secondary pathology reminiscent of Sjögren's syndrome (SS), which is manifested by severe sialadenitis, decreased saliva production, and destruction of submaxillary glands. In humans, SS also correlates with elevated levels of circulating BAFF, as well as a dramatic upregulation of BAFF expression in inflamed salivary glands. A likely explanation for disease in BAFF Tg mice is excessive survival signals to autoreactive B cells, possibly as they pass through a critical tolerance checkpoint while maturing in the spleen. The marginal zone (MZ) B cell compartment, one of the enlarged B cell subsets in the spleen of BAFF Tg mice, is a potential reservoir of autoreactive B cells. Interestingly, B cells with an MZ-like phenotype infiltrate the salivary glands of BAFF Tg mice, suggesting that cells of this compartment potentially participate in tissue damage in SS and possibly other autoimmune diseases. We conclude that altered B cell differentiation and tolerance induced by excess BAFF may be central to SS pathogenesis.

A 338-bp proximal fragment of the glucose transporter type 2 (GLUT2) promoter drives reporter gene expression in the pancreatic islets of transgenic mice.

The high Km glucose transporter GLUT2 is a membrane protein expressed in tissues involved in maintaining glucose homeostasis, and in cells where glucose-sensing is necessary. In many experimental models of diabetes, GLUT2 gene expression is decreased in pancreatic beta-cells, which could lead to a loss of glucose-induced insulin secretion. In order to identify factors involved in pancreatic beta-cell specific expression of GLUT2, we have recently cloned the murine GLUT2 promoter and identified cis-elements within the 338-bp of the proximal promoter capable of binding islet-specific trans-acting factors. Furthermore, in transient transfection studies, this 338-bp fragment could efficiently drive the expression of the chloramphenicol acetyl transferase (CAT) gene in cell lines derived from the endocrine pancreas, but displayed no promoter activity in non-pancreatic cells. In this report, we tested the cell-specific expression of a CAT reporter gene driven by a short (338 bp) and a larger (1311 bp) fragment of the GLUT2 promoter in transgenic mice. We generated ten transgenic lines that integrated one of the constructs. CAT mRNA expression in transgenic tissues was assessed using the RNAse protection assay and the quantitative reverse transcribed polymerase chain reaction (RT-PCR). Overall CAT mRNA expression for both constructs was low compared to endogenous GLUT2 mRNA levels but the reporter transcript could be detected in all animals in the pancreatic islets and the liver, and in a few transgenic lines in the kidney and the small intestine. The CAT protein was also present in Langerhans islets and in the liver for both constructs by immunocytochemistry. These findings suggest that the proximal 338 bp of the murine GLUT2 promoter contain cis-elements required for the islet-specific expression of GLUT2.