Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺), led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.     

Steady-state level of insulin-like growth factor-I (IGF-I) receptor mRNA and the effect of IGF-I on the in vitro culture of caprine preantral follicles

The objectives were to quantify insulin-like growth factor receptor-1 (IGFR-1) mRNA in preantral follicles on Days 0 and 18 of in vitro culture in the presence or absence of FSH, and to evaluate the effects of IGF-I on the rate of normal follicles, antral cavity formation, and in vitro growth and maturation of caprine oocytes on Days 0, 6, 12, and 18 of culture. The expression of IGFR-1 was analyzed using real-time RT-PCR before and after follicle culture. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the presence or absence of bovine IGF-I (50 or 100 ng/mL). At the end of the culture period, the oocytes were submitted to IVM. The expression of IGFR-1 mRNA in preantral follicles cultured in vitro only approached being significantly higher in follicles supplemented with FSH when compared to follicles immediately after recovery (P < 0.06) and cultured without FSH (P < 0.1). There was a higher (P < 0.05) percentage of normal follicles on Days 6, 12, and 18 of culture in IGF-I 50 (97, 92, 67%, respectively) and IGF-I 100 (100, 90, 80%) groups versus the control (90, 64, 36%). In addition, the rate of antrum formation at 6 and 12 d of culture was higher (P < 0.05) in IGF-I groups (IGF-I 50: 72 and 90% and IGF-I 100: 69 and 85%) than the control group (41 and 59%). After 18 d of culture, the percentages of grown oocytes acceptable for IVM were higher (P < 0.05) in follicles cultured in the presence of IGF-I (82 vs 49%). Furthermore, follicles cultured in the presence of IGF-I 50 and IGF-I 100 had higher (P < 0.05) meiotic resumption rates (63 and 66%, respectively) than the control group (11%). In conclusion, treatment with FSH tended to increase IGFR-1 mRNA expression during the in vitro culture of preantral follicles and the addition of IGF-I to the culture medium clearly improved the in vitro development of caprine preantral follicles.     

New records of rhodolith-forming species (Corallinales, Rhodophyta) from deep water in Espírito Santo State, Brazil

Little is known about the diversity of non-geniculate coralline red algae (Rhodophyta, Corallinophycidae) from deep waters in Brazil. Most surveys undertaken in this country have been carried out in shallow waters. In 1994, however, the REVIZEE program surveyed the sustainable living resources potential of the Brazilian exclusive economic zone to depths of 500?m. In the present study, the rhodolith-forming coralline algae from the continental shelf of Espírito Santo State were identified. Samples were taken from 54 to 60?m depth by dredging during ship cruises in 1997. Three rhodolith-forming species were found: Spongites yendoi (Foslie) Chamberlain, Lithothamnion muelleri Lenormand ex Rosanoff and Lithothamnion glaciale Kjellman. These records extend the distribution ranges of these species into Brazilian waters and extend the depth distribution of non-geniculate coralline red algae into Brazilian water to 58?m.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Genetic characterization of European-Zebu composite bovine using RFLP markers

A population of 370 European-Zebu composite beef heifers, consisting of six different breed compositions (A-F), were characterized genetically, using RFLP markers of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) genes. Our objectives were to genetically characterize this population and to determine the structure and the genetic variability of this hybrid herd. The genotypes were determined through PCR, followed by digestion with restriction endonucleases. The PCR-RFLP analysis made it possible to identify the LHR and FSHR genotypes, as well as to characterize the degree of heterozygosis, which was high for all of the breed compositions, for both loci, except for two combinations for LHR (B and C). The observed heterozygosity (Ho) was lower than the expected heterozygosity (He) for compositions C (for LHR) and A and D (for FSHR); however, for the population as a whole, Ho was above He (with a mean of 57 versus 46%, respectively), reflecting the elevated genetic variability in this population and also the informative value of the RFLP markers, which could be useful for population genetic characterization studies. The analysis of the degree of genetic structure of this population, estimated by the Nei's statistic, for both loci, indicated an elevated total genetic diversity (HT = 47%), with most of this variability being due to intrapopulational diversity (HS = 46%), with a low degree of genetic differentiation among the six breed compositions (GST = 1.2%). The estimates generated by the Wright's F statistic indicated a non-endogamic population, with excess heterozygotes (FIT = -0.22), which was also observed at the intrapopulational level (FIS = -0.23). The results gave evidence that the genetic selection applied to this European-Zebu composite population did not affect the expected high genetic variability for this type of crossbreeding, which makes it possible to use these animals to obtain economically valuable productive and reproductive traits.     

Expression of cell-cycle regulator CDK2-associating protein 1 (p12CDK2AP1) in transgenic mice induces testicular and ovarian atrophy in vivo

The novel cell-cycle regulator p12(CDK2AP1) (p12) gene encodes a cyclin-dependent kinase 2 (CDK2) partner that participates in cell-cycle regulation, apoptosis, and proliferation. CDK2 has been implicated in maintenance of gonadal homeostasis, as knockout mice display reproductive abnormalities. To investigate the role of p12 in homeostasis of gonadal tissues in vivo, we generated a transgenic mouse model driven by the human keratin 14 promoter, reported to target transgene expression to gonadal tissues and also stratified epithelia. Overexpression of the transgene was associated with a gonadal atrophy phenotype in mice of both sexes, yet fertility was not impaired. Histological evaluation of testes showed seminiferous tubule degeneration and decreased tubule diameter. Female transgenic mice had small ovaries, with a higher number of atretic follicles/mm(2) as compared to control nontransgenic mice. Also observed was increased germ cell apoptosis in both sexes (TUNEL). These results suggest that overexpression of p12 leads to testicular and ovarian abnormalities, a phenotype closely related to that of cdk2-/- mice. In combination, these observations suggest that the p12/CDK2 signaling pathways are carefully orchestrated to maintain proper gonadal tissue homeostasis. We suggest that the mechanisms of this regulation may be through p12-mediated altered expression of gonadal-specific genes and apoptotic pathways.     

Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.     

Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.