Biofilm formation on intrauterine devices in patients with recurrent vulvovaginal candidiasis

A biofilm is a complex community of surface-associated cells enclosed in a polymer matrix. They attach to solid surfaces and their formation can be affected by growth conditions and co-infection with other pathogens. The presence of biofilm may protect the microorganisms from host defenses, as well as significantly reduce their susceptibility to antifungal agents. Pathogenic microbes can form biofilms on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices (IUDs). The present study was carried out to analyze the presence of biofilm on the surface of intrauterine devices in patients with recurrent vulvovaginal candidiasis, and to determine the susceptibility profile of the isolated yeasts to amphotericin B and fluconazole. Candida albicans was recovered from the IUDs and it was found to be susceptible to the antifungal agents when tested under planktonic growing conditions. These findings indicate the presence of the biofilm on the surface of the IUD as an important risk factor for recurrent vulvovaginal candidiasis.

In vitro infectivity of species of Leishmania (Viannia) responsible for American cutaneous leishmaniasis

There is little available information regarding the infectivity of New World Leishmania species, particularly those from the Amazonian Brazil, where there are six species of the subgenus Viannia causing American cutaneous leishmaniasis (ACL). The aim of this study was to compare, in vitro, the potential infectivity of the following Leishmania (Viannia) spp.: L. (V.) braziliensis from localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL) patients, L. (V.) guyanensis, L. (V.) shawi, L. (V.) lainsoni and L. (V.) naiffi from LCL patients only, in cultured BALB/c mice peritoneal macrophage, as well as the production of NO by the infected cells. The infectivity of parasites was expressed by the infection index and, the nitric oxide (NO) production in the macrophage culture supernatant was measured by the Griess method. It was found that L. (V.) braziliensis from MCL, the more severe form of disease, showed the highest (p <= 0.05) infection index (397), as well as the lowest NO production (2.15 mu M) compared with those of other species. In contrast, L. (V.) naiffi which is less pathogenic for the human showed the lowest infection index (301) and the highest NO production (4.11 mu M). These results demonstrated a negative correlation between the infectivity and the ability of these parasites to escape from the microbicidal activity of the host cell.

Expression of secretory leukocyte proteinase inhibitor in the submandibular glands of AIDS patients

OBJECTIVE: Secretory leukocyte proteinase inhibitor (SLPI) is an endogenous proteinase inhibitor present in mucosal secretions. It also displays antimicrobial activity including anti-human immunodeficiency virus activity. This protease inhibitor is also expressed in submandibular glands (SMG), but there are few data on its expression in AIDS patients with infectious conditions. METHODS: We analyzed the expression of SLPI using immunohistochemistry in submandibular gland samples of 36 AIDS patients [10 with normal histology, 10 with chronic nonspecific sialadenitis, eight with mycobacteriosis, and eight with cytomegalovirus (CMV) infection] and 10 HIV-negative controls. The proteinase inhibitor was quantified using image analysis and expressed as % of positively stained area. RESULTS: There was a higher expression of SLPI in AIDS patients with CMV infection (% of stained area, mean +/- SD: 37.37 +/- 14.45) when compared with all other groups (P = 0.009). There were no significant differences between control subjects (22.70 +/- 9.42%) and AIDS patients without histologic alterations (18.10 +/- 7.58%), with chronic nonspecific sialadenitis (17.13 +/- 5.36%), or mycobacterial infection (21.09 +/- 4.66%). CONCLUSION: Cytomegalovirus infection increases SLPI expression in the SMG of AIDS patients. Our results reveal new insights into the pathogenic association between HIV and CMV in AIDS patients.

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255 Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific lgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49. VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice: these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG. (C) 2011 Elsevier B.V. All rights reserved.

Frequency of thrombophilia determinant factors in patients with livedoid vasculopathy and treatment with anticoagulant drugs - a prospective study

Background Livedoid vasculopathy (LV) is a chronic idiopathic disease characterized by painful purpuric macules on lower extremities. Its exact aetiology remains uncertain, but thrombotic and microcirculatory phenomena have been implicated as possible pathogenic factors. Objectives To assess prospectively the frequency of thrombophilia and to verify the effectiveness of anticoagulant therapy among LV patients. Methods Thirty-four LV patients were tested for prothrombin time, activated partial thromboplastin time, antithrombin activity, protein C and S activity, anticardiolipin antibodies, lupus anticoagulant, prothrombin gene mutation, factor V Leiden mutation, methylenetetrahydrofolate reductase mutation, plasma homocysteine and fibrinogen. Thirteen of these patients were treated with anticoagulant drugs (either warfarin or heparin). Results Of 34 patients, 18 (52%) presented laboratory abnormalities of procoagulant conditions. Positive treatment response to anticoagulant therapy was observed in 11 patients. Improvement of pain was obtained in 1-3 weeks, an average of 1.8 week. Complete healing of the lesions was observed in about 2.3 months. Remission was sustained even after treatment interruption and lasted an average 7.8 months. No severe adverse effects were noticed. Conclusion The authors suggest all patients with diagnosis of LV to be investigated for thrombophilic status. Anticoagulant drugs were well tolerated and seemed to be effective in treating not only LV symptoms but also its ulcerations.

Oral tolerance reduces Th17 cells as well as the overall inflammation in the central nervous system of EAE mice

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory immune response directed against myelin antigens of the central nervous system. In its murine model, EAE, Th17 cells play an important role in disease pathogenesis. These cells can induce blood-brain barrier disruption and CNS immune cells activation, due to the capacity to secrete high levels of IL-17 and IL-22 in an IL-6 + TGF-beta dependent manner. Thus, using the oral tolerance model, by which 200 mu g of MOG 35-55 is given orally to C57BL/6 mice prior to immunization, we showed that the percentage of Th17 cells as well as IL-17 secretion is reduced both in the periphery and also in the CNS of orally tolerated animals. Altogether, our data corroborates with the pathogenic role of IL-17 and IFN-gamma in EAE, as its reduction after oral tolerance, leads to an overall reduction of pro-inflammatory cytokines, such as IL-1 alpha, IL-6, IL-9, IL-12p70 and the chemokines MIP-1 beta, RANTES, Eotaxin and KC in the CNS. It is noteworthy that this was associated to an increase in IL-10 levels. Thus, our data clearly show that disease suppression after oral tolerance induction, correlates with reduction in target organ inflammation, that may be caused by a reduced Th1/Th17 response. Crown Copyright (c) 2010 Published by Elsevier B.V. All rights reserved.

Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries

Conventional karyotyping detects anomalies in 3-15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cutoff for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams-Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome. (C) 2011 Elsevier Masson SAS. All rights reserved.

Understanding the genetic diversity, spatial genetic structure and mating system at the hierarchical levels of fruits and individuals of a continuous Theobroma cacao population from the Brazilian Amazon

Understanding the mating patterns of populations of tree species is a key component of ex situ genetic conservation. In this study, we analysed the genetic diversity, spatial genetic structure (SGS) and mating system at the hierarchical levels of fruits and individuals as well as pollen dispersal patterns in a continuous population of Theobroma cacao in Para State, Brazil. A total of 156 individuals in a 0.56 ha plot were mapped and genotyped for nine microsatellite loci. For the mating system analyses, 50 seeds were collected from nine seed trees by sampling five fruits per tree (10 seeds per fruit). Among the 156 individuals, 127 had unique multilocus genotypes, and the remaining were clones. The population was spatially aggregated; it demonstrated a significant SGS up to 15m that could be attributed primarily to the presence of clones. However, the short seed dispersal distance also contributed to this pattern. Population matings occurred mainly via outcrossing, but selfing was observed in some seed trees, which indicated the presence of individual variation for self-incompatibility. The matings were also correlated, especially within ((r) over cap (p(m)) = 0.607) rather than among the fruits ((r) over cap (p(m)) = 0.099), which suggested that a small number of pollen donors fertilised each fruit. The paternity analysis suggested a high proportion of pollen migration (61.3%), although within the plot, most of the pollen dispersal encompassed short distances (28m). The determination of these novel parameters provides the fundamental information required to establish long-term ex situ conservation strategies for this important tropical species. Heredity (2011) 106, 973-985; doi:10.1038/hdy.2010.145; published online 8 December 2010

Non-Human Leukocyte Antigen Antibodies Reactive with Endothelial Cells Could Be Involved in Early Loss of Renal Allografts

Preformed donor-specific human leukocyte antigen (HLA) antibodies have been associated with allograft dysfunction and failure. However, recipients of HLA-identical kidneys can develop acute humoral rejection, implicating putative pathogenic antibodies that are directed against non-HLA antigens. We investigated the presence of endothelial cell reactive antibodies in 11 patients who experienced early loss of their transplanted kidneys owing to humoral rejection and 1 loss from renal venal thrombosis. We examined the potential efficacy of intravenous immunoglobulin to block the binding of these antibodies, as previously suggested for anti-HLA antibodies.

Application of recombinant antibody library for screening specific antigens in a bovine sperm cell subpopulation

Antibody phage display libraries are a useful tool in proteomic analyses. This study evaluated an antibody recombinant library for identification of sex-specific proteins on the sperm cell surface. The Griffin.1 library was used to produce phage antibodies capable of recognizing membrane proteins from Nelore sperm cells. After producing soluble monoclonal scFv, clones were screened on Simental sperm cells by flow cytometry and those that bound to 40-60% of cells were selected. These clones were re-analyzed using Nelore sperm cells and all clones bound to 40-60% of cells. Positive clones were submitted to a binding assay against male and female bovine leukocytes by flow cytometry and one clone preferentially bound to male cells. The results indicate that phage display antibodies are an alternative method for identification of molecules markers on sperm cells. (C) 2007 Elsevier B.V. All rights reserved.

Genotype and mating type distribution within clinical Cryptococcus neoformans and Cryptococcus gattii isolates from patients with cryptococcal meningitis in Uberaba, Minas Gerais, Brazil

We molecularly characterized 81 cryptococcal isolates recovered from cerebrospinal fluid samples of 77 patients diagnosed between 1998 and 2007 as having cryptococcal meningitis in Uberaba Minas Gerais, Brazil. Fifty-seven (74%) were male with a mean age 35.6 years. Seventy-two (88.9%) of the isolates were from 68 AIDS patients and cryptococcosis was the first AIDS-defining condition in 38 (55.9%) patients. Cryptococcosis and AIDS were simultaneously diagnosed in 25 (65.8%) of these 38 patients. Genotypes were characterized through the use of URA5 restriction fragment length polymorphisms analysis, the genetic variability was determined using PCR-fingerprinting with the minisatellite-specific primer M13, and the mating type and serotypes were established by PCR. Seventy-six of the 81 isolates were Cryptococcus neoformans (93.8%), while the remaining five were C. gattii (6.1%), but all were mating type a. C. neoformans isolates were genotype VNI (serotype A), while C. gattii isolates were VGII. Four of the latter isolates were identical, but only two were from AIDS patients. Six of the nine isolates from non-AIDS patients were VNI. PCR fingerprints of the isolates from two of the three AIDS patients with clinical relapse were 100% identical. The predominance of VNI and mating type a is in accordance with data from other parts of the world. The occurrence of VGII in Minas Gerais indicates a geographical expansion within Brazil.

Transcriptional changes in U343 MG-a glioblastoma cell line exposed to ionizing radiation

Glioblastoma multiforme (GBM) is a highly invasive and radioresistant brain tumor. Aiming to study how glioma cells respond to gamma-rays in terms of biological processes involved in cellular responses, we performed experiments at cellular context and gene expression analysis in U343-MG-a GBM cells irradiated with 1 Gy and collected at 6 h post-irradiation. The survival rate was approximately 61% for 1 Gy and was completely reduced at 16 Gy. By performing the microarray technique, 859 cDNA clones were analyzed. The Significance Analysis of Microarray algorithm indicated 196 significant expressed genes (false discovery rate (FDR) = 0.42%): 67 down-regulated and 97 up-regulated genes, which belong to several classes: metabolism, adhesion/cytoskeleton, signal transduction, cell cycle/apoptosis, membrane transport, DNA repair/DNA damage signaling, transcription factor, intracellular signaling, and RNA processing. Differential expression patterns of five selected genes (HSPA9B, INPP5A, PIP5K1A, FANCG, and TPP2) observed by the microarray analysis were further confirmed by the quantitative real time RT-PCR method, which demonstrated an up-regulation status of those genes. These results indicate a broad spectrum of biological processes (which may reflect the radio-resistance of U343 cells) that were altered in irradiated glioma cells, so as to guarantee cell survival.

Translocations t(X;14)(q28;q11) and t(Y;14)(q12;q11) in T-cell prolymphocytic leukemia

P>We report a case of T-cell prolymphocytic leukemia (T-PLL) in a 41-year-old male. Classical cytogenetic, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) studies of a blood sample obtained at diagnosis revealed the co-existence of t(X;14)(q28;q11), t(Y;14)(q12;q11) and a ring chromosome derivated from i(8)(q10). Immunophenotypic studies revealed involvement of T-cell lineage, with proliferation of CD4(-) CD8(+). The co-existence of two translocations involving both sex chromosomes in a case of T-PLL is rare. Chromosomal instability associated with the disease progression may have allowed the emergence of cell clones with translocations involving the sex chromosomes and the ring chromosome observed.

Transcriptional Response of Peripheral Lymphocytes to Early Fibrosarcoma: A Model System for Cancer Detection Based on Hybridization Signatures

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer. Exp Biol Med 234:802-812, 2009