Rhodotorula spp. isolated from blood cultures: clinical and microbiological aspects

The emergence of less common fungal pathogens has been increasingly reported in the last decade. We describe 25 cases of Rhodotorula spp. isolated from blood cultures at a large Brazilian tertiary teaching hospital from 1996-2004. We also investigated the in vitro activity of four antifungal drugs, using a standardized method. The median age of patients was 43 years. The majority of patients (88%) had a central venous catheter (CVC) and 10 (40%) were recipients of a bone marrow transplant. The episode was classified as a bloodstream infection (BSI) in 80% of the patients. Amphotericin B deoxycholate was the most common antifungal used and CVC was removed in 89.5% of the patients. Death occurred in four patients (17.4%), all classified as BSI. All strains were identified as R. mucilaginosa by conventional methods. Misidentification of the species was observed in 20% and 5% of the strains with the Vitek Yeast Biochemical Card and API 20C AUX systems, respectively. Amphotericin B demonstrated good in vitro activity (MIC(50/90), 0.5 mu g/ml) and the MICs for fluconazole were high for all strains (MIC(50/90), 64 mu g/ml).

De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot

Tetralogy of Fallot (TOF), the most common severe congenital heart malformation, occurs sporadically, without other anomaly, and from unknown cause in 70% of cases. Through a genome-wide survey of 114 subjects with TOF and their unaffected parents, we identified 11 de novo copy number variants (CNVs) that were absent or extremely rare (<0.1%) in 2,265 controls. We then examined a second, independent TOF cohort (n = 398) for additional CNVs at these loci. We identified CNVs at chromosome 1q21.1 in 1% (5/512, P = 0.0002, OR = 22.3) of nonsyndromic sporadic TOF cases. We also identified recurrent CNVs at 3p25.1, 7p21.3 and 22q11.2. CNVs in a single subject with TOF occurred at six loci, two that encode known (NOTCH1, JAG1) disease-associated genes. Our findings predict that at least 10% (4.5-15.5%, 95% confidence interval) of sporadic nonsyndromic TOF cases result from de novo CNVs and suggest that mutations within these loci might be etiologic in other cases of TOF.

Assessment of a New Experimental Model of Isolated Right Ventricular Failure

We assessed a new experimental model of isolated right ventricular (RV) failure, achieved by means of intramyocardial injection of ethanol. RV dysfunction was induced in 13 mongrel dogs via multiple injections of 96% ethanol (total dose 1 mL/kg), all over the inlet and trabecular RV free walls. Hemodynamic and metabolic parameters were evaluated at baseline, after ethanol injection, and on the 14th postoperative day (POD). Echocardiographic parameters were evaluated at baseline, on the sixth POD, and on the 13th POD. The animals were then euthanized for histopathological analysis of the hearts. There was a 15.4% mortality rate. We noticed a decrease in pulmonary blood flow right after RV failure (P = 0.0018), as well as during reoperation on the 14th POD (P = 0.002). The induced RV dysfunction caused an increase in venous lactate levels immediately after ethanol injection and on the 14th POD (P < 0.0003). The echocardiogram revealed a decrease in the RV ejection fraction on the sixth and 13th PODs (P = 0.0001). There was an increased RV end-diastolic volume on the sixth (P = 0.0001) and 13th PODs (P = 0.0084). The right ventricle showed a 74% +/- 0.06% transmural infarction area, with necrotic lesions aged 14 days. Intramyocardial ethanol injection has allowed the creation of a reproducible and inexpensive model of RV failure. The hemodynamic, metabolic, and echocardiographic parameters assessed at different protocol times are compatible with severe RV failure. This model may be useful in understanding the pathophysiology of isolated right-sided heart failure, as well as in the assessment of ventricular assist devices.

Isolated lymphadenitis due to Histoplasma capsulatum diagnosed by fine-needle aspiration biopsy and immunohistochemistry

In the disseminated form of histoplasmosis, isolation and further identification of Histoplasma capsulatum can be performed by several methods, namely, bone marrow aspiration, blood culture, and liver biopsy. Lymph node disease usually is diagnosed by excisional biopsy. Although fungal stains can identify this fungus, detection of specific antigens by immunohistochemistry shows a higher specificity and sensitivity. This approach can use the cell block method when the material is not sent to fungal cultures or fresh staining.

Experimental Model of Isolated Lung Perfusion in Rats: First Brazilian Experience Using the IL-2 Isolated Perfused Rat or Guinea Pig Lung System

Introduction. Lung transplantation has become the mainstay therapy for patients with end-stage lung disease refractory to medical management. However, the number of patients listed for lung transplantation largely exceeds available donors. The study of lung preservation requires accurate, cost-effective small animal models. We have described a model of ex vivo rat lung perfusion using a commercially available system. Methods. Male Wistar rats weighing 250 g-300 g were anesthetized with intraperitoneal sodium thiopental (50 mg/kg body weight). The surgical technique included heart-lung block extraction, assembly, and preparation for perfusion and data collection. We used an IL-2 Isolated Perfused Rat or Guinea Pig Lung System (Harvard Apparatus, Holliston, Mass, United States; Hugo Sachs Elektronik, Alemanha). Results. Preliminary results included hemodynamic and pulmonary mechanics data gathered in the experiments. Conclusion. The isolated rat lung perfusion system is a reliable method to assess lung preservation.

Karyotype variability in KP1(+) and KP1(-) strains of Trypanosoma rangeli isolated in Brazil and Colombia

In the present study, the molecular karyotypes of 12 KP1(+) and KP1(-) Trypanosoma rangeli strains were determined and 10 different molecular markers were hybridized to the chromosomes of the parasite, including seven obtained from T. rangeli [ubiquitin hydrolase (UH), a predicted serine/threonine protein kinase (STK), hexose transporter, hypothetical protein, three anonymous sequences] and three from Trypanosoma cruzi [ubiquitin-conjugating enzyme E2 (UBE2), ribosomal RNA methyltransferase (rRNAmtr), proteasome non-ATPase regulatory subunit 6 (PSMD6)]. Despite intraspecific variation, analysis of the karyotype profiles permitted the division of the T rangeli strains into two groups coinciding with the KP1(+) and KP1(-) genotypes. Southern blot hybridization showed that, except for the hexose transporter probe, all other probes produced distinct patterns able to differentiate the KP1(+) and KP1(-) genotypes. The UH, STK and An-1A04 probes exclusively hybridized to the chromosomes of KP1(+) strains and can be used as markers of this group. In addition, the UBE2, rRNAmtr and PSMD6 markers, which are present in a conserved region in all trypanosomatid species sequenced so far, co-hybridized to the same T. rangeli chromosomal bands, suggesting the occurrence of gene synteny in these species. The finding of distinct molecular karyotypes in KP1(+) and KP1 (-) strains of T rangeli is noteworthy and might be used as a new approach to the study of genetic variability in this parasite. Together with the Southern blot hybridization results, these findings demonstrate that differences at the kDNA level might be associated with variations in nuclear DNA. (c) 2009 Elsevier BY. All rights reserved.

Consequences of lifetime isolated growth hormone (GH) deficiency and effects of short-term GH treatment on bone in adults with a mutation in the GHRH-receptor gene

Growth hormone (GH) influences bone mass maintenance. However, the consequences of lifetime isolated GH deficiency (IGHD) on bone are not well established. We assessed the bone status and the effect of 6 months of GH replacement in GH-naive adults with IGHD due to a homozygous mutation of the GH-releasing hormone (GHRH)-receptor gene (GHRHR). We studied 20 individuals (10 men) with IGHD at baseline, after 6 months of depot GH treatment, and 6 and 12 months after discontinuation of GH. Quantitative ultrasound (QUS) of the heel was performed and serum osteocalcin (OC) and C-terminal cross-linking telopeptide of type I collagen (ICTP) were measured. QUS was also performed at baseline and 12 months later in a group of 20 normal control individuals (CO), who did not receive GH treatment. At baseline, the IGHD group had a lower T-score on QUS than CO (-1.15 +/- 0.9 vs. -0.07 +/- 0.9, P < 0.001). GH treatment improved this parameter, with improvement persisting for 12 months post-treatment (T-score for IGHD = -0.59 +/- 0.9, P < 0.05). GH also caused an increase in serum OC (baseline vs. pGH, P < 0.001) and ICTP (baseline vs. pGH, P < 0.01). The increase in OC was more marked during treatment and its reduction was slower after GH discontinuation than in ICTP. These data suggest that lifetime severe IGHD is associated with significant reduction in QUS parameters, which are partially reversed by short-term depot GH treatment. The treatment induces a biochemical pattern of bone anabolism that persists for at least 6 months after treatment discontinuation.

Cancer/Testis Antigen Expression on Mesenchymal Stem Cells Isolated from Different Tissues

Background/Aims: The expression of cancer/testis antigens (CTAs) on additional normal tissues or stem cells may restrict their use as cancer targets. The objective of the present study was to evaluate the mRNA levels of some CTAs in a variety of tissues. Materials and Methods: mRNA of pericytes, fibroblasts and mesenchymal stem cells (MSCs) derived from adult and fetal tissues, human umbilical vein endothelial cells, MSC-derived adipocytes, selected normal tissues and control cancer cell lines (CLs) were extracted and quantitative polymerase chain reaction was performed for MAGED1, PRAME, CTAG1B, MAGEA3 and MAGEA4. Results: MAGED1 was expressed in all normal tissues and cells evaluated. CTAG1B was expressed at levels comparable to control CLs on MSCs derived from arterial, fetal skin, adipose tissue and saphenous vein, heart, brain and skin tissues. MAGEA4 was detected only in fibroblasts and differentiated adipocytes from MSCs, at levels comparable to the control CLs. Conclusion: The potential use of CTAs in immunotherapy should take into account the potential off-target effects on MSCs.

A novel homozygous splice acceptor site mutation of KISS1R in two siblings with normosmic isolated hypogonadotropic hypogonadism

Context: Loss-of-function mutations of the kisspeptin-1 receptor gene, KISS1R, have been identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). Objective: To investigate KISS1R defects in patients with absent or delayed puberty. Patients: We investigated KISS1R gene defects in a cohort of 99 Brazilian patients with nIHH or constitutional delay of puberty (CDP). Methods: The entire coding region of KISS1R was amplified by PCR followed by automatic sequencing. In addition, screening for KISS1R exonic deletions was performed by multiplex ligation-dependent probe amplification. Results: One novel homozygous KISS1R mutation was identified in two siblings with nIHH. This variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position -2 to -4, and by the insertion of seven nucleotides (ACCGGCT) at the same position, within the 30 splice acceptor site of intron 2 of KISS1R. The brothers who carried this KISS1R mutation had no clinical evidence of pubertal development at the ages of 14 and 20 years. Computational analysis of this indel mutation predicted the generation of an abnormal protein. In addition, a new heterozygous KISS1R variant (p.E252Q) was identified in a male patient with sporadic nIHH. However, in vitro studies of this variant did not demonstrate functional impairment. Only known polymorphisms were identified in patients with CDP. Conclusion: Loss-of-function mutations of KISS1R represents a rare cause of nIHH, and was absent in patients with CDP. We have described a novel KISS1R homozygous splice acceptor site mutation in the familial form of nIHH.

Agglutinin isolated from the red marine alga Hypnea cervicornis J. Agardh reduces inflammatory hypernociception: Involvement of nitric oxide

Hypnea cervicornis agglutinin (HCA), a lectin isolated from the red marine alga has been previously shown to have an antinociceptive effect. In the present study in rats, mechanisms of action of HCA were addressed regarding mechanical hypernociception induced by carrageenan, ovalbumin (as antigen), and also by prostaglandin E(2) in rats. The lectin administered intravenously inhibited carrageenan- and antigen-induced hypernociception at 1,3, 5 and 7 h. This inhibitory effect was completely prevented when lectin was combined with mucin, demonstrating the role of carbohydrate-binding sites. The inhibition of inflammatory hypernociception by HCA was associated with the prevention of neutrophil recruitment to the plantar tissue of rats but was not associated with the inhibition of the release of pro-hypernociceptive cytokines (TNF-alpha, IL-1 beta and CINC-1). HCA also blocked mechanical hypernociception induced by PGE(2), which was prevented by the administration of nitric oxide synthase inhibitors. These results were corroborated by the increased circulating levels of NO metabolites following HCA treatment. These findings suggest that the anti-hypernociceptive effects of HCA are not associated with the inhibition of pro-inflammatory cytokine production. However, these effects seem to involve the inhibition of neutrophil migration and also the increase in NO production. (C) 2010 Elsevier Inc. All rights reserved.

Neuromuscular activity of BaTX, a presynaptic basic PLA(2) isolated from Bothrops alternatus snake venom

We have previously isolated a Lys49 phospholipase A(2) homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SIDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 mu M) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 mu M toxin: 58 +/- 4 and 24 +/- 1 min, respectively; n = 3-8; mean +/- S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 mu M) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 +/- 4%, n=12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 mu M). In addition, a decrease in the organ bath temperature from 37 degrees C to 24 degrees C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity. (C) 2009 Elsevier Inc. All rights reserved.

Isolated osseous ovarian metaplasia: Case report

Background: Some ovarian metaplasias may contain bone or osteoid tissue. The most common tumors presenting these alterations are teratomas and mixed mesodermal tumors with heterologous elements. Case report: We report the case of a woman who, during gynecologic follow-up for chronic anovulation at the age of 31 years, presented a solid ovarian ultrasonographic image with calcifications. After laparoscopy and histological examination it was found to be an isolated ovarian osseous metaplasia. Conclusion: A rarely occurring condition, ovarian osseous metaplasia continues to be of uncertain clinical significance.

Comparisons of the release of vasodilator substances from left and right cardiac chambers of the isolated perfused rabbit heart: Implications for intraventricular thrombus formation

Prostacyclin (PgI(2)) and endothelium-derived nitric oxide (EDNO) are produced by the arterial and venous endothelium. In addition to their vasodilator action on vascular smooth muscle, both act together to inhibit platelet aggregation and promote platelet disaggregation. EDNO also inhibits platelet adhesion to the endothelium. EDNO and PgI(2) have been shown to be released from the cultured endocardial cells. In this study, we examined the release of vasoactive substances from the intact endocardium by using isolated rabbit hearts perfused with physiological salt solution (95% O(2)/5% CO(2), T = 37 degrees C). The right and left cardiac chambers were perfused through separate constant-flow perfusion loops (physiological salt solution, 8 ml min(-1)). Effluent from left and right cardiac, separately, was bioassayed on canine coronary artery smooth muscle, which had been contracted with prostaglandin F(2 alpha_)(2 x 10(-6) M) and no change in tension was exhibit. However, addition of calcium ionophore A23187 (10(-6) M) to the cardiac chambers` perfusion line induced vasodilation of the bioassay coronary ring, 61.4 +/- 7.4% versus 70.49 +/- 6.1% of initial prostaglandin F(2 alpha) contraction for the left and right cardiac chambers perfusate, respectively (mean +/- SEM, n = 10, p > 0.05). Production of vasodilator was blocked totally in the left heart but, only partially blocked in the right heart by adding indomethacin (10(-5) M) to the perfusate, respectively, 95.2 +/- 2.2% versus 41.5 +/- 4.8% (mean +/- SEM, n = 10, p < 0.05). 6-Keto prostaglandin F(1 alpha), measured in the endocardial superfusion effluent was also higher for the left cardiac chambers than for the right at the time of stimulation with the A23187, respectively, 25385.88 +/- 5495 pg/ml (n = 8) versus 13,132.45 +/- 1839.82 pg/ml (n = 8), (p < 0.05). These results showed that cyclooxygenase pathway plays major role in generating vasoactive substances for the left cardiac chamber endocardium; while it is not the main pathway for the right ventricular endocardium at which EDNO and PgI(2) Could act together and potentiate their antithrombogenic activities in isolated perfused rabbit heart. This may be an explanation for the intraventricular thrombus mostly seen in left ventricle rather than in right ventricle as a complication of myocardial infarction. (C) 2009 Elsevier Inc. All rights reserved.

Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.