Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rond?nia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.     

Caudal Brainstem Raphe Nuclei: Neural Substrate for their Involvement in the Expression of Some Biological Rhythms

Nucleus raphe pallidus (RPa) lies ventrally in the caudal brainstem, where it is coextensive rostrally with the nucleus raphe magnus (RMg) and caudally with the nucleus raphe obscurus (ROb). Retrograde neuronal tracing studies of our laboratory, carried out in rats and presented elsewhere, with fluorogold, true-blue or fast-blue, iontophoretically injected or by crystalline deposit, along the RPa extent, displayed many labeled pericaria at the preoptic area (POA), as well as lateral (LH) and dorsomedial (DMH) hypothalamus; paraventricular nucleus (PVN) and dorsal (DR) and median (MnR) raphe nuclei among others structures. In addition, RPa, which projects to the intermediolateral column, has been demonstrated to bear relation to many of the somatic-visceral functions also reported for POA. Iontophoretic injections of PHA-L, an anterograde tracer, in the POA subnuclei, presented terminal and varicose labeled fibers in RPa, as well as in the RMg, ROb, paraventricular thalamic (PVA), PVN and supraoptic nucleus (SO), LH, subparaventricular zone (sPVZ) and locus coeruleus (LC). Interestingly, POA, PVA, PVN, LH and SO have been described as retino- and suprachiasmatic-recipients. Taken together, these neuronal connections between brainstem raphe nuclei and POA, the similarity of functions to which they are related, as well as connections with other retino-suprachiasmatic-recipient structures, suggest that these caudal brainstem raphe nuclei could be part of the output system for the expression of some biological rhythms.     

Parathyroidectomy Improves Survival In Patients with Severe Hyperparathyroidism: A Comparative Study

Background and objectives

Secondary hyperparathyroidism (SHPT) in CKD is associated with an increased risk for mortality, but definitive data showing that parathormone control decreases mortality is still lacking. This study aimed to compare the mortality of patients with severe SHPT submitted to parathyroidectomy(PTX) with those who did not have access to surgery.

Methods

This is a retrospective study in a cohort of 251 CKD patients with severe SHPT who were referred to a CKD-MBD Center for PTX from 2005 until 2012.

Results

Most of our patients had indication of PTX, but only 49% of them had access to this surgical procedure. After a mean follow-up of 23 months, 72 patients had died. Non-survivors were older; more often had diabetes, lower serum 25 vitamin D and mostly had not been submitted to surgery. The relative risk of death was lower in the PTX patients (0.428; 95% CI, 0.28 to 0.67; p<0.0001). After adjustments, mortality risk was dependent on age (1.04; 95% CI, 1.01 to 1.07; p = 0.002), 25 vitamin D (0.43; 95% CI, 0.24 to 0.81; p = 0.006) and no access to PTX (4.13; 95% CI, 2.16 to 7.88; p<0.0001). Results remained the same in a second model using the PTX date as the study start date for the PTX group.

Conclusions

Our data confirms the benefit of PTX on mortality in patients with severe SHPT. The high mortality encountered in our population is significant and urges the need to better treat these patients.     

Anti‐HSV‐1 and HSV‐2 Flavonoids and a New Kaempferol Triglycoside from the Medicinal Plant Kalanchoe daigremontiana

Kalanchoe daigremontiana (Crassulaceae) is a medicinal plant native to Madagascar. The aim of this study was to investigate the flavonoid content of an aqueous leaf extract from K. daigremontiana (Kd), and assess its antiherpetic potential. The major flavonoid, kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 1 ), was isolated from the AcOEt fraction (Kd‐AC). The BuOH‐soluble fraction afforded quercetin 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 2 ) and the new kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside‐7‐O‐β‐d ‐glucopyranoside ( 3 ), named daigremontrioside. The crude extract, Kd‐AC fraction, flavonoids 1 and 2 were evaluated using acyclovir‐sensitive strains of HSV‐1 and HSV‐2. Kd‐AC was highly active against HSV‐1 (EC₅₀ = 0.97 μg/ml, SI > 206.1) and HSV‐2 (EC₅₀ = 0.72 μg/ml, SI > 277.7). Flavonoids 1 and 2 showed anti‐HSV‐1 (EC₅₀ = 7.4 μg/ml; SI > 27 and EC₅₀ = 5.8 μg/ml; SI > 8.6, respectively) and anti‐HSV‐2 (EC₅₀ = 9.0 μg/ml; SI > 22.2 and EC₅₀ = 36.2 μg/ml; SI > 5.5, respectively) activities, suggesting the contribution of additional substances to the antiviral activity.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease

Background

Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations.

Methods and Findings

Our data established that CH displayed increased expression of CD32⁺ and CD56⁺ in monocytes and enhanced frequency of NK Granzyme A⁺ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8⁺ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8⁺ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8⁺ T-cells, as the most reliable biomarkers of potential use for clinical applications.

Conclusions

Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.     

Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A₂, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.