Utilization of a mouse/human chimeric model for long term metabolic testing of human skin.

Until now, ex vivo human skin explant utilization in tissue culture has consisted of limited short-term studies (less than a week). This short timeframe does not allow for the investigation of metabolic responses of complex tissues to specific molecules or compounds. Here, we aim to develop an improved mouse transplantation model that maintains the viability, structure and functionality of the human skin explants for prolonged periods of time. Healthy human skin explants derived from biopsies were grafted onto nude mice and used to perform a toxicological study of the reactivity and functionality of grafted skin explants after one month. Histological observations suggest that the tissue properties and phenotype of the human skin graft are conserved as a result of re-vascularization upon tissue integration. The toxicological test performed shows that the human skin graft reacts to systemic exposure of a xenobiotic metabolic inducer when applied to this mouse model. This mouse/human chimeric model can be effective for the long-term study of human skin reactivity to chemicals as well to study in vivo responses to complex co-exposures.

Effects of a phytogenic, alone and associated with potassium diformate, on tilapia growth, immunity, gut microbiome and resistance against francisellosis.

This work evaluated the effects of dietary supplementation of A-Live (phytogenic) either individually or in combination with Aquaform (potassium diformate, acidifier) on juvenile Nile tilapia (Oreochromis niloticus) growth performance, innate immune parameters, gut microbiome, and resistance against Francisella noatunensis subsp. orientalis challenge. Each experimental group contained 140 fishes (34.3 ± 0.33) in two 150L tanks. The experimental design consisted of five groups: a negative control; treated groups (G1, G2, G3) supplemented with different concentrations of A-Live and Aquaform in the feed; and a positive control (PC) for pathogen infection. Groups G1, G2, G3, and PC were challenged with Francisella spp. after 15 days. After infection, the mortality was significantly lower in groups G1, G2, and G3 (p < 0.01). Furthermore, these groups showed significant increase (p < 0.05) in daily weight gain, feed conversion rate, and specific growth rate. The PC group presented increase (p < 0.05) in the leukocytes and neutrophils number. Innate immunity parameters showed no difference between treatments after infection. Microbiome analysis revealed an increased number of bacteria belonging to the Vibrionaceae family after pathogen infection suggesting a secondary pathogen function of these bacteria. These results validate the beneficial effects of these products in tilapia farming.

Quality control in the different stages of producing red blood cell concentrate from dogs / Controle de qualidade nas diferentes etapas de produção do concentrado de hemácias em cães

The objective of this study was to perform a quality control assessment of red blood cells after standardization of the blood production stages. For this purpose, separation of the blood components to obtain red blood cells, the storage of the blood packets and an evaluation of blood quality were performed. The mean (± SD) volume, globular volume, hemoglobin and hemolysis percentage of the red blood cell concentrate were 299.77±30.08mL, 60.87±2.60%, 20.57±0.93g/DL and 0.09±0.07%, respectively. The means (± SD) of the volume, globular volume, total hemoglobin percentage of hemolysis and hemoglobin per unit of packed red blood cells after the storage period (8.83±6.73 days) were 57.55±3.01%, 20.30±0.89 0, 20±0.12%, and 60.90±7.65. The red blood cell packets were within the parameters of quality control established by Health Ministry legislation in humans and allow us to conclude that the standardization of blood production stages involves the selection of donors until the end of storage and is necessary to produce quality red blood cells. Quality control aims to find possible flaws in the procedures to be repaired, increasing transfusion safety.(AU)

O objetivo deste estudo foi realizar o controle de qualidade do concentrado de hemácias após a padronização das etapas de produção do sangue. Para isso, realizou-se separação de hemocomponentes para obtenção de concentrado de hemácias, armazenamento das bolsas de sangue e avaliação da qualidade delas. Os valores médios (± DP) do volume, do volume globular, da hemoglobina e do percentual de hemólise do concentrado de hemácias foram: 299,77±30,08mL, 60,87±2,60%, 20,57±0,93g/DL e 0,09±0,07%, respectivamente. Os valores médios (± DP) do volume globular, da hemoglobina total percentual de hemólise e da hemoglobina por unidade de concentrado de hemácias após o período de armazenamento (8,83±6,73 dias) foram: 57,55±3,01%, 20,30±0,89 0,20±0,12%, 60,90±7,65. As bolsas de concentrado de hemácias ficaram dentro dos parâmetros de controle de qualidade estabelecidos pela legislação do Ministério da Saúde em humanos e possibilitaram concluir que a padronização das etapas de produção do sangue envolve desde a seleção de doadores até o final do armazenamento e é necessária para produzir concentrado de hemácias com qualidade. O controle de qualidade visa encontrar possíveis falhas nos procedimentos para que estas possam ser reparadas, aumentando, assim, a segurança transfusional.(AU)

Inhibition of sodium glucose cotransporters following status epilepticus induced by intrahippocampal pilocarpine affects neurodegeneration process in hippocampus.

Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, starting from secondary functional disorders due to several insults, including self-sustaining continuous seizures identified as status epilepticus (SE). Although hypoglycemia has been associated with SE, the effect of inhibition of the Na(+)/glucose cotransporters (SGLTs) on hippocampus during SE is still unknown. Here we evaluated the functional role of SGLT in the pattern of limbic seizures and neurodegeneration process after pilocarpine (PILO)-induced SE. Vehicle (VEH, 1µL) or phlorizin, a specific SGLT inhibitor (PZN, 1µL, 50µg/µL), was administered in the hippocampus of rats 30min before PILO (VEH+PILO or PZN+PILO, respectively). The limbic seizures were classified using the Racine's scale, and the amount of wet dog shakes (WDS) was quantified before and during SE. Neurodegeneration process was evaluated by Fluoro-Jade C (FJ-C), and FJ-C-positive neurons (FJ-C+) were counted 24h and 15days after SE. The PZN-treated rats showed higher (p<0.05) number of WDS when compared with VEH+PILO. There was no difference in seizure severity between PZN+PILO and VEH+PILO groups. However, the pattern of limbic seizures significantly changed in PZN+PILO. Indeed, the class 5 seizures repeated themselves more times (p<0.05) than the other classes in the PZN group at 50min after SE induction. The PZN+PILO animals had a higher (p<0.05) number of FJ-C+ cells in the dentate gyrus (DG), hilus, and CA3 and CA1 of hippocampus, when compared with VEH+PILO. The PZN+PILO animals had a decreased number (p<0.05) of FJ-C+ cells in CA1 compared with VEH+PILO 15days after SE induction. Taken together, our data suggest that SGLT inhibition with PZN increased the severity of limbic seizures during SE and increased neurodegeneration in hippocampus 24h after SE, suggesting that SGLT1 and SGLT2 could participate in the modulation of earlier stages of epileptogenic processes.

Isolation and molecular characterization of Leptospira borgpetersenii serovar Hardjo strain Hardjobovis in the urine of naturally infected cattle in Brazil.

Most epidemiologic studies on bovine leptospirosis are based on serological tests that use antibodies against several serotypes, including the serovar Hardjo, which is widespread and considered to be the most adapted to bovine hosts. However, using only serological studies is not sufficient to identify and distinguish species of leptospires. The aim of this study was report the first isolation in Brazil of two strains serovar Hardjo obtained in urine samples from naturally infected cows in a small Brazilian dairy herd and find the genetic species and consequently the type strain Hardjobovis by molecular characterization. Fifteen dairy cows with a history of reproductive failure, such as abortion and infertility, were selected. Urine samples obtained from each animal were immediately seeded in tubes containing Ellinghausen-McCullough-Johnson-Harris culture medium. The identification of the isolates was performed by Multilocus variable-number tandem-repeat analysis (MLVA) technique and phylogenetic analysis of partial sequence of gene sec Y. From the 15 urine samples evaluated, two Leptospira were found and identified as the Londrina 49 and Londrina 54 strains. The MLVA profiles and sequencing of gene sec Y characterized the isolates as L. borgpetersenii serovar Hardjo strain Hadjobovis because it has different genetic pattern of Leptospira interrogans serovar Hardjo strain Hardjoprajitno. Therefore, more studies are needed including isolation and molecular characterization from regional strains to obtain a better knowledge about epidemiology of serovar Hardjo in bovine which may assist in future strategies of prevention and control of bovine leptospirosis.

Development and validation of a bioartificial liver device with fluidized bed bioreactors hosting alginate-encapsulated hepatocyte spheroids.

Acute and acute-on-chronic liver failure are associated to high mortality when transplantation is not possible. The lack of donors has resulted in an important demand for liver support devices. This paper describes the design and validation of a new bioartificial liver (BAL) device including fluidized bed bioreactors hosting alginate-encapsulated hepatocytes spheroids. To ensure the efficacy of the BAL and the safety of the patients, a complex extracorporeal circulation was designed to be compatible with a commercial medical device, the Prismaflex(®) monitor, already used in intensive care units. Preclinical studies on large animal show that the treatment was well tolerated in terms of hemodynamics considerations. A method using non adhesive coating in petri dish led to the production of large amount of viable spheroids in vitro that were further encapsulated to follow up bioartificial liver activity during four days.

Whole-Genome Sequence of Weissella ceti Strain WS08, Isolated from Diseased Rainbow Trout in Brazil.

We report here the complete genome sequence of Weissella ceti strain WS08, an emerging pathogen to farm-raised rainbow trout. The genome of strain WS08 is composed of a circular chromosome with 1,355,853 bp and a G+C content of 40.78%.

In silico prediction of conserved vaccine targets in Streptococcus agalactiae strains isolated from fish, cattle, and human samples.

Streptococcus agalactiae (Lancefield group B; group B streptococci) is a major pathogen that causes meningoencephalitis in fish, mastitis in cows, and neonatal sepsis and meningitis in humans. The available prophylactic measures for conserving human and animal health are not totally effective and have limitations. Effective vaccines against the different serotypes or genotypes of pathogenic strains from the various hosts would be useful. We used an in silico strategy to identify conserved vaccine candidates in 15 genomes of group B streptococci strains isolated from human, bovine, and fish samples. The degree of conservation, subcellular localization, and immunogenic potential of S. agalactiae proteins were investigated. We identified 36 antigenic proteins that were conserved in all 15 genomes. Among these proteins, 5 and 23 were shared only by human or fish strains, respectively. These potential vaccine targets may help develop effective vaccines that will help prevent S. agalactiae infection.

Genetic diversity and new genotyping scheme for fish pathogenic Streptococcus agalactiae.

UNLABELLED: This study aimed to assess the genetic diversity of fish isolates of Streptococcus agalactiae by capsular serotyping, MLST and the pattern of selected virulence genes. Forty-six isolates from Nile tilapia and Amazon catfish were screened by PCR for the twelve virulence genes. The molecular capsular type and sequence type (ST) were determined. Two capsular types (Ia and Ib) and four STs (103, 260, 552 and 553) were identified. The ST-552 and ST-553 represent new allelic combinations. Variable results were found for the genes gbs2018-6, lmb, hylB and cylE. The combined evaluation of serotype, sequence type and pattern of the presence or absence of cylE and hylB allowed the classification of isolates into nine genetic profiles (I-IX). The proposed scheme showed higher discriminatory power and was able to detect evolutionary events missed by MLST analysis. This study provides new information about the genetic diversity of fish pathogenic Strep. agalactiae, and the proposed scheme was shown to be an improved approach to genotyping these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that critical genetic events in Streptococcus agalactiae isolates pathogenic for fish have been missed by serotyping and multilocus sequence typing (MLST). A proposed genotyping scheme based on the evaluation of concatenated data from serotyping, MLST, and the presence/absence of virulence genes was created, and this was able to detect old and recent evolutionary events. It provided a better understanding of the genetic diversity of Strep. agalactiae populations from fish and will contribute to future studies of the molecular epidemiology, pathogenesis and evolutionary aspects of this pathogen.

Characterization of the first coculture between human primary keratinocytes and the dorsal root ganglion-derived neuronal cell line F-11.

The epidermis can be considered as a sensory organ. Sensory neurons of the peripheral nervous system send many primary afferent fibers to the skin, creating a dynamic communication with epidermal cells. However, little is known about the functional interactions between the sensory fibers and the keratinocytes. It is therefore difficult to reproduce these interactions in vitro. We have developed an in vitro model based on the coculture of primary human keratinocytes and the dorsal root ganglion cell line F-11. F-11 cells have been classically used to mimic authentic peptidergic nociceptive neurons. We first investigated the morphological and functional characteristics of F-11 cells cultured in basal keratinocyte medium and then analyzed the influence of keratinocytes on these properties. We found that F-11 cells survived and differentiated well in this medium. Therefore, the addition of neurotrophins did not enhance their survival or differentiation. These neurons expressed sensory neuron markers and were able to release neuropeptides after capsaicin activation. We noted that neuropeptides release were obtained even at a low calcium concentration and that axonal outgrowth was not influenced by external calcium (Ca(2+)) levels. These properties were reproduced when F-11 cells were cocultured with keratinocytes, but they had no significant influence on axonal development or neuropeptide release. In this study, we describe for the first time the culture of F-11 neurons with another cell type. This coculture model in which keratinocytes and neurons are maintained in low Ca(2+) concentrations may be a useful in vitro alternative for studying and characterizing the close communication between keratinocytes and sensory neurons.

Population diversity of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds.

Population diversity was evaluated in strains of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds by PCR-RFLP and sequencing of the 3'-terminal portion of the coagulase gene, and the susceptibility of strains to antimicrobials. The results showed great diversity in S. aureus population studied and the existence of predominant clones that account for most infections. No associations between the predominant types observed in the PCR-RFLP and the forms of presentation of the mastitis or to any of the different patterns of antimicrobial resistance were observed.

Efficacy of Staphylococcus aureus vaccines for bovine mastitis: a systematic review.

Bovine mastitis is the main cause of economic loss in milk production worldwide and Staphylococcus aureus is the agent most frequently associated with the disease. The aim of this systematic review was to assess the efficacy of vaccines for bovine mastitis caused by S. aureus and suggest the immunotherapeutic protocols that have achieved the best and/or most promising results. An electronic search was made of the PubMed and Web of Science databases in November 2009. Only studies that tested vaccines in vivo in cows were included. The experimental design, methodological quality, type of vaccine and results of the studies were analyzed. Twenty-four papers were selected for this review. In general, bacterin-toxoid vaccines, vaccines of DNA-recombinant protein and recombinant protein alone were investigated in the studies selected. This systematic review suggests that vaccines that employ new technologies (DNA and/or recombinant protein vaccines) and some long-standing bacterins have achieved good results, which supports their use in the prevention and control of bovine mastitis caused by S. aureus. However, methodological differences and in some cases, a lack of more severe scientific criteria (such as double blind protocols) hinder the assessment of the effectiveness of these vaccines.

Mechanisms of the sensory effects of tacrolimus on the skin.

BACKGROUND: Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported. OBJECTIVES: To understand the mechanisms underlying these burning sensations. METHODS: We examined the impact of tacrolimus on substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation. Because phosphorylation of TRPV1 (transient receptor potential subtype vanilloid 1) plays a role in the induction of pain, we investigated whether tacrolimus regulates the phosphorylation state of TRPV1. Finally, we used a macropatch to evaluate the impact of tacrolimus on voltage-gated calcium currents of sensory neurons. RESULTS: Tacrolimus was able to induce initial SP release by extracellular calcium influx and inhibited SP release induced by capsaicin after 1, 24 and 72 h of pretreatment. Analysis of TRPV1 phosphorylation by Western blot confirmed the capacity of tacrolimus to favour phosphorylation. An electrophysiological study showed inhibitory effects on calcium currents. CONCLUSIONS: The efficacy of tacrolimus in pruritus, as well as the sensory side-effects, could be explained by a direct effect on neurons through an effect on calcineurin, possibly by a desensitization of TRPV1 and calcium currents through the PIP(2) regulation pathway.

Genotyping of Streptococcus agalactiae strains isolated from fish, human and cattle and their virulence potential in Nile tilapia.

Streptococcus agalactiae (Lancefield group B; GBS) is a pathogen that causes meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. The objective of this study was to characterize S. agalactiae isolated from fish (n=27), cows (n=9), and humans (n=10) using pulsed-field gel electrophoresis (PFGE) and to investigate the virulence of the identified strains in Nile tilapia (Oreochromis niloticus). The PFGE types were determined by dendogram analyses and the in vivo virulence was evaluated by experimental infection (using i.p. and immersion routes) of Nile tilapia. Among the fish strains, 5 different PFGE patterns were observed and 21 strains showed the same genetic pattern. In some farms two or three profiles occurred simultaneously. The bovine and human strains exhibited high genetic diversity and few relationships were established among S. agalactiae strains from the three host origins analyzed. Eight S. agalactiae strains from fish caused high mortality of Nile tilapia. Three bovine strains infected Nile tilapia (by i.p. route) and two of those strains caused clinical signs of meningoencephalitis. All human strains (n=5) infected Nile tilapia (by i.p. route) and meningoencephalitis was induced by one strain (by both i.p. and immersion routes). In conclusion, the analyzed strains from the three natural hosts did not show genetic relatedness, yet some of the bovine and human strains were able to infect fish and cause meningoencephalitis. We suggest that genetic linkage is not a prerequisite for S. agalactiae to cross the host-specific barrier.