Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

BACKGROUND: The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. RESULTS: Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. CONCLUSION: Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases

Diacutaneous fibrolysis versus passive stretching after articular immobilisation : muscle recovery and extracellular matrix remodelling

Introduction Atrophy and muscle shortening due to articular immobilisation are common problems in musculoskeletal rehabilitation. Muscle stretching mechanical stimuli might be considered as the golden standard procedure to improve muscle flexibility in rehabilitation. Muscle stretching generates mechanotransduction, potentiating specific gene expression and promotes sarcomerogenesis and extracellular matrix remodelling on shortened and atrophied muscles. Hypothesis Diacutaneous fibrolysis, like stretching, uses an external force to stress connective and muscle tissues mechanically to treat muscle shortening; thus, it is widely used in clinical practice even if there is no evidence to support it. Considering this subject, we have hypothesised that diacutaneous fibrolysis can generate mechanotransduction, affecting muscle hypertrophy and extracellular matrix remodelling after immobilisation. Evaluation of hypothesis We have designed a laboratory experimental study with a sample of 50 rats. The sample was randomly divided into five groups: Control group (n = 10) with non–immobilised rats; 3–week immobilisation group (n = 10); 3–week immobilisation/3–week non–immobilisation group (n = 10);3–week immobilisation/3–week stretching group (n = 10); and 3–week immobilisation/3–week diacutaneous fibrolysis group (n = 10). All rats had their left tibiotarsal joint immobilised in maximum plantar flexion with the orthotics for 3 consecutive weeks. After the immobilisation period, the intervention groups received their respective intervention on their left triceps suralis for 3 weeks. Dependent variables of the study were sarcomere analysis, polymerase chain reaction, connective tissue density, collagen birefringence and matrix metalloproteinases. Statistical analysis was performed using analysis of variance and Duncan post hoc test was applied for differences between groups. For all calculations, a 5% (p < 0.05) significance level was established. Conclusion If the hypothesis is confirmed, the present study might provide evidence to support the use of this physical therapy resource widely used to treat muscle dysfunctions

Renal tubular dysfunction in patients with American cutaneous leishmaniasis

Renal dysfunction seen in patients with American cutaneous leishmaniasis (ACL) has been attributed to the use of antimonials for treatment. To determine whether ACL itself causes tubular dysfunction, we measured renal function in 37 patients with ACL prior to their treatment and compared results to that in 10 healthy volunteers of similar mean age. None of the patients presented with glomerular dysfunction; however, 27 had a urinary concentrating defect. There was no statistical difference between groups in the pre- and post-desmopressin test of urine osmolality, but the post-test urine osmolality of the controls was significantly higher. Urinary AQP2 levels, determined by western blot of isolated exosomes, were found to be significantly lower in patients than in controls, whereas that of the cotransporter (NKCC2) was significantly higher. A urinary acidification defect (post-test pH greater than 5.50 following calcium chloride) was found in 15 patients. Pretest plasma bicarbonate was below normal in 12 patients as was the pretest plasma pH in 14. Expression of the Na/H exchanger (NHE3), H+-ATPase, and pendrin were all significantly higher in patients with ACL than in controls. A combined urinary concentration and acidification defect was found in 12 patients. Thus, the urinary concentrating defect of ACL may be caused by decreased AQP2, with increased NKCC2 compensatory. Pendrin upregulation may be related to the urinary acidification defect with increased NHE3 and H+-ATPase also compensatory. Hence, ACL can cause asymptomatic renal tubular dysfunction

Renal function evaluation in patients with American Cutaneous Leishmaniasis after specific treatment with pentavalent antimonial

Background: Renal evaluation studies are rare in American Cutaneous Leishmaniasis (ACL). The aim of this study is to investigate whether specific treatment reverts ACL-associated renal dysfunction. Methods: A prospective study was conducted with 37 patients with ACL. Urinary concentrating and acidification ability was assessed before and after treatment with pentavalent antimonial. Results: The patients mean age was 35.6 +/- 12 years and 19 were male. Before treatment, urinary concentrating defect (U/P-osm &lt; 2.8) was identified in 27 patients (77%) and urinary acidification defect in 17 patients (46%). No significant glomerular dysfunction was observed before and after specific ACL treatment. There was no reversion of urinary concentrating defects, being observed in 77% of the patients before and in 88% after treatment (p = 0.344). Urinary acidification defect was corrected in 9 patients after treatment, reducing its prevalence from 40% before to only 16% after treament, (p = 0.012). Microalbuminuria higher than 30 mg/g was found in 35% of patients before treatment and in only 8% after treatment. Regarding fractional excretion of sodium, potassium, calcium, phosphorus and magnesium, there was no significant difference between pre and post-treatment period. Conclusion: As previously described, urinary concentrating and acidification defects were found in an important number of patients with ACL. Present results demonstrate that only some patients recover urinary acidification capacity, while no one returned to normal urinary concentration capacity.Brazilian National Council for Scientific and Technological Development (CNPq)Brazilian National Council for Scientific and Technological Development (CNPq

A“Dirty” Footprint: Macroinvertebrate diversity in Amazonian Anthropic Soils

International audienceAmazonian rainforests, once thought to be pristine wilderness, are increasingly known to have been widely inhabited, modified, and managed prior to European arrival, by human populations with diverse cultural backgrounds. Amazonian Dark Earths (ADEs) are fertile soils found throughout the Amazon Basin, created by pre-Columbian societies with sedentary habits. Much is known about the chemistry of these soils, yet their zoology has been neglected. Hence, we characterized soil fertility, macroinvertebrate communities, and their activity at nine archeological sites in three Amazonian regions in ADEs and adjacent reference soils under native forest (young and old) and agricultural systems. We found 673 morphospecies and, despite similar richness in ADEs (385 spp.) and reference soils (399 spp.), we identified a tenacious pre-Columbian footprint, with 49% of morphospecies found exclusively in ADEs. Termite and total macroinvertebrate abundance were higher in reference soils, while soil fertility and macroinvertebrate activity were higher in the ADEs, and associated with larger earthworm quantities and biomass. We show that ADE habitats have a unique pool of species, but that modern land use of ADEs decreases their populations, diversity, and contributions to soil functioning. These findings support the idea that humans created and sustained high-fertility ecosystems that persist today, altering biodiversity patterns in Amazonia

Fraqueza muscular adquirida na UTI (ICU-AW): efeitos sistêmicos da eletroestimulação neuromuscular

Com os avanços tecnológicos alcançados atualmente na terapia intensiva e maior sobrevida dos pacientes, outros desafios têm surgido para os profissionais de saúde. Dentre alguns, destaca-se a fraqueza muscular adquirida na UTI (ICU-AW), caracterizada por paresia esquelética e respiratória dos músculos promovendo aumento nastaxas de mortalidade e comprometimento da qualidade de vida. Sua incidência varia de 30% a 60% e tem na síndrome da resposta inflamatória sistêmica (SIRS) e na disfunção de múltiplos órgãos (DMO) sua principal etiologia. Outros fatores de risco como a hiperglicemia,o uso de bloqueadores neuromusculares e sedativos, a imobilidade e a própria ventilação mecânica estão entre os mais comuns. Entre as medidas de combate à ICU-AW, está o conceito de mobilização precoce, bem como despertar diário e controle estreito da glicemia. Nesse contexto, a eletroestimulação muscular apresenta-se como recurso de grande valia. Sua principal vantagem está no fato de poder ser empreendida independentemente da cooperação do paciente, epor ser capaz de gerar respostas musculares eficientes, bem como resultados satisfatórios na preservação da massa muscular, condicionamento físico e funcionalidade dos que usam essa ferramenta. Desfechos interessantes têm sido observados em diversos perfis de pacientes, como os de doença pulmonar obstrutiva crônica (DPOC)e traumatismo raquimedular (TRM). No paciente crítico, seu uso tem mostrado redução nos tempos de ventilação mecânica (VM), internação na UTI e maior funcionalidade dos pacientes. A relevância dos efeitos sistêmicos e metabólicos provenientes da eletroestimulação neuromuscular (ENM) tem sido a base para os estudos nos pacientes críticos. Portanto, a ICU-AW é uma realidade no cenário da terapia intensiva e sua prevenção tem dado margem à aparição de novas propostas e ferramentas na prevenção dessas complicações