TLR-2 and TLR-4 agonists favor expansion of CD4+ T cell subsets implicated in the severity of neuromyelitis optica spectrum disorders.

BACKGROUND: High frequency of circulating Th17 cell subsets expressing TLR2, TLR4 and TLR9 was observed in Neuromyelitis optica spectrum disorder (NMOSD) patients, a severe humoral autoimmune disease of the central nervous system. Our objective was to evaluate the direct effects of different TLR ligands on CD4+ T-cells form those patients. METHODS: CD4+ T-cell cultures from NMOSD and healthy individuals were stimulated with different TLR ligands and the cell proliferation and cytokine profile was analyzed by [3H] TdR up take and ELISA/ cytometry, respectively. The plasma levels of CD14 were determined by ELISA. RESULTS: Here, Pam3C (TLR2) and LPS (TLR4) induced significant cell proliferation and IL-6, IL-17 and IL-21 production by CD4+ T-cells from NMOSD. Additionally, while both TLR ligands were more potent in favoring the expansion of TFH-like cells, Pam3C reduced the frequency of IL-10-secreting FoxP3+and FoxP3- CD4+ T-cells. With regard to disease severity, the levels of IL-6, IL-17 and IL-21 produced by CD4+ T-cells, as well as the frequency of TFH-like cells, in response to TLR2 and TLR4 agonists were positively correlated with neurological disabilities and the occurrence of new acute relapses during follow up. Finally, circulating levels of CD14, an indirect marker of microbial translocation, were positively correlated with IL-6, IL-17 and IL-21 release by Pam3C- and LPS-activated CD4+ T-cells. CONCLUSIONS: In summary, our data suggest that microbial antigens may affect NMOSD outcomes by favoring an imbalance between Th17 and TFH-like cells and regulatory T cell subsets.

Sub-Inhibitory Concentrations of Rifampicin Strongly Stimulated Biofilm Production in S. aureus.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus is an important pathogen and a frequent cause of infections associated with biofilm production in implantable medical devices. Biofilm production can be induced by sub-inhibitory concentrations (sub-MICs) of certain antibiotics, but few studies have researched this occurrence in S. aureus. In this study, we investigated the effect of sub-MICs of rifampicin and minocycline on biofilm production by five clinical and five non-clinical S. aureus isolates. METHODS: Microtiter Plate assay and Congo Red Agar Test were used to analyze the biofilm production. The biofilm composition was evaluated by the detachment assay with sodium metaperiodate and proteinase K. RESULTS: Rifampicin sub-MICs induced very high biofilm formation in seven isolates that were non-producers in Tryptic Soy Broth. In one producer isolate, the biofilm formation level was not affected by sub-MICs of this drug. Sub-MICs of minocycline did not induce biofilm production in all isolates tested and in two producer isolates, instead, MIC/2 and MIC/4 inhibited biofilm production. The results of the drugs in combination were similar to those with rifampicin alone. The biofilm matrix was identified as polysaccharide, except for one producer isolate, classified as proteinaceous. Polysaccharide biofilm producer isolates, when grown on Congo Red Agar without sucrose, but with sub-MICs of rifampicin, showed results in agreement with those obtained in Microtiter Plate Test. CONCLUSION: The high biofilm production induced by sub-MICs of rifampicin has potential clinical relevance, because this is one of the drugs commonly used in the impregnation of catheters. In addition, it is used adjunctively to treat certain S. aureus infections.

Evaluation of the influence of the antibiotic ciprofloxacin in the development of an Old World screwworm fly, Chrysomya putoria.

Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae), an Old World screwworm fly, is a species with potential for maggot therapy practice and has been described in myiasis and forensic entomology studies. The objective of the present study was to assess the action of different ciprofloxacin concentrations on the growth and development of C. putoria. First instar maggots of the third generation were raised on 60 g of chicken gizzard homogenate in 65% agar diet and received ciprofloxacin chloridrate. Each concentration of the antibiotic tested (3.33 µg/mL, 6.66 µg/mL, and 13.33 µg/mL) and the control (no antibiotic) were replicated four times (40 maggots/replication). The control received distilled water instead of the antibiotic. Maggots were kept in an acclimatized chamber at 30° C during the day and 28° C at night, with 70 + 10% RH and a 14:10 L:D photoperiod. They were weighed in batches of five and stored in test tubes sealed with nylon fabric and elastic. Microsoft Excel and STAT were used for the analysis. The variation among the maggot weight means and the duration of the maggot stage, pupal stage, and time to total development (neolarvae to adult) were analyzed by Student's t-test (α= 5%). The viabilities and the normality rates were compared using ANOVA, and the expected sex ratio frequency was tested by the chisquared test (χ(2)). There was no significant difference among the four treatments regarding mean individual maggot weight, mean duration of the maggot inoculation until abandonment, the duration of the maggot and pupal stages, and the total duration of all stages. The sex ratios found in the four treatments did not differ from the expected. Only treatment 2 (6.66 µg/mL concentration of ciprofloxacin) differed significantly from the control in maggot and total viability. The antibiotic did not seem to alter C. putoria development in the postembryonic period.

Sterilization of Chrysomya putoria (Insecta: Diptera: Calliphoridae) eggs for use in biotherapy.

Large-scale, quality-controlled laboratory production of fly larvae is needed for biotherapy. The objective of this study was to assess the action of glutaraldehyde on the sterilization of Chrysomya putoria eggs by applying pharmaceutical sterility tests. Egg masses with 0.600 g were divided into three parts of 0.200 g, the eggs were separated using sterile distilled water, and the suspensions obtained were mixed with activated 2% glutaraldehyde solution. After 15-min contact, the suspensions were filtered through Whatman filter paper, and the glutaraldehyde residue obtained in the filtrate was neutralized by rinsing with Tryptone Soy Broth. The treated eggs were placed aseptically on Petri dishes containing gauze moistened with sterile saline solution. About 10% of the sterilized mass was transferred to test tubes containing Tryptone Soy Broth and Fluid Thioglycollate Broth. The tubes were incubated, respectively, at 22.5 and 35.0°C for 14 d to verify egg mass sterility. The plates containing the rest of the eggs (90%) were sealed with plastic film and kept in a climatized chamber at 30°C/d, 28°C per night, 60 ± 10% relative humidity, and under a 12-h light period to assess insect viability and survival. Each experiment was carried out in triplicate using a biological class II safety cabinet. No change in color or turgidity was observed with the agent tested, proving the sterility of the product and that there was no trace of contamination. Forty larvae (in three replications) in the periods of 12, 24, and 48 h after sterilization, when transferred to diet, produced larvae, pupae, and total viability similar to the control (larvae without sterilization). However, for the 72-h treatment, larvae and total viability were significantly lower than for the other treatments. There was no significant difference for the pupal stage. The product tested was shown to be efficacious for use as a sterilizer of C. putoria eggs for all the parameters assessed.

Alternative diets for Chrysomya putoria, an Old World screwworm fly.

Abstract The purpose of this study was to evaluate the post-embryonic development of Chrysomya putoria (Wiedemann 1818) (Diptera: Calliphoridae) reared on a diet of gizzard or gizzard/agar homogenate, with a diet of beef used as the control. Four replicates per treatment were performed (60 mL of each diet). The gizzard (60%), distilled water, and agar homogenate were combined in a blender. Each replicate consisted of 40 newly hatched larvae of C. putoria (5(th) generation). Each glass beaker containing a diet was inserted into a larger flask containing sawdust, which was covered with a nylon cloth held in place by an elastic band. The larvae were weighed and stored in test tubes sealed with a nylon cloth and an elastic band. The average temperature, measured with a thermohygrograph, was 20.6 °C, and the average relative humidity was 67.7%. The variation in the mean weight of mature larvae and in the duration of the larval, pupal, and total stages (newly hatched larvae to imagoes) were analyzed by Student's t-test (α = 5%), while viability was compared by ANOVA. The sex ratio was evaluated by the chi-squared test. The average duration of the period from the larval to imago stage was 8.868 days on the beef diet, 8.676 on the gizzard diet, and 9.067 on the gizzard/agar homogenate diet. Larval survival rates on these diets were 98, 92, and 73%, respectively, while pupal viabilities were 98, 91, and 71%, respectively, and larva-to-imago viabilities were 93, 83, and 64%, respectively. The duration of the pupal period differed significantly between the blowflies reared on the beef and gizzard/agar homogenate diets. The two diets proved to be good alternatives for rearing C. putoria.

Interleucina 10 produzida por gestantes infectadas pelo HIV-1 diminui a replicação viral: efeito amplificado pela terapia anti-retroviral / Interleukin 10 produced by HIV-1 infected pregnant women reduces viral replication: effect enhanced by anti-retroviral therapy

Desde o inicio da epidemia da infecção pelo HIV, o número de mulheres adultas jovens infectadas vem aumentando consideravelmente, fato esse que tem grande impacto na transmissão vertical do virus. Nesse sentido poucos trabalhos tèm avaliado o impacto das citoninas maternas sobre a dinàmica da replicaçao viral durante a gestaçãao. Estudos recentes desenvolvidos pelo nosso grupo demostraram uma relçãó inversa entre a carga viral plasmática e a capacidade de gestantes infectadas pelo HIV-1 em produzir IL-10. Adicionalmente , a neutralização desta citocina aumentou a capacidade replicativa do virus em culturas de células contendo linfocitos T ativados. O estudo aqui reportado teve como objetivo investigar os eventos moleculares relacionados à ação anti-HIV-1 da IL-10 e o impscto da terapia antiretroviral sobre este fenômeno. Nossos resultados demonstram que a capacidade da IL-10 em reduzir a secreção de IL-1 e TNF-a, ambas envolvidas em favorecer a replicação do virus in vitro. Interessantemente, o tratamento anti-retroviral das gestantes elevou a produção sistèmica da IL-10. Esses resultados sugerem que a IL-10 produzida por gestantes infectadas pelo HIV-1 reduz a replicação do virus, e que esse efeito parece ser potencializado pelo tratamento anti-retroviral materno.

Interleucina 10 produzida por gestantes infectadas pelo HIV-1 diminui a replicaþÒo viral: efeito amplificado pela terapia anti-retroviral / Interleukin 10 produced by HIV-1 infected pregnant women reduces viral replication: effect enhanced by anti-retroviral therapy

Desde o inicio da epidemia da infecþÒo pelo HIV, o número de mulheres adultas jovens infectadas vem aumentando consideravelmente, fato esse que tem grande impacto na transmissÒo vertical do virus. Nesse sentido poucos trabalhos tÞm avaliado o impacto das citoninas maternas sobre a dinOmica da replicaþao viral durante a gestaþÒao. Estudos recentes desenvolvidos pelo nosso grupo demostraram uma relþÒó inversa entre a carga viral plasmática e a capacidade de gestantes infectadas pelo HIV-1 em produzir IL-10. Adicionalmente , a neutralizaþÒo desta citocina aumentou a capacidade replicativa do virus em culturas de células contendo linfocitos T ativados. O estudo aqui reportado teve como objetivo investigar os eventos moleculares relacionados O aþÒo anti-HIV-1 da IL-10 e o impscto da terapia antiretroviral sobre este fen¶meno. Nossos resultados demonstram que a capacidade da IL-10 em reduzir a secreþÒo de IL-1 e TNF-a, ambas envolvidas em favorecer a replicaþÒo do virus in vitro. Interessantemente, o tratamento anti-retroviral das gestantes elevou a produþÒo sistÞmica da IL-10. Esses resultados sugerem que a IL-10 produzida por gestantes infectadas pelo HIV-1 reduz a replicaþÒo do virus, e que esse efeito parece ser potencializado pelo tratamento anti-retroviral materno.(AU)

Determinaçao de especies de estafilococos coagulase-negativos de significado clinico / Species identification of coagulase-negative staphylococci of clinical significance

Uma coleçao de 141 amostras de estafilococos coagulase-negativos, isoladas de especimes clinicos, assim como de portadores e ambiente hospitalar, foram analisadas quanto as caracteristicas fisiologicas e propriedades biologicas. Oito especies foram identificadas pelos metodos convencionais segundo Kloos & Schleifer, e corresponderam a S. saprophyticus, S. epidermis, S. cohnil, S. simulans, S. hominis, S. warneri, S. haemolyticus e S. xylosus. Quatorze amostras nao foram identificadas. Atraves da aplicaçao da tecnica de taxonomia numerica se observou uma tendencia para a separaçao em dois grandes grupos, compreendendo S. saprophyticus e S. epidermidis. A partir dai as amostras restantes se identificaram com as outras especies de referencia de diferentes graus de similaridade. O emprego do micrometodo API no estudo de 39 das amostras ja identificadas so permitiu a classificaçao de 17 cepas em especies.

Estudo da virulência de amostras de Pseudomonas aeruginosa: comparaçäo de amostras isoladas de microflora normal e de infecçöes humanas / Virulence of strains of Pseudomonas aeruginosa: comparison of isolated strains of the normal microflora and of human infections

Foram estudadas, comparativamente, 33 amostras de Pseudomonas aeruginosa isoladas de microflora normal do orofaringe e de infecçöes humanas, usando-se marcadores de virulência in vitro. O teste de sensibilidade ao cloreto de benzalcônio, quando empregado isoladamente, foi ineficaz como marcador de virulência e mostrou correlaçäo quando em conjunto com o teste de produçäo de enzimas

Tipagem de amostras de P. aeruginosa do orofaringe de pessoal do CTI / Typing of P. aeruginosa strains from oropharyngeal of personal of ICV

Foram isolados bastonetes de P.aeruginosa do orofaringe de 13(26%) indivíduos estudados. O mais freqüente piocinotipo foi o 624271. A piocinotipagem sucessiva destes bastonetes permitiu classificá-los como flora transitória do orofaringe destes indivíduos