Padronização de técnica manual para obtenção de plasma rico em plaquetas de bovino

To evaluate the biochemical profile and protein concentration of whey from milk samples of healthy Murrah primiparous and pluriparous buffaloes, 30 female buffaloes were analyzed during a complete lactation. The animals were divided into three groups: G1 = 10 primiparous buffaloes, G2 = 10 pluriparous buffaloes with 2-3 lactations and G3 = 10 pluriparous buffaloes with > 3 lactations. The lactation period was divided into: early stage (I: 1-3 months of lactation), intermediate stage (T: 4-6 months of lactation) and final stage (F: 7-9 months of lactation). Before milk sampling, physical examination of the mammary gland, strip cup test and California Mastitis Test (CMT) were performed. After mammary quarters asepsis, 20mL of milk were collected monthly from each mammary quarter, during a complete lactation, in sterilized plastic bottles without preservative, in order to perform microbiological isolation, biochemical profile and protein electrophoresis in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 30mL of milk from each mammary quarter were collect, in sterilized plastic bottles containing preservative bronopol to perform the somatic cell count (SCC). A total of 1,042 milk samples were collected from the experimental groups during lactation, of which 923 samples showed negative reaction to CMT and negative microbiological isolation and were selected to biochemical profile analysis and protein electrophoresis in SDS-PAGE. There were influence of parity order and stage of lactation in biochemical profile and protein concentration of healthy Murrah buffaloes'whey. Primiparous buffaloes (G1) showed higher gamma-glutamyltransferase (GGT: 2,346 U/L), alkaline phosphatase (ALP: 181 U/L), phosphorus (P; 56.6mg/dL), potassium (K; 32.0mg/dL) and alpha-lactalbumin (458mg/dL). Buffaloes with 2-3 lactations (G2) showed higher SCC (70,700 cells/mL) and higher concentrations of total protein (1.55g/dL), albumin (100mg/dL), magnesium (Mg; 8.80mg/dL), chlorides (Cl; 176mg/dL), iron (Fe; 10.7 mu g/dL), sodium (Na; 178mMol/L) and lactoferrin (59.5mg/dL). Bufalloes with > 3 lactations (G3) showed higher concentrations of total calcium (Ca; 41.8mg/dL), ionized calcium (iCa; 2.92mMol/L), immunoglobulin A (IgA; 1.32mg/dL), serum albumin (99.1mg/dL), immunoglobulin G (IgG; 49.7mg/dL) and beta-lactoglobulin (1,068mg/dL). During lactation it was observed increase in SCC, GGT, ALP, total protein, albumin, P, Mg, Cl, Na, lactoferrin, serum albumin, IgG and alpha-lactalbumin, as well as decrease in concentrations of Ca, Fe, iCa, K, IgA and beta-lactoglobulin in buffaloes'whey. The results may be used as reference for buffaloes and to support diagnosis and prognosis of diseases common to lactation periods.

Hydroxyapatite coating deposited on grade 4 titanium by plasma electrolytic oxidation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Incorporation of Ca, P, and Si on bioactive coatings produced by plasma electrolytic oxidation: The role of electrolyte concentration and treatment duration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Low-temperature plasma: an effective approach against Candida albicans biofilm

This study evaluated the antifungal potential of low-temperature plasma (LTP) on a 72-hour Candida albicans biofilm. A growth inhibition zone test was conducted with agar plates inoculated with C. albicans and submitted to LTP and argon application at 3 and 10 mm for 10, 30, 60, 90, and 120 seconds. The groups for biofilm assays were 60 seconds of LTP application with a tip-to-sample distance of 3 mm (LTP-3) and 10 mm (LTP-10); –application of only argon gas for 60 seconds with a tip-to-sample distance of 3 mm (Ar-3) and 10 mm (Ar-10); and no treatment. The C. albicans biofilm was grown on saliva-coated discs. The medium was replaced every 24 hours. Confocal laser scanning microscopy revealed the proportion of live and dead cells, and variable pressure scanning electron microscopy (VPSEM) showed biofilm/cell structure. No inhibition zone was observed for control and either Ar groups. For the LTP groups, a progressively increasing of inhibition zone diameter was observed for different treatment durations. The LTP-3 and LTP-10 groups presented higher proportions of dead cells compared with the Ar-3 and Ar-10 groups. VPSEM revealed cell perforations in the LTP-3 and LTP-10 groups. A short period of LTP exposure demonstrated an antifungal effect on C. albicans biofilm.

Lesão condral do joelho: comparação entre ressonância magnética e vídeo-artroscopia. Efeito da aplicação do plasma rico em plaquetas

Pós-graduação em Bases Gerais da Cirurgia - FMB

Comparison of apoptotic cells between cryopreserved ejaculated sperm and epididymal sperm in stallions

The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5 C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5 C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computerassisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.

Avaliação da bioatividade de revestimentos produzidos sobre Tântalo por oxidação eletrolítica assistida por plasma

Pós-graduação em Ciência e Tecnologia de Materiais - FC

Efeito do filme de carbono DLC aplicado por plasma como recobrimento em Polietileno de Ultra Densidade e Alto Peso Molecular (UHWPE) e planejamento virtual utilizando a técnica de elementos finitos em cirúrgias de próteses totais da ATM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da bioatividade de revestimentos produzidos sobre Tântalo por oxidação eletrolítica assistida por plasma

Pós-graduação em Ciência e Tecnologia de Materiais - FC

Efeito do filme de carbono DLC aplicado por plasma como recobrimento em Polietileno de Ultra Densidade e Alto Peso Molecular (UHWPE) e planejamento virtual utilizando a técnica de elementos finitos em cirúrgias de próteses totais da ATM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

IGFBP7 participates in the reciprocal interaction between acute lymphoblastic leukemia and BM stromal cells and in leukemia resistance to asparaginase

The interaction of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells (BMSCs) has a positive impact on ALL resistance to chemotherapy. We investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL cells upon coculture with BMSCs. Coculture with stromal cells resulted in increased insulin-like growth factor (IGF)-binding protein 7 (IGFBP7) expression by ALL cells. Assays with IGFBP7 knockdown ALL and stromal cell lines, or with addition of recombinant rIGFBP7 (rIGFBP7) to the culture medium, showed that IGFBP7 acts as a positive regulator of ALL and stromal cells growth, and significantly enhances in-vitro resistance of ALL to asparaginase. In these assays, IGFBP7 function occurred mainly in an insulin-and stromal-dependent manner. ALL cells were found to contribute substantially to extracellular IGFBP7 levels in the conditioned coculture medium. Diagnostic BM plasma from children with ALL had higher levels of IGFBP7 than controls. IGFBP7, in an insulin/IGF-dependent manner, enhanced asparagine synthetase expression and asparagine secretion by BMSCs, thus providing a stromal-dependent mechanism by which IGFBP7 protects ALL cells against asparaginase in this coculture system. Importantly, higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (Cox regression model, P = 0.003) in precursor B-cell Ph(-) ALL patients (n = 147) treated with a contemporary polychemotherapy protocol.

Blood transfusions increase circulating plasma free hemoglobin levels and plasma nitric oxide consumption: a prospective observational pilot study

Introduction: The increasing number of reports on the relation between transfusion of stored red blood cells (RBCs) and adverse patient outcome has sparked an intense debate on the benefits and risks of blood transfusions. Meanwhile, the pathophysiological mechanisms underlying this postulated relation remain unclear. The development of hemolysis during storage might contribute to this mechanism by release of free hemoglobin (fHb), a potent nitric oxide (NO) scavenger, which may impair vasodilation and microcirculatory perfusion after transfusion. The objective of this prospective observational pilot study was to establish whether RBC transfusion results in increased circulating fHb levels and plasma NO consumption. In addition, the relation between increased fHb values and circulating haptoglobin, its natural scavenger, was studied. Methods: Thirty patients electively received 1 stored packed RBC unit (n = 8) or 2 stored packed RBC units (n = 22). Blood samples were drawn to analyze plasma levels of fHb, haptoglobin, and NO consumption prior to transfusion, and 15, 30, 60 and 120 minutes and 24 hours after transfusion. Differences were compared using Pearson's chi-square test or Fisher's exact test for dichotomous variables, or an independent-sample t test or Mann-Whitney U test for continuous data. Continuous, multiple-timepoint data were analyzed using repeated one-way analysis of variance or the Kruskall-Wallis test. Correlations were analyzed using Spearman or Pearson correlation. Results: Storage duration correlated significantly with fHb concentrations and NO consumption within the storage medium (r = 0.51, P < 0.001 and r = 0.62, P = 0.002). fHb also significantly correlated with NO consumption directly (r = 0.61, P = 0.002). Transfusion of 2 RBC units significantly increased circulating fHb and NO consumption in the recipient (P < 0.001 and P < 0.05, respectively), in contrast to transfusion of 1 stored RBC unit. Storage duration of the blood products did not correlate with changes in fHb and NO consumption in the recipient. In contrast, pre-transfusion recipient plasma haptoglobin levels inversely influenced post-transfusion fHb concentrations. Conclusion: These data suggest that RBC transfusion can significantly increase post-transfusion plasma fHb levels and plasma NO consumption in the recipient. This finding may contribute to the potential pathophysiological mechanism underlying the much-discussed adverse relation between blood transfusions and patient outcome. This observation may be of particular importance for patients with substantial transfusion requirements.

Detection of human T-cell lymphotropic virus type 1 in plasma samples

Human T-cell lymphotropic virus type 1 (HTLV-1) is-an RNA virus responsible for diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATL). Cell-to-cell contact and Tax-induced clonal expansion of infected cells are the main modes of virus replication, making virus detection during the viremic stage difficult. Consequently, the proviral load is the current virologic marker for disease monitoring, but the mechanisms of progression have not been established yet. Thus, this study investigated the presence of virus in plasma from asymptomatic HTLV-1 carriers and from HAM/TSP patients. Real-time PCR was performed on DNA from 150 plasma samples; 12(8%) had detectable DNA amplification, including 6(4%) asymptomatic HTLV-1 carriers and 14(26%) HAM/TSP patients (p < 0.005). Of the 33 samples submitted for nested PCR, six (18%, p = 0.02) were positive for HTLV-1 RNA in the plasma. Additionally, 26 plasma samples were treated with DNAse enzyme to eliminate any DNA contamination before RNA extraction. Two of them (8%) showed amplification for HTLV-1 (p = 0.5). Therefore, this study described for the first time the detection of free HTLV-1 RNA in plasma from HTLV-1-infected subjects, regardless of their clinical status. Thus, HTLV-1 viral replication does occur in plasma, and other transmission pathways for HTLV-1 should be investigated further. (C) 2011 Elsevier B.V. All rights reserved.

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an "immune exhaustion'', with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28(-)CD57(+)CD8(+) T cells between the groups. However, the frequency of Tim-3(+)CD8(+) and Tim-3(+)CD4(+) exhausted T cells, but not PD-1(+) T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1(+)CD8(+) T cells were directly associated with T cell immune activation in children. The frequency of Tim-3(+)CD8(+) T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.

Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

Background: Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert) as therapeutic agent requires further investigation. Results: Here, we showed that plasmid DNA (pcDNA3) at low doses inhibits the production of IL-6 and TNF-alpha by lipopolysaccharide (LPS)-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 mu g of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP) drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP), a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO) production. Conclusion: Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.