Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined ill 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values. (C) 2011 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: Role of interleukin-8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production. © 2008 The Authors

Viability and cell cycle analysis of equine fibroblasts cultured in vitro

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO2 until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD®) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% × 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawin

Role of TLR2 and TLR4 in Human Neutrophil Functions Against Paracoccidioides brasiliensis

In paracoccidioidomycosis, a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), studies have focused on the role of neutrophils that are involved in primary response to the fungus. Neutrophil functions are regulated by pro- and anti-inflammatory cytokines. The molecular mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of toll-like receptors (TLR). We aimed at evaluating TLR2 and TLR4 expression on human neutrophils activated with GM-CSF, IL-15, TNF-alpha or IFN-gamma and challenged with a virulent strain of P. brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H(2)O(2) and IL-6, IL-8, TNF-alpha and IL-10 production by activated and challenged cells. All cytokines increased TLR2 and TLR4 expression. Pb18 also increased TLR2 expression inducing an additional effect to that of cytokines. on the contrary, it inhibited TLR4 expression. All cytokines increased neutrophil fungicidal activity and H(2)O(2) production, but this process was not associated with TLR2 or TLR4. Neutrophils activation with GM-CSF and TNF-alpha resulted in a significative increase in IL-8 production, while IL-15 and IFN-gamma have no effect. Pb18 alone also increased IL-8 production. None of the cytokines activated neutrophils for IL-10 release. This cytokine was only detected after Pb18 challenge. Interestingly, IL-8 and IL-10 production involved TLR2 and mainly TLR4 modulation. Our data suggest that Pb18 uses TLR4 to gain access to human neutrophils. This interaction results in IL-8 and IL-10 production that may be considered as a pathogenic mechanism in paracoccidioidomycosis.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Avaliação das subclasses IgG1 e IgG3 na doença hemolítica perinatal

A doença hemolítica perinatal (DHPN) ainda é um problema clínico. Nenhum teste isolado prediz, com segurança, a gravidade do quadro hemolítico. O objetivo do presente estudo foi determinar as subclasses de anticorpos IgG1 e IgG3 por citometria de fluxo no soro de 42 gestantes isoimunizadas e correlacionar os dados obtidos com a gravidade da DHPN. A distribuição dos fetos ou neonatos segundo a gravidade do quadro hemolítico evidenciou 13 casos com doença leve, 16 casos com doença moderada e 13 com doença grave. As subclasses foram detectadas em 33/42 (79%) amostras. A subclasse IgG1, isoladamente, foi evidenciada em 14/33 (42,4%) casos. Na relação entre gravidade da doença e subclasses de IgG, observou-se que IgG1 isolada foi encontrada em todos os grupos, e os valores da mediana de intensidade de fluorescência (MIF) foram significativamente mais altos nas formas mais graves da DHPN (p<0,01). Contrariamente, os valores da MIF para IgG3 se apresentaram mais homogêneos em todas as categorias (p=0,11). A presença de IgG3 parece, portanto, estar mais associada à hemólise leve. A associação das subclasses IgG1 e IgG3 está relacionada à situação clínica mais grave, o que se deve, possivelmente, à presença de IgG1 associada. Apesar dos altos valores para IgG1 e a associação de IgG1 com IgG3 indicarem maior gravidade da DHPN, sugere-se que outras variáveis sejam analisadas conjuntamente, uma vez que os relatos existentes na literatura, até o momento, não dão suporte para seu uso como instrumento exclusivo de avaliação de gravidade e prognóstico da doença.The hemolytic disease of the newborn (HDN) continues to be a clinical problem in spite of prophylaxis. To date, none of the available tests, developed to predict the severity of HDN, has provided complete reliability. The objective of the present study was to determine the IgG1 and IgG3 subclasses in 42 isoimmunized pregnant women, and to correlate them with clinical severity of hemolytic disease. The IgG subclasses were determined employing flow cytometry. According to the clinical severity of HDN, fetuses and newborn babies were classified as 13 mild, 16 moderate and 13 severe cases. The IgG subclasses were detected in 33 of the 42 pregnant women. of these, IgG1 was predominant in 72.7% of the cases; either isolated (42.4%) or in association with IgG3 (30.3%). IgG1 was present in all the three clinical severity categories, however, its values were significantly higher in cases with greater clinical severity of HDN (p<0.01). on the other hand, the distribution of IgG3 values within each group was not statistically significant (p=0.11). IgG3 seems to be more associated with the mild hemolytic form of the disease, whereas the association of IgG1 and IgG3 suggested a clinically more severe form of HDN. It is possible, however, that the severity in these cases is related to the presence of IgG1. These results suggest that IgG1 and IgG3 isotypes should be included in multi-parametric protocols for the evaluation of clinical severity of HDN, as International literature does not give support to the use of IgG subclass determination alone as a reliable indicator to predict severity or prognosis of the disease

Relationship among Short and Long Term of Hypoinsulinemia-Hyperglycemia, Dermatophytosis, and Immunobiology of Mononuclear Phagocytes

Dermatophytes are fungi responsible for causing superficial infections. In patients with diabetes mellitus (DM), dermatophytosis is usually more severe and recurrent. In the present study, we aimed to investigate the influence of short and long term hypoinsulinemia-hyperglycemia (HH) during experimental infection by Trichophyton mentagrophytes as well as alterations in the mononuclear phagocytes. Our results showed two distinct profiles of fungal outcome and immune response. Short term HH induced a discrete impaired proinflammatory response by peritoneal adherent cells (PAC) and a delayed fungal clearance. Moreover, long term HH mice showed low and persistent fungal load and a marked reduction in the production of TNF-α by PAC. Furthermore, while the inoculation of TM in non-HH mice triggered high influx of Gr1+ monocytes into the peripheral blood, long term HH mice showed low percentage of these cells. Thus, our results demonstrate that the time of exposure of HH interferes with the TM infection outcome as well as the immunobiology of mononuclear phagocytes, including fresh monocyte recruitment from bone marrow and PAC activity