In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases

The correct diagnosis of renal allograft rejection may be difficult using only clinical and/or histopathological criteria. Immunological assays should be considered in order to evaluate the phenotype of inflammatory infiltrate in renal allograft biopsies. Immunohistochemical studies were performed to detect mononuclear cells, CD4 and CD8 T lymphocytes, B lymphocytes, macrophages, null cells, and positive cells for interleukin-2 receptors. A total of 41 allograft biopsies classified into three groups were studied: acute cellular rejection (28 biopsies/22 patients), borderline (7 biopsies/5 patients) and control (6 biopsies/6 patients). In the rejection group (RG), increased cellularity was found mainly at the tubulo-interstitial level. Expression of CD8 positive cells was higher in RG when compared to borderline (BG) and control (CG) groups, respectively (0.9 vs. 0.0 vs. 0.35 cells/mm2; p < 0.001). Expression of macrophages was not statistically significant among the three groups (RG = 0.6 vs. BG = 0.2 vs. CG = 0.0 cells/mm2; p < 0.02). In the BG, CD4 + cells predominated (BG = 0.2 vs. RG = 0.05 vs. CG = 0.0 cells/mm2; p < 0.05). Clinically these patients were treated as cases of acute rejection. The numbers and different types of infiltrating cells did not correlate with patient's clinical outcome. Copyright © Informa Healthcare.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP > AP > DP > VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma. © 2006 International Federation for Cell Biology.

Paracoccidioidomycosis: No genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay

Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis. As infectious diseases can cause DNA damage, the authors aimed at analyzing DNA breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. The results suggested that paracoccidioidomycosis does not cause genotoxicity.

Changes in blood parameters of hybrid tambacu fish parasitized by Dolops carvalhoi (Crustacea, Branchiura), a fish louse

Measurement of blood parameters has been used for many years as a tool for monitoring the health offish. The present study investigated the effects of a natural infestation of Dolops carvalhoi Lemos de Castro, 1949, on red blood cells, thrombocytes and white blood cell counts, as well as plasma glucose and serum electrolyte levels in hybrid tambacu (Piaractus mesopotamicus × Colossoma macropomum). Parasitized fish showed low haematocrit and magnesium levels and increases in MCHC, plasma glucose levels, serum protein, sodium and chloride levels, number of monocytes and PAS-positive granular leukocytes (PAS-GL), when compared with values in control fish. This study is the first to report changes in fish physiology caused by D. carvalhoi infestation, and the results obtained indicate that a mild infection can lead to important osmoregulatory disturbances in hosts.

Influence of sex, age, and fasting on blood parameters and body, bursa, spleen and yolk sac weights of broiler chicks

The effects of water and feed fasting for 24, 48 and 72 hours post-hatching on blood parameters (mean corpuscular volume, MCV; red blood-cell, RBC; hematocrit, HCT; hemoglobin, HGB; plasma glucose, CGP; plasma total protein, PP, and differential leukocytes count), and on body, liver, spleen, bursa, and yolk sac weights were analyzed. Erythrogram data were obtained with a blood cell counter. Total plasma protein and plasma glucose were determined by using the Bradford method (1976) and a glucose PAP liquiform kit (Labtest, cat. n. 84), respectively. Specific leukocyte counts were carried out on blood smears stained with Rosenfeld solution. According to the obtained data, water and feed post-hatching fasting reduced MCV values, which also were lower in males than that in females. Fasting for 48 hours promoted an increase in PP, while fasting for 72 hours reduced HCT. Chicks submitted to fasting presented lower body weights as compared to fed chicks, but their liver weight did not increase between 48 and 72 hours of age. Fasting decreased spleen weight, but bursa and yolk sac weight were not affected. Data showed that female and male chicks react in a similar way to post-hatching fasting, which affects body weight, liver and spleen weight, and HCT and PP values. Moreover, 72 hours of fasting affected more intensely HCT and MCV values.

Avoiding leukocyte contamination and early platelet activation in platelet-rich plasma.

The objective of this study was to describe a new platelet-rich plasma (PRP) protocol with a reduced concentration of leukocytes and intact platelets. We collected 8 mL of venous blood (VB) from marginal ear veins of 10 male New Zealand white rabbits in acid dextrose citrate Vacutainer tubes. Tubes were centrifuged at 302g for 10 minutes. All plasma was collected in plastic tubes to avoid buffy-coat contamination and centrifuged at 2862g for 5 minutes. A 10% calcium chloride activator (10 PRP:2 CaCl2) was added to the lower third of this plasma (PRP), and the PRP gel was obtained. Mean platelet count was 317.7 x 10(3) +/- 39.9/microL in VB and 1344.9 x 10(3) +/- 347.5/microL in PRP. Leukocyte counts were 3.96 x 10(3) +/- 2.01/microL and 0.46 x 10(3) +/- 0.45/microL in VB and PRP, respectively. Mean platelet enrichment was 327.4 +/- 97.8%. All differences were statistically significant (P > .05). This protocol is practical and reproducible, resulting in a high concentration of intact platelets to help tissue repair and low levels of leukocytes.

Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - Part I: Clinical evaluation, hematology and myelogram

The present study aimed at evaluating clinical and laboratory aspects during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. The clinical signs of ataxia, sedation, flaccid paralysis, mydriasis, eyeball paralysis, mandible ptosis, sialorrhea, vomiting and diarrhea observed in the dogs were very similar to those observed in humans. The decrease in hemoglobin, hematocrit, erythrocyte, platelet and fibrinogen levels, prolongation of clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT), as well as hypocellularity in the bone marrow characterized anemia, thrombocytopenia and blood incoagulability, as well as hypofibrinogenemia and decreased bone-marrow activity. Important bleeding was not observed. Increased numbers of leukocytes and neutrophils and decreased numbers of lymphocytes and eosinophils characterized an acute inflammatory response and stress caused by generalized pain. The employed antiophidic serum was effective and all animals survived.

Metodologia para quantificação de leucóciotos totais em peixe Oreochromis Niloticus

Haematological investigation is an important part of disease diagnosis. The techniques used for mammals are generally applicable for fishes with slight modification. The presence of nucleated erythrocytes and thrombocytes in fish may cause some confusion in the identification of blood cells, mainly in total leukocytes count. This work evaluated two different methods (direct and indirect) of leukocytes counting. Specimens of tilapia were divided randomly into four groups of 48 fish each. Each group was further randomized into three replicate experiments of 16 fish per replicate in 40 L aquarium and maintained for 10 days. Counting of leukocytes in peripheral blood was determined in six fishes per group at the times: 0, 3, 7 and 10 days. The direct method in Neubauer chamber showed a large dispersion of data in regard to the average, the opposite was observed in indirect method determined in blood smears that showed more homogeneity among groups. Due to this factor the indirect method can be considered more accurate than the direct method for counting leukocytes in fishes.

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.

Tratamento hepatoprotetor favorece a resposta leucocitária de ratos wistar intoxicados por CCL4

The aims of this investigation was to evaluate the effect of hepatoprotective treatments with a compound prepared by the association of N-Acetyl DL-Methionine (5%) + Choline chloride (2%) + Caffeine (1%) + Thiamine hydrochloride (1%) + Nicotinamida (0,5%)+ Pyridoxine hydrochloride (0.04%), administered through intramuscular (IM) route, at doses of 0.2, 0.6 and 1.0 mL/kg of BW, through the study of leukocytes responses in rats submitted to acute intoxication with CCl4. 147 females were randomized into 21 groups, performing five different treatments, which were evaluated seven animals in four periods: two, four, six and eight days after CCl4-induced intoxication. In this study, it was observed absolute eosinophilia and monocytosis in animals untreated and treated with the lowest dose of 0.2 mL. These responses were significantly better in animals treated with 0.6 and 1.0 mL/ kg BW. The untreated animals showed thrombocytopenia, when compared to treated animals. Absolute neutropenia and lymphocytosis was observed in all rats intoxicated with CCl4, there is no difference among treatments. The analysis of white blood cells demonstrated that the hepatoprotective treatments favored the leukocyte response, by act beneficially on the population of these cells, supporting the hypothesis that these events may reduce the deleterious effects in liver tissue after intoxication by CCl4.

Diferentes fontes proteicas em rações de leitões sobre atividade da tripsina e parâmetros sangüíneos

Were used 64 piglets submitted eight treatments: ration with skim milk (SM), three rations with crescent levels of swine plasma (SP), three rations with whole egg (WE) and a ration with high inclusion of soybean meal (SB). Were monitored the blood parameters (BP) in pigs at 27 and at 34 days of age. The piglets were slaugther at 28 and at 35 days of age, for collections pancreas and posterior mensurements of absolut (AW) and relative weigth (RW) of pancreas and trypsin activity (TA). Treatments not influencied AW and TA. Significant effect of the crescent levels was verified of SP, with lineal reduction of the leukocytes and increase of the globular volume, to the 27 days; while to the 34 days, lineal increase of the hematias was observed. At 27 days, animals feds rations with crescent levels of SP have inferior percentage of eosinophils than others that consumed crescent levels of WE. The utilization of SP promoted smaller stimulus to the immune reply, while the use of WE promoted larger humoral reply of the piglets.

Female sex hormones mediate the allergic lung reaction by regulating the release of inflammatory mediators and the expression of lung E-selectin in rats

Background: Fluctuations of estradiol and progesterone levels caused by the menstrual cycle worsen asthma symptoms. Conflicting data are reported in literature regarding pro and anti-inflammatory properties of estradiol and progesterone.Methods: Female Wistar rats were ovalbumin (OVA) sensitized 1 day after resection of the ovaries (OVx). Control group consisted of sensitized-rats with intact ovaries (Sham-OVx). Allergic challenge was performed by aerosol (OVA 1%, 15 min) two weeks later. Twenty four hours after challenge, BAL, bone marrow and total blood cells were counted. Lung tissues were used as explants, for expontaneous cytokine secretion in vitro or for immunostaining of E-selectin.Results: We observed an exacerbated cell recruitment into the lungs of OVx rats, reduced blood leukocytes counting and increased the number of bone marrow cells. Estradiol-treated OVx allergic rats reduced, and those treated with progesterone increased, respectively, the number of cells in the BAL and bone marrow. Lungs of OVx allergic rats significantly increased the E-selectin expression, an effect prevented by estradiol but not by progesterone treatment. Systemically, estradiol treatment increased the number of peripheral blood leukocytes in OVx allergic rats when compared to non treated-OVx allergic rats. Cultured-BAL cells of OVx allergic rats released elevated amounts of LTB4 and nitrites while bone marrow cells increased the release of TNF-α and nitrites. Estradiol treatment of OVx allergic rats was associated with a decreased release of TNF-α, IL-10, LTB4 and nitrites by bone marrow cells incubates. In contrast, estradiol caused an increase in IL-10 and NO release by cultured-BAL cells. Progesterone significantly increased TNF- α by cultured BAL cells and bone marrow cells.Conclusions: Data presented here suggest that upon hormonal oscillations the immune sensitization might trigger an allergic lung inflammation whose phenotype is under control of estradiol. Our data could contribute to the understanding of the protective role of estradiol in some cases of asthma symptoms in fertile ans post-menopausal women clinically observed. © 2010 de Oliveira et al; licensee BioMed Central Ltd.

Bactérias e resposta inflamatória no útero de cadelas, após inseminação artificial

The present study aimed to obtain information about the uterine inflammatory response (number of polymorphonuclear neutrophilic granulocytes - PMNs) in bitches after artificial insemination (AI) and identify the uterine microflora present after the following treatments: insemination using semen with extender (n=6), insemination with fresh semen (n=6) and no inseminated (n=6). The percentage of PMNs on the endometrial surface and within histological sections was evaluated together with the presence of aerobic bacteria in the uterine lumen. For endometrial cytology, there was no significative difference on the number of inflammatory cells between bitches not inseminated (3.05 ± 1.74 PMNs) and those inseminated with fresh semen (3.55 ± 1.51 PMNs); There was a significative difference in both groups compared to the inseminated with semen plus extender (7.80 ± 1.67 PMNs) (p<0.05). Histology showed that there was no significative difference on the number of inflammatory cells between bitches not inseminated (87.72 ± 35.2 PMNs) and those inseminated with fresh semen (122.97 ± 43.31 PMNs); however, it was observed differences in both groups compared to those inseminated with semen plus extender (171.94 ± 42.74 PMNs) (p<0.05). Eight animals, randomly distributed in the groups, showed the presence of Staphylococcus sp and Proteus sp., in the microbiological exam. The extender for semen, with Tris, is a potent inducer of uterine inflammation, and positive uterine cultures may be obtained during estrus without inflammation or uterine infection.

Mast cells modulate the inflammatory process in endotoxin-induced uveitis

Purpose: To investigate the role of mast cells and annexin-A1 (Anxa1) in endotoxin-induced uveitis (EIU). Methods: EIU was induced by injection of lipopolysaccharide (LPS) into the paws of rats, which were then sacrificed after 24 and 48 h. To assess EIU in the absence of mast cells, groups of animals were pretreated with compound 48/80 (c48/80) and sacrificed after 24 h after no treatment or EIU induction. The eyes were used for histological studies and the aqueous humor (AqH) pool was used for the analysis of transmigrated cells and Anxa1 levels. In inflammatory cells, Anxa1 expression was monitored by immunohistochemistry. Results: After 24 h, rats with EIU exhibited degranulated mast cells, associated with elevated numbers of infiltrating leukocytes and the high expression of Anxa1 in the AqH and the neutrophils. After 48 h of EIU, the mast cells were intact, indicating granule re-synthesis, and there was a reduction of neutrophil transmigration and an increase in the number of mononuclear phagocytic cells in ocular tissues. Anxa1 expression was decreased in neutrophils but increased in mononuclear phagocytic cells. In the animals pretreated with c48/80 and subjected to EIU, mast cells responded to this secretagogue by degranulating and few transmigrated neutrophils were observed. Conclustions: We report that mast cells are a potential source of pharmacological mediators that are strongly linked to the pathophysiology of EIU, and the endogenous protein Anxa1 is a mediator in the homeostasis of the inflammatory process with anti-migratory effects on leukocytes, which supports further studies of this protein as an innovative therapy for uveitis. © 2011 Molecular Vision.

Determinação da toxicidade do selênio por meio de análises hematológicas em Oreochromis niloticus

Selenium (Se) is described as an essential micronutrient and participates in different biological functions, as the antioxidant defense systems maintenance and regulation. However, when in high concentrations, Se may cause toxic effects as well as hematological changes in fish. The aim of the present study was to determine the toxicity of selenium in the form of sodium selenate (Na 2Se 6+O 4) in Oreochromis niloticus based on hematological parameters, after exposure to different concentrations (0.01, 0.14 and 1.4 mg Se 6+ L -1). The erythrocytic and leukocytic series were examined over 14 days at intervals of 0, 3, 5, 7,10 and 14 days. The erythrocytic series showed significant alterations in the first 7 days, including the control group. Neutrophils and monocytes showed variations in the first 3 days at a concentration of 1.40 mgSe 6+ L -1 characterizing an acute response. The total number of leukocytes was different in relation to time zero on all Se concentrations. The thrombocyte count also differed statistically from time zero and control in the first 3 days at 0.14 mgSe 6+ L -1. These results indicate that different concentrations induce an acute response with diminution of total leukocytes, neutrophilia, monocytosis and thrombocytosis.