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.

Systemic leukopenia, evaluation of laminar leukocyte infiltration and laminar lesions in horses with naturally occurring colic syndrome

The present study was aimed at identifying laminar lesions and leukocyte infiltration in hoof laminar tissue of horses with colic syndrome and its correlation with the total leukocyte count before death. Six healthy horses were used as control group (CG), and eighteen horses with lethal gastrointestinal disease were divided into two groups: leukopenic group (LG) with seven leukopenic horses, and non-leukopenic group (NLG) with 11 horses with total leukocyte count within reference range for the species. Leukocyte infiltration was examined by immunohistochemistry. Laminar lesions were observed in both LG and NLG, with no differences in severity between them. LG showed increase of the leukocyte infiltration in the hoof laminar tissue, when compared to CG and NLG. Horses with severe colic syndrome (LG and NLG) developed intense laminar lesions without clinical signs of laminitis, with increased leukocyte infiltration. However, the LG demonstrated an even higher increase of leukocyte infiltration compared to both CG and NLG.

Systemic inflammatory response indicators in rabbits (Oryctolagus cuniculus) experimentally infected with sporulated oocysts of Eimeria stiedai (Apicomplexa: Eimeriidae)

Hemograms and acute-phase proteins in adult male New Zealand White rabbits that had been experimentally infected orally with sporulated oocysts of Eimeria stiedai were evaluated over a 28-day period. Fifty animals were used, divided into two groups: group A infected with 1 x 10(4) sporulated oocysts of E. stiedai and group B inoculated with distilled water. on the seventh day after infection, the infected animals presented anemia and leukocytosis with neutrophilia and monocytosis. Protein fractionation by means of electrophoresis identified 19 acute-phase proteins with molecular weights ranging from 24 to 238 kD. Ceruloplasmin, transferrin and haptoglobin showed high levels on the seventh day after infection, with gradual increases in their concentrations until the end of the experimental period. Thus, from the data of the present study, E. stiedai is considered to be a pyogenic etiological agent for which the infection level can be monitored through the leukocyte count and serum concentrations of ceruloplasmin, transferrin and haptoglobin, and these can be recommended as complementary tests.

Obstrução intestinal no idoso

RACIONAL: O envelhecimento da população é realidade sentida na vivência diária, levando à necessidade associada de prover cuidados médicos eletivos e de emergência a um número cada vez maior de idosos. OBJETIVO: Avaliar os aspectos clínicos, terapêuticos e evolutivos de pacientes idosos com obstrução intestinal atendidos no Hospital das Clínicas da Faculdade de Medicina de Botucatu - UNESP. MÉTODOS: Análise retrospectiva de pacientes internados no período de janeiro de 2002 a dezembro de 2006. Foram estudados 50 pacientes, 24 homens e 26 mulheres, com idade média de 74,7 ± 7,4 anos, analisando-se a diferenciação entre obstrução funcional e orgânica; a freqüência das causas obstrutivas; a mortalidade; a incidência dos sinais e sintomas; e correlação laboratorial. RESULTADOS: O quadro obstrutivo intestinal teve as seguintes causas: brida (38%), câncer de colon (24%), hérnias (12%), impactacão fecal (12%), carcinomatose (10%) e volvo da sigmóide (4%). A mortalidade foi de 18% associada à complicações infecciosas em todos os pacientes. CONCLUSÕES: a) A distinção entre a obstrução intestinal funcional e orgânica oferece dificuldade no idoso; b) o câncer do colon constitui-se em importante causa de obstrução intestinal com quadro obstrutivo já na primeira manifestação clínica desse tumor; c) bridas e hérnias são igualmente importantes na etiologia da obstrução intestinal; d) os sinais clínicos obstrutivos são poucos evidentes no idoso e a leucometria é mais fidedigna para este diagnóstico.

Monitoramento de parâmetros laboratoriais em gatos sem raça definida durante o período neonatal

O objetivo do presente estudo foi à monitoração dos parâmetros laboratoriais como hemograma, enzimas hepáticas alanina aminotransferase (ALT) e gama-glutamiltransferase (GGT), glicemia e proteinograma sérico, e avaliar o efeito da idade em gatos sem raça definida durante a fase neonatal. Vinte gatos machos e fêmeas foram utilizados a partir do terceiro dia de vida até o 38º dia de idade. As amostras de sangue foram colhidas semanalmente e as análises laboratoriais (hemograma, enzimas hepáticas, glicemia e proteinograma sérico) realizadas no 3º, 10º, 17º, 24º, 31º e 38º dia de idade. Os resultados exibiram efeito significativo da idade sobre a contagem total de eritrócitos, concentração de hemoglobina, volume globular, volume corpuscular médio, concentração de hemoglobina corpuscular média, leucócitos totais, neutrófilos, eosinófilos e basófilos. Nenhum efeito foi observado em células como linfócitos, monócitos ou na concentração sérica de glicose. A análise das modificações ocorridas nos parâmetros laboratoriais durante a fase neonatal reflete o desenvolvimento fisiológico do filhote e contribui para o conhecimento do processo adaptativo em gatos neonatos durante o primeiro mês de vida, sendo útil para a avaliação clínica, diagnóstico e tratamento das doenças neonatais.

Efeito do sítio de venopunção nos parâmetros hematológicos em tigre-d'água-americano, Trachemys scripta elegans

O objetivo do presente estudo foi realizar a comparação entre dois sítios de coleta sanguínea em 24 exemplares de tigre-d'água-americano (Trachemys scripta elegans) oriundos de um criadouro comercial, localizado no município de Antonina, litoral do Paraná, Brasil. Os animais foram submetidos a contenção física e as venopunções foram realizadas no seio supraocciptal e na veia coccígea dorsal. As amostras heparinizadas foram identificadas e refrigeradas para posterior análise laboratorial. A contagem total de eritrócitos e leucócitos foi realizada pela técnica de hemocitometria. O hematócrito (Ht) e a hemoglobina (Hb) foram determinados pelo método de microhematócrito e cianometahemoglobina, respectivamente. A proteína plasmática total (PPT) foi determinada por refratometria e a contagem diferencial de leucócitos foi realizada através da técnica de Shilling. Houve diferença significativa no número de leucócitos e no valor da proteína plasmática total, e em ambos os casos os valores encontrados nas amostras provenientes da veia coccígea dorsal foram inferiores. A diferença encontrada no número de leucócitos provavelmente foi devido à contaminação por linfa, que também justifica o menor valor na concentração da proteína plasmática total. Conclui-se que é mais indicada a venopunção no seio supraocciptal quando comparado a veia coccígea dorsal.

The influence of leukocytospermia on the outcomes of assisted reproductive technology

Background: It is not well established whether the increased number of leukocytes in the seminal fluid impairs the outcomes of assisted reproductive technology (ART). This investigation analysed the outcomes of the intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) cycles in couples in which the male partner exhibited leukocytospermia.Methods: A total of 100 cycles in 100 couples were included in this study. For the ICSI or IMSI procedures, the patients were divided into two groups according to the presence or absence of leukocytospermia and then matched by (female) age:- ICSI: Group I (n = 25): Leukocytospermia - semen samples with a leukocyte count of greater than or equal to 1 x 10(6)/mL; and Group II (n = 25): Non-leukocytospermia - semen samples with a leukocyte count < 1 x 10(6)/mL.- IMSI: Group I (n = 25): Leukocytospermia; and Group II (n = 25): Non-leukocytospermia.The endpoints included the rates of fertilisation, implantation, clinical pregnancy, miscarriage, ongoing pregnancy and live birth. Student's t-tests, Mann-Whitney tests and Chi-square tests were performed, and P < 0.05 was considered significant.Results: The data from the ICSI groups showed that leukocytospermia did not have a negative influence on the rates of fertilisation (Group I: 57.9+/-30.2%, Group II: 61.9+/-27.7%; P = 0.74), implantation (Group I: 12.3%; Group II: 13.5%; P = 0.93), clinical pregnancy (Group I: 24%; Group II: 24%; P = 1.0), miscarriage ( Group I: 0, Group II: 0), ongoing pregnancy (Group I: 24%; Group II: 24%; P = 1.0), or live births (Group I: 24%; Group II: 24%; P = 1.0). Similarly, the data from the IMSI groups also showed that the leukocytospermia did not have a negative influence on the rates of fertilisation (Group I: 67.6+/-24.6%, Group II: 59.5+/-28.1%; P = 0.36), implantation (Group I: 17.5%; Group II: 16.7%; P = 0.90), clinical pregnancy (Group I: 28%; Group II: 24%; P = 1.0), miscarriage (Group I: 14.3%; Group II: 0; P = 0.33), ongoing pregnancy (Group I: 24%; Group II: 24%; P = 1.0), or live births (Group I: 24%, 6/25; Group II: 24%, 6/25; P = 1.0).Conclusions: The results indicate that the leukocytospermia may not have a negative effect on the outcomes of ICSI or IMSI cycles. Nevertheless, it seems that it is necessary to more precisely determine the effects, if any, of seminal leukocytes on fertilisation and implantation processes. Such efforts will help to establish a more reliable leukocyte threshold, which could eventually demonstrate whether there is a negative influence on the ART procedures.

Effects of Inhaled Nitric Oxide on Oxidative Stress and Histopathological and Inflammatory Lung Injury in a Saline-Lavaged Rabbit Model of Acute Lung Injury

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

Hematological changes in sheep inoculated with natural and Cobalt60-irradiated Crotalus durissus terrificus venom (Laurenti, 1768)

Natural (NV) and Cobalto60-irradiated (IrV) Crotalus durissus terrificus venom were used to evaluate serum production capacity of sheep and possible hematological and biochemical effects. Freeze-dried venom aliquots were diluted in acidified saline solution (NaCl 150 mM, pH 3.0) and irradiated by a Cobalt 60 source at a dose of 5.54 x 102 Gy/h and a concentration of 2.000 Gy. Twelve sheep were divided into two groups of six animals. One group received irradiated venom (IrV) and the other natural venom (NV). Three antigen doses (venom) were administered at monthly intervals. Blood samples were collected weekly for analysis of serum neutralization potency and capacity, complete blood count (CBC), total plasma protein, fibrinogen, albumin, and globulin. At the end of the experiment, the animals were challenged with a LD50 for sheep and showed no signs of envenoming. The two groups did not present clinical alterations. Results of the total leukocyte count did not present interaction or time factor effect for both groups, but there was a different action between them, with the NV group presenting more cells than the IrV group. The leukocyte increase to 13,000/ml indicates that slight leukocytosis occurred in the week after the first inoculation in the NV group. There was no statistically significant difference between groups in the absolute count of segmented neutrophils, eosinophils, and lymphocytes but there were statistically significant oscillations in values at the different collecting times. The NV group presented an increase in the absolute neutrophil count after the first inoculation that persisted for 5 weeks. In the IrV group, the increase in neutrophils occurred only in the first week returning to normal in the following weeks. The alterations in the neutrophil count are indicative of systemic inflammatory response related to cytokine release; response was more marked in the NV group, showing its greater toxicity.

Efeitos do treinamento físico de alta intensidade sobre os leucócitos de ratos diabéticos

Estudos têm demonstrado que o exercício físico regular melhora as condições do diabetes, facilitando a captação periférica da glicose e o metabolismo de glicogênio, proteínas, etc. Por outro lado, pouco se conhece sobre os efeitos do exercício intenso em diabéticos, principalmente com relação ao sistema imune desses organismos. O presente estudo teve como objetivo verificar os efeitos de um treinamento físico de alta intensidade sobre a contagem total e diferencial de leucócitos em ratos diabéticos. Ratos machos jovens Wistar foram distribuídos em quatro grupos: controle sedentário (CS), controle treinado (CT), diabético sedentário (DS) e diabético treinado (DT). O diabetes foi induzido por aloxana (35mg/kg de peso corporal). Durante seis semanas os animais dos grupos CT e DT realizaram um protocolo de treinamento físico, que consistiu na realização de quatro séries de 10 saltos (intercaladas por um minuto de intervalo) em piscina, com o nível da água correspondendo a 150% do comprimento corporal e sobrecarga equivalente a 50% da massa corporal dos animais. Ao final do período experimental, amostras de sangue foram coletadas para a contagem total e diferencial dos leucócitos. Os resultados foram avaliados estatisticamente por ANOVA com um nível de significância de 5%. A glicemia foi aumentada entre os diabéticos e a insulinemia diminuída. Não foram observadas diferenças significativas na contagem diferencial dos linfócitos, neutrófilos, eosinófilos e contagem total de leucócitos entre os grupos estudados. Houve aumento dos monócitos entre os treinados (CS = 10,0 ± 4,5, CT* = 25,4 ± 7,9, DS = 19,75 ± 7,4, DT* = 25,8 ± 4,4%). O peso relativo do timo foi reduzido pelo treinamento e pelo diabetes (CS = 125,0 ± 37,7, CT* = 74,6 ± 8,2, DS* = 47,5 ± 12,2, DT* = 40,1 ± 16,9mg/100g). Esses resultados permitem concluir que o treinamento físico de alta intensidade não alterou o estado geral do diabetes, mas aumentou os monócitos, o que pode representar um efeito positivo sobre a resposta imunológica desses animais.

Haematological alterations of Leporinus macrocephalus (Osteichtyes: Anostomidae) naturally infected by Goezia leporini (Nematoda: Anisakidae) in fish pond

Estudou-se efeito da infecção por Goezia leporini Martins & Yoshitoshi, 2003 (Nematoda: Anisakidae) sobre as características hematológicas de Leporinus macrocephalus (Osteichthyes:Anostomidae) cultivado. Palidez das brânquias, rins, fígado e coração, pontos negros nos rins e acúmulo de líquido na cavidade visceral, estômago e intestinos foram observados. O conteúdo da vesícula biliar tinha aparência pálida e translúcida. Observaram-se alta e moderada correlações positivas entre número de nematóides e peso do peixe estimadas dentro dos grupos de peixe de 0-100g e 100-200g, respectivamente. As extensões sangüíneas revelaram variações no tamanho (anisocitose) e forma (poiquilocitose) dos eritrócitos, bem como eritrócitos em divisão. Não houve alteração (P>0,05) na contagem total de eritrócitos, de leucócitos, na taxa de hemoglobina e nos percentuais de trombócitos e monócitos. A infecção provocou redução (P<0,05) no percentual de hematócrito, no volume corpuscular médio, na concentração de hemoglobina corpuscular média e no percentual de linfócitos, e aumento (P<0,05) no percentual de neutrófilos e eosinófilos no sangue circulante de peixes infectados. Este é o primeiro relato no Brasil que relaciona hematologia e infecção por nematóides em peixes cultivados.

Characterization and quantification of blood cells from the umbilical cord of dogs

Background: Models for the study of hematopoietic stem cells in dogs provide important information for bone marrow transplantation in humans. Recent studies have reported the importance of human umbilical cord blood (UCB) as an alternative to allogenic bone marrow for hematopoietic reconstitution. However, there are no studies on the UCB cells of dogs. Objective: the aim of this experiment was to characterize and quantify the blood cells of the umbilical cord of dogs. Methods: the blood of the umbilical cord of 20 neonatal dogs, delivered at term, with a median gestation time of 58 days, was collected with a 5-mL syringe containing EDTA. Total RBC, WBC, and platelet counts, HCT, hemoglobin (Hgb) concentration, and RBC indices were determined using an automatic cell counter. The differential leukocyte count was determined manually in blood smears stained with May-Grunwald-Giemsa. Reticulocyte percentages were determined on blood smears stained with brilliant cresyl blue and counterstained with May-Grunwald Giemsa. Results: the MCHC and numbers of RBCs, WBCs, neutrophils, and eosinophils in UCB were lower as compared with reference values for the peripheral blood of healthy neonatal and adult dogs; whereas, the MCV and reticulocyte percentages were higher. Conclusion: Erythrocyte macrocytosis and hypochromasia in UCB were consistent with marked reticulocytosis and indicative of high erythropoietic activity. The results of this study are an important first step in the characterization of UCB from neonatal dogs.

An animal model for the study of Chamomilla in stress and depression: pilot study

The behavioral and hematological effects of treatment with Chamomilla 6cH in mice subjected to experimental stress are described. Swiss mice were randomly divided into pairs, one animal was inoculated with Ehrlich's tumor, the other was treated daily with Chamomilla 6cH or control or received no treatment. After 7 days, the animals were observed in an open-field arena and blood samples taken. Mice who cohabitated with a sick cage-mate showed a decrease in their general activity, but those treated with Chamomilla 6cH were less severely affected (p = 0.0426). No hematologicall changes were observed.In a second experiment, the forced swimming test was applied to mice pre-treated with Chamomilla 6cH, controls were: water, 10% ethanol or amitriptyline. Only the amitriptyline and ethanol treated groups showed significant excitatory behavior (p = 0.0020), Chamomilla 6cH treated animals' scores intermediate between water control and ethanol or amitriptyline. A decrease in the leukocyte count was observed in the amitriptyline and Chamomilla 6cH treated groups (p = 0.039). These data suggest that treatment with Chamomilla 6cH is related to the recovery of basal behavioral conditions in mice subjected to stressful conditions.

Endogenous corticosteroids and insulin in acute inflammation

Carrageenin-induced inflammatory responses in the hindpaws of rats were quantitated by measuring: (1) alterations in volumes of the paws; and (2) alterations in concentration of dye, previously injected intravenously, which was recovered in perfusates from the paws. The inflammatory response in one paw was attenuated by previously inducing an inflammatory response in the contralateral paw. The effect was abolished by pretreatment with insulin. Indexes of adrenal activity were increased after the induction of the inflammatory response and they were not attenuated by pretreatment with insulin. Adrenal hyperactivity was characterized by increased serum corticosterone concentration, decreased adrenal ascorbic acid content, and reduced number of circulating eosinophils. It is concluded that inflammatory stimuli which lead to alterations in microvessels depend on a facilitatory effect of insulin. This effect is antagonized by glucocorticoids released in enhanced concentrations after the application of noxious stimuli. Therefore, endogenous insulin and glucocorticoids act as modulators of inflammatory responses.

Correlation between cell-mediated immunity and clinical forms of paracoccidioidomycosis

Cellular immune response to specific and non-specific stimulants was investigated, both in vivo and in vitro, in 29 healthy controls and in 53 previously untreated patients with the chronic isolated organic form (CIOF), the chronic mixed form (CMF) and the acute progressive form (APF) of paracoccidioidomycosis. The study included skin tests to Paracoccidioides brasiliensis antigen (PbAg) and phytohaemagglutinin (PHA), DNCB sensitization, determination of T lymphocytes and complement rosette-forming cells, lymphocyte transformation and leucocyte migration inhibition tests using PbAg and PHA. Patients displayed staggered cutaneous response to PHA and to PbAg, with marked decrease in intensity in the APF group. DNCB sensitization test and proliferative response of lymphocytes to PHA and PbAg were severely depressed in most of the patients. Leucocyte migration inhibition indices to PbAg were highly positive, while response to PHA was slightly decreased regardless of the clinical form. The number of T lymphocytes was reduced in most of patients and in them the number of complement-rosette forming cells was normal. The distribution of patients according to a suppression index, based in the results of the tests employed, revealed a tendency towards an increased degree of cellular immunosuppression from the least severe (CIOF) to the most severe (APF) clinical form of the disease. On the whole, the present study demonstrated a gamut of immunological reactivity in paracoccidioidomycosis. © 1985.