The inhibition of microtubule dynamics instability alters lipid homeostasis in TM4 Sertoli cells.

Sertoli cells (SC) structurally support and transport nutrients to germ cells during spermatogenesis facilitated by an active cytoskeleton. Chemical perturbation of SC microtubule (MT) dynamics instability leads to premature germ cell exfoliation demonstrating that this process is essential for male fertility, yet the effects of MT damaging drugs on SC lipid metabolism have been less explored. The aim of this study was to advance our understanding of how adequate SC MT dynamicity is needed to finely tune lipid homeostasis. To elucidate the role of MT dynamics instability on the latter, we suppressed MT dynamicity by long-term exposures to 10 nM of nocodazole (NCZ) on TM4-SC cultures. Inhibition of MT dynamics instability affected the distribution of [3H] arachidonate on TM4-SC. Triacylglycerols (TAG) exhibited a higher proportion of the [3H] label, with significantly lower percentages in the mitochondrial phospholipid cardiolipin, and notably, also in phosphatidylethanolamine. A noteworthy and progressive accumulation of lipid droplets during the period of exposure to NCZ was accompanied by increased TAG levels but not cholesterol levels in TM4-SC. NCZ-exposed cells reduced their mitochondrial membrane potential and increased ROS production without triggering apoptosis, had a compromised autophagic flux, and lost their transferrin expression. Although SC morphology was preserved, the NCZ-exposed cells displayed alteration of the normal organization of microfilaments (f-actin) and intermediate filaments (vimentin). Our findings suggest that a preserved MT dynamicity is essential in the maintenance of lipid and fatty acids homeostasis in SC, and thus highlights a novel target in these cells for drugs that impair MT dynamicity.

Pesquisa de eosinófilos em secreçäo nasal no Hospital Universitário de Florianópolis / A research on eosinophils in nasal secretion at the Florianópolis University Hospital

A caracterizaçäo de uma rinite depende de vários exames que incluem desde a anamnese até outros exames complementares, para a definiçäo da etiologia da rinite. é definido como rinite, um hiperfuncionamento nasal e inflamaçäo tecidual, que leva a congestäo nasal, rinorréia, obstruçäo nasal, prurido e espirros. As rinites podem ser classificadas quanto ao tipo como: näo-alérgica e näo-infecciosa, alérgica, alérgica aguda, alérgica sazonal, alérgica perene, infecciosa. Nesta pesquisa, analisamos 97 casos de exames, quanto à sua parte citológica, onde encontramos apenas 7 casos de eosinofilia. Na maior parte (46 exames), verificamos ausência de eosinófilos, seguido de 37 exames com menos de 10 porcento de presença de eosinófilos. Quanto ao sexo, outro item observado, observamos a equivalência de casos, ou seja, 50 exames para o sexo masculino e 47 para o sexo feminino

Análise citológica do líquido pleural no Hospital Universitário de Florianópolis / Cytologic analysis of the pleural fluid at the University Hospital of Florianópolis

A pleura consiste de uma cavidade que separa os pulmões e a porção interior da parede torácica, é revestida por uma membrana serosa com uma divisão visceral e outra parietal. Separando estas membranas, está o líquido pleural, o qual serve como um lubrificante para o livre movimento dos pulmões para as trocas de volume que apresentam durante a respiração. Este líquido é um ultrafiltrado do plasma, incolor e relativamente acelular em condições normais. Nesta pesquisa em particular, tivemos como objetivo, avaliar apenas os exames citológicos e comparar estes resultados com a revisão bibliográfica. O trabalho desenvolvido, compreendeu o período de março de 1997 a fevereiro de 1999, no total de 141 exames verificados. Observamos um predomínio de análises do líquido pleural em pacientes do sexo masculino (41%), seguido de 36% de pacientes do sexo feminino. Quanto ao tipo celular (citologia diferencial), considerando neutrófilos, mononucleares, células mesoteliais e eosinófilos, foi na faixa de 1 a 10% do total dos líquidos que as mesmas apareceram em números superiores ao relatado na literatura (neutrófilos/40, mononucleares/39, células mesoteliais/50, eosinófilos/16, célula/número de exames respectivamente). Ao verificarmos a citologia global, as hemácias/mm³, apresentaram o seu maior índice entre 1.001-10.001 hem/mm³ (46%), considerando-se valores normais. Acima dos valores normais citados na literatura, temos as células nucleadas/mm³, entre 1.001-10.001 células nucleadas/mm³ (38%). Para dados conclusivos, deveríamos continuar o acompanhamento das análises citológicas.

Rationale for the use of a low molecular weight heparin during hemodialysis with polysulphone membrane in uremic patients.

We carried out an open, randomized, cross-over study to test the rationale for use of a low molecular weight heparin during hemodialysis with polysulphone membranes. Ten uremic patients (6 men, 4 women, mean age 50 +/- 11 years, range 31-70) in chronic hemodialysis underwent paired filtration dialysis with either low molecular weight heparin or unfractionated heparin, then crossed over to the other anticoagulant treatment. We measured the following: anti-factor Xa and anti-factor IIa activity in patients' plasma and in the ultrafiltrate, activated partial thromboplastin time and bleeding time before, during and after hemodialysis. Some low molecular weight fractions of heparin were lost in the ultrafiltrate during hemodialysis, while higher molecular weight fractions were retained. The activated partial thromboplastin time during dialysis and the bleeding time after dialysis were more prolonged with unfractionated heparin than with the low molecular weight heparin (p < 0.01 and p < 0.05 respectively). Our data indicate that the use of a low molecular weight heparin determines a less pronounced prolongation of the activated partial thromboplastin time during dialysis and prevents a post-dialytic increase in bleeding time.

Hemostatic parameters and platelet activation by flow-cytometry in normal pregnancy: a longitudinal study.

Nineteen pregnant women with uncomplicated pregnancies were studied during the first, second, and third trimesters. We measured the following hemostatic parameters: prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin III, protein C, protein S, platelet number and volume. Platelet function was examined by a cytofluorimetric method, using an anti-GPM-140 antibody which is directed against a platelet alpha granule membrane protein. Activated platelets were expressed as a percentage of the GMP-140-positive platelets over total platelets. Fibrinogen levels showed a steady increase during pregnancy; conversely prothrombin time, activated partial thromboplastin time, protein C, and antithrombin III showed no significant modifications and remained within the reference range. There was a decrease of protein S activity throughout pregnancy, although protein S antigen did not follow this trend. The decrease occurred early in pregnancy and persisted during the second and third trimesters, reaching a stable plateau. We observed no platelet volume change or activation: the percentage of activated platelets was within the normal reference range, even in late pregnancy.

Pharmacokinetics of monoclonally-purified and recombinant factor VIII in patients with severe von Willebrand disease.

A monoclonally-purified factor VIII (FVIII) concentrate, containing little von Willebrand factor (vWF), was infused to 11 patients with severe von Willebrand disease and unmeasurable levels of plasma vWF. In comparison with the historical data obtained infusing hemophiliacs in the same conditions, monoclonally-purified FVIII had a significantly shorter half-life and faster clearance from plasma but similar in vivo recovery and volume of distribution. Two additional patients with severe von Willebrand disease were also infused with recombinant FVIII totally devoid of vWF. Half-life was very short and in vivo recovery low, with a larger volume of distribution than for monoclonally-purified FVIII. We conclude that in patients with severe von Willebrand disease the small amounts of vWF contained in the monoclonally-purified FVIII concentrate are not sufficient to stabilize infused FVIII, nor to support the normal circulation of endogenous FVIII that these patients produce at a normal rate.

Comparison of four virus-inactivated plasma concentrates for treatment of severe von Willebrand disease: a cross-over randomized trial.

Until recently, cryoprecipitate has been the treatment of choice in patients with severe von Willebrand disease (vWD) because it can transiently correct low plasma levels of factor VIII coagulant activity (FVIII:C) and shorten or normalize the prolonged bleeding time (BT), the two laboratory hallmarks of the disease. However, cryoprecipitate may still transmit blood-borne viruses, whereas the development of virucidal methods have rendered plasma concentrates containing FVIII:C and von Willebrand factor (vWF) safer. To establish their potential usefulness in the treatment of vWD, we compared the effect of four virus-inactivated concentrates on FVII:C and vWF plasma levels and the BT (template method) in 10 patients with severe vWD using a crossover randomized design. The concentrates were an intermediate-purity, pasteurized FVIII-vWF concentrate; an intermediate-purity, dry-heated FVIII-vWF concentrate; a solvent/detergent-treated vWF concentrate, containing little FVIII; and a high-purity solvent/detergent-treated FVIII-vWF concentrate. All concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed after the vWF concentrate. The effect of concentrates on the BT, however, was less uniform and satisfactory. The pasteurized FVIII-vWF concentrate transiently corrected, completely or partially, the BT in 8 of 10 patients, the dry-heated and solvent/detergent FVIII/vWF concentrates in five, whereas in no patient did the vWF concentrate correct the BT according to the criteria used in this study. These effects on the BT were not related to the plasma levels of ristocetin cofactor activity-attained postinfusion (100 U/dL or more in the majority of patients) or to the multimeric structure of vWF in concentrates (defective in larger multimers in all cases). In conclusion, even though virus-inactivated concentrates can be used to increase FVIII:C levels in patients with severe vWD, none of the concentrates studied by us consistently normalizes the BT in a sustained fashion.

Effects of subcutaneously administered dermatan sulfate (MF 701) on the coagulation and fibrinolytic parameters of healthy volunteers.

Eight healthy volunteers were given single subcutaneous doses of dermatan sulfate (DS, 100, 200 and 400 mg), heparin (5,000 IU) and placebo in random order. Wash-out between treatments was greater than or equal to 10 days. Serial blood samples were taken before and up to 24 hours after treatment to measure coagulation and fibrinolytic parameters. Thrombin generation was significantly inhibited by DS and heparin as compared to placebo. The effect of DS was dose-dependent. Peak inhibition after 200 mg DS was comparable to that of 5,000 IU heparin, but lasted longer. A small, bordeline significant prolongation of APTT was observed after 400 mg DS and heparin. The changes in PAI and fibrinolytic activities were those of the circadian variation. No changes were seen in the other parameters tested. In conclusion, single s.c. doses of DS (200, or 400 mg) inhibit ex vivo thrombin generation equally or more than 5,000 IU heparin and for a longer time. The effect of both treatments on fibrinolysis is negligible.

In vitro effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production.

We studied the in vitro effects of picotamide (N,N' bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B2 (TxB2) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/l) inhibited platelet aggregation, the release of ATP and TxB2 production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/l) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB2 production both under stirring and non-stirring conditions, whereas the pure thromboxane A2 receptor antagonist BM13177 (0.5 mmol/l) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB2 production, whereas BM13177 inhibits the potentiation of TxB2 production due to TxA2/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA2/PE antagonism; (ii) inhibition of thromboxane synthase.

Subcutaneous desmopressin (DDAVP) shortens the prolonged bleeding time in patients with liver cirrhosis.

The intravenous infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) shortens the prolonged bleeding time in patients with congenital or acquired bleeding disorders, including patients with uremia or liver cirrhosis. We carried out a double-blind, placebo-controlled crossover study in ten patients with liver cirrhosis to evaluate whether or not their prolonged bleeding times could be shortened by subcutaneous injections of DDAVP (0.3 microgram/kg), a more practical route of administration than intravenous infusions. One hour after DDAVP injection the bleeding time was significantly shortened (p less than 0.05). After 4 h, however, the bleeding time shortening was no longer statistically significant. There was no bleeding time change after placebo. Plasma levels of von Willebrand factor antigen (vWF:Ag) did not significantly increase after DDAVP or placebo. The study shows that subcutaneous DDAVP is an alternative method for short-term shortening of the bleeding time in liver cirrhosis.