The adenosine A1 receptor antagonist SLV320 reduces myocardial fibrosis in rats with 5/6 nephrectomy without affecting blood pressure.

BACKGROUND AND PURPOSE: Myocardial fibrosis is an unwanted effect associated with chronic renal failure. The adenosine system is involved in cardiac and renal function. Therefore, we investigated the novel selective adenosine A(1) receptor antagonist SLV320 focusing on its potential in preventing cardiomyopathy in rats with 5/6 nephrectomy. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats were allocated to 4 groups of 12 rats each: 5/6 nephrectomy (5/6 NX), 5/6 NX plus SLV320 (10 mg kg(-1) d(-1) mixed with food), sham and sham plus SLV320. Study duration was 12 weeks, blood pressure was assessed repeatedly. At study end kidney function was assessed, blood samples and hearts were taken for histology/immunohistochemistry. Pharmacological properties of SLV320 were assessed using receptor binding and enzyme assays and in vivo. KEY RESULTS: SLV320 is a selective and potent adenosine A(1) antagonist in vitro (Ki=1 nM) with a selectivity factor of at least 200 versus other adenosine receptor subtypes. Functional A(1) antagonism was demonstrated in vivo. In rats with 5/6 NX SLV320 significantly decreased albuminuria by about 50%, but did not alter glomerular filtration rate (GFR). SLV320 normalized cardiac collagen I+III contents in 5/6 NX rats. SLV320 prevented nephrectomy-dependent rise in plasma levels of creatinine kinase (CK), ALT and AST. Blood pressure did not differ between study groups. CONCLUSION: SLV320 suppresses cardiac fibrosis and attenuates albuminuria without affecting blood pressure in rats with 5/6 nephrectomy, indicating that selective A(1) receptor antagonists may be beneficial in uraemic cardiomyopathy.

Long-chain fatty acid-binding to albumin: re-evaluation with directly measured concentrations.

In studies on uptake of fatty acids (FA) into organs, the unbound (or free) fatty acid fraction is commonly calculated from the concentration bound to albumin and from published binding constants. However, there is some dispute on the methods used for determining those binding constants. We developed a method allowing direct measurement of unbound FA by extending the previous studies of Svenson et al. [1] and Reed et al. [2]. Albumin was coupled to a solid phase (Sepharose 4B), loaded with FA and equilibrated with an aqueous solution. Laurate, palmitate and oleate concentrations in the aqueous phase were determined at different molar ratios of FA to albumin (r) and at different temperatures. FA albumin-binding constants (Ki) increase with chain length and decrease with temperature, in accordance with data obtained by others. However, the unbound concentrations measured are markedly lower than those obtained from binding constants, and the resulting Ki values markedly higher. This difference is presumed to result from (1) our direct measurement of unbound FA and (2) utilizing different more physiological conditions. Recalculating kinetic parameters from published FA uptake data, we found considerably different Km and Vmax values compared to the original data. Thus, the FA-binding characteristics measured in this study may influence the interpretation of FA uptake substantially.

Phenylarsine oxide and hydrogen peroxide stimulate glucose transport via different pathways in isolated cardiac myocytes.

The aim of this study was to investigate the stimulating effects of sulfhydryl reagents on glucose transport in isolated rat heart muscle cells and to compare them with the action of insulin. Low concentrations of the sulfhydryl oxidants hydrogen peroxide (H2O2) and diamide (5-100 microM), but also of phenylarsine oxide (PAO) (0.5-3 microM), that is known to specifically react with vicinal SH-groups, stimulated the rate of 2-deoxy-D-glucose uptake by a factor of 4 to 8 in these cells, while higher concentrations were inhibitory. The stimulating effects of H2O2 or diamide, and, to a significantly lesser extent, those of PAO or insulin, were depressed in cells pretreated with the sulfhydryl-alkylating agent N-ethylmaleimide (56-100 microM). H2O2 raised the Vmax and lowered the Km of 3-O-methyl-D-glucose uptake, while PAO or insulin solely increased Vmax. The increase in glucose transport caused by H2O2 was antagonized by the beta-adrenergic agonist isoprenaline (1 microM) or by a membrane-permeant cyclic AMP analog, whereas the effects of PAO or insulin were not altered. The action of H2O2 was additive with the stimulation induced by the protein phosphatase inhibitors okadaic acid (1 microM) or vanadate (6 mM), whereas the responses to PAO or insulin were reduced in the presence of these agents. Finally, H2O2 and PAO, but not insulin, acted additively with the protein kinase C ligand phorbol myristate acetate (0.8 microM) and with phospholipase C (0.03 units/ml). We conclude that, in cardiac myocytes, H2O2, on the one hand, and PAO (and possibly insulin), on the other hand, stimulate glucose transport via at least two distinct, SH-dependent pathways. These pathways, in turn, differ from a protein kinase C- and from a phospholipase C-mediated mechanism.

Acute stimulation of glucose transport by histamine in cardiac microvascular endothelial cells.

The purpose of the present work was to study the acute regulation of glucose uptake in cultured cardiac endothelial cells (CEC). Two types of potential stimuli were considered: (1) agents that are known to acutely stimulate glucose transport (i.e., within minutes) in fat and muscle tissues and (2) agents that influence endothelial cell function. Among the former agents, neither insulin, nor catecholamines (adrenaline, dopamine, phenylephrine), nor serotonin affected the rate of glucose transport in CEC, while SH-group reagents (phenylarsine oxide, diamide or menadione) were inhibitory. Among the factors of the second group that were tested (heparin, ADP, histamine, bradykinin), histamine was found to stimulate glucose transport in CEC by 10-50%. This effect was concentration-dependent (with an EC50 value approximately equal to 12 microM) and reached a maximum within 5 min upon histamine addition. This stimulation of glucose transport was suppressed by pyrilamine (100 nM), a specific H1-receptor antagonist, but not by cimetidine (100 microM), a H2-selective antagonist. Northern blot and Western blot analysis of CEC extracts revealed the presence of the ubiquitous glucose transporter isoform GLUT1 mRNA and protein, but not of the 'insulin-regulatable' isoform GLUT4. In conclusion, this is the first report on an acute stimulation of glucose transport in cardiac endothelial cells, in particular, and in an insulin-unresponsive cell type, in general. The effect of histamine is most likely mediated by H1-receptors and cannot be accounted for by a recruitment of GLUT4.

Changes of hemostasis, endogenous fibrinolysis, platelet activation and endothelins after percutaneous transluminal coronary angioplasty in patients with stable angina.

OBJECTIVES: This study investigated parameters of endogenous fibrinolysis, activation of coagulation and platelets, and endothelin levels before and after elective percutaneous transluminal coronary angioplasty (PTCA) in patients with stable coronary artery disease (CAD). BACKGROUND: Abrupt vessel closure is a serious short-term complication after PTCA and is often unforeseeable. Detailed insight into the effect of PTCA on hemostasis, platelets and the release of vasoconstrictive substances, which are among the mainly discussed mechanisms of abrupt vessel closure, is needed to enhance the safety of coronary intervention. METHODS: Plasma levels of markers of platelet activity, coagulation, endogenous fibrinolysis and endothelins were determined in 20 patients with stable CAD undergoing elective PTCA. The blood specimens were drawn before, immediately after, 1 h after intervention and on the next morning. RESULTS: All patients showed an initially uncomplicated PTCA. Regarding the efficacy of anticoagulation after receiving 15.000 IU heparin during PTCA, two groups were compared. In eight patients with ineffective anticoagulation production of thrombin and platelet activation directly after and 1 h after PTCA was significantly higher compared with 12 patients with effective anticoagulation. Despite the strong activation of coagulation, only a low fibrinolytic response could be observed. Endothelins rose significantly after PTCA in both groups but stayed longer on higher levels in patients with distinct thrombin generation. Three of the eight patients without sufficient heparin treatment suffered abrupt vessel closure. CONCLUSIONS: Initially uncomplicated dilation of coronary arteries leads to systemically measurable activation of coagulation and platelets in patients with ineffective doses of heparin and release of endothelins in all patients. Therefore, individual adjustment of anticoagulation and platelet inhibition in combination with effective antivasospastic substances are needed in every patient before, during and after initially uncomplicated PTCA to prevent this serious complication.

Action of metformin on glucose transport and glucose transporter GLUT1 and GLUT4 in heart muscle cells from healthy and diabetic rats.

The effects of the antidiabetic drug metformin on glucose transport were investigated in freshly isolated heart muscle cells from healthy and streptozotocin-diabetic rats. In vivo treatment of diabetic rats with metformin failed to affect the basal and insulin-stimulated rate of glucose transport measured in isolated cells. In vitro exposure to therapeutic concentrations (< or = 10(-4) M) of metformin did not influence glucose transport, even upon incubation times up to 5 h or in the presence of high glucose (20 nM). In contrast, higher metformin concentrations produced an 8- to 12-fold increase in glucose uptake (with a lag of 90 min, and a maximum at 180 min and approximately 5 mM). In the presence of submaximal insulin concentrations (< or = 3.10(-10) M), the effects of metformin (5 mM) and of insulin were more than additive, whereas, at saturating insulin concentrations (10(-8) M), partial additivity was observed. Like insulin, metformin caused an approximately 1.6-fold increase in the content of both glucose transporter isoforms GLUT1 and GLUT4 in the plasma membrane of cardiac myocytes, with a corresponding decrease in an intracellular membrane fraction. cAMP-elevating treatments depressed the metformin-, but not the insulin-dependent glucose uptake, by 20-30%. In myocytes from diabetic rats, the rate of metformin-activated glucose transport was similar to that of cells from control animals, whereas basal and insulin-stimulated transport were substantially diminished. Finally, metformin (5 mM) induced a slight depression of oxygen consumption and energy metabolism of myocytes (as determined by measuring their level of energy-rich phosphates) comparable to the effects of hypoxia in rat hearts. In conclusion, these data do not provide evidence in favor of the hypothesis that glucose uptake by muscle tissue represents the site of metformin's therapeutic action in vivo. On the other hand, the large, insulin-independent effect of metformin at high concentrations (approximately mM) in vitro may be related to the action of hypoxia and occurs through a redistribution of glucose carriers from an intracellular locus to the plasma membrane. The mechanism (or signal) involved in metformin's action is likely to differ from that triggered by insulin and is not impaired in the diabetic state.

The endosomal compartment is an insulin-sensitive recruitment site for GLUT4 and GLUT1 glucose transporters in cardiac myocytes.

In nonstimulated cardiomyocytes, the glucose transporter GLUT4 is confined to intracellular vesicles forming at least two populations: a storage pool enriched in GLUT4 (pool 1) and an endosomal pool containing both GLUT4 and GLUT1 (pool 2). We have now studied the dynamics of these pools in response to insulin or the mitochondrial inhibitor rotenone in rat cardiomyocytes. Rotenone recruited GLUT4 and GLUT1 to the cell surface from endosomal pool 2 without affecting pool 1. Kinetic experiments were consistent with rotenone acting on an intracellular compartment that is in close connection with the plasma membrane. In contrast, insulin caused rapid, complete depletion of GLUT4 from pool 1 and reduced the GLUT1 content of pool 2 by approximately 50%, whereas, surprisingly, no net decrease in GLUT4 occurred in this pool. Subsequent insulin withdrawal resulted in slow replenishment of pool 2 with GLUT1 and of pool 1 with GLUT4. When pool 1 was still largely depleted of GLUT4, a second insulin challenge did reduce GLUT4 in pool 2 and stimulated glucose transport to the same extent as the first insulin treatment. In conclusion, the storage pool is the primary source of GLUT4 in response to insulin, but not to rotenone. In addition, the endosomal compartment is an important recruitment site of both GLUT1 and GLUT4 when the storage pool is either unaffected (rotenone) or depleted (by a previous insulin challenge). GLUT4 mobilized by insulin from the storage pool may pass through an intermediary (possibly endosomal) compartment on its way to the cell surface.

Characterization of two distinct intracellular GLUT4 membrane populations in muscle fiber. Differential protein composition and sensitivity to insulin.

A major objective for the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 glucose carriers in the muscle fiber. In this report, we provide functional and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin; thus, one showed a marked decrease in GLUT4 levels but the other did not. They also showed a markedly different protein composition as detected by quantitative vesicle immunoisolation analysis. The GLUT4 pool showing no response to insulin contained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin, whereas only VAMP2 was found in the insulin-recruitable GLUT4 pool. SDS-PAGE and further silver staining of the immunoprecipitates revealed discrete polypeptide bands associated to the insulin-sensitive pool, and all these polypeptide bands were found in the insulin-insensitive population. Furthermore, some polypeptide bands were exclusive to the insulin-insensitive population. The presence of cellubrevin and SCAMP proteins, endosomal markers, suggest that the insulin-insensitive GLUT4 membrane population belongs to an endosomal compartment. In addition, we favor the view that the insulin-sensitive GLUT4 membrane pool is segregated from the endosomal GLUT4 population and is undergoes exocytosis to the cell surface in response to insulin. Intracellular GLUT4 membranes obtained from skeletal muscle contain cellubrevin, and VAMP2 and GLUT4-vesicles from cardiomyocytes also contain cellubrevin. This suggests that vSNARE proteins are key constituents of GLUT4 vesicles. The presence of the tSNARE protein SNAP25 in skeletal muscle membranes and SNAP25 and syntaxin 1A and syntaxin 1B in cardiomyocyte plasma membranes further suggest a role of the SNAREs in GLUT4 trafficking in muscle.

Possible involvement of alanine and pyruvate in the regulation of glucose transport in heart muscle cells.

In isolated rat heart muscle cells, addition of L-alanine (1.5 mmol/l) or of L-valine (3 mmol/l) resulted in either a ca 1.5- or 1.3-fold increase in glucose transport, resp. half-maximal stimulation was observed in the presence of L-alanine, but not of L-valine, within a physiological plasmatic range of concentrations. D-Alanine (1.5 mmol/l) was ineffective and the stimulating effect of L-alanine could be prevented by an excess of L-serine (15-30 mmol/l). L-Alanine produced an increase in 3-O-methyl-D-glucose transport Vmax (from 44.6 to 81.5 pmol.s-1.mg protein-1) without affecting the Km (12.2 in control vs 12.8 mmol/l in alanine-treated cells). Pyruvate (1.5 mmol/l) inhibited glucose transport by 20% and prevented the stimulating action of L-alanine (1.5 mmol/l). These results suggest that the effect of L-alanine in cardiac myocytes occurs through the interaction with an intracellular site and that both alanine and pyruvate may play a role in the regulation of glucose transport in these cells.

Regulation of glucose transport, and glucose transporters expression and trafficking in the heart: studies in cardiac myocytes.

Cardiac muscle is characterized by a high rate of glucose consumption. In the absence of insulin, glucose transport into cardiomyocytes limits the rate of glucose utilization and therefore it is important to understand the regulation of glucose transporters. Cardiac muscle cells express 2 distinct glucose transporters, GLUT4 and GLUT1; although GLUT4 is quantitatively the more important glucose transporter expressed in heart, GLUT1 is also expressed at a substantial level. In isolated rat cardiomyocytes, insulin acutely stimulates glucose transport and translocates both GLUT4 and GLUT1 from an intracellular site to the cell surface. Recent evidence indicates the existence of at least 2 distinct intracellular membrane populations enriched in GLUT4 with a different protein composition. Elucidation of the intracellular location of these 2 GLUT4 vesicle pools in cardiac myocytes, their role in GLUT4 trafficking, and their relation to insulin-induced GLUT4 translocation needs to be addressed.

Antithrombotic treatment in stable coronary syndromes: long-term intermittent urokinase therapy in end-stage coronary artery disease and refractory angina pectoris.

Interventions that modify lipid metabolism and blood coagulation have been shown to favourably influence the natural course of coronary artery disease in terms of the primary prevention and treatment of acute cardiovascular events. Various findings suggest that such interventions may also preserve and enhance myocardial perfusion in the chronic stage of the disease. Long-term intermittent urokinase therapy was developed for patients with end-stage coronary artery disease and refractory angina pectoris. A dose of 500,000 IU of urokinase given intravenously as a bolus three times a week for of 12 weeks reduced symptoms by 70% and was accompanied by objective improvements in myocardial perfusion and an increase of ergometric exercise capacity. The possible therapeutic mechanisms of long-term intermittent urokinase therapy-improvement of rheological blood properties mediated by fibrinogen reduction, thrombolysis of non-occlusive subclinical thrombi, and regression of atherosclerotic plaques-are discussed in the context of other antithrombotic approaches.

Highly insulin-responsive isolated rat heart muscle cells yielded by a modified isolation method.

Freshly isolated adipocytes or cardiac myocytes appear to be subject to unspecific stimulation during isolation and subsequent handling, e.g. with respect to glucose transport. We have developed a modified procedure that yields rat cardiomyocytes with a very low basal, i.e. non stimulated hexose uptake rate (ca. 3 pmol * s-1 * mg protein-1 at 1 mM sugar), as compared to data reported by others. This low value correlates with the reported oxygen consumption of non-beating, isolated rat hearts, when these are perfused with glucose as the only substrate. The basal rate of glucose uptake in our quiescent cardiomyocytes is slightly lower than the value measured by others in beating rat hearts in vivo. Insulin (10 nM) stimulates 2-deoxy-D-glucose uptake 8- to 20-fold and 3-O-methyl-D-glucose uptake 14- to 20-fold, as compared to control. This insulin effect is markedly larger than that usually observed in isolated cardiomyocytes, but it is similar in magnitude to the stimulation of glucose transport reported for isolated, perfused rat hearts. In these cells, new stimulatory effects on the glucose transport, e.g. that of sulfhydryl reagents like phenylarsine oxide, become apparent. We conclude that the cardiomyocytes obtained by this modified method exhibit a basal glucose transport rate that is close to physiological values. These cells represent a new highly responsive model to detect and to investigate the effects of glucose transport stimulators (insulin, contraction etc.).

Alanine and hyperosmolarity are responsible for the stimulation of cardiomyocyte glucose transport by samples containing a glucose tolerance factor.

Low-molecular-weight, cationic samples, that were previously reported to contain a glucose tolerance factor, were obtained by partial purification from yeast extract. These samples increased the rate of glucose transport in isolated cardiomyocytes 2.0- to 2.5-fold. A further purification by gel filtration led to the separation of two active components that were identified as (i) L-alanine and (ii) an elevated osmolarity. Moreover, the effect of partially purified fractions (before gel filtration) (i) was decreased upon alanine depletion with alanine dehydrogenase and (ii) was mimicked by the additive action of alanine and of a hyperosmolar medium. These findings indicate that the effect of this partially purified material is not accounted for by a putative glucose tolerance factor. Interestingly, alanine elicited its effect at concentrations that correspond to physiological plasma values, which suggests that this amino acid might be involved in the regulation of glucose transport in cardiomyocytes. Furthermore, the effect of alanine was prevented by DL-cycloserine (1 mM) or aminooxyacetate (1 mM), but not by cycloheximide (35 microM), indicating that (a) transamination reaction(s), but not protein synthesis, is required.

Inhibition of glucose transport by cyclic GMP in cardiomyocytes.

Recent evidence points to a potential role of cyclic GMP (cGMP) in the control of cardiac glucose utilization. The present work examines whether the glucose transport system of cardiac myocyte is a site of this cGMP-dependent regulation. Treatment of isolated rat cardiomyocytes (for 10 min) with the membrane-permeant cGMP analogue 8-(4-chlorophenylthio)-cGMP (8-p-CPT-cGMP, 200 microM) caused a decrease in glucose transport in non-stimulated (basal) myocytes, as well as in cells stimulated with insulin or with the mitochondrial inhibitor oligomycin B by up to 40%. An inhibitory effect was also observed with another cGMP analogue (8-bromo-cGMP), and in cells stimulated by hydrogen peroxide or anoxia. In contrast, 8-p-CPT-cAMP (200 microM), or the beta-adrenergic agonist isoprenaline (which increases cAMP levels) did not depress glucose transport, and even potentiated the effect of insulin. Blockade of endogenous cGMP formation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) significantly increased basal and insulin-dependent glucose transport (by 25%), whereas addition of the guanylate cyclase activator 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazol (YC-1, 30 microM) produced a depression of glucose transport (by 20%). Confocal laser scanning microscopic studies revealed that cGMP partially prevents the insulin-induced redistribution of the glucose transporter GLUT4 from intracellular stores to the cell surface. These observations suggest that the glucose transport system of cardiomyocytes represents a metabolic target of inhibition by cGMP, and that this regulation occurs at the level of the trafficking of glucose transporters.

The effect of anoxia on cardiomyocyte glucose transport does not involve an adenosine release or a change in energy state.

The action of anoxia on glucose transport was investigated in isolated resting rat cardiomyocytes. Incubation of these cells in the absence of oxygen for 30 min resulted in a 4- to 5-fold increase in glucose transport (with a lag period of 5-10 min). Up to 40 min of anoxia failed to alter the cellular concentrations of ATP, phosphocreatine, and creatine. Adenosine deaminase (1.5 U/ml), the A1-adenosine receptor antagonist 1,3-diethyl-8-phenylxanthine (1 microM), or the A2-selective antagonist 3,7-dimethyl-1-propargylxanthine (20 microM) had no effect on anoxia-dependent glucose transport. Moreover, adenosine (10-300 microM, added under normoxia) did not stimulate glucose transport. Wortmannin (1 microM) did not influence the effect of anoxia, but completely suppressed that of insulin. On the other hand, the effects of anoxia and insulin were not additive. These results indicate (i) that the effect of anoxia on cardiomyocyte glucose transport is not mediated by a change in energy metabolism, nor by an adenosine release; (ii) that it probably does not involve a phosphatidylinositol 3-kinase, in contrast to the effect of insulin, and (iii) that the signal chains triggered by anoxia or insulin may converge downstream of this enzyme, or, alternatively, that anoxic conditions may impair the action of the hormone.

Heart-type fatty acid binding protein (hFABP) in the diagnosis of myocardial damage in coronary artery bypass grafting.

OBJECTIVES: Heart-type fatty acid binding protein (hFABP) is an intracellular molecule engaged in the transport of fatty acids through myocardial cytoplasm and has been used as a rapid marker of myocardial infarction. However, its value in the evaluation of perioperative myocardial injury has not yet been assessed. METHODS: 32 consecutive patients undergoing coronary artery bypass grafting were included in a prospective, randomized study using standardized operative procedures and myocardial protection. Three patients with perioperative myocardial infarction were added. Serial blood samples were taken preoperatively, before ischemia, 5 and 60 min after declamping, 1 and 6 h postoperatively and on postoperative days 1, 2 and 10 and were tested for hFABP, creatine kinase isoenzyme MB (CKMB) and troponin I (TnI). RESULTS: Hospital mortality was zero. The kinetics of the biochemical parameters revealed a typical pattern for each marker. In routine patients, hFABP levels peaked as early as 1 h after declamping, whereas CKMB and TnI peaked only 1 h after arrival in the intensive care unit. Patients with perioperative infarction displayed peak levels some hours later in all marker proteins. Peak serum levels of hFABP correlated significantly with peak levels of CKMB (r=0.436, P=0.011) and TnI (r=0.548, P=0.001), indicating the degree of myocardial damage. CONCLUSIONS: hFABP is a rapid marker of perioperative myocardial damage and peaks earlier than CKMB or TnI. The kinetics of marker proteins in serial samples immediately after reperfusion is more suitable for the detection of perioperative myocardial infarction than a fixed cut-off level.

Enhancing filtration rates by the use of blood flow around the capillaries of plasmafilters: an in vitro study.

As the low clearance rate of plasmaseparation limits its use in the treatment of patients suffering from liver failure, sepsis or MOF, we intend to develop strategies for a plasmaseparation unit which increases plasmafiltration rates. Our first question focused on whether commercially available plasmaseparation filters, and in particular their membranes, are suitable for the inversion of blood and plasma compartments. This experimental study was performed using in vitro systems. Commercially available plasmafilters PF2000N (Gambro) and Plasmaflo (Asahi) were compared in both their normal operating mode with blood flow through the capillary lumen, and in the inverse mode. Inverse mode means that blood flows through the outer space of the capillaries while plasma was obtained from the lumen. Heparinised porcine blood (5 I.U./ml) was used in a heated, recirculating in vitro circuit. Our main results were that the normal use of both filter types Plasmaflo and PF2000N enabled maximal blood flows (Qb) of 200 ml/min and filtration rates (Qf) of 25-40 ml/min. Operating the filters in the inverse mode enabled Qb up to 500 ml/min and Qf up to 100 ml/min. Hemolysis, platelet counts and coagulation parameters did not differ significantly regardless of whether the normal or inverse mode was used. The tested plasmafiltration membranes appear to be suitable for use in inverse mode. Although in our experiments, hemocompatibility tests did not indicate severe problems induced by the module geometry, the development of a module specially constructed for blood flow outside of the hollow fibers appears to be necessary in order to minimise shunts and low perfusion areas.

The Rivalta's test as a diagnostic variable in feline effusions--evaluation of optimum reaction and storage conditions.

OBJECTIVE: The Rivalta's test is used to diagnose feline infectious peritonitis (FIP) in cats with effusion. Only little information on the influence of sample storage and reaction conditions on test results is available, and diagnostic sensitivity and specificity to diagnose FIP vary considerably between few available studies. This study determined the influence of storage of effusion, modifications on reaction conditions, and inter-observer variation. MATERIAL AND METHODS: The Rivalta's test was repeated up to 21 days after storage at room temperature, in the refrigerator, or freezer. The test was performed by two independent, blinded investigators. It was also performed using different volumes of acetic acid, different acids, and different kinds of water. RESULTS: Even after storage for 21 days, test results were comparable. While inter-observer variation revealed substantial disagreement, different modifications in performance showed no major influence on test outcome. CONCLUSION: The Rivalta's test seems to be a very robust test concerning storage conditions. Modifications in reaction condition also do not substantially influence outcome. However, the test is subjective and depends on the evaluating person.

Randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis.

BACKGROUND: Currently there is no drug proven to effectively treat cats with feline infectious peritonitis (FIP). HYPOTHESIS: Propentofylline (PPF) can decrease vasculitis, and therefore prolong survival time in cats with FIP, and increase their quality of life. ANIMALS: Twenty-three privately owned cats with FIP. METHODS: Placebo-controlled double-blind trial. FIP was confirmed by histology or immunostaining of feline coronavirus (FCoV) antigen in effusion or tissue macrophages or both. The cats were randomly selected for treatment with either PPF or placebo. All cats received additional treatment with glucocorticoids, antibiotics, and low molecular weight heparin according to methods. RESULTS: There was no statistically significant difference in the survival time of cats treated with PPF (8 days, 95% CI 5.4-10.6) versus placebo (7.5 days, 95% CI 4.4-9.6). The median survival time of all cats was 8 days (4-36 days). There was neither a difference in quality of life (day 7, P = .892), in the amount of effusion (day 7, P = .710), the tumor necrosis factor-alpha (TNF-α) concentration (day 7, P = .355), nor in any other variable investigated in this study, including a complete blood count, and a small animal biochemistry profile. CONCLUSIONS AND CLINICAL IMPORTANCE: This study did not detect an effect of PPF on the survival time, the quality of life, or any clinical or laboratory parameter in cats with FIP. Therefore, PPF does not appear to be an effective treatment option in cats with a late stage of the disease FIP.