Native plasma-derived FVIII/VWF complex has lower sensitivity to FVIII inhibitors than the combination of isolated FVIII and VWF proteins. Impact on Bethesda assay titration of FVIII inhibitors.

Sensitivity to FVIII inhibitors of the native plasma-derived (pd) FVIII/VWF complex vs. the complexes formed after exogenous FVIII infusion in the haemophilic patient has not been thoroughly studied. The role of VWF in the interaction of FVIII with inhibitors was studied in vitro using different combinations of VWF and FVIII concentrates. Normal plasma, pdFVIII/VWF and isolated FVIII (recombinant FVIII, B-domain deleted and pdFVIII) were used. Titre (BU) was kinetically determined (up to 2 h) in serial dilutions of inhibitor IgG (purified from a pool of plasmas with inhibitors) mixed with VWF and then incubated with the different FVIII. Inhibitor was also added to previously mixed VWF+FVIII. Residual FVIII:C was determined. TGA assays were performed with FVIII-deficient plasma spiked with the FVIII-VWF mixtures with/without an ESH-8 antibody. Inhibitor titres for plasma and pdFVIII/VWF were comparable at all time points. Titres for all concentrates of isolated FVIII were significantly higher than those for plasma or pdFVIII/VWF (1.4-1.9 fold) even after preincubation with VWF. At t = 0 h, titres for plasma or pdFVIII/VWF were unquantifiable, but were detectable for isolated FVIII (0.6-1.6 BU). In contrast to pdFVIII/VWF, the decrease in thrombin generation parameters by isolated FVIII in the presence of ESH-8 was significant (P < 0.01) even when previously combined with VWF. In conclusion, VWF protection against FVIII inhibitor activity might be higher with native pdFVIII/VWF complex than with the corresponding compound formed from the isolated proteins. Bethesda assay titration using different FVIII concentrates would be advisable to guide the treatment of inhibitor patients.

Effect of temperature on plasma freezing under industrial conditions.

The European Pharmacopoeia monograph on Human plasma for fractionation does not define the freezing process time but does define the freezing temperature (- 30 degrees C or below). Initial freezing conditions are crucial for the quality of plasma. These conditions were intended to preserve labile proteins such as fVIIl, but they can also be considered favourable for the plasma quality in general. This study evaluates the way the industrial plasma freezing affects labile coagulation factors. We have studied the freezing of plasma in industrial-size chambers at temperatures close to - 30 degrees C, - 25 degrees C and - 20 degrees C, and the possible differences between performing the freezing process in a chamber or in a freezer, in order to elucidate whether or not these parameters affect the quality of plasma. For this study, plasma bottles were frozen in industrial chambers set at - 30 degrees C, - 25 degrees C and - 20 degrees C, and in a freezer set at - 20 degrees C. The freezing rates were followed by means of probes in plasma control bottles. From this plasma, coagulation factors (fVIII, fIX and fibrinogen) were analysed before and after freezing, and cryoprecipitate was obtained in some cases. Statistically significant differences exist in fVIII:C recovery in thawed plasma between freezing at - 30 degrees C and at - 20 degrees C (n = 11; 85.4 +/- 4.3 % versus 74.6 +/- 6.0 % (chamber) or 79.3 +/- 6.3 % (freezer)). There is no difference between - 30 degrees C and - 25 degrees C, or between freezing at - 20 degrees C in a chamber or in a freezer. No significant loss of activity in thawed plasma is observed for fIX and fibrinogen at - 25 degrees C or - 20 degrees C versus - 30 degrees C. The fVIII and vWF recovery in cryoprecipitates does not show differences (464.2 IU fVIII/ml at - 30 degrees C, 446.7 IU fVIII/ml at - 25 degrees C, and 475.8 IU fVIII/ml at - 20 degrees C). The results obtained from this study support that plasma might also be frozen at - 25 degrees C or below without any impact on its quality, and that sporadic and short term deviations, from - 30 degrees C or below up to - 25 degrees C, in the currently required freezing temperature, would not have an effect on the labile factors recovery.

Effects of herbicide on the kidneys of two Venezuelan cultured fish: Caquetaia kraussii and Colossoma macropomum (Pisces:Ciclidae and Characeae)

The use of chemical pesticides and herbicides has increased environmental pollution and affected ichthyofauna in the watersheds where they are used.We studied the effect of an herbicide, triazine, on the kidneys of two species (Caquetaia kraussii and Colossoma macropomum )widely found in Caribbean and South American rivers.In Venezuela,these species are abundant and have a high aquaculture potential because they may be cultured and reproduced in captivity.Four kidney samples from juveniles of each species exposed to the herbicide were examined by Transmission Electron Microscopy.Kidney tubule alterations included loss of plasmalemma and cell interdigitations, misshaped mitochondria,decrease in rough endoplasmic reticulum and free polysomes,and the presence of autophagic vacuoles and primary lysosomes.These alterations at the cellular level may explain fish behaviour in terms of kidney tubule pathology,and relative amounts and conditions of organelles within affected cells

Effects of herbicide on the kidneys of two Venezuelan cultured fish: Caquetaia kraussii and Colossoma macropomum (Pisces: Ciclidae and Characeae).

The use of chemical pesticides and herbicides has increased environmental pollution and affected ichthyofauna in the watersheds where they are used. We studied the effect of an herbicide, triazine, on the kidneys of two species (Caquetaia kraussii and Colossomna macropomum) widely found in Caribbean and South American rivers. In Venezuela, these species are abundant and have a high aquaculture potential because they may be cultured and reproduced in captivity. Four kidney samples from juveniles of each species exposed to the herbicide were examined by Transmission Electron Microscopy. Kidney tubule alterations included loss of plasmalemma and cell interdigitations, misshaped mitochondria, decrease in rough endoplasmic reticulum and free polysomes, and the presence of autophagic vacuoles and primary lysosomes. These alterations at the cellular level may explain fish behaviour in terms of kidney tubule pathology, and relative amounts and conditions of organelles within affected cells.

Ecophysiological behavior of Caquetaia kraussii (Pisces: Cichlidae) exposed to different temperatures and salinities

Tropical river sardine, Caquetaia kraussii, captured from La Aguá lagoon (Sucre State, Venezuela) were acclimatized for four weeks at 22, 24, 30 and 32 degrees C and at 0, 5, 10, 15 and 17@1000 salinity. To evaluate effects of thermal response to acclimatization level, the fish were transferred suddenly from lower temperatures (22 and 24 degrees C) to higher ones (32 and 30 degrees C) respectively. Then thermal resistance time was measured at the lethal temperature of 40.9 degrees C for 30 days. We considered that acclimatization process completed when resistance time was stabilized at the new temperature regime. For the saline effect, the concentrations of sodium and potassium were measured in the tissues at each treatment: gills, white muscle, gut and heart. The results showed that thermal tolerance increased rapidly in 3 h with a 6 degrees C rise in temperature (from 24 to 30 degrees C) and in 24 h with a 10 degrees C rise (22 to 32 degrees C). With decreasing temperatures, the acclimatization level reached its lowest in 11 days with a 6 degrees C decreases (from 30 to 24 degrees C) and in 14 days with a 10 degrees C decrease (32 to 22 degrees C). Caquetaia kraussii regulates as much sodium as potassium in gills and white muscle tissues at all salinity levels tested; however, gut and heart tissues showed significantly different regulations among salinities examined.

Ecophysiological behavior of Caquetaia kraussii (Pisces: Cichlidae) exposed to different temperatures and salinities.

Tropical river sardine, Caquetaia kraussii, captured from La Aguá lagoon (Sucre State, Venezuela) were acclimatized for four weeks at 22, 24, 30 and 32 degrees C and at 0, 5, 10, 15 and 17@1000 salinity. To evaluate effects of thermal response to acclimatization level, the fish were transferred suddenly from lower temperatures (22 and 24 degrees C) to higher ones (32 and 30 degrees C) respectively. Then thermal resistance time was measured at the lethal temperature of 40.9 degrees C for 30 days. We considered that acclimatization process completed when resistance time was stabilized at the new temperature regime. For the saline effect, the concentrations of sodium and potassium were measured in the tissues at each treatment: gills, white muscle, gut and heart. The results showed that thermal tolerance increased rapidly in 3 h with a 6 degrees C rise in temperature (from 24 to 30 degrees C) and in 24 h with a 10 degrees C rise (22 to 32 degrees C). With decreasing temperatures, the acclimatization level reached its lowest in 11 days with a 6 degrees C decreases (from 30 to 24 degrees C) and in 14 days with a 10 degrees C decrease (32 to 22 degrees C). Caquetaia kraussii regulates as much sodium as potassium in gills and white muscle tissues at all salinity levels tested; however, gut and heart tissues showed significantly different regulations among salinities examined.

Actividad antimicrobiana de extractos orgánicos aislados de Aplysina fistularis (Demospongiae: Aplysinidae) / Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)

Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The minimal inhibitory concentration (MIC) and toxic activity of extract were determined. Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E. coli, while CHCl3 fraction inhibited E. coli and S. aureus growth. The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria. Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E. coli and S. aureus. In Sf2 EtOAc/MeOH, MIC was moderate for S. aureus (MIC > 256 g/ml). F4 CHCl3/MeOH produced a high inhibition in S. aureus (MIC = 0.125 g/ml) and for E. coli (MIC > 16 g/ml). F10 CHCl3/MeOH showed a moderate activity against S. aureus (MIC > 128 g/ml) and low activity against E. coli (MIC = 512 g/ml). F10 CHCL3/MeOH did no present toxic activity against Artemia salina. The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml. LC50 in both cases was 548.4 and 243.4 microg/ml respectively. Secondary metabolites of medium polarity obtained from A. fistularis have a wide spectrum of anti bacterial activity. Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.

Efecto de la alimentación sobre la condición fisiológica del mejillón Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN / Food effect on the physiological condition of the Perna viridis mussel (Bivalvia: Mytilidae), using the RNA/DNA quotient

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.

Efecto de la temperatura de aclimatación sobre el crecimiento instantáneo de Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN / Effects of acclimation temperature on the instant growth of Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA quotation

Temperature affects growth rate in aquatic organisms. This can be evaluated in short term using biochemical indexes (RNA/DNA and Protein/DNA). The effect of acclimatization temperature on the instantaneous growth and physiological condition of Perna viridis was studied in organisms collected in La Esmeralda, Sucre State (Venezuela) and taken to the laboratory, where groups of 100 organisms (size 3.0 - 3.5 cm, anteroposterior measurement) were acclimatized at 15, 20, 26 or 28 degrees C during four weeks. Later they were kept in a 60 liters aquarium for another six weeks under the same conditions. Each week, ten organisms per group were extracted to measure concentrations of RNA, DNA (by a fluorometric method with ethidium bromide) and proteins (by a colorimetric method), in tissues (digestive gland, adductor muscle and gills). Protein concentration was greater and highly significant at 15 degrees C for all studied tissues. The opposite was obtained with the RNA/DNA and Protein/DNA ratios: the greatest increase was observed at the highest temperature (28 degrees C) for all tissues. At the lowest temperature there was a tendency to reduce both indexes with time. Greater instantaneous growth can be expected at higher temperatures and 28 degrees C was optimal for growth in these specimens.

[Effects of acclimation temperature on the growth of Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio]. / Efecto de la temperatura de aclimatación sobre el crecimiento instantáneo de Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN.

Temperature affects growth rate in aquatic organisms. This can be evaluated in short term using biochemical indexes (RNA/DNA and Protein/DNA). The effect of acclimatization temperature on the instantaneous growth and physiological condition of Perna viridis was studied in organisms collected in La Esmeralda, Sucre State (Venezuela) and taken to the laboratory, where groups of 100 organisms (size 3.0 - 3.5 cm, anteroposterior measurement) were acclimatized at 15, 20, 26 or 28 degrees C during four weeks. Later they were kept in a 60 liters aquarium for another six weeks under the same conditions. Each week, ten organisms per group were extracted to measure concentrations of RNA, DNA (by a fluorometric method with ethidium bromide) and proteins (by a colorimetric method), in tissues (digestive gland, adductor muscle and gills). Protein concentration was greater and highly significant at 15 degrees C for all studied tissues. The opposite was obtained with the RNA/DNA and Protein/DNA ratios: the greatest increase was observed at the highest temperature (28 degrees C) for all tissues. At the lowest temperature there was a tendency to reduce both indexes with time. Greater instantaneous growth can be expected at higher temperatures and 28 degrees C was optimal for growth in these specimens.

[Food effect on the physiological condition of the mussel Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio]. / Efecto de la alimentación sobre la condición fisiológica del mejillón Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN.

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.

[Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)]. / Actividad antimicrobiana de extractos orgánicos aislados de Aplysina fistularis (Demospongiae: Aplysinidae).

Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The minimal inhibitory concentration (MIC) and toxic activity of extract were determined. Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E. coli, while CHCl3 fraction inhibited E. coli and S. aureus growth. The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria. Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E. coli and S. aureus. In Sf2 EtOAc/MeOH, MIC was moderate for S. aureus (MIC > 256 g/ml). F4 CHCl3/MeOH produced a high inhibition in S. aureus (MIC = 0.125 g/ml) and for E. coli (MIC > 16 g/ml). F10 CHCl3/MeOH showed a moderate activity against S. aureus (MIC > 128 g/ml) and low activity against E. coli (MIC = 512 g/ml). F10 CHCL3/MeOH did no present toxic activity against Artemia salina. The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml. LC50 in both cases was 548.4 and 243.4 microg/ml respectively. Secondary metabolites of medium polarity obtained from A. fistularis have a wide spectrum of anti bacterial activity. Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.

von Willebrand factor contained in a high purity FVIII concentrate (Fanhdi) binds to platelet glycoproteins and supports platelet adhesion to subendothelium under flow conditions.

BACKGROUND AND OBJECTIVE: There is evidence suggesting that von Willebrand factor (VWF) from high purity factor VIII concentrates could be of clinical use in the management of patients suffering from VWD. We analyzed structural and functional characteristics of VWF present in a high purity factor VIII concentrate VWFHPC (Fanhdi). The multimeric structure, the ability to bind to platelet GP Ib/IX or GP IIb/IIIa, and the capacity of VWFHPC to promote platelet adhesion on injured vessels were investigated and compared with that present in standard plasma cryoprecipitates [VWFCRYO]. DESIGN AND METHODS: Binding studies were carried out by incubating radiolabeled VWF and washed platelets, which were activated with either ristocetin (1 mg/mL; for GP Ib/IX), or thrombin (2.5 U/mL; for GP IIb/IIIa). Platelet adhesion was assessed in a perfusion system (shear rate = 800 s-1, 10 min) in which the source of VWF was added (at 0.4 or 0.8 U/mL VWF:Ag) to washed platelets and red cells suspended in a human albumin solution. The deposition of platelets onto the perfused subendothelial surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). RESULTS: The VWFHPC (152 Units VWF:RCof/mg protein; VWF:RCof/VWF:Ag = 0.97), lacked only a small proportion of high-molecular-weight multimers present in VWFCRYO. Binding affinities (Kd values, nM) of VWFHPC were similar to those of VWFCRYO (5.3 +/- 0.86 vs 5.2 +/- 0.95, for GP Ib/IX; and 11.6 +/- 2.7 vs 15.4 +/- 1.7 for GPIIb-IIIa). A slightly, though not significantly, higher binding capacity for these receptors (Bmax values, molecules/pit) was obtained for VWFHPC. The %SC in perfusions in the presence of albumin was < 10%. Addition of VWFHPC or VWFCRYO significantly increased the %SC, with values of 27.1 +/- 4.9 and 17.5 +/- 2.8%, respectively with 0.4 U/mL (p < 0.004 and p < 0.02 vs albumin); and 30.8 +/- 4.9% and 20.03 +/- 4.1%, respectively, at 0.8 U/mL (p < 0.001 and p < 0.02 vs albumin). INTERPRETATION AND CONCLUSIONS: Our data show that VWF present in the high purity FVIII concentrate Fanhdi retains the functional capacity to bind to GPs Ib/IX and IIb/IIIa and to promote platelet adhesion onto exposed subendothelium.

[No ELISA detectable alterations in immunogenicity following dry-hear treatment (72 hours at 80 degrees C) of FANHDI]. / Ausencia de alteraciones detectables por ELISA en la inmunogenicidad de FANHDI debidas al proceso de calentamiento en seco (72 horas a 80 degrees C).

OBJECTIVE: Neoantigen formation during heat treatment (HT) of factor VIII:von Willebrand Factor (FVIII:vWF) concentrates may induce an immune response against the modified protein, which may also affect the native protein. We present a comparative in vitro study on the immunogenicity of a dual virally inactivated (solvent-detergent and 80 degrees C 72 hours) high purity FVIII:vWF concentrate (Fanhdi) versus the same product without heat treatment. MATERIAL AND METHODS: For this purpose rabbit antisera were prepared using both Fanhdi and the same product from which the human albumin, used as stabilizer, had been removed (these were both HT products). Also, antisera were prepared against the same products made without the dry-heat treatment step (non-HT products). Antisera were analysed by Elisa. Mixtures of antisera with increasing amounts of product (incubation-absorption in liquid phase) were assayed in plates coated with HT and non-HT products. RESULTS: The binding of antibodies against HT products to ELISA plates coated with HT products, could be blocked (in a saturable manner) with non-HT products, following liquid phase incubation. These results strongly suggest the absence of neonantigens. Furthermore, the binding of antibodies against non-HT products to ELISA plates coated with non-HT products, could be blocked (also in a saturable manner) with HT products. This result indicates that there is no epitope loss. CONCLUSIONS: The data obtained in these studies suggest that the heat treatment of viral inactivation as applied in Fanhdi, does not give rise to any major alteration in immunogenicity of the product. The data from clinical and drug surveillance studies carried out with Fanhdi do not show any indication of an increase in the frequency of inhibitors.

Functional compensation of the low platelet count by increased individual platelet size in a patient with May-Hegglin anomaly presenting with acute myocardial infarction.

Platelets are known to play a crucial role in normal hemostasis as well as in thrombus formation at sites exposed to blood flow, as in coronary thrombosis. Thus, low platelet count is a strong negative risk factor for the occurrence of arterial thrombosis, such as occurs in acute myocardial infarction. We encountered a patient with May-Hegglin anomaly, presenting with acute myocardial infarction in his sixth decade, even though his platelet counts had always been less than 50 x 10(3)/microl. We investigated the characteristics of his platelets under the effect of shearing and found that shear-induced platelet aggregation and binding of soluble von Willebrand factor (vWF) to platelets could be induced, even when the patient's platelet count was less than 10 x 10(3)/microl, but that virtually no aggregation or vWF binding by normal platelets could be induced by shearing when platelet counts were less than 50 x 10(3)/microl. We conclude that the low platelet counts in a patient with May-Hegglin anomaly can be functionally compensated for by larger individual platelets, in view of the vWF-dependent platelet thrombus formation occurring under the effect of blood flow and that that is why most patients with May-Hegglin anomaly do not have a bleeding tendency, even though their platelet counts are very low.

Identification of human nonpancreatic-type ribonuclease by antibodies obtained against a synthetic peptide.

An antibody that recognizes human nonpancreatic-type ribonuclease was obtained by immunizing a rabbit with a 14-residue synthetic peptide corresponding to the N-terminal sequence of eosinophil-derived neurotoxin which is identical to human liver ribonuclease. This amino acid sequence is unique to this protein. The anti N-peptide antibody was purified by protein A-Sepharose and by using ELISA and SDS-PAGE immunoblot techniques, the antibody reactivity against EDN and partially purified nonpancreatic-type ribonucleases from human plasma and urine was observed. Cross-reactivity with bovine pancreatic ribonuclease A and other proteins was not detected. In addition, the activity of the nonpancreatic-type ribonuclease was not affected by the antibody. The immune response was elicited without the need for a carrier protein showing that the N-terminal sequence of nonpancreatic ribonuclease contains a specific epitope. This antibody can be used for the immunological identification of both the native and denatured forms of this type of enzyme.