In vitro germination and reserve mobilization of Vriesea friburgensis Mez.

Studies on the germination and establishment of plants are key pieces to understanding the reproductive success of plants. This work aimed to describe in vitro germination and reserve mobilization in the bromeliad Vriesea friburgensis through morphological, histochemical, and biochemical analysis. The conditions used in this study for the in vitro germination are adequate. From the third day of in vitro inoculation, a uniform germination of 98% was obtained, exhibiting a high physiological quality of the seeds and a high potential to produce seedlings (94%). There is early reserve mobilization, which began in the imbibition phase. The accumulated reserves in the endosperm cytoplasm are degraded by hydrolytic enzymes provided by the aleurone layer. It is possible that compounds in the cell walls of the endosperm contribute to a lesser extent in mobilization. Additionally, it was observed that starch accumulation in the cotyledon increases when the seedling has formed. Results from this study provide insights for future studies on ecology, seed technology, and conservation in this species. This study contributes to the limited knowledge of the dynamics of reserves during germination and seedling establishment in Bromeliaceae. To the best of our knowledge, this is the first study with this approach in the genus Vriesea.

Parainfluenza-3 and bovine respiratory syncytial virus: intraherd correlation adjusted for sensitivity and specificity / Parainfluenza-3 y virus respiratorio bovino sincitial: correlación intrahato ajustado por la sensibilidad y especificidad

Objective. The purpose of this study was to compare the intra-class correlation coefficients (ICC) and design effects (D) estimates adjusted or unadjusted for sensibility (Se) and specificity (Sp) of the diagnostic tests using a Bayesian procedure. Materials and methods. Sera from 232 animals from 44 randomly selected herds, to detect antibodies against parainfluenza-3 virus (PIV3) from non-vaccinated dual-purpose cattle from Colima Mexico, were used. Only 176 animals from 33 herds were used to evaluate the presence of the bovine respiratory syncytial virus (BRSV). Results. The ICC and D values adjusted and unadjusted for PIV3 were 0.33, 2.73, 0.32, and 2.71, respectively. For BRSV the values were 0.31, 2.64, 0.28 and 2.49. Conclusions. The adjusted or unadjusted ICC and D estimates were similar because of the high Se and Sp of the diagnostic tests and the relatively high prevalence of the diseases here studied.

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Defibrotide: properties and clinical use of an old/new drug.

The drug named defibrotide (DFT) has been studied for many years. It has been shown to possess many activities: profibrinolytic, antithrombotic-thrombolytic, antiischemic (heart, liver, kidney, skin, brain), antishock, antiatherosclerotic, antirejection and anti-angiogenic. The previously displayed activities, as antithrombotic, profibrinolytic and anti-inflammatory, suggested its use in vascular disorders, as in the treatment of peripheral obliterative arterial disease and in thrombophlebitis. Some years after, the use of DFT in hepatic veno-occlusive disease has been also proposed. Even if DFT was considered for long time a multi-target drug, now it could be considered on the whole as a drug able to protect endothelium against activation. The present work reviews the more important experimental and clinical studies performed to detect DFT effects.

Aclimatação de gengibre (Zingiber officinale Roscoe) e a relação com carboidratos endógenos / Acclimation of ginger (Zingiber officinale Roscoe) and its relation to endogenous carbohydrates

O objetivo deste trabalho foi determinar as variações dos teores de açúcares solúveis totais e de reserva nas diferentes fases de desenvolvimento de mudas de gengibre. Foram avaliados os parâmetros de sobrevivência, teores de açúcares solúveis totais e de reserva na fase de micropropagação, aclimatação e cultivo em campo. A sobrevivência das plantas no processo de aclimatação aos 60 dias foi de 100 por cento, enquanto que as plantas em condições de campo apresentaram um índice de sobrevivência de 80 por cento aos 60 dias de cultivo. Os teores médios de amido, no primeiro ano de cultivo, foram superiores nas plantas micropropagadas, tanto nas folhas (303,19±0,17 mgGlu gMF-1 mgGlu gMF-1) quanto nas raízes (3341,59±1,24 mgGlu gMF-1). Foram observados altos teores médios de amido nos rizomas (164,91±2,4 mgGlu gMF-1) e gemas (190,88±0,25 mgGlu gMF-1). Os teores médios de açúcares solúveis totais apresentaram um decréscimo nas plantas in vitro com valores médios de 86,56±0,55 mgGlug MF-1 e 94,26±0,40 mgGlu gMF-1 para folhas e raízes, respectivamente. Obteve-se um aumento nesses valores nas plantas aclimatadas em casa de vegetação, tanto nas folhas (168,22±0,77 mgGlu gMF-1) quanto nas raízes (189,68±0,70 mgGlu gMF-1), sendo superadas pelos teores médios de folhas e raízes das plantas cultivadas a campo, cujos valores foram 227,51±0,8 e 183,97±0,32 mgGlu gMF-1, respectivamente. No segundo ciclo de cultivo observou-se um aumento nos teores médios de amido nos rizomas, gemas e raízes, com valores de 210,87±0,85 mgGlu gMF-1 , 203,45±0,91 mgGlu gMF-1 e 201 ±0,69 mgGlu gMF-1 e um declínio nos teores médios nas folhas (41,55±0,88 mgGlu gMF-1) com uma queda gradativa até os 240 dias de cultivo. Porém, em relação à quantidade de açúcares solúveis totais as folhas apresentaram-se com teores médios altos no período inicial, com um declínio ao longo do desenvolvimento em casa de vegetação. O oposto foi verificado para as raízes (225,29±0,75 mgGlu gMF-1), rizoma (250,08±0,93 mgG...

The aim of this work was to determine the variations in total soluble sugar and starch levels during the different development stages of ginger seedlings. Survival parameters, as well as total soluble sugar and starch levels, were evaluated during micropropagation, acclimation and field cultivation phases. The survival of plants was 100 percent after 60 days of acclimation, whereas under field conditions such value was 80 percent after the same period. In the first year of cultivation, mean starch levels were higher in micropropagated plants, both in leaves (303.19 ± 0.17 mgGlu gFM-1) and in roots (3341.59 ± 1.24 mg Glu gFM-1). High mean starch levels were detected in rhizomes (164.91 ± 2.4 mg Glu gFM-1) and buds (190.88 ± 0.25 mgGlu gMF-1). The mean levels of total soluble sugars were lower in plants in vitro: 86.56 ± 0.55 mgGlu gFM-1 and 94.26 ± 0.40 mgGlu gFM-1 in leaves and roots, respectively. Higher values were obtained for plants acclimatized in greenhouse, both in leaves (168.22 ± 0.77 mgGlu gFM-1) and roots (189.68 ± 0.70 mgGlu gFM-1); these values were inferior to those detected in leaves and roots of plants grown in the field, which were 227.51 ± 0.8 and 183.97 ± 0.32 mgGlu gFM-1, respectively. In the second cultivation cycle, mean starch levels in rhizomes, buds and roots increased to 210.87 ± 0.85 mgGlu gFM-1, 203.45 ± 0.91 mgGlu gFM-1 and 201 ± 0.69 mgGlu gFM-1, respectively, whereas in leaves these values progressively decreased (41.55 ± 0.88 mgGlu gFM-1) until 240 days of cultivation. However, leaves presented high mean total soluble sugar levels in the initial period, with a posterior decline over the development in greenhouse. The opposite was observed in roots (225.29 ± 0.75 mgGlu gFM-1), rhizomes (250.08 ± 0.93 mgGlu gFM-1) and buds (225.75 ± 0.80 mgGlu gFM-1). Based on the high survival rate of acclimatized plants and the higher levels of total soluble sugars and starch in early stages of cultivation, acclimation is recommended to assure...

Antithrombin activity of an algal polysaccharide.

In an effort to reduce the risks of a possible iatrogenic transmission of bovine spongiform encephalitis (BSE) through the use of bovine-derived medicinal products, we patented in the USA in 1999 a polysaccharide from brown algae, endowed with interesting pharmacological activities: (a) concentration-dependent inhibition of thromboplastin or cephalin-kaolin-induced thrombin generation from platelets, (b) concentration-dependent inhibition of thrombin-induced platelet aggregation, (c) thrombin has hypotensive effect, which was blunted and zeroed by our fucansulfate in a dose-dependent way, (d) when aortae are stimulated with thrombin, they become stickier for polymorphonucleated leukocytes (PMNs); our fucansulfate decreased concentration-dependently, PMNs sticking to autologous rabbit aortae, (e) dose-dependent inhibition of thrombin-induced thrombosis. All the above data suggest that our fucansulfate could be a heparin substitute endowed with antithrombotic and anti-inflammatory activities, devoid or the problems caused to heparin by its animal origin, i.e., possible prion protein contamination.

Defibrotide normalizes cardiovascular function hampered by established atherosclerosis in the rabbit.

In a previous paper we gave evidence that chronic oral defibrotide antagonizes the noxious effect of developing atherosclerosis in the cardiovascular system. In the present paper we give evidence that defibrotide is still capable of exerting beneficial effects on cardiovascular function once atherosclerosis is established. In fact, there was statistically significant amelioration by defibrotide infusion in the following, all of which were hampered by established atherosclerosis: in rabbit aorta relaxation to acetylcholine, prostaglandin E2, and 6-keto-prostaglandin F1alpha generation from rabbit aortas, rabbit heart left ventricular end-diastolic pressure, coronary perfusion pressure, and left ventricular developed pressure, vasopressor activity of acetylcholine and endothelin-1 on coronary perfusion pressure, and 6-keto-prostaglandin F1alpha generation from the rabbit heart. Since prostacyclin takes part in NO generation, is cellular protective, and inhibits 5-lipoxygenase product synthesis, its increase, caused by defibrotide, could explain defibrotide cardioprotective activity. Prostacyclin activity could be backed by prostaglandin E2, another cardioprotective prostaglandin.

Chronic oral defibrotide counteracts hypercholesterolemia noxious effects on cardiovascular function in the rabbit.

The aim of the present work was to assess if the cardioprotective drug defibrotide could counteract the hypercholesterolemia noxious effects on cardiovascular function. Aortas and hearts from normal- or cholesterol-fed rabbits, treated or not with chronic oral defibrotide (100 mg/kg/day) for 45 days, were used in in vitro tests throughout the experiment. Hypercholesterolemia worsened: aorta stickiness toward polymorphonuclear leukocytes, aorta relaxation to acetylcholine, heart left ventricular end-diastolic pressure and coronary perfusion pressure, heart left ventricular diastolic pressure, acetylcholine and endothelin-1 activity on coronary perfusion pressure, and heart generation of 6-Keto-prostaglandin F1alpha. Oral defibrotide counteracted and/or obliterated the above hypercholesterolemia noxious effects. Particularly, oral defibrotide counteracted the parameters associated with early endothelial cell disfunction: that is, increased adherence of leukocytes to endothelium and endothelial vasorelaxation induced by acetylcholine, which acts through the release of endothelium-derived relaxing factor. These activities of defibrotide are probably exerted through the increased generation of prostacyclin. The fact that acetylcholine induced vasorelaxation is partially protected by oral defibrotide points to a partial rescue of endothelial ability to generate endothelium-derived relaxing factor, as acethylcoline acts through the release of endothelium-derived relaxing factor, by defibrotide itself. Defibrotide's endothelial protection could, in turn, explains why defibrotide protected cardiovascular function. This is not surprising as, in a few cases, endothelial dysfunction, observed in hypercholesterolemia, was found to be prevented or reversed, pharmacologically, by PN-2001-10, a calcium channel blocker, dipyridamole, and lovastatin.

Pharmacokinetics, absorption, distribution and disposition of [125I]-oligotide following intravenous or oral administration in the rat.

Oligotide (O) was labelled with 125I. The radiolabelled compound ([125I]-Oligotide ([125I]-O)) retained the biological activity of parent O. Following single intravenous administration the half lives of radioactivity associated with O and/or O related components in plasma were 9-10 min and 9-10 h for alpha and beta phases respectively. Following single oral administration the half life of radioactivity associated with O and/or O related components in plasma was 11.45-12.76 h for beta fase. Following multiple oral administration once daily for 7 days, the half life of radioactivity associated with O and/or O related components following the 7th dose was 10-12 h for beta phase. The areas under plasma total radioactivity versus time curves were dose-dependent. Following single intravenous administration the major proportion of the administered dose was excreted via urine, while following single oral administration excretion via urine and faeces accounted for similar proportions of the administered dose. Following both single and oral administration the levels of radioactive components derived from [125I]-O in organs examined were generally highest in highly perfused organs.

Correlation between serum HCV RNA and aminotransferase levels in patients with chronic HCV infection.

Cross-sectional studies on the correlation between serum hepatitis C virus (HCV) RNA and alanine aminotransferase (ALT) levels in patients with chronic hepatitis C have yielded conflicting results. We conducted a longitudinal study to examine the correlation between HCV viremia and serum ALT levels in individual patients over time. Serial samples (mean 9) from 25 patients with chronic HCV infection, including interferon-treated and untreated immunocompetent and immunosuppressed patients, collected over a period of 1-4.8 years (mean 2.6 years) were tested for HCV RNA and ALT levels using a highly reproducible quantitative (bDNA) assay. A significant correlation was found between serum HCV RNA and ALT levels in the patients who received IFN therapy, but no correlation was observed in the untreated patients. Among the untreated patients, the immunosuppressed patients had significantly higher HCV RNA levels (39 +/- 4 vs 3.6 +/- 8 Meq/ml, P < 0.0001) but significantly lower ALT (56 +/- 11 vs 97 +/- 12 units/liter, P = 0.03) levels when compared to the immunocompetent ones. In summary, we found no correlation between serum HCV RNA and ALT levels in chronic hepatitis C patients who are not receiving interferon therapy. Immunosuppression results in higher HCV RNA but lower ALT levels.

Effect of human immunodeficiency virus infection on hepatitis C virus infection in hemophiliacs.

Chronic liver disease due to hepatitis C virus (HCV) infection is a major problem in hemophiliacs. Recent reports suggested that hemophiliacs coinfected with hepatitis C virus and human immunodeficiency virus (HIV) have an increased incidence of liver failure but the mechanism of accelerated liver injury is not clear. We tested plasma from 100 hemophiliacs for anti-HCV by second generation ELISA, anti-HIV by EIA, and HCV RNA and HIV RNA by branched DNA and polymerase chain reaction assays to determine if hemophiliacs coinfected with HCV and HIV have higher HCV RNA levels and more active liver disease. Seventy-nine (79%) patients were anti-HCV positive, of whom 85% were HCV RNA positive. None of the anti-HCV-negative patients had detectable HCV RNA in plasma. Forty-two (42%) patients were anti-HIV positive, of whom 47% had detectable HIV RNA. All the anti-HIV-positive patients were also anti-HCV positive. The prevalence of both anti-HCV and anti-HIV increased significantly with age. There was no difference in HCV RNA levels between anti-HIV-positive and anti-HIV-negative patients (mean: 21 +/- 4 vs 18 +/- 5 Meq/ml), although HCV RNA levels were significantly higher in anti-HIV-positive patients with CD4 counts < 200/mm3 (P = 0.008). There was an inverse correlation between HCV RNA levels and CD4 counts but no correlation was found between HCV RNA and serum aminotransferase levels. We found a high prevalence of HCV and HIV coinfection in our hemophiliacs. Hepatitis C virus replication appears to be increased in patients with severe immunodeficiency secondary to progressive HIV infection. However, there was no correlation between HCV RNA and serum ALT level, suggesting that HCV is not directly cytopathic.

Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response.

An integrated view of the activities of defibrotide.

Defibrotide is a polydeoxyribonucleotide sodium salt extracted from mammalian organs. Its mean molecular weight is 15,000-30,000 daltons. Defibrotide contains aptamers, i.e., single-stranded polynucleotides with a well-defined base sequence and composition (5'-GGTTGG-ATT-GGTTGG-3' and 5'-GGTTGG-ATC-GGTTGG-3') that bind thrombin. For the time being, these aptamers are the only ones that have been identified in defibrotide, but there may also be other aptamers that bind proteins other than thrombin. Defibrotide has no anticoagulant activity, but in some other aspects it probably parallels heparin. Heparin is a sulfated polysaccharide sodium salt with a mean molecular weight ranging from 5,000 to 40,000 daltons extracted from mammalian organs. It contains disaccharide sequences of well-defined structure and frequency. Heparin binds an array of proteins, enzymes, and growth factors and shows inhibitory or stimulatory or protective activity. Defibrotide has a spectrum of interesting pharmacological properties that make this drug very useful for the treatment of arterial and venous thrombotic diseases. In fact, defibrotide has profibrinolytic, antithrombotic-thrombolytic, anti-ischemic, and antiatherosclerotic activity and protective activity in shock. What have all the above activities to do with a single drug? The explanation is that atherosclerosis, myocardial, renal, and liver ischemia, hemorrhagic and septic shock, and shock induced by occlusion of splanchnic artery and thrombosis are different facets of the same entity: the polyhedric inflammatory process.

Study on pharmacokinetics of radioactive labelled defibrotide after oral or intravenous administration in rats.

Defibrotide (D) was labelled with 125I or with 32P. The radiolabelled compounds ([125I]-Defibrotide ([125I]-D), [32P]-Defibrotide ([32P]-D) retained the same profibrinolytic activity, in vitro, as the parent drug, suggesting that the labelling procedures had not modified the pharmacological properties of D and hence that its chemical structure was not affected significantly. After single intravenous or oral administration of [125I]-D or [32P]-D the pharmacokinetic parameters for the two labels were generally in good agreement (75%). t 1/2 alpha was in the range of minutes while t 1/2 beta was in the range of hours. Bioavailability, following single oral administration of [125I]-D or [32P]-D, was in the range of 58-70%. These data suggest that D, in spite of its macromolecular nature, is absorbed, after oral administration, fairly well.

Is defibrotide's activity on leukocytes adenosine-receptor mediated? An "in vitro"--"ex vivo" appraisal.

Defibrotide both "in vitro" and "ex vivo" decreases PMN adhesion to nylon columns. This activity is blunted by 8-phenyltheophylline, a known blocker of adenosine A1/A2 receptors, suggesting that Defibrotide acts on that kind of receptors. Indomethacin, a blocker of PGI2 synthesis, did not cancel DFT's activity, suggesting that PGI2 is not involved in DFT's activity, at least under our experimental conditions. Non-PMN leukocytes behaved differently "ex vivo" than PMNs. DFT's activity was not blunted by 8-phenyltheophylline, suggesting that DFT acts on this kind of cell through a different mechanism. The "in vitro" experiment did not confirm "ex vivo" studies suggesting that in the "in vitro" experiment "something" is missing that is present in "ex vivo" experiments.

Effect of chronic administration of hydrocortisone on the induction and evolution of acute pancreatitis induced by cerulein.

The effects of chronic administration of hydrocortisone (10 mg/kg/day) on the development and evolution of acute pancreatitis induced by supramaximal stimulation with cerulein were examined in the rat. In these circumstances the potentially therapeutic effect of L-364,718, a CCK-receptor antagonist, was assayed. Administration of hydrocortisone over 7 days did not increase the severity of edematous acute pancreatitis induced by cerulein, since the reduction in pancreatic secretion, the hyperamylasemia and the increase in the levels of hematocrit and fluid in the pancreatic tissue were similar in rats with acute pancreatitis treated and untreated with hydrocortisone previously. When hydrocortisone was administered chronically, before administration of supramaximal doses of cerulein, a spontaneous regression of acute pancreatitis occurred. However, when hydrocortisone administration was continued after inducing pancreatitis, pancreatic recovery was prevented, observing a significantly depressed acinar secretion and elevated values of hematocrit and tissue fluid (edema). L-364,718 administration proved to be detrimental in the evolution of edematous acute pancreatitis when the rats had been treated chronically with hydrocortisone because the blockade exerted on secretion prevented the draining of enzymes stored in excess by hydrocortisone administration.

Action of CCK on CDE diet-induced acute pancreatitis in rats treated with hydrocortisone.

The present work studies the effect of previous hydrocortisone administration (10 mg/kg/day) over 7 days on the later development of diet-induced acute pancreatitis in the rat. Acute pancreatitis was induced by feeding a diet deficient in choline and supplemented with 0.5% ethionine (CDE diet) over 10 days. Hydrocortisone pretreatment exacerbated CDE-induced acute pancreatitis. There was a significant increase in serum amylase, pancreatic edema, and haematocrit levels and an insignificant decrease in pancreatic mass in rats pretreated with hydrocortisone. Pancreatic enzyme secretion was strongly reduced in the rats subjected to acute pancreatitis, and although the drop in enzyme levels did not reach statistical significance, the values of secretion were even further reduced in the animals treated with hydrocortisone, pointing to the absence of pancreatic functionality. This effect can be attributed to enzyme storage elicited by previous hydrocortisone administration; activated intracellularly, these enzymes could aggravate the pathology. Administration of the cholecystokinin octapeptide (CCK-8) (10 micrograms/kg/day) during the development of acute pancreatitis in animals pretreated with hydrocortisone substantially improved the general state of the animals' pancreases. There was a significant decrease in serum amylase, pancreatic edema and haematocrit levels in rats injected with CCK, which was accompanied by an increase in pancreatic functionality. Conversely, the administration of L-364,718 (0.1 mg/kg/day), a CCK antagonist, did not improve pancreatic functionality and did not appreciably affect the general state of the organ. It is concluded that in rats with storage levels increased by hydrocortisone administration that are subjected to acute pancreatitis, the secretagogue effect of CCK is more beneficial than the repose of the gland induced by L-364,718.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligotide, a new single-stranded oligodeoxyribonucleotide, preserves postsynaptic beta-adrenergic and cholinergic receptor functions in rabbit hearts after acute infarction.

Acute myocardial infarction was induced in New Zealand male albino rabbits. 3 days after surgery, the mortality rate and plasma CPK activity were reduced by 64% and 66% respectively by intravenous infusion of Oligotide (32 mg/kg/h for 6h), a single-stranded oligodeoxyribonucleotide of mammalian origin. Perfused hearts, obtained from Oligotide-treated rabbits 3 days after surgery, showed a strength of contraction in the range of that of hearts of sham-operated animals and an ameliorated postsynaptic beta-adrenergic and cholinergic receptor function. In addition, in these hearts, "ex vivo" treatment with Oligotide resulted in almost complete preservation of both adrenergic and cholinergic responses to their specific agonists. These data suggest that Oligotide by protecting the hearts from the ischemic damage and possibly by restricting left ventricular tissue necrosis may have normalized the biological responses of this organ to isoproterenol and preserved the integrity of coronary endothelial-dependent relaxant function.

Defibrotide's activity on leukocytes and platelets in rabbits with diet-induced atherosclerosis.

Oral Defibrotide decreased leukocyte and platelet counts, raised by cholesterol diet, and the area (%) of aorta endothelial surface involved in atherosclerosis. Frequency of intimal thickening in blood vessels of kidneys and hearts and in cardiac valves was reduced by oral Defibrotide by 47%, 29% and 17%. It is suggested that oral Defibrotide reduced the involvement of the aorta in the atherosclerotic process by acting on leukocytes and platelets, to both reduce their number and deactivate them.

Effects of defibrotide on leukocytosis in rabbits with diet-induced atherosclerosis.

The antithrombotic drug Defibrotide (DFT) (a polydeoxyribonucleotide with a mean MW of 20,000 Daltons) reduces the number of leukocytes and platelets in thrombi. Because leukocytes and platelets are of importance in the genesis of endothelial lesions leading to atherosclerosis, DFT was given to challenge leukocytosis in rabbits with diet-induced atherosclerosis (0.25% cholesterol for 16 weeks). After 9 weeks of cholesterol feeding and at the end of experiment, oral DFT (60 mg/Kg per day) had decreased the leukocyte count raised by the cholesterol diet. Leukocyte stickiness and leukocyte differential counts were not modified by either oral cholesterol or by oral cholesterol plus oral DFT. At the end of experiment, oral DFT had normalized the platelet count increased by cholesterol diet. The red blood cell count decreased by oral cholesterol at 9 weeks and at the end of experiment was normalized by DFT. The % of aortae endothelial surface involved in the atherosclerotic process was decreased by oral DFT. The frequencies of intimal thickening in blood vessels of kidneys and hearts and in cardiac valves were reduced by oral DFT by 47%, 29% and 17%, although these reductions were not statistically significant. It is suggested that DFT, by preventing the increase in the number of leukocytes and platelets and deactivating them, as demonstrated in papers already published, was able to counteract against the atherosclerotic process.

Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA.

There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides. Enzyme-labelled probes are bound to the arms of the bDNA and light output from a chemiluminescent substrate is directly proportional to the amount of starting HCV RNA. Appropriate standards provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal. In testing a standardized, coded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all six infectious sera. Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commercial seroconversion panels. In acute cases, 10(7)-10(8) molecular equivalents per ml (eq per ml) of HCV RNA were detected prior to peak alanine aminotransferase (ALT) activity and then rapidly declined to non-detectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chronic course was characterized by diminished, fluctuating and sometimes non-detectable levels of HCV RNA. In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when assayed by PCR. In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship between viral replication and subsequent hepatocellular injury. In testing 50 blood donors whose anti-HCV reactivity was confirmed by a recombinant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81%), while PCR was positive in 45 (90%); the overall concordance between bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive donor samples was 96%.(ABSTRACT TRUNCATED AT 400 WORDS)