Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 g/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.     

Paratuberculosis in free-ranging fallow deer in Spain

Paratuberculosis was diagnosed in a population of approximately 1,000 free-ranging fallow deer (Dama dama) sampled from 1997-98 in the Regional Hunting Reserve of El Sueve (Asturias, Spain). Five of eight animals observed with diarrhea were diagnosed as having paratuberculosis on the basis of gross lesions at postmortem examination and histopathology. In two deer, Mycobacterium avium subsp. paratuberculosis was cultured and identified by polymerase chain reaction. Indirect enzyme-linked immunosorbent assay and immunodiffusion tests were used to evaluate sera from 33 adult deer from this population. All fallow deer tested were seronegative.     

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

Previous studies have demonstrated that phenolic compounds, including genistein (4',5,7-trihydroxyisoflavone) and resveratrol (3,4',5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in phi X-174 plasmid DNA. H(2)O(2)/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H(2)O(2)/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H(2)O(2) were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Altering the expression kinetics of VP5 results in altered virulence and pathogenesis of herpes simplex virus type 1 in mice

While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U(L)38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.     

The role of astroglia on the survival of dopamine neurons

Glial cells play a key role in the function of dopamine (DA) neurons and regulate their differentiation, morphology, physiological and pharmacological properties, survival, and resistance to different models of DA lesion. Several studies suggest that glial cells may be important in the pathogenesis of Parkinson’s disease (PD), a common neurodegenerative disorder characterized by degeneration of the nigrostriatal DA system. In this disease the role of glia could be due to the excessive production of toxic products such as nitric oxide (NO) or cytokines characteristic of inflammatory process, or related to a defective release of neuroprotective agents, such as small antioxidants with free radical scavenging properties or peptidic neurotrophic factors.     

Pre-implantational timetable of embryonal development of Myocastor coypus (Coypu)

The purpose of the present study was to determine the chronology of the pre-implantation embryonic development in Myocastor coypus (coypu). It was carried out by daily colpocytological examination and controlled mating of 33 females. Oocytes and embryos were obtained by flushing from day 0 to day 10 post-coitus (p.c.). On day 1 p.c., oocytes predominated whereas on day 2 p.c. zygotes were predominant. The cleavage period was from day 3 to day 6 p.c.. Morulae were collected from day 6 to day 9 p.c., whereas blastocysts were collected on days 8 and 9. From oviduct flushing, the embryos in the zygote stage and up to the morula stage with less than a 30-cell stage were recovered. Embryos in the morula stage with 30 or more cells and up to the growing blastocyst stage were collected from the flushing of hemiuteri.     

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft?Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, were port an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at ～4 °C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85%glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchangedmechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.     

Postnatal maturation in nitric oxide-induced pulmonary artery relaxation involving cyclooxygenase-1 activity

The maturation in the vasodilator response to nitric oxide (NO) in isolated intrapulmonary arteries was analyzed in newborns and 15- to 20-day-old piglets. The vasodilator responses to NO gas but not to the NO donor sodium nitroprusside increased with age. The inhibitory effects of the superoxide dismutase inhibitor diethyldithiocarbamate and xanthine oxidase plus hypoxanthine and the potentiation induced by superoxide dismutase and MnCl(2) of NO-induced vasodilatation were similar in the two age groups. Diphenyleneiodonium (NADPH oxidase inhibitor) potentiated the response to NO, and this effect was more pronounced in the older animals. The nonselective cyclooxygenase inhibitors indomethacin and meclofenamate and the preferential cyclooxygenase-1 inhibitor aspirin augmented NO-induced relaxation specifically in newborns, whereas the selective cycloxygenase-2 inhibitor NS-398 had no effect. The expressions of alpha-actin, cycloxygenase-1, and cycloxygenase-2 proteins were similar, whereas Cu,Zn-superoxide dismutase decreased with age. Therefore, the present data suggest that the maturational increase in the vasodilatation of NO in the pulmonary arteries during the first days of extrauterine life involves a cycloxygenase-dependent inhibition of neonatal NO activity.     

In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep.

The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied. HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties. After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm. HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient. The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein. Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV.