Perinatal and long-term effects of maternal uterine artery adenoviral VEGF-A165 gene therapy in the growth-restricted guinea pig fetus.

Uterine artery application of adenoviral vascular endothelial growth factor A165 (Ad.VEGF-A165) gene therapy increases uterine blood flow and fetal growth in experimental animals with fetal growth restriction (FGR). Whether Ad.VEGF-A165 reduces lifelong cardiovascular disease risk imposed by FGR remains unknown. Here, pregnant guinea pigs fed 70% normal food intake to induce FGR received Ad.VEGF-A165 (1×1010 viral particles, n = 15) or vehicle ( n = 10), delivered to the external surface of the uterine arteries, in midpregnancy. Ad libitum-fed controls received vehicle only ( n = 14). Litter size, gestation length, and perinatal mortality were similar in control, untreated FGR, and FGR+Ad.VEGF-A165 animals. When compared with controls, birth weight was lower in male but higher in female pups following maternal nutrient restriction, whereas both male and female FGR+Ad.VEGF-A165 pups were heavier than untreated FGR pups ( P < 0.05, ANOVA). Postnatal weight gain was 10-20% greater in female FGR+Ad.VEGF-A165 than in untreated FGR pups, depending on age, although neither group differed from controls. Maternal nutrient restriction reduced heart weight in adult female offspring irrespective of Ad.VEGF-A165 treatment but did not alter ventricular wall thickness. In males, postnatal weight gain and heart morphology were not affected by maternal treatment. Neither systolic, diastolic, mean arterial pressure, adrenal weight, nor basal or challenged plasma cortisol were affected by maternal undernutrition or Ad.VEGF-A165 in either sex. Therefore, increased fetal growth conferred by maternal uterine artery Ad.VEGF-A165 is sustained postnatally in FGR female guinea pigs. In this study, we did not find evidence for an effect of maternal nutrient restriction or Ad.VEGF-A165 therapy on adult offspring blood pressure.

Maternal Uterine Artery Adenoviral Vascular Endothelial Growth Factor (Ad.VEGF-A165) Gene Therapy Normalises Fetal Brain Growth and Microglial Activation in Nutrient Restricted Pregnant Guinea Pigs.

Fetal growth restriction (FGR) is associated with uteroplacental insufficiency, and neurodevelopmental and structural brain deficits in the infant. It is currently untreatable. We hypothesised that treating the maternal uterine artery with vascular endothelial growth factor adenoviral gene therapy (Ad.VEGF-A165) normalises offspring brain weight and prevents brain injury in a guinea pig model of FGR. Pregnant guinea pigs were fed a restricted diet before and after conception and received Ad.VEGF-A165 (1 × 1010 viral particles, n = 18) or vehicle (n = 18), delivered to the external surface of the uterine arteries, in mid-pregnancy. Pregnant, ad libitum-fed controls received vehicle only (n = 10). Offspring brain weight and histological indices of brain injury were assessed at term and 5-months postnatally. At term, maternal nutrient restriction reduced fetal brain weight and increased microglial ramification in all brain regions but did not alter indices of cell death, astrogliosis or myelination. Ad.VEGF-A165 increased brain weight and reduced microglial ramification in fetuses of nutrient restricted dams. In adult offspring, maternal nutrient restriction did not alter brain weight or markers of brain injury, whilst Ad.VEGF-A165 increased microglial ramification and astrogliosis in the hippocampus and thalamus, respectively. Ad.VEGF-A165 did not affect cell death or myelination in the fetal or offspring brain. Ad.VEGF-A165 normalises brain growth and markers of brain injury in guinea pig fetuses exposed to maternal nutrient restriction and may be a potential intervention to improve childhood neurodevelopmental outcomes in pregnancies complicated by FGR.

Treatment of BRONJ with ozone/oxygen therapy and debridement with piezoelectric surgery.

OBJECTIVE: Bisphosphonate related osteonecrosis of the jaw (BRONJ) is progressive bone destruction in the maxillofacial region of patients under current or previous treatment with Bisphosphonates. The present case series study aimed to evaluate if ozone/oxygen therapy and debridement with piezoelectric surgery may improve the treatment of BRONJ. PATIENTS AND METHODS: The treatment modality of the patients included ozone/oxygen mixture from medical oxygen. The protocol for ozone/oxygen mixture therapy appointments was set as twice a week for 10 weeks, for a total of 20 applications for each patient. The evaluation of the lesions was based on the clinical and radiologic parameters. The primary outcome was the necrotic lesion reduction during ozone/oxygen therapy sessions and up to the end of follow up periods. The healing of the lesion was taken as a positive result. The level of significance was taken as p <0.05. RESULTS: A total of 14 patients affected by osteonecrosis were included. The mean follow-up of the patients was 14.3 months. The overall success rate after treatment was 64.2%. CONCLUSIONS: According to the results, ozone/oxygen therapy and debridement with Piezoelectric surgery for BRONJ treatment is a safe procedure with successful outcomes.

Enhanced arboviral transmission by mosquitoes that concurrently ingested microfilariae.

Infection, dissemination, and transmission of an arbovirus in mosquitoes are enhanced by concurrent ingestion of microfilariae. Ingestion of Rift Valley fever virus alone infected only 64 percent of female Aedes taeniorhynchus. Of these, only 5 percent of refeeding mosquitoes actually transmitted virus. In contrast, ingestion of the same amount of virus from concurrently microfilaremic (Brugia malayi) gerbils resulted in 88 percent infection and 31 percent transmission. Enhanced transmission of virus may be attributed to increased transit of virus across the midgut wall. Endemic filariasis may promote arbovirus transmission in nature.

Comparison of a grain-based diet supplemented with synthetic vitamin E versus a lucerne (alfalfa) hay-based diet fed to lambs in terms of carcass traits, muscle vitamin E, fatty acid content, lipid oxidation, and retail colour of meat.

Dietary supplementation of vitamin E (VitE) in a synthetic or natural form was examined. Forty-eight lambs were assigned (n = 16) to either a grain-based diet at moderate (MOD, 42 mg∙kg-1 VitE as all-rac α-tocopheryl acetate) or supranutritional (SUP, 285 mg∙kg-1 of vitE) levels of synthetic VitE or a lucerne hay-based diet (LUC; 37 mg∙kg-1 VitE) for 8 weeks. Meat from the LUC group had lower muscle n-6 and PUFA levels compared to meat from the MOD and SUP groups. Despite a similar VitE intake, muscle VitE was higher for LUC compared to MOD, while SUP lambs showed the highest VitE. Lipid oxidation did not differ between groups. For fresh meat, redness tended to be higher in LUC fed lambs than the other two groups, but brownness formation was only lower than the SUP group. For aged meat colour stability, redness tended to be higher in lambs fed SUP and LUC, whereas highest browning occurred in the MOD group.

Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.

Draft Genome Sequences of Eight Crimean-Congo Hemorrhagic Fever Virus Strains.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a geographically widespread RNA virus with a high degree of genomic diversity that complicates sequence-based diagnostics. Here, we sequenced eight CCHFV strains for improved assay design and deposition into FDA-ARGOS, the FDA's pathogen database for development and verification of next generation sequencing assays.

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.

Affinity labeling of histidine and lysine residue in the adenosine deaminase substrate binding site.

1. Adenosine deaminase was inactivated by 9-(4-bromoacetamidobenzyl)-adenine (I) and 9-(2-bromoacetamidobenzyl)adenine (II), two affinity labels. 2. The stoichiometry of the reaction with reagent II is reported: 1 mol reagent is bound per mol inactive enzyme. Amino acid analysis of the 6 N HCl hydrolyzate of the inactive enzyme identified CM-histidine as the main alkylation product. This is the first evidence of the presence of a histidine in the active site region. 3. The alkylation rate and involved amino acid residues were studied for both reagents I and II, at pH 8 and 5.5. The particular reactivity of a lysine near or in the active site is discussed.

Mitogenic and haemagglutinating properties of a lectinpurified from Hura crepitans seeds.

A lectin from the seeds of Hura crepitans has been purified to homogeneity by affinity chromatography on acid-treated Sepharose CL-6B, followed by elution with D-galactose. The lectin is a glucosamine-containing glycoprotein with a molecular weight of 120 00, as determined by sucrose density gradient centrifugation, and consists of identical subunits with molecular weights of 30 000. The amino acid composition and total neutral sugar content are given. The Hura lectin agglutinates directly erythrocytes from several species, without specificity for human blood groups. In all cases, with the exception of pig erythrocytes, agglutination was enhanced by neuraminidase. Agglutination was inhibited, in decreasing order of potency, by N-acetyl-D-galactosamine, by D-galactose and galactose-containing oligosaccharides. The lectin has mitogenic activity for purified human T lymphocytes but not for B lymphocytes, and the activity is still evident at a concentration as low as 10 ng/ml. The specific mitogenic activity increases throughout the purification process.

Purification and partial characterization of a mitogenic lectin from Vicia sativa.

From the seeds of Vicia sativa a lectin has been purified by affinity chromatography on Sephadex G-100, followed by specific elution with D-glucose. The lectin is a glycoprotein with a molecular weight of 70 000. The aminoacid composition and the total sugar content have been determined. This lectin agglutinates horse, rabbit and human erythrocytes, with no specificity for human blood groups, but does not agglutinate calf and sheep erythrocytes. The agglutinating activity is inhibited by mono-, di-, and trisaccharides with a pyranosyl residue whose free hydroxyl group in position 4 has the configuration of glucose, and by fructose. The lectin has mitogenic activity on human peripheral blood lymphocytes.

Purification and partial characterization of a mitogenic lectin from the latex of Euphorbia marginata.

A lectin was purified from the latex of Euphorbia marginata by affinity chromatography on acid-treated Sepharose 6B and elution with lactose. The lectin is a glycoprotein composed of two identical subunits with M(r) 30,000, approx. The haemagglutinating activity of the lectin is not specific for any human blood group, and is inhibited by galactose and galactose-containing sugars and by gentiobiose. The lectin is strongly mitogenic for human T-lymphocytes and induces the release of interleukin-1 beta and tumor necrosis factor-alpha from cultured mononuclear cells.

Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

Purification and partial characterization of a lectin from the seeds of Trichosanthes kirilowii Maximowicz.

A lectin was purified from the seeds of Trichosanthes kirilowii, belonging to the family Cucurbitaceae, growing in China. The lectin is a glycoprotein of 57 kDa, consists of two subunits with apparent molecular masses of 37 and 25 kDa, is specific for galactose, and is not mitogenic for human lymphocytes.

Rapid and sensitive immunomagnetic-electrochemiluminescent detection of staphyloccocal enterotoxin B.

Sensitive, rapid and reproducible detection of staphyloccocal enterotoxin B (SEB) in a range of different biological matrices was achieved using the ORIGEN((R)) Immunoassay System (Igen, Inc). The homologous immunoassay format consisted of a double antibody sandwich in which a biotinylated capture antibody, pre-bound to streptavidin-coated paramagnetic beads, was used to bind antigen from test samples. A detector antibody, labeled with ruthenium (II) tris-bipyridal chelate, was added and, when bound to the bead immunocomplex, generated light in the presence of an excess of tripropylamine. The light was detected and measured by the ORIGEN analyzer. The sensitivity of this assay was 1 pg of enterotoxin per ml of serum, urine, tissue, or buffer and was highly reproducible. Concentration curves generated from SEB standards produced consistently wide linear ranges (0.1-100 ng/ml), making quantitation possible with only two dilutions of sample (undiluted and 1:1000). The assay used 50 microl of sample per test and required a 30 min incubation period in addition to a 1 min per tube reading time (50 tubes maximum). This assay was significantly better in terms of sensitivity, linear range, and assay time than the standard microplate enzyme-linked immunosorbent assay and should permit early SEB detection in clinical samples, food, and environmental samples.

Antigenic and molecular characterization of hantavirus isolates from China.

Hemorrhagic fever with renal syndrome (HFRS) is caused by certain viruses in the genus Hantavirus, family Bunyaviridae, and is a major public health problem in China. By using molecular and serological tests, we characterized 15 hantaviruses isolated either from patients with HFRS or from rodents captured in endemic areas of China. By cross plaque-reduction neutralization tests performed with rabbit immune sera, we identified two serologically distinct groups of viruses, comprised of those related to Hantaan virus, and those related to Seoul virus. To study the genetic relationships among these viruses, we amplified a 330 base pair region of the medium (M) genome segment of each isolate by reverse transcription and polymerase chain reaction (PCR) and compared the nucleotide sequences to those of other, well-characterized hantaviruses. In addition, we PCR-amplified and analyzed the entire coding region of the small (S) genome segment of each isolate by restriction enzyme digestion with a battery of enzymes. The results of our genetic analyses of both the M and S segments of these isolates confirmed our serological data, indicating that Hantaan and Seoul viruses co-circulate in endemic disease regions of China. We constructed a phylogenetic tree based on multiple alignment of the partial M segment sequences. The resulting dendrogram distinguished three genetic subtypes of Hantaan viruses and one type of Seoul virus.

Phosphatidylinositol metabolism in lymphocytes of chronic alcoholic patients after anti-CD3 stimulation.

The immunological alterations observed in chronic alcoholic patients may be due to alterations of signal transduction across the lymphocyte membrane. Upon binding of mitogens or antigens to specific plasma membrane receptors, the activation of phospholipase C leads to the hydrolysis of inositol phospholipids, producing inositol phosphates and diacylglycerol. One of the early events in lymphocyte activation is an increase of intracellular calcium concentration, due to both an influx from extracellular fluid and a release from intracellular stores mediated by inositol phosphates. In this study we verified whether the diminished mobilization of intracellular calcium, previously observed in alcoholics, is caused by alteration in phosphoinositide turnover. We evaluated total inositol phosphate production in peripheral blood lymphocytes after anti-CD3 stimulation, comparing control subjects and alcoholic patients. Lymphocyte activation generated inositol phosphates in both controls and alcoholics, but to a different extent, inositol phosphate production being significantly higher in controls than in alcoholics. This reduction in inositol phosphate production could be accounted either to an inhibition of PLC activity or to a modified affinity of phospholipase C for its own substrates, i.e., phosphoinositides, which fatty acid composition has been previously demonstrated to be greatly different in alcoholics in comparison to healthy subjects.

Diamine oxidase plasma activities after treatment with heparin and jejunal morphometry in untreated coeliac disease.

Diamine oxidase plasma concentrations after treatment with heparin were measured and compared with the surface to volume ratio of jejunal biopsy samples assessed by a morphometric technique in patients with untreated and treated coeliac disease and in biopsied controls. As expected, enzyme activity was significantly lower in patients with untreated coeliac disease than in patients on a gluten-free diet and in biopsied controls. No difference was found between treated patients and biopsied controls. There was a significant overall correlation between plasma enzyme activity and surface to volume ratio of jejunal mucosa, although two untreated patients without an overt malabsorption syndrome but with a very low surface to volume ratio had normal enzyme activity. This study shows that in coeliac disease plasma diamine oxidase activity after treatment with heparin does not always mirror the extent of the jejunal lesions, particularly in those patients with minimal or unrelated symptoms who would benefit most from a valid screening test to identify their condition.

Potential for mosquito transmission of attenuated strains of Rift Valley fever virus.

Studies were conducted to determine if two attenuated strains of Rift Valley fever (RVF) virus could be transmitted by Culex pipiens mosquitoes. Both strains (RVF MP 12 and T1) replicated in and were transmitted by female Cx. pipiens after intrathoracic inoculation. Mosquitoes also became infected with and transmitted the RVF MP12 strain after ingesting virus from a blood-soaked cotton pledget. However, because of the low viremias produced in infected animals, it is unlikely that mosquitoes would become infected by feeding on an animal inoculated with either of these viruses. Although both strains were transmitted by mosquitoes after intrathoracic inoculation, there was no evidence of reversion to a virulent virus.

Increased mosquito feeding on Rift Valley fever virus-infected lambs.

Experiments were conducted to determine whether or not mosquitoes exhibited an increase in feeding on Rift Valley fever (RVF) virus-infected lambs as compared to uninfected ones. Overall, when given a choice between a viremic lamb, or its uninfected twin, 65% (1,264/1,943) of the engorged female Culex pipiens obtained blood from the viremic lamb. This excess of feedings on viremic lambs was demonstrated regardless of the age of the lambs, or the time period between inoculation of the lambs and exposure to mosquitoes. With 3-day-old and 6- to 8-week-old lambs, 66% of the mosquitoes obtained blood from the viremic lamb. At 28, 48, and 52 hours after infection, the percentages of mosquitoes feeding on viremic lambs were 72, 68, and 64, respectively. Temperature, as measured by the difference in temperature of the viremic lamb minus that of the control lamb, was positively correlated with mosquito feedings on young lambs (3 days old), but not on older lambs (6 to 8 weeks old). When Cx. pipiens and Aedes taeniorhynchus were simultaneously given the opportunity to feed on 3-week-old lambs, a significantly higher percentage of engorged Cx. pipiens (94/108, 87%) fed on the viremic lamb than did engorged Ae. taeniorhynchus (187/350, 53%). The data generated by this study (e.g., an excess of feedings on the viremic lambs) warrant further study to determine the significance of this observation under field conditions.