Tracking novel adenovirus in environmental and human clinical samples: no evidence of endemic human adenovirus type 58 circulation in Córdoba city, Argentina.

In recent years, several types of human adenovirus (HAdV) have arisen from the recombination between two or more previously known HAdV types, but their epidemiology is poorly understood. In this study, we investigated the circulation of HAdV-58, a recently described HAdV isolated from an HIV-positive patient in Córdoba city, Argentina. For this purpose, a 30-month survey was conducted to study the presence of this type of adenovirus in sewage samples collected at the inlet from a wastewater treatment plant in Córdoba city, Argentina. Complementarily, the virus was sought in stools of HIV-positive patients. Although HAdVs were detected in human stool samples and in a high percentage of sewage samples, no evidence of HAdV-58 circulation was detected. We suggest that there is no endemic circulation of HAdV-58 in the geographical local area. The trend is that the number of identified HAdVs increases over time. In this context, understanding the current circulating HAdVs may be biologically relevant.

MRI measurement of the canine auditory pathways and relationship with brainstem auditory evoked responses.

The objective of this study was to determine direct measurements of auditory pathways by magnetic resonance imaging (MRI) during the growth period of healthy Beagles, and to discover how canine brainstem auditory evoked response (BAER) latencies vary in relation to these MRI measurements. Eighty healthy Beagles were tested at eight, 16 and 52 weeks of age (stages 1, 2, 3, respectively) with BAER and brain MRI. The BAER interpeak latency (IPL) II-V and brain MRI neural generators of BAER waves II and V were identified. A linear distance was calculated in millimeters in order to determine the approximate length of auditory pathways. Sensory nerve conduction velocity (SNCV) of the auditory pathway between peak II and peak V was calculated for each group. A significant difference was observed between brain MRI distances among the three stages. Mean BAER IPL II-V were not significantly different between the three stages. The progressive growth of the skull and brain witnessed by the progressive increased distance of the MRI auditory pathways between peak II and peak V was not associated with a progressive maturation of the BAER IPL II-V. The SNCV of the auditory pathway between peak II and peak V was 6.14 m/sec for group 1; 6.76 m/sec for group 2; and 7.32 m/sec for group 3.

Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts.

We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P=0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 x 10(6) to 365 x 10(6) hMSCs in PL versus a variable number from 2.7 x 10(6) to 31 x 10(6) in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.

Erythropoietin therapy and preoperative autologous blood donation in children undergoing open heart surgery.

We assessed the feasibility and efficacy of subcutaneous erythropoietin alpha (EPO) therapy and preoperative autologous blood donation (ABD) in children undergoing open heart surgery. Thirty-nine children were treated consecutively with EPO (100 U x kg(-1) s.c. three times a week in the 3 weeks preceding the operation and i.v. on the day of surgery) and two ABDs were made (Group 1). As controls to compare transfusion requirements, 39 consecutive age-matched patients who had undergone open heart surgery during the two preceding years were selected (Group 2). In a mean time of 20 (SD 5) days, 96% of scheduled ABDs were performed and only three mild vasovagal reactions were observed. The mean volume of autologous red blood cells (RBC) collected was 6 (1) ml x kg(-1) and the mean volume of autologous RBC produced as a result of EPO therapy before surgery was 7 (3) ml x kg(-1), corresponding to a 28 (11)% increase in circulating RBC volume. The mean volume of autologous RBC collected was not different from that produced [6 (1) vs 7 (3) ml x kg(-1), P=0.4]. Allogenic blood was administered to three out of 39 children in Group 1 (7.7%) and to 24 out of 39 (61.5%) in Group 2. Treatment with subcutaneous EPO increases the amount of autologous blood that can be collected and minimizes allogenic blood exposure in children undergoing open heart surgery.

Transfusion-transmitted virus infection in HIV-1-seropositive patients.

OBJECTIVES: To investigate the prevalence, persistence and genome heterogeneity of transfusion-transmitted (TTV) in HIV-1-infected patients, a group at high risk both of contracting blood-borne viruses and having viral persistence relating to immunodepression. METHODS: Plasma samples from 238 HIV-1 seropositive subjects and 226 healthy blood donors were examined for TTV-DNA both by polymerase chain reaction (PCR) using primers from the conserved regions in the N22 clone and PCR using primers deduced from the untranslated region (UTR). Direct DNA sequencing and phylogenetic analysis were used to characterize 27 TTV isolates from HIV-1 patients or healthy controls. RESULTS: Using PCR with the UTR primers, TTV DNA was detected in a very high percentage (> 80%) of samples both from HIV-1 seropositive subjects and from blood donors. Using PCR with N22 primers, shown to detect viral strains associated with hepatitis of unknown etiology, TTV DNA was found in 103 of 238 (43.3%) HIV-1-infected patients and in 22 of 226 (9.7%) blood donors. There was no difference in the prevalence of the TTV DNA in HIV seropositive subjects with regard to clinical features related to immunosuppression, markers of HCV infection or intravenous drug use; presence of TTV DNA was associated significantly only with male gender (P = 0.003). Persistent or intermittent viremia was detected in plasma samples taken up over a period of 19 months in all (15 of 15) HIV-infected patients tested. CONCLUSIONS: The persistence and high frequency of infection detected by PCR with N22 primers in HIV-1 seropositive patients suggest that further clinical investigation of immunocompromised hosts will provide information to clarify the pathogenic role of TTV.

Comparison of performance of six different cell separators in collecting peripheral blood mononuclear cells.

We compared the efficacy of six different cell separators in collecting peripheral mononuclear cells to be used for autologous or homologous peripheral stem cell transplantation. The product obtained with the Dideco Vivacell cell separator showed a low percentage of mononuclear cells (38%) in the final product and a high platelet efficiency (38%). The Baxter CS3000 Plus cell separator required the longest time to load and prime the kit (18 min), it showed a high MNC efficiency (68%), with the highest percentage of MNC in the final product, the highest platelet efficiency (45%), a low red blood cell contamination in the final product (2.7 mL), the highest extracorporeal volume (450 mL) and a high percentage of technical failures (15%). The product obtained with the Fresenius AS104 cell separator with P1Y kit showed the highest final volume (297 mL), the lowest platelet efficiency (12%) and the lowest extracorporeal volume (230 mL). The same cell separator with C4Y kit showed a lower MNC efficiency (52 vs 60%) and a higher percentage of MNC in final product (63 vs 41%). The platelet contamination in final product was the lowest (18 x 10(9)/100 mL). The Haemonetics MCS3p cell separator required the lowest time to load and prime the kit (5 min), it showed the highest MNC efficiency (71%). The blood volume processed per hour (1328 mL) and the percentage of MNC in final product was lowest (32%), the extracorporeal volume (450 mL) was the highest. The Cobe Spectra cell separator allowed to process the highest blood volume per hour (3383 mL) and the final product had the lowest red blood cell contamination (2.3 mL/100 mL). The Dideco Excel cell separator required the longest time to load and prime the kit (18 min), the lowest MNC efficiency (38%), the highest platelet contamination in final product. Furthermore this machine showed the highest percentage of technical failure (20%). None of the six instruments have all the required preconditions and the ideal cell separator for peripheral stem cell apheresis at present is not available on the market.

HCV transmission in family members of subjects with HCV related chronic liver disease.

To investigate the risk of sexual and intrafamilial transmission of HCV, 220 family members of 76 patients (index cases) with chronic type C viral liver disease were tested for serological markers of HCV. Of the family members, 129 were offspring, 64 sexual partners, 15 parents and 12 siblings of the index cases. Anti-HCV was tested in all the household contacts; HCV-RNA was tested in antibody positive samples. The serologic markers of HCV were tested in a control group of 168 family members of 81 patients with chronic hepatitis unrelated to HCV. The overall prevalence of anti-HCV was 8.2% compared to 0.6% in the control group (p < 0.001). Sexual partners were anti-HCV positive more frequently than the other contacts (20% vs 2.2%; p < 0.001), without any difference in males or females. No correlation was observed between the occurrence of HCV infection in contacts and age, severity of liver disease or risk factor for the acquisition of HCV in the index cases. Seven of the 18 (39%) anti-HCV positive family contacts had bio-chemical evidence of chronic liver disease, histologically confirmed in the 6 patients who underwent a liver biopsy. Liver chemistry was normal in all the HCV-negative contacts. Ten of the 18 anti-HCV positive contacts (55%) were HCV-RNA positive, Genotypes were the same (1b) in 4 of the 7 viremic couples of subjects: in 3 of the 6 couples of sexual partners and in the only mother/son couple. These data suggest the occurrence of intraspousal transmission of HCV, while intrafamiliar acquisition of HCV in non-sexual contacts seems to be rare.

Insulin regulation of glucose turnover and lipid levels in obese children with fasting normoinsulinaemia.

To evaluate the early metabolic alterations induced by obesity, we studied glucose turnover and lipid levels in obese children with fasting normoinsulinaemia. Two experimental protocols were carried out. Protocol I consisted of a euglycaemic glucose clamp at two rates of insulin infusion. Protocol II was similar to protocol I except for a variable lipid infusion used to maintain basal non-esterified fatty acid (NEFA) levels. During protocol I, the glucose disappearance rates were lower in obese children, while no differences were found in hepatic glucose release. NEFA response to insulin was not substantially altered in obese children either at low or high insulin infusion. During protocol II, the NEFA clamp induced a 25% reduction in peripheral insulin sensitivity in control children whereas no changes were observed in obese children. Interestingly, lipid infusion in control children was not sufficient to reproduce the same degree of insulin resistance observed in obese children, suggesting that NEFA are only one of the determinants of insulin resistance at this stage of obesity. In conclusion, the present study provides a portrait of glucose metabolism and lipid levels in normoinsulinaemic obese children. Our results document that peripheral insulin resistance is the first alteration at this stage of obesity, whereas an increase in insulin secretion and a defect in the inhibition of hepatic glucose release by insulin may develop at a later stage. In addition, primarily receptor and post-receptor defects and some alterations of NEFA metabolism are likely to coexist in the induction of insulin resistance at this stage of obesity.

Intravenous infusion of diarginylinsulin, an insulin analogue: effects on glucose turnover and lipid levels in insulin-treated type II diabetic patients.

Diarginylinsulin is an intermediate in the conversion of proinsulin to insulin and is usually present in small amounts in vivo in humans. This study was designed to evaluate the following in insulin-treated type II diabetic patients: (1) the feasibility of an overnight intravenous infusion of diarginylinsulin, as compared with an overnight intravenous infusion of short-acting insulin, and the degree of early morning glycemic control; and (2) the effects of diarginylinsulin and human insulin on hepatic glucose production (HGO) in the postabsorptive state and on the glucose turnover rate and peripheral insulin sensitivity during an euglycemic hyperinsulinemic clamp. Diarginylinsulin and regular human insulin maintained a comparable degree of normoglycemia during the night, without significant glucose increases in the morning. Free-diarginylinsulin and free-insulin concentrations were not significantly different, and (HGO) was 2.1 +/- 0.5 versus 2.1 +/- 0.4 mg/kg/min with diarginylinsulin and regular human insulin, respectively (NS). During the euglycemic clamp, glucose infusion rate per unit of diarginylinsulin or human insulin infused (M/I ratio) was similar, and HGO was equally suppressed with diarginylinsulin and regular human insulin. No significant differences were seen in NEFA and triglyceride levels. In conclusion, these results indicate that diarginylinsulin is as potent as regular human insulin; it is normalizes HGO in the postabsorptive state; and its hepatic and peripheral actions on glucose and lipids are comparable to those of human insulin during an euglycemic hyperinsulinemic clamp.

Determination of plasma [6,6-2H2]glucose enrichment by a simple and accurate gas chromatographic-mass spectrometric method.

An improved method for the evaluation of glucose turnover rate in humans, using a prime-continuous infusion of [6,6-2H2]glucose, was developed. Deproteinization of plasma and conversion of glucose into the aldononitrile pentaacetate derivative are the only sample manipulations required prior to the gas chromatographic-mass spectrometric analysis. In six normal adults (prime = 5 mg kg-1; continuous infusion = 0.05 mg kg-1 min-1) the hepatic glucose output calculated at steady state by the procedure described here was 2.1 +/- 0.2 mg kg-1 min-1, the isotopic enrichment being determined with a coefficient of variation of ca. 2%. In six additional subjects, with half of the above-mentioned doses of labelled glucose, the mean hepatic glucose output was exactly the same (3.2% coefficient of variation for the isotopic enrichment measurement). The method described allows the hepatic glucose output to be precisely determined with savings both of time and of labelled glucose.

[Hypothalamo-pituitary-adrenal function in liver cirrhosis of viral etiology]. / Studio della funzione ipotalamo-ipofiso-surrenalica nella cirrosi epatica di origine virale.

With the aim of evaluating the glucocorticoid function and the role of the adrenal gland in hypogonadism and feminization of cirrhotic patients, we examined 11 patients with virus-induced liver cirrhosis and 8 normal subjects as controls. In each subject serum levels of cortisol (C), progesterone (P), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), delta 4-androstenedione (A), estrone (E1), testosterone (T), luteinizing hormone (LH) were assayed in basal conditions and after adrenocorticotropic hormone (ACTH) stimulation. Serum levels of ACTH, C, E1, estradiol (E2), T were assayed in basal condition and after dexamethasone suppression test. Moreover, a circadian study of ACTH, C and corticosteroid-binding globulin (CBG) was performed, with blood samples drawn at 8:00 and 20:00 on two consecutive days. Our results demonstrate that in cirrhosis: 1) normal levels of C, when metabolism is altered and CBG levels are reduced, are maintained by inhibition of ACTH secretion; 2) circadian rhythmicity of the pituitary-adrenal axis is well preserved; 3) in non-alcoholic cirrhosis, too, there is a reduction of androgens (T, DHEA, DHEAS, A) and a rise of estrogens (E2 and, more markedly, E1) and P; 4) in cirrhotic men E1 is mainly of adrenal origin and contributes, through negative feedback on LH secretion, to low levels of T.

[Evaluation of the activity of the bladder detrusor muscle and analysis of bladder function in subjects with diabetes mellitus under insulin treatment]. / Valutazione dell'attività del muscolo detrusore della vescica ed analisi della funzione vescicale in soggetti affetti da diabete mellito in trattamento insulinico.

The present report concerns investigations of detrusor muscle adrenergic innervations in patients affected by bladder neuropathy secondary to diabetes without obstructive disturbances. Detrusor contractile activity evoked by NE is markedly reduced which can probably be attributed to receptor deficit. Urodynamic evaluation demonstrated a prevalence of sensory peripheral neuropathy than a motor conduction abnormality. In vitro study demonstrated that motor conduction abnormality of detrusor contractile activity is present early without bladder disturbances. Therefore early urodynamic measurements are necessary to evaluate bladder dysfunction and neuropathy in diabetic patients.