Low human immunodeficiency virus type 1 (HIV-1) DNA burden as a major cause for failure to detect HIV-1 DNA in clinical specimens by PCR.

To determine the sensitivity of a nested PCR procedure for detecting human immunodeficiency virus type 1 DNA in clinical specimens, 553 peripheral blood mononuclear cell samples obtained from 268 human immunodeficiency virus type 1-seropositive subjects were assayed by use of two independent primer sets for each sample. Overall, 1,088 of 1,106 (98.37%) reactions were positive. Investigation of the negative reactions showed that a low viral burden in some infected subjects, rather than primer-template mismatches, was the primary cause for the false-negative PCR results.     

The Vi capsular antigen of Salmonella enterica serotype Typhi reduces Toll-like receptor-dependent interleukin-8 expression in the intestinal mucosa

Human infections with nontyphoidal Salmonella serotypes, such as S. enterica serotype Typhimurium, are characterized by a massive neutrophil influx in the colon and terminal ileum. In contrast, neutrophils are scarce in intestinal infiltrates of typhoid fever patients. Here, we show that in S. enterica serotype Typhi, the causative agent of typhoid fever, expression of the Vi capsular antigen reduced expression of the neutrophil chemoattractant interleukin-8 (IL-8) in host cells. Capsulated bacteria elicited IL-8 expression in polarized human epithelial cells (T84) and human macrophage-like cells (THP-1) in vitro at significantly reduced levels compared to noncapsulated bacteria. Experiments with a human cell line (HEK293) transfected with human Toll-like receptors (TLRs) demonstrated that in the presence of TLR5 or TLR4/MD2/CD14, a noncapsulated serotype Typhi mutant was able to induce the expression of IL-8, while this host response was significantly reduced when cells were infected with the capsulated serotype Typhi wild type. The relevance of these in vitro observations for the interaction of serotype Typhi with its human host was further studied ex vivo using human colonic tissue explants. Expression of IL-8 was detected in human colonic tissue explants infected with serotype Typhimurium or a noncapsulated serotype Typhi mutant. In contrast, infection with the serotype Typhi wild type did not elicit IL-8 expression in colonic tissue explants. Collectively, these data suggest that the scarcity of neutrophils in intestinal infiltrates of typhoid fever patients is due to a capsule-mediated reduction of TLR-dependent IL-8 production in the intestinal mucosa.     

Right ventricular cardiomyopathy and sudden death in young people

From 1979 to 1986, we conducted postmortem studies of 60 persons under 35 years of age who had died suddenly in the Veneto Region of northeastern Italy. Unexpectedly, we found that 12 subjects--7 males and 5 females ranging in age from 13 to 30 years--had morphologic features of right ventricular cardiomyopathy. This disorder had not been diagnosed or suspected before the subjects died. In five cases, sudden death was the first sign of disease; the remaining seven subjects had a history of palpitation, syncopal episodes, or both, and in five of those seven, ventricular arrhythmias had previously been recorded on electrocardiographic examination. Ten of the subjects had died during exertion. At autopsy, the subjects' heart weights were normal or moderately increased. Two main histologic patterns were identified--a lipomatous transformation or a fibrolipomatous transformation of the right ventricular free wall (6 cases each); in all cases, the left ventricle was substantially spared. Signs of myocardial degeneration and necrosis, with or without inflammatory infiltrates, were occasionally observed. These findings indicate that right ventricular cardiomyopathy, the cause of which is still unknown, may be more frequent than previously thought. At least in this area of Italy, it may represent an important cause of sudden death among young people.     

Sarcomeric determinants of striated muscle relaxation kinetics

Ca²⁺ is the primary regulator of force generation by cross-bridges in striated muscle activation and relaxation. Relaxation is as necessary as contraction and, while the kinetics of Ca²⁺-induced force development have been investigated extensively, those of force relaxation have been both studied and understood less well. Knowledge of the molecular mechanisms underlying relaxation kinetics is of special importance for understanding diastolic function and dysfunction of the heart. A number of experimental models, from whole muscle organs and intact muscle fibres down to single myofibrils, have been used to explore the cascade of kinetic events leading to mechanical relaxation. By using isolated myofibrils and fast solution switching techniques we can distinguish the sarcomeric mechanisms of relaxation from those of myoplasmic Ca²⁺ removal. There is strong evidence that cross-bridge mechanics and kinetics are major determinants of the time course of striated muscle relaxation whilst thin filament inactivation kinetics and cooperative activation of thin filament by cycling, force-generating cross-bridges do not significantly limit the relaxation rate. Results in myofibrils can be explained well by a simple two-state model of the cross-bridge cycle in which the apparent rate of the force generating transition is modulated by fast, Ca²⁺-dependent equilibration between off- and on-states of actin. Inter-sarcomere dynamics during the final rapid phase of full force relaxation are responsible for deviations from this simple model.     

Enterotoxicity and cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin in in vitro systems

Vibrio parahaemolyticus is a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of V. parahaemolyticus. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysin's enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.     

Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs

Summary The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genusTombusvirus andCarmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Database under the accession number X85215.     

Postzygotic instability of the myotonic dystrophy p[AGC]n repeat supported by larger expansions in muscle and reduced amplifications in sperm

We have analysed the [AGC] expansion in leucocytes, muscle and sperm from 17 individuals affected by myotonic dystrophy (DM). Skeletal muscle showed a larger repeat number than leucocytes in the same patient. A similar degree of expansion was detected in differently affected muscles of a single patient. The germline mutation ( 350 repeats) was expanded in somatic cells of the progeny in all patients examined. Our results provide evidence of an early postzygotic instability of the [AGC] repeat in DM.     

Glucose tolerance and insulin secretion in pregnancy

The authors have studied the behaviour of plasma glucose, insulinaemia and insulin-glucose ratio in 2 groups of pregnant women with BMI values > or = or <26, respectively. Each group was divided into 3 subgroups on the basis of an oral glucose tolerance test (OGTT) response: GIGT (gestational impaired glucose tolerance), GD (gestational diabetes), and C (normal controls). Data from non-obese pregnant women demonstrate that both basal and OGTT-stimulated glucose levels were significantly different in all subgroups. The total insulin amount in the GIGT and GD subgroups is quite similar to or greater than the controls, but with a significant reduction of the insulin-glucose ratio. In GD also an absolute deficiency of insulin rise at 30 min during the glucose load, as in subjects with Type 2 diabetes mellitus (T2DM), was observed. The behaviour of these parameters in obese pregnant women seems to be similar, even though with some significant differences: in these subjects, there is a less clear-cut differentiation among all subgroups, and the appearance of gestational diabetes is not accompanied by a significant decrease of insulin secretion at 30 min. Our data seem to demonstrate that insulin resistance with an inadequate hyperinsulinaemia is a common factor for the alterations of carbohydrate metabolism during pregnancy. Only in the non-obese patients with gestational diabetes, is there an absolute defect of early insulin response to the glucose load, as it is seen in T2DM.     

Pertussis toxin does not affect the adenosine-induced inhibition of the efferent function of cardiac capsaicin-sensitive nerves

Summary The negative inotropic effect of adenosine (1–100 M) was abolished in isolated guinea-pig atria obtained from pertussis toxin-pretreated guinea pigs electrically driven at 4 Hz. However, the inhibitory effect of the same concentrations of adenosine on the cardiac response to stimulation of non-adrenergic non-cholinergic (NANC), capsaicin-sensitive sensory nerves, was not modified by the toxin.These results suggest that, while pertussis toxin-sensitive G proteins are involved in the negative inotropic effect of adenosine, they do not mediate the inhibitory effect of adenosine on cardiac NANC neurotransmission.