Recommendations for the use of cardiovascular tests in diagnosing diabetic autonomic neuropathy.

Despite its prevalence, clinical and prognostic impact, diabetic autonomic neuropathy, is widely under-diagnosed. The need for training and expertise to perform the cardiovascular tests (usually the task of diabetologists) is one possible reason. The availability of computer-assisted systems has allowed a wider diffusion of testing, but has also highlighted the need for an adequate knowledge of physiopathological backgrounds for their correct application and interpretation. The recommendations presented here were developed by the Neuropathy Study Group of the Italian Society of Diabetology and then endorsed by the Italian Association for the Study of Neurovegetative System, to promote the widespread adoption of good clinical practice in diabetic cardiovascular autonomic testing by outlining main evidence-based aspects, i.e. which tests, how to perform them, adequate interpretation of the results and their diagnostic use, confounding conditions that can impact on tests reliability. Therefore, these recommendations include the essential aspects of the physiopathological substrate of the tests, the controversial points in their analysis, their diagnostic characteristics, as well as safety. Detailed information is given on the physiological (age, weight, body position, resting heart rate and blood pressure, respiratory pattern, exercise, meals, acute blood glucose changes) and pathophysiological confounding factors, with emphasis on the effects of drugs. Instructions on how to perform the tests and interpret their results are also considered together with indications of candidate patients and periodicity of testing. A patient instruction sheet on why and how to perform the tests is included. Finally, the specific requirements for computerized systems to perform and evaluate cardiovascular tests are provided.

E-cadherin negatively regulates neoplastic growth in non-small cell lung cancer: role of Rho GTPases.

Non-small cell lung cancers (NSCLC) that express the cell surface adhesion protein E-cadherin may carry a better prognosis than E-cadherin-negative tumors. Here, we found substantial inhibition of anchorage-independent growth in soft agar and cell migration in each of four NSCLC lines stably transfected with E-cadherin. The inhibitory effects were independent of the EGFR and beta-catenin/Wnt-signaling pathways. However, E-cadherin expression was associated with an adhesion-dependent reduction in the activity of Rho family proteins, RhoA in two lines and Cdc42 in the other two. The reduction of RhoA activity was dependent on DLC-1 Rho-GAP and p190 Rho-GAP and associated with an increase in a membrane-associated p190 Rho-GAP/p120 Ras-GAP complex. In parental cells with high levels of RhoA-GTP, siRNA-mediated knock-down of RhoA reduced cell migration and agar growth in a manner analogous to E-cadherin. In parental cells with high levels of Cdc42-GTP, transfection of a Cdc42 dominant-negative mutant reduced cell growth and migration similarly to cells expressing E-cadherin. Thus, E-cadherin can negatively regulate cell proliferation and migration in NSCLC by reducing the level of the predominant active form of Rho family protein, RhoA or Cdc42. These proteins can be considered downstream effectors of E-cadherin and might represent therapeutic targets in some NSCLC.

The influence of autonomic neuropathy on hypotension during hemodialysis.

UNLABELLED: Many patients with chronic renal failure experience profound hypotension during hemodialysis. This phenomenon may be caused by hypovolemia, autonomic or cardiac dysfunction, vascular resistance defects or vasoactive substances such as nitric oxide or adrenomedullin. The aim of the study was to evaluate the influence of autonomic neuropathy on the occurrence of hypotension during hemodialysis. Fifty-three patients with chronic renal failure on maintenance hemodialysis underwent a standard battery of cardiovascular tests for the diagnosis of autonomic neuropathy. The study population was then divided into two groups, with (AN+) and without (AN-) autonomic neuropathy. Blood pressure (BP) was recorded before, during and after hemodialysis for 10 consecutive dialysis sessions and the mean value was calculated. RESULTS: Of the patients, 38% were AN+. During hemodialysis, systolic BP was lower in AN+ than in AN- patients at the third hour (110.0 +/- 17.5 vs 128.0 +/- 26.2; p = 0.049). Systolic BP reduction during hemodialysis was greater in AN+ than AN-patients (-21.2 +/- 10.9 vs -13.5 +/- 10.6% change, p = 0.013). CONCLUSION: The presence of autonomic neuropathy is associated with a more severe BP fall during hemodialysis. Routine evaluation of autonomic function may be helpful in defining patients at risk for dialysis-induced hypotension.

Detection of the negative-strand hepatitis C virus RNA in tissues: implications for pathogenesis.

The replication of hepatitis C virus (HCV) RNA is believed to occur via its transcription into a complementary, genomic-length RNA, the so-called negative-strand HCV RNA. This is based on the comparison with the replication of other members of the Flaviviridae family. Detection of the negative-strand HCV RNA in human tissues by semi-quantitative, strand-specific RT-PCR has contributed to the understanding of the HCV cell tropism and of the pathogenesis of HCV-associated disease manifestations. In particular, it was shown that the levels of intrahepatic HCV RNA are not correlated to the extent of the necroinflammation, but that a significant correlation was found with the liver steatosis. These results suggest that most liver disease associated with HCV infection is mediated by the host immune response. However, in some patients, most notably those infected with HCV genotype 3, HCV may cause a cytopathic effect, consisting in the lipid accumulation within hepatocytes.

Are there any subgenomic forms of hepatitis C virus RNA in the liver?

BACKGROUND: Hepatitis C virus has a single stranded positive RNA genoma. Although believed to replicate via semi-conservative transcription of a negative-stranded, genomic-length RNA intermediate, detailed steps of its replicative cycle are unknown. AIMS: To quantify some of intrahepatic hepatitis C virus RNA forms, as inferred from comparison with replication of other members of the Flaviviridae family. PATIENTS AND METHODS: Genomic and negative-stranded hepatitis C virus RNA were semi-quantitated by strand-specific reverse transcriptase-polymerase chain reaction at both their 5' and 3' ends in liver of 10 patients with recurrent hepatitis C after liver transplantation. RESULTS: Our data are consistent with the existence of hitherto unrecognized, very large amounts (up to approximately 10,000 fold the amount of the replication intermediate proper) of subgenomic hepatitis C virus RNAs of genomic polarity, starting in the 5' untranslated region, of unknown length. Similarly, subgenomic RNAs of negative polarity, starting in the 3' untranslated region, may also be produced, albeit to a less extent. We found no correlation between the amount of these forms and any clinical, histological or virological feature. However, the number of subgenomic RNA molecules of negative polarity tended to be inversely correlated with viraemia (r = 0.7, p = 0.058), suggesting their possible role in controlling rate of virion production. CONCLUSIONS: Hepatitis C virus replication results in transcription of huge amounts of subgenomic RNAs both of genomic and negative polarity, which may either regulate translation of excess structural antigens of hepatitis C virus, or play the role of defective RNAs interfering with viral replication. A revised model of hepatitis C virus RNA replication is proposed.

Liver steatosis in chronic hepatitis C: a morphological sign suggesting infection with HCV genotype 3.

AIMS: To identify factors associated with liver steatosis in chronic hepatitis C. METHODS AND RESULTS: Occurrence and severity of liver steatosis in 254 chronic hepatitis C patients were compared with presence of alcohol abuse, body mass index (BMI) >26, history of intravenous drug addiction and hepatitis C virus (HCV) genotype. Steatosis was found in 109 (43%) patients. The occurrence of steatosis was significantly associated with ongoing alcohol abuse (P=0.03) or HCV genotype 3 (P= 0.003), but not with BMI >26. A moderate to severe steatosis was present in 60% of patients infected with HCV genotype 3, irrespective of the presence of alcohol abuse, BMI >26 or history of intravenous drug addiction. Using a multivariable stepwise logistic regression analysis, infection with genotype 3 had an odds ratio (OR) of 10 (95% confidence interval (CI)=4.56-22) for a liver steatosis, whereas the presence of a cirrhosis at histology had an OR=0.256 (95% CI=0.07-0.92). CONCLUSIONS: A moderate to severe degree of steatosis of the liver is a morphological sign suggestive of infection with HCV genotype 3, independent of other risk factors of a fatty liver, but it may disappear at late stages of the disease.

Nosocomial outbreak of multiple bloodborne viral infections.

In resource-limited countries, nosocomial transmission of bloodborne pathogens is a major public health concern. After a major outbreak of human immunodeficiency virus (HIV) infection in approximately 400 children in 1998 in Libya, we tested HIV, hepatitis C virus (HCV), and hepatitis B virus (HBV) markers in 148 children and collected epidemiological data in a subgroup of 37 children and 46 parents. HIV infection was detected in all children but one, with HCV or HBV coinfection in 47% and 33%, respectively. Vertical transmission was ruled out by analysis of parents' serology. The children visited the same hospital 1-6 times; at each visit, invasive procedures with potential blood transmission of virus were performed. HIV and HCV genotypic analyses identified a HIV monophyletic group, whereas 4 clusters of HCV sequences were identified. To our knowledge, this is the largest documented outbreak of nosocomial HIV transmission.

Sympathetic nervous system, diabetes, and hypertension.

Hypertension is twice as frequent in diabetic patients than in the general population. Its prevalence is higher in Type 2 than in Type 1 diabetes: in the former, the onset of hypertension often precedes the diagnosis of diabetes, whereas, in the latter it is strictly related to the presence of nephropathy. Sympathetic nerve overactivity is crucial in the pathogenesis of hypertension in diabetes. It can be related to the activation of the renin-angiotensin-aldosterone (RAA) system in Type 1 diabetic patients with chronic renal failure, or to a condition of insulin resistance/hyperinsulinemia in Type 2 patients with the metabolic syndrome. In patients with early autonomic neuropathy, vagal impairment can lead to a relative predominance of sympathetic activity in the sympatho-vagal balance. In these patients, the onset of hypertension is frequently preceded by reduced nocturnal dipping. Sympathetic overactivity stimulates RAA activity, promotes sodium reabsorption, and increases heart rate, stroke volume and peripheral vascular resistance, thus inducing hypertension and increasing cardiovascular risk. A number of drugs acting either directly or indirectly on sympathetic activity are available for the treatment of hypertension in diabetic subjects. Opinions on the potential advantages of the metabolic profile of some of these drugs are as yet conflicting.

Autonomic nervous function in de novo parkinsonian patients in basal condition and after acute levodopa administration.

The aims of this study were to assess autonomic nervous function in subjects with recently diagnosed Parkinson's disease (de novo patients) and to evaluate its changes following acute levodopa administration. In 13 patients (8 males, 5 females) and 13 age-matched control subjects, three cardiovascular autonomic function tests (Deep Breathing, Valsalva, Lying to Standing) were performed, the QT interval was calculated on a 12-lead electrocardiogram, and the response of plasma norepinephrine to standing was assessed in basal conditions. The cardiovascular tests and the measurement of the QT interval were repeated in de novo Parkinsonian patients 90 minutes after the administration of levodopa 200 mg per os. The results of the Deep Breathing and Valsalva tests were worse and the QT interval longer in patients than in control subjects (although the differences were not statistically significant). The heart rate increase at 30 seconds after standing up was significantly higher in Parkinsonian patients than in the control group. The response of plasma norepinephrine to standing was similar in both groups. Levodopa administration produced a slight improvement in the Deep Breathing test, a shortening of the QT interval and increased tachycardia on standing. Our data suggest that a mild subclinical impairment of parasympathetic function can be a feature of de novo Parkinsonian patients and that levodopa therapy could have a beneficial effect on this autonomic dysfunction.

Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3.

BACKGROUND/AIMS: Patients infected with the hepatitis C virus (HCV) often have liver steatosis, suggesting the possibility of a viral cytopathic effect. The aim of this study was to correlate the occurrence and severity of liver steatosis with HCV RNA type, level and sequence of the core-encoding region. METHODS: We scored the liver steatosis in 101 HCV-infected individuals carefully selected to exclude other risk factors of a fatty liver. Results were compared with HCV RNA genotype and level in serum and liver. In selected patients, we assessed the effect of antiviral therapy on steatosis and the relationship between nucleocapsid sequence heterogeneity and fat infiltration. RESULTS: Steatosis was found in 41 (40.6%) patients, irrespective of sex, age or route of infection. HCV genotype 3 was associated with higher steatosis scores than other genotypes. A significant correlation between steatosis score and titer of intrahepatic HCV RNA was found in patients infected with genotype 3, but not in those infected with genotype 1. In selected patients, response to alpha-interferon was associated with the disappearance of steatosis. Analysis of the nucleocapsid of 14 HCV isolates failed to identify a sequence specifically associated with the development of steatosis. CONCLUSIONS: We provide virological and clinical evidence that the steatosis of the liver is the morphological expression of a viral cytopathic effect in patients infected with HCV genotype 3. At variance with published evidence from experimental models, the HCV nucleocapsid protein does not seem to fully explain the lipid accumulation in these patients.

A novel hepatitis C virus (HCV) subtype from Somalia and its classification into HCV clade 3.

Hepatitis C virus (HCV) sequences from throughout the world have been grouped into six clades, based on recently proposed criteria. Here, the partial sequences and clade assignment are reported for three HCV isolates from chronic hepatitis C patients from Somalia, for whom conventional assays failed to identify the genotype. Phylogenetic analysis of the sequences of the core, envelope 1 and part of the non- structural 5b regions suggests that all three isolates belong to a distinct HCV genetic group, tentatively classified as subtype 3h. This novel HCV subtype shows the highest sequence similarity with HCV isolates from Indonesia. Despite the fact that these patients were infected with HCV clade 3, none of them responded to standard interferon treatment.

Helicobacter pylori infection in type 2 diabetic patients.

BACKGROUND AND AIM: A high prevalence of Helicobacter pylori (Hp) infection in diabetic patients has been described in recent years. This study investigates its prevalence in type 2 diabetics and its correlation with the degree of metabolic control and the presence of chronic complications. METHODS AND RESULTS: Forty-one consecutive type 2 diabetics (21 males, 20 females aged 46-78, mean 62) and 31 age-matched controls participated. Hp infection was assessed by means of the 13C-urea breath test. Fasting glucose and glycated haemoglobin (HbA1c) levels were measured to evaluate metabolic control. Chronic complications were assessed by means of albumin excretion rate (AER), fundoscopy, vibratory perception threshold (VPT), ECG, clinical history of coronary, cerebral or peripheral arteriopathy, foot examination and cardiovascular autonomic function tests. A higher prevalence of Hp infection was found in diabetic than in control women (80% vs 37.5%; p < 0.05), whereas there was no difference between males. A higher prevalence correlated with macroangiopathy and neuropathy and higher BMI, blood pressure, fasting glucose and HbA1c values. By contrast, microangiopathy was significantly more prevalent (p < 0.05) in Hp negative (85%) than in Hp positive patients (48%). CONCLUSIONS: There is a high prevalence of Hp infection in type 2 diabetic women. The absence of microangiopathy may be a predisposing factor: microvascular changes in the gastric mucosa may create an unfavourable environment for the establishment or survival of Hp.

Hemochromatosis gene mutations in chronic hepatitis C patients with and without liver siderosis.

Chronic hepatitis C is often associated with liver iron overload, which may affect the long-term prognosis and the response to antiviral treatment. The occurrence of hemochromatosis (HFE) mutations were studied to determine whether may contribute to the liver iron overload of chronic hepatitis C patients. The prevalence of two HFE mutations (C282Y and H63D) in 120 chronic hepatitis C patients was determined and the findings were correlated with clinical, histological and virological features. Hepatic iron was determined semiquantitatively by a histochemical hepatic iron index, defined as the ratio of a histochemical staining score to the patient's age, after correction for heterogeneous lobular iron distribution. Serum hepatitis C virus (HCV) RNA was measured by bDNA assay and typed by restriction fragment length polymorphism. Liver HCV RNA was measured by a semi-quantitative strand-specific reverse transcription-polymerase chain reaction (RT-PCR). Excess liver iron was stained in the liver of 36 patients (30%). Siderotic patients had the same geographic origin, serum and liver HCV RNA levels and H63D and C282Y mutations frequency as non-siderotic patients. However, siderotic patients were older (P = 0.015), more frequently males (P = 0.02), less frequently infected with HCV genotype 3 (P = 0.037) and had a higher liver fibrosis score (P = 0.008). The liver iron content did not correlate with the serum or liver HCV RNA titers. Ten of the 36 patients with liver siderosis had neither a history of excess alcohol intake, multiple transfusions, or HFE mutations. In conclusion, the pathogenesis of the liver iron overload in chronic hepatitis C patients cannot be fully explained by the occurrence of HFE mutations. The exact mechanism of iron accumulation in these patients therefore remains unexplained.

Failure of T lymphocytes from elderly humans to enter the cell cycle is associated with low Cdk6 activity and impaired phosphorylation of Rb protein.

Cell cycle analyses of activated T lymphocytes from elderly humans generally show that the proportion of noncycling cells increases with age. T cells that are not definitively blocked in G0 usually strain to traverse the G1 phase and may still be arrested at the G1/S boundary. The molecular mechanisms underlying these cell cycle arrests are unknown. Because G0/G1 and G1/S transitions are regulated in part by cyclin-dependent kinase Cdk6, we investigated the possibility that a loss of activity of this kinase is implicated in the age-related dysfunction of the cell cycle in its initial phases. G0/G1 and G1/S blocks were first confirmed by [(3)H]uridine and [(3)H]thymidine incorporation studies in anti-CD3 activated T lymphocytes derived from elderly donors. In the same cell preparations, in vitro phosphorylation of recombinant truncated Rb protein by immunoprecipitated Cdk6 was significantly decreased. The reduced Cdk6 activity was not attributable to a low level of the protein since a 24-h activation resulted in a comparable expression of the kinase in T cells from young and old individuals. However, at least two other mechanisms might be incriminated in the loss of Cdk6 activity: (1) a poor induction of the associated cyclin D2 upon anti-CD3 stimulation and (2) a delayed downregulation of the Cdk inhibitor p27 following cell activation. The low Cdk6 activity observed in T lymphocytes from the elderly was associated with a defective phosphorylation of the endogenous Rb protein and an increased sequestration of the E(2)F-1 transcription factor, possibly resulting in early cell cycle arrest.

Lack of hepatitis C virus replication intermediate RNA in diseased skin tissue of chronic hepatitis C patients.

The extent of extrahepatic hepatitis C virus (HCV) replication seems to be low-level and confined to cells of hematopoietic lineage. However, given the spectrum of extrahepatic manifestations associated with HCV, several tissues other than the liver have been suggested as targets of HCV replication and damage. The presence and level of HCV RNA were examined in 19 skin tissue samples from patients chronically infected with HCV and referred for lichen ruber planus (n = 11) or cutaneous vasculitis associated with mixed cryoglobulinemia (n = 8). Serum HCV RNA was quantitated and genotyped by assays that are available commercially. Tissue HCV RNA of genomic- and minus-strand polarity was titrated by a strand-specific semiquantitative RT-PCR. Low titers of genomic-strand HCV RNA were found in three skin specimens from patients with cutaneous vasculitis due to mixed cryoglobulinemia, but in none with lichen ruber planus. The replication intermediate HCV RNA was not detected in any of the skin tissues examined, independent of the serum HCV RNA level or genotype. It is concluded that the occurrence of cutaneous vasculitis and lichen ruber planus in chronic hepatitis C patients is unlikely to be due to HCV replication in the skin.

Detection of genomic- and minus-strand of hepatitis C virus RNA in the liver of chronic hepatitis C patients by strand-specific semiquantitative reverse-transcriptase polymerase chain reaction.

Studies aimed at correlating the intrahepatic hepatitis C virus (HCV)-RNA level and anatomo-clinical features have been difficult because of sensitivity and specificity shortcomings of available techniques. We titered the genomic- and minus-strand HCV RNAs by a strand-specific, semiquantitative, genotype-independent reverse-transcriptase polymerase chain reaction (RT-PCR) in the liver tissue of 61 patients with chronic hepatitis C. Findings were correlated with the levels of HCV RNA in the serum, the HCV genotype, the expression of intrahepatic HCV antigens, the histological activity (using separate scores for the lobular and the portal/periportal necroinflammatory activity and for the fibrosis), and the response to interferon alfa (IFN-alpha) treatment. Genomic- and minus-strand HCV RNA were detected in 59 and 57 liver specimens, respectively. The HCV-RNA level in the serum correlated with the genomic-strand, but not with the minus-strand, HCV-RNA titer in the liver. No correlations were found between either strand of the intrahepatic HCV RNA and the level of expression of HCV antigens in the liver, or with the grading/staging of the underlying liver disease. The response to IFN-alpha treatment could be predicted by the serum HCV-RNA level and genotype, but not by the intrahepatic level of genomic- or minus-strand HCV RNA. These results suggest that, although the detection of the minus-strand HCV RNA reliably identifies the presence of replicating HCV in its target organ, the quantitative measurement of viremia remains the clinically meaningful "golden standard" for assessing the level of HCV replication.