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.

Susceptibility to apoptosis of T lymphocytes from elderly humans is associated with increased in vivo expression of functional Fas receptors.

We recently showed that mature T lymphocytes derived from elderly humans were more susceptible to activation-induced cell death than similar cells from young individuals. Because this excessive apoptosis is unrelated to either the age-associated decrease in IL-2 production, a differential Bcl-2 expression or to a modification of the antioxidant pathway, we examined the possibility that the Fas receptor (FasR) is directly implicated in the generation of the unwarranted death signal. We investigated the expression and the function of FasR on T lymphocyte populations from healthy young and elderly individuals. We found that the frequency of FasR+ T cells increases as a function of age. The FasR expressed at the surface of freshly isolated T lymphocytes from elderly donors appear to be fully functional since their ligation by a cytocidal IgM anti-Fas mAb leads to a significant increase in DNA fragmentation in this cell population. Conversely, exposure of T cells derived from aged individuals to an antagonistic anti-FasR mAb partially prevents the age-related increase in apoptotic cell death. The population of FasR+ T lymphocytes is essentially constituted of previously activated CD45RO+ cells and also includes recently activated lymphocytes bearing the CD25 and CD69 activation markers. The accumulation of chronically and recently in vivo activated T-cells with age probably contributes to the amplification of the process of Fas-mediated cell death in T lymphocytes isolated from senescent organisms.

Excessive apoptosis of mature T lymphocytes is a characteristic feature of human immune senescence.

Recent evidence suggests that apoptotic deletion of activated mature lymphocytes is an essential physiological process implicated in both the regulation of the immune response and the control of the overall number of immunocompetent cells. Tightly interrelated signaling mechanisms convey either activation or death messages, achieving the necessary equilibrium between cell proliferation and cell deletion. During the course of aging, numerous alterations of these signaling pathways may shift the balance toward cell death. In the present investigation, the reduced DNA synthesis of anti-CD3 activated T lymphocytes isolated from elderly individuals is associated with an important and early cell deletion from the cultures. Visualization of DNA fragmentation in the remaining activated cells argues in favour of the apoptotic nature of the cell deletion. Quantification of histone-associated DNA fragments shows that the apoptotic process is greatly amplified in activated lymphocytes derived from senescent organisms. Further analysis reveals that IL-2 deprivation does not play a significant role in the age-related increase in apoptosis. Partial correction of this excessive apoptosis by products that bypass the early steps of the signaling cascade suggests that transmembrane signaling defects are involved in this process. Exploration of the antioxidant pathway reveals that the increased susceptibility of lymphocytes from senescent organisms to apoptosis is not explained by a decreased Bcl-2 expression and is not influenced by a modification of the intracellular concentration of glutathione (GSH).

Age-related tyrosine-specific protein phosphorylation defect in human T lymphocytes activated through CD3, CD4, CD8 or the IL-2 receptor.

Although transmembrane signaling defect has been recognized as one of the major functional alterations involved in immune senescence, its biochemical nature as well as its precise molecular localization are still unknown. The available data indicate that an early step in the signaling cascade may be affected during the aging process. Because protein tyrosine kinases (PTK) are ubiquitously implicated in the initiation of physiological signals, they appear as prime candidates for age-related changes. The present investigation examined the effect of age on the activity of PTK associated with CD3, CD4, CD8 or the IL-2 receptor (IL-2R) in human T lymphocytes. By comparison with cells derived from young individuals, anti-CD3-activated T lymphocytes from elderly donors were more susceptible to herbimycin A, a PTK inhibitor known to prevent signal transduction by the T cell antigen receptor. This increased sensitivity of cells from senescent organisms to PTK inhibitors is most likely related to a lesser PTK activity since a significant decrease in the tyrosine phosphorylation of particular endogenous substrates was observed as a consequence of either CD3, CD4, CD8 or IL-2R activation. However, no age-related difference in tyrosine phosphorylation could be demonstrated when T cells were activated by pervanadate, a pharmacological activator of PTK. These results suggest that the intrinsic activity of the enzymes is preserved and that the age-associated defect in PTK activation occurs as a consequence of an upstream biochemical alteration. The defect in PTK activation could be the primary cause for the dysfunction of various components of the signaling cascade observed during the course of aging.

Autonomic function and autoantibodies to autonomic nervous structures, glutamic acid decarboxylase and islet tyrosine phosphatase in adolescent patients with IDDM.

Recent studies have linked autoimmunity to nervous tissue structures and diabetic autonomic neuropathy, but data on the early stage of IDDM and on the natural history of this association are not available. For this reason, we investigated autonomic nervous function, and the presence of autoantibodies to sympathetic and parasympathetic nervous structures, to glutamic acid decarboxylase (GAD) and tyrosine phosphatase (IA-2/ICA512) in 85 adolescents with insulin-dependent diabetes mellitus (IDDM) (mean age 14.7+/-1.6 yr, mean duration of diabetes 6.8+/-3.5 yr), and 45 age and sex-matched healthy subjects. Nervous tissues autoantibodies were detected using an indirect immunofluorescent complement-fixation technique, with monkey adrenal gland, rabbit cervical ganglia and vagus nerve as substrates. GAD and IA-2/ICA512 autoantibodies were detected by radioimmunoprecipitation assay. Seven patients (8%) had anti-vagus nerve autoantibodies, 7 other patients (8%) had anti-cervical ganglia autoantibodies, while all controls were negative (P < 0.05). Anti-adrenal medulla antibodies were detected in 16 patients (19%) and in 2 control subjects (P<0.02). None of the patients had autonomic symptoms. When patients were divided according to the presence or absence of autoantibodies, values of the cardiovascular tests (deep breathing, 30:15 ratio, Valsalva ratio) were similar in the two groups and similar to those in healthy subjects. However, when considered together, patients positive for one or more autoantibody showed a trend for lower values of deep breathing test and 30:15 ratio test, compared with healthy control subjects, which failed to reach conventional significance values (P=0.17 and P=0.07, respectively). No correlation was found between cardiovascular parameters and metabolic control or diabetes duration. There was no association between autoimmunity to nervous tissue structures and presence of GAD and IA-2/ICA512 Ab, and no correlation between these two autoantibodies and values of cardiovascular tests. Our data indicate that autonomic dysfunction is not a characteristic of young diabetic patients, but that autoantibodies against autonomic nervous structures are present during the first 1 to 15 yr of diabetes. GAD and tyrosine phosphatase appear to be excluded as target autoantigens within autonomic structures. Follow-up studies are required to evaluate future autonomic dysfunction and symptoms in these patients, and to establish whether the subtle autonomic dysfunction detected and/or the nervous tissue autoantibodies, are predictive of the development of this complication.

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.

[Analysis of seminal fluid: modern aspects of an old examination]. / L'analisi del liquido seminale: moderni aspetti di un vecchio esame.

Semen analysis stands as the most widely employed test for the diagnosis of male infertility. Subjectivity of evaluation and intra-individual variations of sperm concentration and motility are major limitations of this technique. Intra-individual variations are due to spontaneous circannual rhythmicity, to collection artifacts or to several environmental, physiological and pathological factors. The diagnostic and prognostic usefulness of semen analysis is related to strict compliance with the guidelines recently suggested by the World Health Organization. In recent years, the development of computerized systems provides an objective and rapid method for semen analysis, suitable for the study of more sophisticated parameters of sperm motility. Electron microscopy should be performed for the evaluation of ultrastructural abnormalities of spermatozoa in men with infertility of uncertain origin.

[Diabetes, obesity, hypertension and the autonomic nervous system]. / Diabete, obesità, ipertensione e sistema nervoso vegetativo.

Functional changes of the autonomic nervous system may represent a common pathophysiologic factor in the association between non insulin-dependent diabetes, obesity, and essential hypertension. In all these conditions a number of sympathetic and/or parasympathetic dysfunctions consistent with autonomic neuropathy or simply with functional adaptations to haemodynamic changes have been reported. Autonomic neuropathy is a well known diabetic complication which is responsible for some clinical aspects of different severity. Subtle sympathetic and parasympathetic abnormalities possibly affecting thermogenesis have been shown in obese people. An increased sympathetic activity has been proposed as one of the pathogenetic mechanisms of essential hypertension. Finally, the association between diabetes, obesity, hypertension and sympathetic overactivity could be explained by a common trans-membrane ionic disturbance with an increase of intracellular calcium and a decrease of both intracellular magnesium and pH.

[Systemic lupus erythematosus in an elderly man with hepatic involvement, skin hyperpigmentation and increase in tumor markers]. / Lupus eritematoso sistemico in uomo anziano con interessamento epatico, iperpigmentazione cutanea e aumento dei markers tumorali.

A previously healthy 62 years old man was admitted for weight loss, weakness and pigmentation of skin with increased tumor markers and altered hepatic tests. The final diagnosis was Systemic Lupus Erythematosus, despite clinical appearance. High doses of glucocorticoids were used and a sudden improving of the disease was obtained.

Autonomic nervous activity in obese subjects before and after caloric restriction.

The heart rate response to deep breathing (DB test) and standing (30:15 r test) and the blood pressure response to standing (LS test) and sustained handgrip (HG test) were assessed in 19 obese subjects and 15 age matched lean controls. The results of DB, 30:15 r and LS tests were not significantly different in both groups. The diastolic blood pressure increase during handgrip was significantly higher in obese than in control subjects. After a period of caloric restriction the tests were repeated in 9 patients who had obtained a weight loss of at least 5 kg: a significant decrease in heart rate, diastolic blood pressure and 30:15 r results was observed, whereas the caloric restriction did not cause significant variations in the results of DB, LS and HG tests. Our results suggest that in obese patients some autonomic nervous changes can occur before and after weight loss.

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.

[ACE inhibitors and vagal activity: the effect of captopril and lisinopril on cardiovascular reflexes]. / ACE-inibitori e attività vagale: effetto di captopril e lisinopril su alcuni riflessi cardiovascolari.

The influence of the ACE-inhibitors captopril and lisinopril on parasympathetic activity in normotensive subjects was evaluated. Three cardiovascular tests which explored chiefly parasympathetic function (deep breathing, lying to standing and Valsalva test) were performed in 10 normotensive volunteers (mean age 26.1 years) in both basal conditions and after four days of treatment with either captopril (25 mg twice a day) or lisinopril (20 mg once a day). Mean blood pressure was not influenced by captopril, whereas it was significantly lowered with lisinopril (from 94.4 +/- 6.8 to 88.7 +/- 5.7 mmHg; p < 0.05). Neither drug interfered with heart rate or with the results of the deep breathing and Valsalva tests. The 30/15 ratio, an index of heart rate variability during the lying to standing test, significantly worsened after assumption of both captopril (from 1.37 +/- 0.18 to 1.21 +/- 0.14; p < 0.05) and lisinopril (from 1.31 +/- 0.17 to 1.20 +/- 0.11; p < 0.05). Although our subjects had a lisinopril-induced drop in blood pressure, their heart rate remained steady. This finding confirms previous studies reporting the lack of reflex tachycardia during ACE-inhibition. The slight effect of ACE-inhibitors on the results of deep breathing and Valsalva tests suggests that such drugs do not directly stimulate vagal activity; the significant decrease of the 30/15 ratio may be due to a functional impairment of the baroreflex mechanism.

[Libido-related changes in the elderly]. / Le modificazioni della libido nell'anziano.

Sexual desire can be influenced in elderly men by a number of factors such as hormonal abnormalities, socio-cultural conditions, chronic diseases, drugs. Testosterone decrease per se is not sufficient to impair sexual function in the elderly; social and psychological factors are probably of relevance as it can be observed in people living in nursing homes. Diabetes may have a major impact on sexual desire. Drugs usually associated with impairment of libido (psychotropic drugs, anti-hypertensives) are frequently used by elderly people; alcohol is a leading cause of sexual dysfunction, particularly in unfavourable social conditions.

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.

Over-estimation of the number of complement receptor type 1 (CR1) on erythrocytes.

The number of CR1 on erythrocytes, as measured by monoclonal antibodies, remains undefined because of the repetitive structure of CR1 and the presence of different types of structural CR1 alleles. We studied the number of CR1 per erythrocyte using two monoclonal antibodies, E11 and 3D9, which recognize different sites on CR1. The number of binding sites was higher for E11 than for 3D9 (ratio E11/3D9: 1.9 +/- 0.4, n = 17); however, this ratio was not affected by CR1 numbers or alleles. Partial digestion with papain of CR1 on erythrocytes abolished the binding of 3D9. It reduced the binding of E11 to one-third of its initial value (0.35 +/- 0.03; n = 13) using cells with different CR1 numbers or alleles. By immunoblotting, a unique 75-kDa stump of CR1 remained attached to the erythrocytes for every allele studied. Taken together, these results suggest that the number of CR1 has been over-estimated in the past using E11 by a factor of 2, or more probably 3. However, the over-estimation of CR1 number has been identical for erythrocytes bearing different CR1 numbers or alleles.