Giovanni Mario Pes

Inflammatory biomarkers in blood of patients with acute brain ischemia

Although many failed surrogate markers are provided in the literature, inflammation may contribute to the outcome of ischemic stroke. In 50 consecutive patients with acute ischemic stroke, in the absence of symptoms and signs of concomitant infection, we evaluated a panel of biomarkers reported to be variably associated with brain ischemia, and correlate their serum level with the brain lesion volume and clinical outcome. Infarct size was calculated on computed tomography (CT) scans by means of the Cavalieris method. Neurological impairment was scored by using the Glasgow Coma Scale, Glasgow Outcome Scale and National Institutes of Health (NIH) scales at stroke onset and 3‐month follow‐up. Some markers showed a direct significant correlation with both initial and final NIH scale and with infarct size, particularly tumor necrosis factor alpha (TNF‐α) (P = 0.002), intercellular adhesion molecule‐1 (P < 0.01) and matrix metalloproteinase‐2/9 (P = 0.001). In contrast to previous reports, interleukin‐6 (IL‐6) serum level showed a significant inverse correlation with both final neurological impairment and infarct size (P < 0.001). This novel finding allows us suggesting that IL‐6, in the context of a complex pro‐inflammatory network occurring during stroke, is associated with neuroprotection rather than neurotoxicity in patients with ischemic brain injury.

Reduced intravenous glutathione in the treatment of early parkinson's disease

1. Several studies have demonstrated a deficiency in reduced glutathione (GSH) in the nigra of patients with Parkinsons Disease (PD). In particular, the magnitude of reduction in GSH seems to parallel the severity of the disease. This finding may indicate a means by which the nigra cells could be therapeutically supported. 2. The authors studied the effects of GSH in nine patients with early, untreated PD. GSH was administered intravenous, 600 mg twice daily, for 30 days, in an open label fashion. Then, the drug was discontinued and a follow-up examination carried-out at 1-month interval for 2-4 months. Thereafter, the patients were treated with carbidopa-levodopa. 3. The clinical disability was assessed by using two different rating scale and the Webster Step-Second Test at baseline and at 1-month interval for 4-6 months. All patients improved significantly after GSH therapy, with a 42% decline in disability. Once GSH was stopped the therapeutic effect lasted for 2-4 months. 4. Our data indicate that in untreated PD patients GSH has symptomatic efficacy and possibly retards the progression of the disease.

Do men and women follow different trajectories to reach extreme longevity

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in pro-inflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human life-span, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years.

AKEntAnnos. The Sardinia Study of Extreme Longevity

This paper describes an epidemiological study performed in all centenarians living in Sardinia, a large island located in the Mediterranean sea, 120 Km from the Italian coast. Due to its longstanding isolation, low immigration rate, high endogamy and rather uniform life-style, Sardinia offers an ideal setting in which to study the genetic traits associated with extreme longevity and successful aging. A total of 233 potentially eligible centenarians were traced in the entire territory. Of these, 66 died prior to being interviewed, 11 were not found and unknown, and 15 refused to be interviewed. A multidi-mensional home interview was administered to 141 centenarians, and an equivalent number of 60-year-old controls matched for gender and area of residence. Furthermore, 41 living siblings of the centenarians, and 41 age- and sex-matched controls for these siblings were also studied. The prevalence of centenarians was 13.56 per 100 000, and the female/male ratio was approximately 2. Prevalence and female/male ratio were consistent across the four Sardinian municipalities and are, respectively, higher and lower than those reported in other population-based surveys. A number of methodological problems confronted in doing the field work, and plans for future analysis of this rich dataset are discussed.

Association between longevity and cytokine gene polymorphisms. A study in Sardinian centenarians

Background and aims: Human longevity seems to be directly correlated with optimal functioning of the immune system, suggesting that some genetic determinants of longevity reside in those polymorphisms for the immune system genes which regulate immuneinflammatory responses, in particular cytokine gene polymorphisms. The frequency of − 174C single nucleotide polymorphism (SNP) in the promoter region of the interleukin(IL)-6 gene is increased in Italian male centenarians. Moreover, the frequency of − 1082G SNP at the 5′ flanking region of the IL-10 gene coding sequence is increased among male centenarians, and that of +874A SNP at the interferon (IFN)- γ gene was found more frequently in female centenarians. These findings indicate that different alleles at different cytokine gene codings for pro- (IL-6, IFN- γ) or anti-inflammatory (IL-10) cytokines may affect the individual life-span expectancy, influencing the type and intensity of immune-inflammatory responses against environmental stressors. Methods: In the present study, we analyzed these IL-6, IL-10 and IFN- γ gene polymorphisms in 112 (36 male, 76 female) centenarians from the island of Sardinia, whose population shows a genetic background quite different from that of mainland Italy, as well as in 137 sixty-year-old controls from the same geographic area. Results: No significant differences were observed on analyzing IL-6, IL-10 and IFN- γ polymorphism frequencies among centenarians and controls, either on the whole and when the data were analyzed according to gender. Conclusions: These data indicate that gene polymorphisms of cytokines playing a major regulatory role in the inflammatory response do not affect life expectancy in the Sardinian population. Thus, cytokine/longevity associations have a population-specific component, being affected by the population-specific gene pool as well as by gene-environment interactions, behaving as survival rather than longevity genes.

p53 Codon 72 Polymorphism and Longevity: Additional Data on Centenarians from Continental Italy and Sardinia

In a previous letter (Bonafe et al. 1999) we tested the hypothesis that polymorphic variants of p53 have an impact on human longevity, by comparing p53 codon 72 allelic and genotypic frequency distributions between young people and centenarians. A nonsignificant difference emerged between the groups, and several explanations were offered. Following the reply letter of Sun et al. (in this issue), we would like to argue with some of their comments and to provide new data regarding centenarians from continental Italy and Sardinia.

Number of autoantibodies and HLA genotype, more than high titers of glutamic acid decarboxylase autoantibodies, predict insulin dependence in latent autoimmune diabetes of adults

OBJECTIVE In latent autoimmune diabetes of adults (LADA), the progression into insulin-dependent diabetes is usually faster than in type 2 diabetes (T2D) but the factors influencing this progression are not completely known. In this study, we searched for sensitive markers associated with early development of insulin dependence. DESIGN The screening of 5568 T2D patients for glutamic acid decarboxylase autoantibodies (GAD65Ab) identified 276 LADA patients (M=131; F=145) and in 251 of them, tyrosine phosphatase-2 (IA-2Ab) and thyroperoxidase autoantibodies (TPOAbs), some clinical features and genotype variation of the main type 1 diabetes (T1D) disease susceptibility loci (HLA-DRB1 and HLA-DQB1) were analyzed. RESULTS Four years after the diagnosis of diabetes, high GAD65Ab titer was not significantly associated with faster progression toward insulin deficiency (P=0.104). Patients with GAD65Ab and TPOAb or IA-2Ab or triple positivity for both islet and TPOAbs (GAD65Ab/IA-2Ab/TPOAb) showed a significantly faster disease progression (P=0.002). Among 104 TPOAb-positive LADA patients, 10 received replacement therapy (l-thyroxine), 43 showed high TSH levels (62.7% developed insulin dependence), and 3 had hyperthyroidism treated with methimazole. Multivariate analysis revealed a significant effect on disease progression only for TPOAb (P=0.022), female gender (P=0.036), low body mass index (BMI; P=0.001), and T1D high/intermediate risk HLA-DRB1/DQB1 genotypes grouped (P=0.020). CONCLUSIONS High GAD65Ab titers per se are not a major risk factor for disease progression in LADA, while the number of positive autoantibodies and HLA DRB1-DQB1 genotypes at high risk for T1D are significant predictors. Moreover, clinical characteristics such as low BMI and female gender are more likely to identify patients who will require insulin therapy within 4 years of diagnosis.

Ultrarapid capillary electrophoresis method for the determination of reduced and oxidized glutathione in red blood cells

We describe a very rapid high‐performance capillary electrophoresis method for the separation and quantification of reduced (GSH) and oxidized (GSSG) glutathione in red blood cells. Two procedures for sample preparation have been compared, Microcon‐10 membrane filtration and acid precipitation. The separation is obtained in an uncoated capillary using a high ionic strength borate buffer at pH 7.8. The intra‐assay coefficients of variation (CVs%) are 1.53 and 1.66 for GSH and GSSG, respectively. The run is shorter than 90 s and the migration time is highly reproducible both for GSH (CV% 0.22) and GSSG (CV% 0.17). When the filtration step is used only GSH is found, whereas both GSH and GSSG are detectable after acid precipitation, suggesting that GSSG revealed after acid treatment may be an artefact due to GSH oxidation. Because of its good analytical performance this method could be used for routine red blood cell glutathione measurement in healthy or pathological conditions.

Y chromosome binary markers to study the high prevalence of males in Sardinian centenarians and the genetic structure of the Sardinian population

We have analyzed a sample of 40 centenarians and 116 young controls from Sardinia, with a set of new Y chromosome binary markers, to evaluate if Y chromosome genes are involved in the high prevalence of males among centenarian Sardinians (1/2 vs. 1/4 in other populations studied). The results indicate that none of the seven lineages that account for >97% of the Y chromosome diversity in Sardinia provide an advantage with respect to the extreme longevity. However, our results, although based on the male-specific Y chromosome polymorphisms, give a clear profile of the pattern of genetic variability in Sardinia. Indeed they indicate that the Sardinian population had two main founder populations that have evolved in isolation for at least the last 5,000 years. These findings set the stage for future studies on longevity and other complex traits in Sardinia.

Association between the HFE mutations and longevity: a study in Sardinian population

Hereditary hemochromatosis is an HLA-linked inherited disease characterised by inappropriately high absorption of iron by the gastrointestinal mucosa. The cysteine-to-tyrosine substitution at codon 282 of the HFE encoding gene sequence is responsible for the disease, although other variants, as H63D and S65C, may modify the affinity of the protein for transferrin receptors. We have recently reported that C282Y mutation is significantly increased in very old (>90 years) Sicilian women, suggesting a role in attainment of longevity. In addition, an increase of H63D polymorphism was also observed in these women but the difference was not significant. To validate and extend these results we investigated the distribution of these three common HFE gene mutations in Sardinian centenarians and controls. DNA samples, obtained from 61 controls and 57 Centenarians, were typed for HFE polymorphisms using sequence specific primers. Among the controls, none was heterozygous for the C282Y mutation, 15 were heterozygous for H63D mutation and one for S65C. Among the centenarians, none was heterozygous for the C282Y mutation whereas 25 were heterozygous for H63D mutation and four for the S65C mutation. No significant differences were observed in frequencies of the different alleles between young and centenarians both on the whole and when the data were analysed according to gender. However, there was a trend for an increased frequency of H63D allele in centenarian women (24 vs. 17%, i.e. 19/80 vs. 13/78). It is noteworthy that the cumulative frequency of H63D mutations in Centenarian and very old women from Sardinia and Sicily is 22 vs. 11%, i.e. 30/136 vs. 23/210, P=0.008. These findings are consistent with the hypothesis that there may be a survival difference for centenarian women, among carriers and non-carriers of alleles involved in iron sparing.