Waterborne Norovirus outbreak at a seaside resort likely originating from municipal water distribution system failure.

In May 2016 a Norovirus (NoV) gastroenteritis outbreak involved a high school class visiting a seaside resort near Taormina (Mascali, Sicily). Twenty-four students and a teacher were affected and 17 of them showed symptoms on the second day of the journey, while the others got ill within the following 2 days. Symptoms included vomiting, diarrhoea and fever, and 12 students required hospitalisation. Stool samples tested positive for NoV genome by Real-Time polymerase chain reaction assay in all 25 symptomatic subjects. The GII.P2/GII.2 NoV genotype was linked to the outbreak by ORF1/ORF2 sequence analysis. The epidemiological features of the outbreak were consistent with food/waterborne followed by person-to-person and/or vomit transmission. Food consumed at a shared lunch on the first day of the trip was associated to illness and drinking un-bottled tap water was also considered as a risk factor. The analysis of water samples revealed the presence of bacterial indicators of faecal contamination in the water used in the resort as well as in other areas of the municipal water network, linking the NoV gastroenteritis outbreak to tap water pollution from sewage leakage. From a single water sample, an amplicon whose sequence corresponded to the capsid genotype recovered from patients could be obtained.

Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity.

Hepatitis B immunity in teenagers vaccinated as infants: an Italian 17-year follow-up study.

We assessed the persistence of hepatitis B surface antigen antibody (anti-HBs) and immune memory in a cohort of 571 teenagers vaccinated against hepatitis B as infants, 17 years earlier. Vaccinees were followed-up in 2003 and in 2010 (i.e. 10 years and 17 years after primary vaccination, respectively). When tested in 2003, 199 vaccinees (group A) had anti-HBs <10 mIU/mL and were boosted, 372 (group B) were not boosted because they had anti-HBs ≥10 mIU/mL (n = 344) or refused booster (n = 28) despite anti-HBs <10 mIU/mL. In 2010, 72.9% (416/571) of participants had anti-HBs ≥10 mIU/mL (67.3% in group A vs. 75.8% in group B; p 0.03). The geometric mean concentrations (GMCs) were similar in both groups. Between 2003 and 2010, anti-HBs concentrations in previously boosted individuals markedly declined with GMC dropping from 486 to 27.7 mIU/mL (p <0.001). Fifteen vaccinees showed a marked increase of antibody, possibly due to natural booster. In 2010, 96 individuals (37 of group A and 59 of group B) with anti-HBs <10 mIU/mL were boosted; all vaccinees of the former group and all but two of the latter had an anamnestic response. Post-booster GMC was higher in group B (895.6 vs. 492.2 mIU/mL; p 0.039). This finding shows that the immune memory for HBsAg persists beyond the time at which anti-HBs disappears, conferring long-term protection.

Molecular study of receptor for advanced glycation endproduct gene promoter and identification of specific HLA haplotypes possibly involved in chronic fatigue syndrome.

The receptor for advanced glycation end product (RAGE) is thought to play an important role in inflammation. Chronic fatigue syndrome (CFS) is a long-lasting fatigue that compromises at least 50% of a subject's daily activities without other known cause. Immune dysfunction has been implicated and an association with a peculiar genetic cytokine profile, predisposing to an immunomodulatory response of inflammatory nature, was found. The aim of this study is to analyse RAGE polymorphisms and HLA-DRB1 alleles in seventy-five Italian CFS patients and 141 controls matched for age, sex and ethnicity. These two groups underwent genomic study for RAGE 374T/A and 429C/T promoter polymorphisms; moreover, 46 patients and 186 controls were typed for HLA-DRB1 at low resolution molecular level. Of these, 31 patients and 99 controls also underwent high resolution analysis to define the HLA-DRB1*11 and DRB1*13 alleles. The haplotypes RAGE-374T, DRB1*04; RAGE-374T, DRB1*09; RAGE-374T, DRB1*11; RAGE-374A, DRB1*13; RAGE-429T, DRB1*04 and RAGE-429C, DRB1*11 were significantly more frequent in CFS patients, whereas RAGE-429C, DRB1*07 would seem protective. A significantly lower frequency of DRB1*1104 (5.4% vs 12.9% p=0.04, OR=0.39) and a significantly higher frequency of HLA-DRB1*1301 (13.0% vs 5.1% p=0.006, OR= 2.79) were found in CFS patients. A synergic effect was observed with RAGE polymorphism. The OR values strengthened in the following cis combinations: RAGE-374A, HLA-DRB1*1104 (OR=0.27) and RAGE-374A, HLADRB1*1301 (OR=6.23). HLA haplotypes rather than single alleles of RAGE or of DRB1 genes seem to be involved in CFS, probably including a subregion of major interest.

HLA haplotypes and birth weight variation: is your future going to be light or heavy?

Birth weight is known to be a direct indicator of perinatal mortality and a clear predictor of adult pathologies too. It has been correlated with several causes of mortality in adulthood: low birth weight with diabetes, nephropathy and cardiovascular diseases and high birth weight with autoimmune diseases and cancer. In genome-wide studies, an extended human leucocyte antigen (HLA) region has been linked to birth weight variation. We focused our attention on the HLA haplotypes marked by HLA-A, HLA-B and HLA-DRB1 polymorphisms in 1206 healthy Caucasian newborns belonging to the Cord Blood Bank of Pavia (Italy) and their mothers, aiming to investigate the association between this restricted HLA region and birth weight variation. In our study, the HLA-B*38;DRB1*13 haplotype showed an ascending trend among centiles addressing to the high foetal weight. The HLA-A*02;B*15 haplotype showed a descending trend among centiles addressing to the low foetal weight. Besides the acknowledged correlation between the HLA-A*02 and HLA-B*15 alleles (as well as low birth weight) and type I diabetes and between the HLA-B*38 and HLA-DRB1*13 alleles (as well as high birth weight) and several autoimmune diseases, we cannot predict if our babies, healthy at birth, will suffer from these pathologies during life. Nevertheless, our data point to the HLA telomeric end for markers linked to the low birth weight and to the HLA centromeric end for markers linked to the high birth weight, thus limiting the region involved in birth weight variation, which still represents a useful predictor of disease risk in adulthood.

Demographic and clinical aspects of an Italian patient population with chronic fatigue syndrome.

OBJECTIVE: The purpose of this study was to investigate demographic and clinical aspects of a group of Italian patients with Chronic Fatigue Syndrome (CFS) which have not yet been described, in order to compare them with International literature, and to better define certain clinical aspects of the syndrome with respect to the Fukuda et al. case definition. METHODS: A detailed questionnaire was sent to patients with certified CFS diagnosed in a referral center and the data were collected two weeks later. RESULTS AND CONCLUSIONS: Besides persistent fatigue, a clinical syndrome with infectious, neurological and rheumatological characteristics is outlined from the data. Demographic characteristics of Italian patients are very similar to those described in international literature. Therapy has yet to be validated with evidence-based studies in Italy. Studies on the prevalence of CFS in Italy are lacking and would be useful to better define the syndrome in this Mediterranean population.

Inflammation and atherosclerosis: the role of TNF and TNF receptors polymorphisms in coronary artery disease.

Inflammation plays an important role in the pathogenesis of atherosclerosis and coronary syndromes; moreover, various lines of evidence suggest that genetic factors contribute significantly to the risk of coronary artery disease (CAD). Through its effects on endothelial function, coagulation, insulin resistance and lipid metabolism, the proinflammatory cytokine TNF could be involved in cardiovascular pathophysiology. The aim of our study is to analyze whether TNF gene promoter (-308 G/A; -857 G/A) and TNF receptor polymorphisms (TNFR1 MspA1 I exon 1 and TNFR2 Nla III exon 6) show involvement in CAD predisposition in a group of Italian patients compared with healthy controls. Genotyping was performed by PCR-RFLP. Consecutive Italian patients with angiographically proven CAD (n= 248) were compared with controls (n=241), matched for age, sex and geographical origins. CAD patients showed a higher frequency of the TNF -308 A allele than healthy controls (p=0.046). After stratification according to risk factors for CAD, our analysis revealed that CAD patients with diabetes (p=0.042) and CAD patients without hypertension (p= 0.0495) displayed a higher frequency of the TNF -308 AA genotype compared with healthy controls. Our data stress the inflammatory nature of CAD and show a possible involvement of TNF -308G/A promoter polymorphisms in the predisposition to the development of this disease. The less frequent A allele seems to be a predisposing factor for development of CAD in particular pathological settings associated with the disease itself, such as diabetes.

The -374T/A variant of the rage gene promoter is associated with clinical restenosis after coronary stent placement.

Upregulation of the receptor for advanced glycation end products (RAGE) may play a crucial role in neointimal formation upon vessel injury. The -374T/A variant of the RAGE gene promoter, which has been associated with an altered expression of the cell-surface receptor, could exert a protective effect toward the development of vascular disease. The aim of this study is to determine the impact of this common genetic variant in the occurrence of clinical in-stent restenosis after coronary stent implantation. The -374T/A polymorphism of the RAGE gene promoter was evaluated by PCR-RFLPs in 267 patients with coronary artery disease who underwent coronary stent implantation and a subsequent coronary angiography 6-9 months later for suspected restenosis. In-stent restenosis was assessed by means of quantitative angiography. Carriers of the -374AA genotype showed a significantly reduced risk of developing restenosis after percutaneous transluminal intervention than non-carriers. To determine whether the protective effect of the homozygous AA genotype toward clinical restenosis was independent of potential confounders, we performed multivariable logistic regression analysis. After allowance for clinical and biochemical risk factors and stent length, the AA genotype remained significantly associated with a reduced prevalence of in-stent restenosis. No relation was evident between the RAGE genotype and established cardiovascular risk factors. In conclusion, the -374AA genotype of the RAGE gene promoter could be associated with a reduced risk of in-stent restenosis after coronary stent implantation.

Distribution of killer cell immunoglobulin-like receptors genes in the Italian Caucasian population.

BACKGROUND: Killer cell immunoglobulin-like receptors (KIRs) are a family of inhibitory and activatory receptors that are expressed by most natural killer (NK) cells. The KIR gene family is polymorphic: genomic diversity is achieved through differences in gene content and allelic polymorphism. The number of KIR loci has been reported to vary among individuals, resulting in different KIR haplotypes. In this study we report the genotypic structure of KIRs in 217 unrelated healthy Italian individuals from 22 immunogenetics laboratories, located in the northern, central and southern regions of Italy. METHODS: Two hundred and seventeen DNA samples were studied by a low resolution PCR-SSP kit designed to identify all KIR genes. RESULTS: All 17 KIR genes were observed in the population with different frequencies than other Caucasian and non-Caucasian populations; framework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present in all individuals. Sixty-five different profiles were found in this Italian population study. Haplotype A remains the most prevalent and genotype 1, with a frequency of 28.5%, is the most commonly observed in the Italian population. CONCLUSION: The Italian Caucasian population shows polymorphism of the KIR gene family like other Caucasian and non-Caucasian populations. Although 64 genotypes have been observed, genotype 1 remains the most frequent as already observed in other populations. Such knowledge of the KIR gene distribution in populations is very useful in the study of associations with diseases and in selection of donors for haploidentical bone marrow transplantation.

Hierarchy of baby-linked immunogenetic risk factors in the vertical transmission of hepatitis C virus.

Mother-to-infant transmission of Hepatitis C Virus (HCV) represents the major cause of pediatric HCV infection today. Immunogenetic influence has been poorly investigated and mainly confined to HLA-class II serological polymorphisms. Among 290 parities, 135 from Pavia and 155 from Bergamo, of HCV-RNA-infected Italian women, 21 babies (7.24%) were HCV-RNA positive at birth and steadily positive over 20 months of life. All the 21 infected babies and 44 randomly selected uninfected ones, born to HCV-RNA+ mothers but steadily negative for HCV-RNA during a follow-up of 2 years, and their mothers were investigated for HLA-G, -C, -DRB1, -DQA1 and -DQB1 genomic polymorphisms. Among the different covariates, HLA-Cw*07, -G*010401, -DRB1*0701, -DRB1*1401 and homozygosity for HLA-G 14bp deletion can be considered as risk factors for HCV vertical transmission. On the contrary, protection was conferred by the HLA-DQB1*06, -G*0105N, -Cw*0602, DRB1*1104 and -DRB1*1302 alleles. Our initial question was: has the immunogenetic profile any role in the protection of the fetus growing in an infected milieu and, if so, is it independent from the other non-immunogenetic parameters? The answer to both questions should be yes.

A first study of cytokine genomic polymorphisms in CFS: Positive association of TNF-857 and IFNgamma 874 rare alleles.

OBJECTIVE: In the past two years we have developed a biological bank of genomic DNA, cDNA, serum and red blood cells of Italian patients with certified CFS from the two Italian referral centers for the syndrome. Recent studies have shown an imbalance in cytokine production in disease states similar to Chronic Fatigue Syndrome (CFS), such as sickness behavior, both in animals and in humans. However we notice that serum cytokine concentrations are often inconstant and degrade rapidly. With this in mind, we investigated cytokine gene polymorphisms in 80 Italian patients with CFS in order to ascertain whether in this group of patients it is possible to describe a genetic predisposition to an inflammatory response. METHODS: We analyzed the promoter polymorphisms of IL-10, IL-6 and the IFNgamma 874 T/A polymorphism in intron 1 with a PCR-SSP method (Cytogen One Lambda Inc. Canoga Park, CA, U.S.A) in 54 patients and TNF-308 G/A and -857 C/T promoter polymorphisms with a PCR-RFLP method (in 54 and 80 patients respectively). RESULTS: There is a highly significant increase of TNF -857 TT and CT genotypes (p = 0.002) among patients with respect to controls and a significant decrease of IFN gamma low producers (A/A) (p = 0.04) among patients with respect to controls. CONCLUSIONS: We hypothesize that CFS patients can have a genetic predisposition to an immunomodulatory response of an inflammatory nature probably secondary to one or more environmental insults of unknown nature.

Non classical HLA genes and non-HLA genes in a population of infants at familial risk of atopy.

AIM: We investigated on parental history and IgE serum level in 2588 consecutive newborns to individuate babies "at risk" of atopy at birth and we analysed the polymorphisms of class III region to evaluate the association with immunogenetic markers of HLA: C4A, C4B, LTA, RAGE and TNFA genes; we performed TNF and IgE receptor (FCERB1) physiologically related gene polymorphisms. RESULT: 791 babies/2588 (30.6%) were considered "at risk" for atopy and followed-up: 400 had familial history of atopy (at least one parent or sibling), 256 had IgE >0.35 kUA/l at birth and during the follow-up and 135 were positive for both conditions. The allele C4B2 was significantly more frequent in the sample of babies at risk (22.1% vs 10%, p< 0.001). Furthermore, the mean value of IgE at birth in babies carrying the allele C4B2 was 2.26 KUA/l versus 0.74 KUA/l in those not carrying this allele (p=0.01). No significant association emerged for RAGE at the centromeric end of class III region and for LTA, TNFA at the telomeric one. TNFRI, TNFRII and FCERB1 gene polymorphisms also seemed not implicated. CONCLUSION: Our study confirms that HLA class III region seems involved in familial predisposition to atopy, and C4B gene probably acts as a marker of a more restricted subregion.

HLA and hypocomplementemia: the disadvantage of carrying the HLA-B35 and the silent alleles of the C4 complement component.

Hypocomplementemia is an extremely complex phenomenon: we devoted our attention to its immunogenetic basis, particularly to the HLA haplotypes involved and to the study of C4 polymorphic genes. With this in mind we analyzed a group of unrelated patients with hypocomplementemia and 15 families suffering from specific C4 deficiency. Firstly, we performed a population analysis in order to identify a statistically significant association: HLA-B35 and C4BQ0 alleles, in the total group of hypocomplementemic individuals, seem to be associated with the primary disease. Secondly, we defined HLA haplotypes clear-cut segregation in the hypocomplementemic families and we identified the most common HLA haplotypes carrying B35 and C4 null allele associated with this condition. With the aid of correspondence analysis and the Transmission Disequilibrium Test (TDT), we measured the strength of this association. In this work, mainly through family analysis, we envisaged a potentially interesting genomic trait, within HLA, close to B locus, that seems to be involved in hypocomplementemia itself and perhaps in hypocomplementemia-related disorders.

Antigen-specific T cell response in infants after recombinant hepatitis B virus vaccination at birth: evaluation of T helper lymphocyte diversity.

Recombinant hepatitis B virus antigen (rHBsAg)-specific CD4+ T cell clones (TCC) were isolated and expanded from the peripheral blood of nine children vaccinated at birth against the hepatitis B (HB) virus. Four of them responded with protective antibody production (responders), three subjects were unable to produce detectable antibody levels even after revaccination (nonresponders), and two infants produced antibodies only after revaccination (slow responders). TCC were then characterized for their ability to produce cytokines known to be important for T cell expansion (interleukin-2, IL-2) and/or effector functions (IL-4, IFN-gamma, IL-10). Results demonstrated that the frequency of rHBsAg-specific TCC in the samples of nonresponders was comparable to or higher than that in the samples of responders. Nevertheless, the majority of TCC obtained from responders or from slow responders before revaccination displayed the T helper 1 (T(H1))-dominant phenotype, while the majority of TCC obtained from nonresponders were nonpolarized T lymphocytes. After revaccination, the distribution of the different T(H) subsets in slow responders was heterogeneous. Overall, our present data suggest that an absence or delay in developing an rHBsAg-specific antibody response to vaccination is not associated with the capacity to generate an Ag-specific T cell response. However, compared to responders, nonresponding infants react to the rHBsAg vaccination with a reduced capacity to expand and differentiate toward polarized T(H) cells.

["Herd immunity" after administration of Pneumococcal conjugate vaccine and its impact on vaccination campaigns]. / "Herd immunity" della vaccinazione antipneumococcica ed impatto delle campagne vaccinali.

Parallel to the diffusion of conjugated pneumococcal vaccine in children, several studies have shown a reduction of the incidence of invasive pneumococcal disease both in the vaccinated and in the unvaccinated. Such an observation is not a surprise if one considers that a similar phenomenon has taken place also after the mass introduction of Hib vaccine, at its turn a conjugated polysaccharide vaccine. Another observation is that also the carriage of vaccinal pneumococci was reduced in both the vaccinated and the unvaccinated, while in some cases the carriage of non-vaccinal serotypes, definitely less pathogenic, could be increased.

Family study of non-responsiveness to hepatitis B vaccine confirms the importance of HLA class III C4A locus.

Non-responsiveness to hepatitis B virus (HBV) vaccine in adults is strongly associated with HLA-C4AQ0,DRB1*0301,DQB1*02 haplotype. This association was also demonstrated in neonates who failed to mount a humoral response to challenge with HBV vaccine. About 4% of vaccinated newborns do not reach a protective antibody level (> or =10 mIU/ml) at seroconversion and 0.4% is a non-responder even after receiving a fourth dose of vaccine (true non-responders (TNR)); while 3.6% achieved an antibody level > or =10 mIU/ml (slow responders (SR)) only when reboostered with the fourth dose. In the present study we extend the vaccination and HLA typing to 91 family members of probands to understand better the possible parent-to-child transmission of this trait. A transmission disequilibrium test (TDT), performed in 27 families, showed that the C4AQ0 allele was almost always transmitted to probands, both TNRs and SRs. Although not statistically significant, the highest LOD score was obtained with C4A locus: 1.58. These results suggest the presence of a region regulating immune response against HBV vaccination near to or coincident with the C4A locus.

Polymorphic markers in MHC class II/III region: a study on Italian patients with myasthenia gravis.

With an Italian case series of 81 Italian patients and 130 controls, we analysed associations between myasthenia gravis (MG) and genetic polymorphisms in the MHC class II/III region. Increases in the frequency of the TNF-B*1, C4A*Q0, C4B*1, DRB1*03 supratype, which is likely part of the 8.1 ancestral haplotype, were maximal in females with early onset (EO) MG vs. controls [p<0.05, relative risk (RR)=9.9]. These patients showed neither a significantly high frequency of thymic hyperplasia, nor high levels of serum anti-acethylcholine receptor antibodies. The DRB1*03 allele was absent in patients with thymoma; however, in comparison with controls, occurrence of this marker was frequent in MG patients (p<0.005; RR=6.2), more frequent in females (p<0.005; RR=7.8) and most frequent in EOMG female patients (p<0.005; RR=15.1). Analysis of the TNF-B*1, C4A*Q0, C4B*1, DRB1*03 supratype and its recombinants showed that the MHC region between C4 and TNF might contain genes that influence susceptibility to MG in females. Polymorphic markers within the supratype, e.g. TNF-B*1 and C4A*Q0, might contribute to pathogenetically significant abnormalities in immune responses in a subset of female MG patients. The combined effect of other intervening genes cannot be excluded.

Paternal HLA genotype and offspring sex ratio.

For twenty years, W.H. James has been proposing that the sex hormone level of both parents could control at least a quota of the secondary sex ratio variation at the time of conception. Observations supporting this hypothesis have come from investigations on some diseases related to the human leukocyte antigen (HLA). In the present study on 1102 healthy Italian families, we investigated the potential effect on the offspring sex ratio of HLA-B alleles on the basis of a genetic model. We defined three subsets of HLA-B alleles and hypothesized a locus (L) with three alleles, L(H), L(N), L(B15), on the basis of the positive, neutral, or negative effect on the testosterone level. According to the genetic model and the dominance relation L(H) > L(B15) > L(N), six genotypic and three phenotypic classes (H, N, B15) can be expected. We found a significantly high number of daughters (66%) born to fathers carrying the B15 phenotype. This result suggests an effect of the HLA-B15 allele on the secondary sex ratio, mediated by a low testosterone level.