Raffaele D'Amelio

Cross sectional retrospective study of prevalence of atopy among Italian military students with antibodies against hepatitis a virus

Abstract Objective: To investigate the working hypothesis that common infections occurring early in life prevent atopy. Design: Cross sectional, retrospective study of young Italian men with results for hepatitis A serology and atopy. Setting: Air force school for military students in Caserta, Italy. Subjects: 1659 male students aged 17-24, most of whom (90%) were from central and southern Italy. Main outcome measures: Skin sensitisation and specific IgE antibodies to locally relevant airborne allergens; diagnosis of respiratory allergy (asthma or rhinitis, or both); hepatitis A seropositivity. Results: 443 of the 1659 subjects (26.7%) were positive for hepatitis A virus antibody. Atopy was less common among seropositive than seronegative subjects according to skin sensitization (weal reaction ≥3 mm) to one or more allergens (21.9% (97/443) v 30.2% (367/1216), P<0.001); polysensitisation (sensitive to three or more allergens) (2.7% (12/443) v 6.4% (78/1216), P<0.01); high specific IgE concentration (9.7% (43/443) v 18.4% (224/1216), P<0.00005); and lifetime prevalence of allergic rhinitis or asthma, or both (8.4% (37/443) v 16.7% (203/1216), P<0.001). Hepatitis A seropositivity remained inversely associated with atopy after adjusting for fathers education, the number of older siblings, and the area of residence (based on the number of inhabitants). The prevalence of atopy was constantly low among seropositive subjects, whatever the number of older siblings; by contrast, it increased with a decreasing number of older siblings among seronegative subjects. Conclusion: Indirect but important evidence is added to the working hypothesis as common infections acquired early in life because of the presence of many older siblings (among seronegative subjects) or because of unhygienic living conditions (among seropositive subjects) may have reduced the risk of developing atopy. Key messages Young men with antibodies to hepatitis A virus had a lower prevalence of atopy and atopic respiratory diseases, and this was independent of the number of older siblings and other relevant risk factors The prevalence of atopy was as low in seronegative as in seropositive subjects only when they had three or more older siblings Among seropositive subjects the prevalence of atopy was low, whatever the number of older siblings Common infections acquired early in life because of the presence of many older siblings (among seronegative subjects) or because of unhygienic living conditions (among seropositive subjects) may have reduced the risk of development of atopy This study adds indirect but important evidence to the hypothesis that improvements in hygiene and reduced recirculation of common infections may be a major cause of the increasing prevalence of atopy and atopic diseases in Western countries

Long-term immunogenicity of hepatitis B vaccination and policy for booster: an Italian multicentre study

BACKGROUND Universal anti-hepatitis-B vaccination of infants and adolescents was implemented in Italy in 1991. We undertook a multicentre study in previously vaccinated individuals to assess the duration of immunity and need for booster, over 10 years after vaccination. METHODS In 1212 children and 446 Italian Air Force recruits vaccinated as infants and adolescents, respectively, we measured the concentrations of antibodies to hepatitis-B surface antigen (anti-HBs) and the presence of antibodies to hepatitis-B core antigen (anti-HBc) at enrollment; postimmunisation values were not available. Individuals positive for anti-HBc were tested for hepatitis B surface antigen (HBsAg) and hepatitis B viral DNA. Individuals with anti-HBs concentrations at 10 IU/L or more were regarded as protected; those with antibody less than 10 IU/L were given a booster dose and retested 2 weeks later. Individuals showing postbooster anti-HBs concentrations of less than 10 IU/L were offered two additional vaccine doses and retested 1 month after the third dose. FINDINGS Protective anti-HBs concentrations were retained in 779 (64%, 95% CI 61.6-67) children and 398 (89%, 86.4-92.1) recruits. We recorded antibody amounts of less than 10 IU/L in 433 children (36%, 33-38.4) and 48 (11%, 7.9-13.6) recruits. One child and four recruits were positive for anti-HBc, but negative for HBsAg and hepatitis B viral DNA. Antibody concentrations were higher in recruits than in children (geometric mean titre 234.8 IU/L vs 32.1 IU/L, p=0.0001). 332 (97%) of 342 children and 46 (96%) of 48 recruits who received a booster showed an anamnestic response, whereas ten (3%) children and two (4%) recruits remained negative for anti-HBs or had antibody concentrations of less than 10 IU/L. Prebooster and postbooster antibody titres were strongly correlated with each other in both groups. All individuals given two additional vaccine doses (eight children and two recruits) showed anti-HBs amounts of more than 10 IU/L at 1 month after vaccination. INTERPRETATION Strong immunological memory persists more than 10 years after immunisation of infants and adolescents with a primary course of vaccination. Booster doses of vaccine do not seem necessary to ensure long-term protection.

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

BackgroundThe genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary.ResultsGenotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats.ConclusionIn a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

Could Autoimmunity Be Induced by Vaccination

Autoimmune reactions to vaccinations may rarely be induced in predisposed individuals by molecular mimicry or bystander activation mechanisms. Autoimmune reactions reliably considered vaccine-associated, include Guillain-Barré syndrome after 1976 swine influenza vaccine, immune thrombocytopenic purpura after measles/mumps/rubella vaccine, and myopericarditis after smallpox vaccination, whereas the suspected association between hepatitis B vaccine and multiple sclerosis has not been further confirmed, even though it has been recently reconsidered, and the one between childhood immunization and type 1 diabetes seems by now to be definitively gone down. Larger epidemiological studies are needed to obtain more reliable data in most suggested associations.

Modulation of human concanavalin A-induced lymphocyte proliferative response by physiological concentrations of β-endorphin

The effects of physiological concentrations of beta-endorphin on the proliferative response to concanavalin A of human peripheral blood mononuclear cells from a large series of healthy donors are reported. These effects are also compared with those obtained employing beta-endorphin and phytohemagglutinin under the same experimental conditions. The donors (32), aged between 20 and 48 years, chosen among military personnel of the Italian Air Force, underwent clinical and laboratory investigations to exclude any detectable disturbance in their psychophysical fitness. Our results show that beta-endorphin is not mitogenic per se and is unable to modify the response of mononuclear cells to phytohemagglutinin irrespective of the concentration of opioid or mitogen used. beta-Endorphin is also unable to alter the PBMC response to low concentrations of concanavalin A, but significantly increases such a response when higher concentrations of concanavalin A and concentrations of beta-endorphin similar to those found in human plasma under physiological conditions are used. The effect is not reverted by naloxone, the specific opiate antagonist. When the activity of beta-endorphin on the mononuclear cell response to concanavalin A is examined at the single donor level, it is noted that some of the donors fail to show the opioid-dependent increase. The baseline levels of the response to concanavalin A of such subjects, compared to those of the donors whose response is augmented by the opioid, are significantly higher, thus demonstrating that beta-endorphin can selectively modulate concanavalin A-induced mitogenesis with a behavior depending on the individual characteristics of the donors response. The process involves non-opioid cell receptors.

Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

BackgroundAnthrax and plague are diseases caused by Bacillus anthracis and Yersinia pestis respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination.ResultsThirty-nine B. anthracis and ten Y. pestis strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA). Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer) and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced.ConclusionIn this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of B. anthracis and Y. pesti s. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.

Evaluation of the overall degree of sensitization to airborne allergens by a single serologic test: Implications for epidemiologic studies of allergy

In most epidemiologic studies of respiratory allergy, the overall degree of sensitization is usually measured by indices based on skin prick test reactions to a panel of relevant airborne allergens. In the present study we used a single assay (CAP-Phadiatop, Pharmacia, Uppsala, Sweden), which is based on the reaction between serum-specific IgE and a mixture of locally relevant inhalant allergens to measure the degree of sensitization to inhalants in the sera of 1815 young Italian men, in whom a complete diagnostic workup for respiratory allergy was performed in parallel. The intensity of reaction in the CAP-Phadiatop assay was highly correlated with: (1) an allergy index (p < 0.005), based on skin prick test reactivity to seven relevant inhalant allergens (Dermatophagoides pteronyssinus, mixed grass pollens, cat epithelium, Parietaria judaica, Olea europaea, Artemisia vulgaris, Alternaria tenuis); (2) the concentration of specific IgE to the same allergens, measured by CAP-RAST (p < 0.001), in both monosensitized and polysensitized subjects; (3) the rate of respiratory allergic diseases (p < 0.0001). Therefore CAP-Phadiatop intensity of reaction is a reliable indicator of the overall degree of sensitization to inhalant allergens. Furthermore, as demonstrated by relative operating characteristic analysis, it has an acceptable detectability power in identifying subjects with symptomatic expression of the atopy status [P(A) = 0.948]. The evaluation of the degree of sensitization to inhalants by a single and semiautomated assay represents a step forward in the standardization of procedures for epidemiologic studies of inhalant allergic diseases and for mass screening programs, which are aimed at identifying predictive markers of disease susceptibility.

Are Anti-Infectious Vaccinations Safe and Effective in Patients with Autoimmunity?

Vaccinations have been traditionally considered a risk factor for the induction/reactivation of autoimmune diseases. A Medline search through key words “vaccinations and autoimmune diseases” from 1947 through December 2009 was conducted. Until now, vaccination effects in autoimmune diseases have only been studied in over 5000 patients. Vaccinations generally did not induce worsening of disease activity in patients with multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, insulin-dependent-diabetes-mellitus, chronic arthritis in children, vasculitides, and myasthenia gravis, whereas immunogenicity, although protective, was generally lower than in normal controls, depending on disease severity and immunosuppressive therapy. Data are very poor on the efficacy.

Characterization of Specific Immune Complexes in HIV‐Related Disorders

Eighty‐seven seropositive subjects with HIV (human immunodeficiency virus) infection together with 20 normal controls with no history of any illness were investigated for the presence of circulating immune complexes (CIC) by the conglutinin binding assay (KgBA) and further studied for isotype characterization of CIC. Six out of 87 patients showing very high values for immune complexes (CIC) were studied for the presence of free antigen. In 3 out of 6 (1, IVcl; 1, III; I, IVa) we could detect by ultracentrifugation analysis the presence of specific HIV (p15) anti‐HIV (anti‐p15) and gp41‐anti‐gp41 CIC. Evidence in favour of this finding is supported by: (1) the presence of specific CIC (p15‐anti‐p15 or gp41‐anti‐gp41) seen only at pH 7.2; (2) the apparent presence of free antigen and specific HIV antibodies were only at pH 4.0. The relevance of this finding lies in the attempt to explain the occurrence of false seronegativity seen occasionally in symptomatic patients. Thus, the presence of CIC might perhaps interfere in the routine assay (i.e. ELISA) making the diagnosis difficult. All these considerations will have to be taken into account in the future handling of this disease.

Clinical and immunological response to typhoid vaccination with parenteral or oral vaccines in two groups of 30 recruits

The clinical and immunological responses to typhoid vaccination with parenteral and oral vaccines in two groups of 30 adult male subjects were studied. Specific anti-Salmonella typhi cell-mediated immunity and total or specific anti-lipopolysaccharide faecal immunoglobulin (Ig) A titres in vaccinated subjects were monitored. Cellular antibacterial activity was significantly increased only in orally vaccinated subjects. Serum arming activity and inhibition experiments suggested an IgA-dependent cellular cytotoxicity in those orally vaccinated. In these subjects, a total and anti-lipopolysaccharide faecal IgA increase was observed lasting up to 8 months after completion of the vaccination schedule. In parenteral vaccinated subjects, an early onset transitory increase of IgM rheumatoid factor was observed. Oral vaccine was well tolerated and free of side effects, whereas 65% of parenterally vaccinated subjects reported side effects such as fever, headache, malaise and local tenderness in the injection site.