The change in Ig regulation from children to adults disconnects the correlation with the 3'RR hs1.2 polymorphism.

BACKGROUND: In the immune system, the serum levels of immunoglobulin (Ig) increase gradually during ageing. Through B cell development, the Ig heavy chain expression is modulated by a regulatory region at the 3' of the constant alpha gene (3'RR), in single copy in rodents and, due to a large duplication, in two copies in apes. The human 3'RR1 and 3'RR2 are both characterized by three enhancers, the central of which, namely hs1.2, is highly polymorphic. Human hs1.2 has four different variants with unique binding sites for transcription factors (e.g. NF-kB and SP1) and shows variable allelic frequencies in populations with immune disorders. In previous works, we have reported that in several autoimmune diseases the *2 allele of hs1.2 is genetically associated to high level of IgM in peripheral blood. In subjects with altered levels of circulating Ig, an increased level was associated to *2 allele of hs1.2 and low levels corresponded to high frequency of *1 allele. RESULTS: We have correlated the allelic frequencies of hs1.2 with IgM, IgG and IgA serum concentrations in two cohorts of healthy people of different age and after three years follow-up in children homozygous for the allele. Here we show that when the expression levels of Ig in children are low and medium, the frequencies of *1 and *2 alleles are the same. Instead, when the Ig expression levels are high, there is a significantly higher frequency of the allele *2. The follow-up of children homozygous for *1 and *2 alleles showed that the increase or decrease of circulating Ig was not dependent on the number of circulating mature B cells. CONCLUSIONS: These data support the idea that under physiologic condition there is a switch of regulative pathways involved in the maturation of Ig during ageing. This mechanism is evidenced by hs1.2 variants that in children but not in adults participate to Ig production, coordinating the three class levels.

Genetic polymorphisms and risk of recurrent wheezing in pediatric age.

BACKGROUND: Wheezing during early life is a very common disorder, but the reasons underlying the different wheezing phenotypes are still unclear. The aims of this study were to analyse the potential correlations between the risk of developing recurrent wheezing and the presence of specific polymorphisms of some genes regulating immune system function, and to study the relative importance of the associations of different viruses and genetic polymorphisms in causing recurrent episodes. METHODS: The study involved 119 otherwise healthy infants admitted to hospital for a first episode of wheezing (74 of whom subsequently experienced recurrent episodes) and 119 age- and sex-matched subjects without any history of respiratory problem randomly selected from those attending our outpatient clinic during the study period. All of the study subjects were followed up for two years, and 47 single nucleotide polymorphisms (SNPs) in 33 candidate genes were genotyped on whole blood using an ABI PRISM 7900 HT Fast Real-time instrument. RESULTS: IL8-rs4073AT, VEGFA-rs833058CT, MBL2-rs1800450CT and IKBKB-rs3747811AT were associated with a significantly increased risk of developing wheezing (p = 0.02, p = 0.03, p = 0.05 and p = 0.0018), whereas CTLA4-rs3087243AG and NFKBIB-rs3136641TT were associated with a significantly reduced risk (p = 0.05 and p = 0.04). IL8-rs4073AT, VEGFA-rs2146323AA and NFKBIA-rs2233419AG were associated with a significantly increased risk of developing recurrent wheezing (p = 0.04, p = 0.04 and p = 0.03), whereas TLR3-rs3775291TC was associated with a significantly reduced risk (p = 0.03). Interestingly, the study of gene-environment interactions showed that rhinovirus was significantly associated with recurrent wheezing in the presence of IL4Ra-rs1801275GG and G (odds ratio [OR] 6.03, 95% confidence interval [CI]: 1.21-30.10, p = 0.03) and MAP3K1-rs702689AA (OR 4.09, 95% CI: 1.14-14.61, p = 0.03). CONCLUSIONS: This study shows a clear relationship between the risk of wheezing and polymorphisms of some genes involved in the immune response. Although further studies are needed to confirm the results, these findings may be useful for the early identification of children at the highest risk of developing recurrent episodes and possibly subsequent asthma.

Human rhinovirus infection in children with cystic fibrosis.

Nasopharyngeal swabs obtained from 78 pediatric patients with cystic fibrosis (CF), including 47 with acute pulmonary exacerbation and 31 in a stable clinical condition, were evaluated for 17 respiratory viruses. Human rhinovirus (HRV) was the most frequently detected virus in patients with pulmonary exacerbation and in those who were clinically stable (21.3% vs. 12.9%; P = 0.52). HRV-A was the main RV detected in patients with pulmonary exacerbations. However, no prevalence of particular HRV-A subtypes was found. This study highlights that RV is frequently found in the respiratory secretions of patients with CF and the impact of HRV-A appears higher than that of the other HRV types in patients with pulmonary exacerbations.

Impact of a mixed bacterial lysate (OM-85 BV) on the immunogenicity, safety and tolerability of inactivated influenza vaccine in children with recurrent respiratory tract infection.

It is known that the immunogenicity and efficacy of conventional inactivated influenza vaccines (IIVs) are not completely satisfactory in children. The aim of this prospective, randomised, single-blind study was to compare the immune response to, and the effectiveness and safety of, an IIV (Fluarix, GlaxoSmithKline Biologicals, Rixensart, Belgium) administered to 68 children aged 36-59 months affected by recurrent respiratory tract infections (RRTIs) who were vaccinated with (n=33) or without (n=35) the mixed bacterial lysate OM-85 BV (Broncho-vaxom, Vifor Pharma, Geneva, Switzerland). OM-85 BV had no effect on seroconversion or seroprotection rates, geometric mean titres, or dendritic cells, which were not significantly different between the two groups. Moreover, OM-85 BV did not significantly increase the pool of the memory B cells that produce IgG and IgM antibodies against the influenza antigens. However, respiratory morbidity was significantly lower in the children treated with OM-85 BV (p<0.05), thus confirming its positive effect on the incidence of RRTIs. There was no difference in the incidence of adverse events between the two groups. These findings show that the immune response of children to influenza vaccine is not significantly influenced by the administration of OM-85 BV. However, the use of OM-85 before and at the same time as IIV seems to reduce respiratory morbidity, and seems to be safe and well tolerated.

Human rhinoviruses and severe respiratory infections: is it possible to identify at-risk patients early?

Molecular methods of viral screening have demonstrated that human rhinoviruses (HRVs) are associated with lower respiratory tract infections (LRTIs, including bronchiolitis and pneumonia), exacerbations of chronic pulmonary disease and the development of asthma. Patients with severe chronic diseases are at greater risk of developing major clinical problems when infected by HRVs, particularly if they are immunocompromised or have a chronic lung disease. Analysing the characteristics of HRVs does not provide any certainty concerning the risk of a poor prognosis and, although viremia seems to be associated with an increased risk of severe HRV infection, the available data are too scanty to be considered conclusive. However, a chest x-ray showing alveolar involvement suggests the potentially negative evolution of a bacterial superinfection. There is therefore an urgent need for more effective diagnostic, preventive and therapeutic measures in order to prevent HRV infection, and identify and treat the patients at highest risk.

Detection of norovirus in respiratory secretions in children with respiratory tract infection.

To evaluate whether norovirus (NoV) can be a possible cause of respiratory tract infection, 562 nasopharyngeal samples collected from children admitted for influenza-like illness were tested for NoV. Three (0.5%) were positive NoV GII.4. The data show that NoV can be found in the respiratory secretions of children with respiratory symptoms and that respiratory involvement can precede gastrointestinal manifestations.

Phylogenetic analysis of human rhinovirus isolates collected from otherwise healthy children with community-acquired pneumonia during five successive years.

In order to evaluate the circulation of the different human rhinovirus (HRV) species and genotypes in Italian children with radiographically confirmed community-acquired pneumonia (CAP), a nasopharyngeal swab was obtained from 643 children admitted to hospital because of CAP during five consecutive winter and early spring seasons (2007-2012). Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify HRV, and the HRV-positive samples were used for sequencing analysis and to reconstruct the phylogenetic tree. HRV was identified in 198 samples (42.2%), and the VP4/VP2 region was successfully amplified in 151 (76.3%). HRV-A was identified in 78 samples (51.6%), HRV-B in 14 (9.3%) and HRV-C in 59 (39.1%). Forty-seven (31.1%) of the children with HRV infection were aged <1 year, 71 (47.0%) were aged 1-3 years, and 33 (21.9%) were aged ≥4 years. Blast and phylogenetic analyses showed that the HRV strains were closely related to a total of 66 reference genotypes, corresponding to 29 HRV-A, 9 HRV-B and 28 HRV-C strains. Nucleotide variability was 37% between HRV-A and HRV-B, 37.3% between HRV-A and HRV-C, and 39.9% between HRV-B and HRV-C. A number of sequences clustered with known serotypes and, within these clusters, there were strains circulating during several seasons. The most frequently detected genotypes were HRV-A78 (n=17), HRV-A12 (n=9) and HRV-C2 (n=5). This study shows that, although it is mainly associated with HRV-A, pediatric CAP can also be diagnosed in subjects infected by HRV-C and, more rarely, by HRV-B. Moreover, a large number of genotypes may be involved in causing pediatric CAP and can be different from year to year. Although the prolonged circulation of the same genotypes can sometimes be associated with a number of CAP episodes in different years.

Complete genome characterization of enterovirus 104 circulating in Northern Italy shows recombinant origin of the P3 region.

Human enterovirus 104 (EV-C104) is a member of the Human Enterovirus species C (Family Picornaviridae, Genus Enterovirus) and has been associated with mild respiratory syndromes. At present, only two EV-C104 complete genome sequences from strains detected in Switzerland and Japan have been deposited in GenBank. In this study a complete genome analysis of seven Italian EV-C104 strains was carried out. In addition, VP1 sequence analysis was performed in an additional 5 Italian strains (for a total of 12 strains). The genome length of the seven strains was 7406 nucleotides (nt). The seven genomes showed 91.0-96.9% nucleotide identity with respect to other available EV-C104 complete genomes. The P1 and P2 regions of the Italian strains were closely related to EV-C104 identified in Switzerland, while the P3 region was closely related to the EV-C117 strain. In addition, bootscan analysis showed the presence of one putative recombination breakpoint between the P2 and P3 regions. Based on the trees constructed with partial VP1/2A nucleotide sequences, as well as the 3D partial coding region tree, the Italian strains appear to form a single and independent cluster together with the EV-C104 Japanese strain. In conclusion, a complete phylogenetic analysis of the relationship between EV-C104 and other known HEV-C strains was achieved. In addition, the recombinant origin of EV-C104, which has circulated in Italy and Japan, was demonstrated.

Pneumococcal bacterial load colonization as a marker of mixed infection in children with alveolar community-acquired pneumonia and respiratory syncytial virus or rhinovirus infection.

BACKGROUND: The main aim of this study was to evaluate whether nasopharyngeal Streptococcus pneumoniae colonization in children with alveolar community-acquired pneumonia (CAP) and respiratory syncytial virus (RSV) or rhinovirus (RV) infection indicates a mixed lung infection. METHODS: The nasopharyngeal secretions of 530 children with radiographically confirmed CAP were tested using the Luminex x TAG respiratory virus panel fast assay. Real-time polymerase chain reaction for the autolysin-A (LytA) and wzg (cpsA) genes of S. pneumoniae was performed on the RSV- and RV-positive samples. RESULTS: Sixty-five of the 126 RSV-positive children (51.6%) were colonized with S. pneumoniae. Mean bacterial load was significantly higher in the patients with alveolar involvement (4.54±1.47 log10 DNA copies/mL vs. 3.75±1.62 log10 DNA copies/mL; P=0.04). Serotypes 5 and 19A were almost exclusively identified in the children with RSV and alveolar CAP, although the difference was statistically significant only for serotype 19A (P=0.03). Eighty-three of the 134 RV-positive children (61.9%) were colonized with S. pneumoniae and again mean bacterial load was significantly higher in the patients with alveolar involvement (4.21±1.37 log10 DNA copies/mL vs. 3.41±1.47 log10 DNA copies/mL; P=0.03). Serotypes 1, 5 and 19A were more frequently identified in the children with RV and alveolar CAP, although the difference was statistically significant only for serotype 5 (P=0.04). CONCLUSIONS: In children with alveolar CAP and RSV or RV infection, the determination of nasopharyngeal pneumococcal bacterial load and identification of the serotypes can contribute to the diagnosis of mixed lung infection.

Genome characterisation of enteroviruses 117 and 118: a new group within human enterovirus species C.

The more than 120 genotypes of human enteroviruses (HEVs) reflect a wide range of evolutionary divergence, and there are 23 currently classified as human enterovirus C species (HEV-C). Two new HEV-C (EV-C117 and EV-C118) were identified in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study, and the present paper describes the characterisation of the complete genome of one EV-C117 strain (LIT22) and two EV-C118 (ISR38 and ISR10) strains. The EV-C117 and EV-C118 5'UTR sequences were related to those of EV-C104, EV-C105 and EV-C109, and were slightly shorter than those of other HEV A-D species. Similarity plot analyses showed that EV-C117 and EV-C118 have a P1 region that is highly divergent from that of the other HEV-C, and phylogenetic analyses highly supported a monophyletic group consisting of EV-C117, EV-C118, EV-C104, EV-C105 and EV-C109 strains. Phylogenetic, Simplot and Bootscan analyses indicated that recombination was not the main mechanism of EV-C117 and EV-C118 evolution, thus strengthening the hypothesis of the monophyletic origin of the coding regions, as in the case of other HEV-C. Phylogenetic analysis also revealed the emergence of a new group within HEV-C that is divided into two subgroups. Nucleotide and amino acid identity in VP1 sequences have been established as useful criteria for assigning new HEV types, but analysis of the complete P1 region improves resolution.

Influenza C virus­associated community-acquired pneumonia in children.

To evaluate the impact of influenza C (ICV) infection in children with community-acquired pneumonia (CAP), all of the children consecutively seen during 4 influenza seasons with respiratory symptoms and radiographically confirmed CAP were prospectively evaluated. ICV was identified in the respiratory secretions of five of 391 patients (1·3%). In children with ICV-associated CAP, clinical data were similar to those observed in children with IAV-associated CAP and worse than those observed in children with IBV-associated. The phylogenetic tree showed that the sequenced strains clustered in two of the six ICV lineages. These findings highlight that ICV can be a cause of CAP of children and that this can be severe enough to require hospitalization.

Full Genome Sequence of a Novel Human Enterovirus C (EV-C118) Isolated from Two Children with Acute Otitis Media and Community-Acquired Pneumonia in Israel.

The new enterovirus C strain EV-C118 belongs to the human enterovirus C species of the Picornaviridae family. We report the complete genome sequence of this strain, which was identified in respiratory specimens of two children hospitalized in Israel because of acute otitis media and community-acquired pneumonia who were enrolled in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study.

Impact of vitamin D administration on immunogenicity of trivalent inactivated influenza vaccine in previously unvaccinated children.

As vitamin D (VD) has a significant regulatory effect on innate and adaptive immunity, the aim of this prospective, randomized, single-blinded, placebo-controlled study was to measure the impact of VD administration on the immune response to trivalent influenza vaccination (TIV). A total of 116 children (61 males, 52.6%; mean age 3.0 ± 1.0 y) with a history of recurrent acute otitis media (AOM), who had not been previously vaccinated against influenza, were randomized to receive daily VD 1,000 IU or placebo by mouth for four months. All of them received two doses of TIV (Fluarix, GlaxoSmithKline Biologicals) one month apart, with the first dose administered when VD supplementation was started. There was no difference in seroconversion or seroprotection rates, or antibody titers, in relation to any of the three influenza vaccine antigens between the VD and placebo groups, independently of baseline and post-treatment VD levels. The safety profile was also similar in the two groups. These data indicate that the daily administration of VD 1,000 IU for four months from the time of the injection of the first dose of TIV does not significantly modify the antibody response evoked by influenza vaccine.

Impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses.

BACKGROUND: Little is known about the prevalence of viral infections in children with community-acquired pneumonia (CAP). OBJECTIVES: To describe the clinical and virological data collected from children with radiographically confirmed CAP in whom 17 respiratory viruses were sought in respiratory secretion samples during the acute phase of the disease. PATIENTS AND METHODS: The study involved 592 children with radiographically confirmed CAP whose respiratory secretion samples were tested using the Luminex xTAG Respiratory Virus Panel Fast assay, which simultaneously detects influenza A virus, influenza B virus, respiratory syncytial virus (RSV)-A and -B, parainfluenzavirus-1, -2, -3, and -4, adenovirus, human metapneumovirus, coronaviruses 229E, NL63, OC43, and HKU1, enterovirus/rhinovirus, and bocavirus. A real-time PCR assay was used to identify the rhinovirus in the enterovirus/rhinovirus-positive samples. RESULTS: A total of 435 children (73·5%) were positive for at least one virus: the most frequently detected was RSV, which was found in 188 (31·7%), followed by rhinovirus (n = 144, 24·3%), bocavirus (n = 60, 10·1%), influenza viruses (n = 57, 9·6), and hMPV (n = 49, 8·2%). Viral co-infections were found in 117 children (19·7% of the enrolled children; 26·9% of those with viral infections). Marginal differences were found between the infections owing to a single virus. Co-infections showed radiographic evidence of alveolar pneumonia significantly more frequently than single infections (OR 1·72, 95% CI 1·05-2·81). CONCLUSIONS: The findings of this study highlight the importance of respiratory viruses (mainly RSV and rhinovirus) in children with CAP and show the characteristics of both the single infections and co-infections associated with the disease.

Virtual quantification of influenza A virus load by real-time RT-PCR.

BACKGROUND: The pan-influenza A real-time RT-PCR detection assay developed by the Centers for Disease Control and Prevention (CDC) during the 2009 pandemic is widely utilized. A quantitative version of the assay may be useful to monitor influenza A infection and response to treatment. OBJECTIVES: To prove in principle the possibility that a virtual quantification tool (VQT) would allow conversion of CDC real-time RT-PCR cycle threshold (Ct) values in virus RNA copy number. STUDY DESIGN: A plasmid carrying the CDC real-time RT-PCR target region of the influenza A Matrix (M) gene was generated. In a multicenter study, a set of 5 ten-fold dilutions (equivalent to 1×10(2) to 1×10(6)copies/reaction) were prepared and distributed to the 4 participating virology laboratories and then amplified to generate a virtual quantification standard curve. Clinical samples (n=120) were quantified in parallel by interpolation with locally generated standard curves and using the VQT. RESULTS: A total of 40 standard curves were obtained by the participating centers (ten from each center). The intra- and inter-laboratory variability showed a coefficient of variation (CV) ≤5%. Influenza A virus quantification in 120 respiratory samples showed a significant correlation between interpolation with locally generated standard curves and the VQT (R(2)=0.9655). Bland Altman analysis showed that the majority (no. 111, 92.5%) of clinical samples had <0.5 log(10) variation. CONCLUSIONS: VQT proofs the concept that qualitative results from real-time RT-PCR assays can be converted into quantitative determination of virus load in clinical samples without running standard curves in parallel.

A novel human enterovirus C (EV-C118) identified in two children hospitalised because of acute otitis media and community-acquired pneumonia in Israel.

We report the discovery of a novel enterovirus C (EV-C118) identified in two Israeli children hospitalised for acute otitis media and community-acquired pneumonia. The highest pair-wise sequence identity scores with the EV-C109 and EV-C117 reference strains were, respectively, 63.5% and 63.6% nucleotide identity, and 82.5% and 79.9% amino acid identity.

Complete genome sequence of a novel human enterovirus C (HEV-C117) identified in a child with community-acquired pneumonia.

The new enterovirus C-117 strain belongs to the human enterovirus C species in the Picornaviridae family. We describe the characterization of the complete genome of this strain identified in a respiratory specimen of a child enrolled in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study evaluating the etiology of community-acquired pneumonia (CAP).

Role of polymorphisms of toll-like receptor (TLR) 4, TLR9, toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A genes in malaria susceptibility and severity in Burundian children.

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries. As genetic variations in the toll-like receptors (TLRs)-signalling pathway have been associated with either susceptibility or resistance to several infectious and inflammatory diseases, the supposition is that single nucleotide polymorphisms (SNPs) of TLR2, TLR4, TLR9, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A could modulate malaria susceptibility and severity. METHODS: This study was planned to make a further contribution to solving the problem of the real role of the most common polymorphisms of TLR4, TLR9, TIRAP and FCGR2A genes in modulating the risk of malaria and disease severity in children from Burundi, Central Africa. All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, between February 2011 and September 2011, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears. In children with malaria and in uninfected controls enrolled during the study period in the same hospital, blood samples were obtained on filter paper and TLR4 Asp299Gly rs4986790, TLR9 G1174A rs352139, T-1486 C rs187084 TLR9 T-1237 C rs5743836, TIRAP Ser180Leu rs8177374 and the FCGR2A His131Arg rs1801274 polymorphisms were studied using an ABI PRISM 7900 HT Fast Real-time instrument. RESULTS: A total of 602 patients and 337 controls were enrolled. Among the malaria cases, 553 (91.9%) were considered as suffering from uncomplicated and 49 (8.1%) from severe malaria. TLR9 T1237C rs5743836CC was associated with an increased risk of developing malaria (p=0.03), although it was found with the same frequency in uncomplicated and severe malaria cases. No other differences were found in all alleles studied and in genotype frequencies between malaria cases and uninfected controls as well as between uncomplicated and severe malaria cases. CONCLUSIONS: TLR9 T1237C seems to condition susceptibility to malaria in Burundian children but not its severity, whereas none of the assessed SNPs of TLR4, TIRAP and FCGR2A seem to influence susceptibility to malaria and disease severity in this population.

Molecular signatures of amyotrophic lateral sclerosis disease progression in hind and forelimb muscles of an SOD1(G93A) mouse model.

AIMS: This study utilized proteomics, biochemical and enzymatic assays, and bioinformatics tools that characterize protein alterations in hindlimb (gastrocnemius) and forelimb (triceps) muscles in an amyotrophic lateral sclerosis (ALS) (SOD1(G93A)) mouse model. The aim of this study was to identify the key molecular signatures involved in disease progression. RESULTS: Both muscle types have in common an early down-regulation of complex I. In the hindlimb, early increases in oxidative metabolism are associated with uncoupling of the respiratory chain, an imbalance of NADH/NAD(+), and an increase in reactive oxygen species (ROS) production. The NADH overflow due to complex I inactivation induces TCA flux perturbations, leading to citrate production, triggering fatty acid synthase (FAS), and lipid peroxidation. These early metabolic changes in the hindlimb followed by sustained and comparatively higher metabolic and cytoskeletal derangements over time precede and may catalyze the progressive muscle wasting in this muscle at the late stage. By contrast, in the forelimb, there is an early down-regulation of complexes I and II that is associated with the reduction of oxidative metabolism, which promotes metabolic homeostasis that is accompanied by a greater cytoskeletal stabilization response. However, these early compensatory systems diminish by a later time point. INNOVATION: The identification of potential early- and late-stage disease molecular signatures in an ALS model: muscle albumin, complex I, complex II, citrate synthase, FAS, and phosphoinositide 3-kinase functions as diagnostic markers and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), Sema-3A, and Rho-associated protein kinase 1 (ROCK1) play the role of disease progression markers. CONCLUSION: The differing pattern of cellular metabolism and cytoskeletal derangements in the hind and forelimb identifies the potential dysmetabolism/hypermetabolism molecular signatures associated with disease progression, which may serve as diagnostic/disease progression markers in ALS patients.