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Background aims. The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. Methods. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. Results. Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. Conclusions. We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements. [ABSTRACT FROM AUTHOR]

Abstract: Specific combinations of killer immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA) class I molecules characterized by a particular residue 80 are significantly associated with outcomes in different pathologic conditions, such as autoimmunity, pathogenic infection, cancer, and reproductive failure. Thus, a simplified method for HLA typing used in association with the analysis of KIR genotype (Kirotype) is of particular interest to extend the analysis of larger series. Here, we describe a quick and inexpensive method that allows use of pyrosequencing, a helpful subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I80 or -Bw4 T80, HLA-C1, or -C2 groups and HLA-A allotypes sharing Bw4+ epitope or the rare HLA-B allotypes displaying the C1 motif. In particular, this analysis is focused on the amino acids around residue 80, known to be relevant in defining the affinity of KIR/HLA interaction and in the functional effects. This method was demonstrated to have good sensitivity, specificity, and reproducibility of detection and it was validated using a panel of HLA-typed International Histocompatibility Workshop (IHW) cell lines and clinical isolates. Using an allele quantitative acquisition mode, the method permitted us to obtain an accurate sequencing as required in heterozygous and/or homozygous sample definition. [Copyright &y& Elsevier]

Abstract: To analyze 67 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolated from pediatric hospital infections, we used multilocus variable-number tandem-repeat DNA sequence-based techniques, targeting the protein A polymorphic X region and the clumping factor B complete R domain. We define a “clfB similarity score” and then compare the double loci analysis of closely related MRSA isolates with pulsed-field gel electrophoresis (PFGE). We found an endemic clone (MLST-ST8, spa-t008, SCCmecIV, ClfB lineage 1) able to originate 3 possible outbreaks and a second clone (MLST-ST152, spa-t355, SCCmecV, ClfB lineage 4) responsible for limited cases of MRSA infections, indicating that the combination of spa and clfB-lineage typing is useful to trace MRSA pediatric outbreaks. [ABSTRACT FROM AUTHOR]

Surface expression of human leukocyte antigen (HLA)‐class I molecules is critical for modulating T/natural killer lymphocytes' effector functions. Among HLA molecules, HLA‐C, the most recently evolved form of class I antigens, is subjected to both transcriptional and multiple post‐transcriptional regulation mechanisms affecting its cell surface expression. Among the latter a region placed in the 3′ untranslated region of HLA‐C transcript contains the single nucleotide polymorphism (SNP) rs67384697 "G‐ins/del" that has been found to be strictly associated with surface levels of HLA‐C allomorphs because of the effect on the binding site of a microRNA (Hsa‐miR‐148a). Higher expression of HLA‐C has been proved to influence HIV‐1 infection via a better control of viremia and a slower disease progression. More importantly, the analysis of SNP rs67384697 "G‐ins/del" combined with the evaluation of the HLA‐Bw4/‐Bw6 C1/C2 supratype, as well as the killer immunoglobulin‐like receptor genetic asset, has proved to be pivotal in defining the status of Elite Controllers in the Caucasian population. Here we describe a new reliable and fast method of allele‐specific real‐time PCR to monitor the integrity/disruption of the binding site of the microRNA Hsa‐miR‐148a in a high‐throughput format that can be easily applied to studies involving large cohorts of individuals. [ABSTRACT FROM AUTHOR]

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia. [ABSTRACT FROM AUTHOR]

Background: Hirschsprung disease (HSCR) is a congenital anomaly of the enteric nervous system. Abnormal microbiome composition was reported in HSCR patients. In this study, we addressed and analyzed microbiome modifications with relation tosurgery and HSCR associated enterocolitis (HAEC). Methods: The faecal microbiome of 31 HSCR patients (overall 64 samples) was analyzed. HAEC was diagnosed and classified according to a combination of Pastor's and Elhalabi's criteria. Stool samples were analyzed by 16S sequencing (7 out of 9 polymorphic regions). Compositional and relative abundance profiles, as well as the functional potentials of the microbial community, were analyzed with the marker gene sequencing profiles using PICRUSt. Results: The relative abundance of Bacteroidetes showed a severe decrease with slow recovery after surgery. Conversely, Proteobacteria transiently increased their abundance. Noteworthy, a strong linkage has been found between Proteobacteria descendants and HAEC occurrences. The inferred functional analysis indicated that virulence factors and fimbriae or pili might be associated with HAEC. Conclusions: Our study, addressing microbiome dynamics, demonstrated relevant changes after surgical manipulation. Alpha-diversity analyses indicated that surgery deeply affects microbiome composition. Proteobacteria and Enterobacteriaceae seem to play a pivotal role in HAEC occurrences. Several virulence factors, such as fimbriae or pili, might explain the HAEC-predisposing potential of selected microbiomes. These results suggest some innovative therapeutic approaches that deserve to be tested in appropriate clinical trials. [ABSTRACT FROM AUTHOR]

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/β2 microglobulin [β2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to β2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to β2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication. [ABSTRACT FROM AUTHOR]

Abstract: Background: Human cytomegalovirus (HCMV) is an opportunistic pathogen especially for immuno-suppressed subjects that might develop pharmacological resistance in patients undergoing prolonged antiviral treatment. Ganciclovir (GCV) is the drug used as first choice therapy in affected children and a GCV-resistant phenotype is mainly linked to mutations of the viral protein kinase UL97. Objectives: Here a new quantitative pyrosequence (PSQ) method is presented that allows detection and quantification of the viral species carrying the more frequent UL97 mutations responsible for GCV resistance in clinical samples (>80% of known cases). Study design: The system has been validated using two independent approaches (cloning and sequencing of UL-97 gene fragments and real-time PCR) and clinical samples derived from 3 pediatric patients. Results: The UL97 pyrosequencing analysis has indicated a significant increase of mutant viruses carrying the H520Q and C592G mutations. In particular, the H520Q viral mutation, known to increase GCV resistance (IC50=10) increased around 5 times during hospitalization. In addition, C592G (known to have IC50=2.9) also increased 3 times. Conclusions: PSQ is a quick, cheap, high throughput and sensitive analysis method to detect GCV-associated resistance mutation useful to follow antiviral therapy in perinatal CMV-infection as well as in immune-suppressed patients. [Copyright &y& Elsevier]

In a prospective study of acute pharyngitis in Italian children, 69 (38.3%) of 180 isolates of Streptococcus pyogenes were resistant to macrolides. S. pyogenes was eradicated in 12 (63.1%) of 19 patients with erythromycin-resistant S. pyogenes treated with clarithromycin and in 22 (88%) of 25 patients with erythromycin-susceptible strains. The constitutive-resistant phenotype was correlated with failure of macrolide treatment. [ABSTRACT FROM AUTHOR]

Gut microbiota plays a role in human health and autoimmunity. Among environmental factor linked to type 1 diabetes (T1DM) pathogenesis, gut microbiota impairment seems to be involved. We evaluated gut microbial fingerprinting in 31 pediatric patients with new-onset T1DM and 25 healthy children using multiple polymorphic region of the 16S rRNA. We performed machine learning and metagenome functional analyses to identify significant taxa and metabolic pathways and correlate with clinical and metabolic parameters. Inclusion criteria were living in Northern Italy, born from Caucasian parents, singleton birth, personal history negative for acute/chronic gastrointestinal diseases and/or antibiotic or probiotics administration. Different Bacteroidetes species i.e., B.stercoris (q=1,473E-4), B.intestinalis (q=0.010), B.fragilis (q=0.0452) were significantly more frequent in patients as well as B.bifidum, Gammaproteobacteria, Holdemania, Synergistetes and their descendants. Abundance of the Synergistetes (q=0.001), Synergistia (q=4.798E-5), Synergistales (q=2.64E-4) and Synergistaceae (q=0.0017) was reported. Among Deltaproteobacteria descendants, the Desulfovibrionales (q=0.0016), Desulfovibrionaceae (q=0.016) and the Bilophila genus (q=0.036) were significantly lower in patients as well as B.vulgatus, Deltaproteobacteria, Parasutterella and Lactobacillus, Turicibacter genera. BMI-SDS, insulin autoantibodies, glycemia, HbA1c, Tanner and age were the most significant positively or negatively parameters related to specific clusters of taxa. The supervised analyses confirmed the importance of B. stercoris in T1DM patients and a relevant role of Synergistetes and its descendants. The robustness and coherence of our results underline the relevance of studying microbioma using multiple polymorphic regions, different types of analysis and approaches within each analysis. Disclosure: G. d'Annunzio: Consultant; Self; Sandoz. R. Biassoni: None. M. Squillario: None. E. Ugolotti: None. A. Barla: None. G. Piccolo: None. N. Minuto: None. M. Maghnie: None. [ABSTRACT FROM AUTHOR]