Paola Montanari

Evaluation of IFN-γ polymorphism +874 T/A in patients with recurrent tonsillitis by PCR Real Time Mismatch Amplification Mutation Assay (MAMA Real Time PCR)

Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP)+874∗T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donors blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p<0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p=0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.

Crying Time and RORγ/FOXP3 Expression in Lactobacillus reuteri DSM17938-Treated Infants with Colic: A Randomized Trial

Objectives To evaluate crying time, retinoid‐related orphan receptor‐&ggr; (ROR&ggr;) and forkhead box P3 (FOXP3) messenger RNA levels (transcription factors that can modulate T cell responses to gut microbes), and to investigate gut microbiota and fecal calprotectin in infants treated with Lactobacillus reuteri for infantile colic. Study design A double‐blind, placebo‐controlled randomized trial was conducted in primary care in Torino from August 1, 2015 to September 30, 2016. Patients suffering from infantile colic were randomly assigned to receive daily oral L reuteri (1 × 108 colony forming unit) or placebo for 1 month. Daily crying times were recorded in a structured diary. FOXP3 and ROR&ggr; messenger RNA in the peripheral blood was assessed with real‐time TaqMan reverse transcription polymerase chain reaction. Gut microbiota and fecal calprotectin were evaluated. Results After infants with colic were supplemented with L reuteri DSM 17938 for 30 days, crying times were significantly shorter among infants with colic in the probiotic group compared with infants in the placebo group (74.67 ± 25.04 [IQR = 79] minutes /day vs 147.85 [IQR = 135] minutes /day [P = .001]). The FOXP3 concentration increased significantly (P = .009), resulting in decreased ROR&ggr;/FOXP3 ratios: 0.61 (IQR = 0.60) at day 0 and 0.48 (IQR = 0.28) at day 30 (P = .028). Furthermore, the probiotic increased the percentage of Lactobacillus (P = .049) and decreased fecal calprotectin (P = .0001). Conclusions Infants with colic treated with L reuteri for 30 days had a significantly decreased crying time and an increased FOXP3 concentration, resulting in a decreased ROR&ggr;/FOXP3 ratio. The treatment reduced fecal calprotectin. Trial registration ClinicalTrials.gov: NCT00893711.

Comparison of Two Available RNA Extraction Protocols for microRNA Amplification in Serum Samples

microRNAs play a critical role in many biological processes such as cell proliferation and maturation, apoptosis, regulation of chronic inflammation and development of cancer.

Human Endogenous Retrovirus Expression in Primary Cutaneous T-Cell Lymphomas.

Background: Mycosis fungoides (MF) and Sézary syndrome (SS) are the most frequent cutaneous T-cell lymphomas (CTCL). Human endogenous retroviruses (HERVs) were reverse transcribed and integrated into primate chromosomal DNA, becoming noninfectious, although various stimuli may reactivate them. HERV expression seems to be impaired in several human diseases but limited data regarding CTCL are available. Objective: To evaluate the endogenous retroviral transcription profile in CTCL and their expression among disease clinical stages. Methods: Peripheral blood mononuclear cells from 42 MF/SS patients were analyzed. Total RNA was extracted and amplified with reverse transcription polymerase chain reaction. Results were compared with those obtained in a cohort of 20 healthy donors. Results: HERVs were significantly overexpressed in MF/SS patients compared with healthy donors. No differences were found between early and advanced CTCL stages. Conclusion: HERVs can act as promoters in MF/SS pathogenesis. It remains to link HERV hyperexpression to the outcome in CTCL patients.

HPyV6, HPyV7 and TSPyV DNA sequences detection in skin disease patients and healthy subjects

The discovery, from 2007, of eight new human polyomaviruses (HPyVs) has revived interest in the Polyomaviridae family and their association with human diseases and cancer. In particular, HPyV6 and HPyV7 were discovered in skin swabs of healthy donors and TSPyV was discovered in a heart transplant recipient affected by virus‐associated Trichodysplasia Spinulosa (TS), a rare skin disease, exclusively found in immunocompromised patients.

Molecular detection of human parechovirus in under-Five-Year-Old Children with gastroenteritis.

Abstract Background Currently, RT-PCR is used widely and considered to be a convenient, useful, and powerful method for molecular diagnosis, to detect pathogens from clinical specimens. Objectives In this work we describe the development of an in-house Real-time Taqman PCR assay for quantification of HPeV in stool specimens. Study designs A total of 137 fecal specimens previously screened for rotavirus and adenovirus were tested for HPeV virus. Results A total of 11 out of 137 (8%) episodes of acute gastroenteritis were associated with HPeV genomic detection with median viral load 14678±28927 genomes/mg fecal specimens. There was no significant difference in the detection rate between male and female (54.5% (6/11) vs. 45.5% (5/11). Among the 11 HPeV-positive cases, 2 were also positive for other viral pathogens, including rotavirus (n=2). Conclusion In conclusion, the development of a laboratory designed Real Time PCR TaqMan assay for quantitative detection of HPeV and the optimization and standardization of this assay using stool of children with acute gastroenteritis are described.

Changes in the Messenger RNA Expression of Toll-Like Receptors 2 and 4 in Healthy Infants According to Age

Background Toll‐like receptors (TLRs) are potentially useful indicators of several pediatric disease states. Here, we explore the mechanisms by which inflammation is regulated by interactions between microbiota and the host. Little data are available regarding the expression of TLRs in postnatal healthy infants. TLR 2 and TLR4 are extracellular TLRs that act as innate immune receptors by recognizing a wide range of endogenous ligands and microorganisms. Methods The aim of this study was to use real‐time polymerase chain reaction to investigate the expression of the messenger RNAs (mRNAs) of TLR2 and TLR4 in blood samples obtained from healthy full‐term infants and toddlers. Results We analyzed the mRNA expression levels of TLRs in 88 healthy term children separated according to age. The median expression level of TLR2 was 1.49 ± 1.10 arbitrary units (AU) (n = 25) in infants younger than 3 months, 0.67 ± 0.72 AU (n = 25) in infants aged between 3 and 12 months, and 0.03 ± 0.02 AU (n = 38) in infants older than 12 months. The median expression level of TLR4 was 1.25 ± 0.79 AU (n = 25) in infants younger than 3 months, 0.75 ± 0.54 AU (n = 25) in infants aged 3 to 12 months, and 0.44 ± 0.28 AU (n = 38) in infants older than 12 months. There was difference in the mRNA expression level of TLR2 and TLR4 between infants aged 0 to 3 and 3 to 12 months and those aged more than 1 year (p < 0.0001 and p < 0.0001, respectively) Conclusion We found that the expression levels of TLR2 and TLR4 were associated with age. In particular, we observed that their expression increased during the suckling period and then clearly decreased once the infants reached 1 year of age (p < 0.001). These findings could be related to microbial colonization and the immune system.

Polymorphisms in Lep and Lepr Genes in Infants: Correlation with Serum Leptin Values in the First 6 Months of Life

ABSTRACT Objective: Because several studies indicate that polymorphisms in leptin (Lep) and leptin receptor (Lepr) genes play a central role in determining obesity, we analyzed 2 single nucleotide polymorphisms (SNPs) in the Lep gene (Lep G2548A and A19G) and one in the Lepr gene (Lepr A668G) to verify the effect of the 3 SNPs on leptin concentrations in infancy. Methods: We enrolled 80 healthy Caucasian infants under 6 months of age, who were genotyped for the 3 SNPs with amplification refractory mutation system–mismatch amplification mutation assay (ARMS-MAMA) real-time polymerase chain reaction (PCR). Serum leptin values were measured with a radioimmunoassay method. Statistical significance was set at p < 0.05. Results: There were no significant differences between individually analyzed leptin polymorphisms Lep G2548A and A19G and serum leptin levels (p > 0.05). Because we found that Lep G2548A and A19G are in linkage disequilibrium on chromosome 7, we performed the haplotype analysis for Lep G2548A and Lep A19G. We obtained higher serum leptin levels in infants with the GG/GG haplotype (p < 0.05). Regarding receptor, we found higher leptin levels in GG-genotype infants for Lepr A668G (p < 0.001). Considering the 3 SNPs together, we found higher serum leptin values in GG/GG-GG infants (LepG2548A/A19G-Lepr A668G; p < 0.001). Conclusion: We obtained higher serum leptin levels in infants with the GG genotype for Lepr A668G, with haplotype GG/GG for Lep G2548A/A19G, and with GG/GG-GG (LepG2548A/A19G-Lepr A668G); thus, it seems that the genotype GG could be a protector against obesity development in infancy and adulthood. Moreover, these data confirm that not variations in the Lep gene as well as in the Lepr gene could play a role in weight gain. Further studies are needed to evaluate the role of genetics and the environment in a predisposition toward obesity later in life.

Aichivirus in Children with Diarrhea in Northern Italy

Objective: Since its discovery, Aichivirus (AiV) A has been detected, with an incidence of 0.9–4.1%, primarily when studying outbreaks of diarrhea in children or young adults. In this paper, we report the first detection of AiV in Piedmont, Italy, in pediatric patients. Methods: A total of 159 fecal specimens (from 96 males and 63 females) previously screened for rotaviruses, adenoviruses, noroviruses, human parechoviruses, saliviruses, and sapoviruses were collected from infants and children with acute gastroenteritis. Results: The most commonly detected virus was norovirus GII (33.80%), fol lowed by rotavirus (21.30%), astrovirus (18.87%), boca virus (13.92%), sapovirus (10.90%), parechovirus (8%), norovirus GI (6.70%), adenovirus (1%), and salivirus (0.52%). Real-time polymerase chain reaction detected AiV A in 1 (0.62%) case subjects. AiV A was detected in monoinfection only in January. Conclusions: Our results indicate that AiV may be associated with a limited number of diarrhea cases in pediatric patients.

Mismatch Amplification Mutation Assay Real-Time PCR Analysis of the Leptin Gene G2548A and A19G Polymorphisms and Serum Leptin in Infancy: A Preliminary Investigation

Leptin is a hormone that regulates food intake and energy metabolism. Its coding gene (LEP) is one of the most promising candidates for obesity. Although some studies have detected associations of different single nucleotide polymorphisms (SNPs) in the LEP gene with serum leptin levels and obesity-related traits, the results are still conflicting. We investigated two SNPs to find relationships with leptin concentrations. Thirty healthy Caucasian infants younger than 6 months were genotyped for the SNPs G2548A and A19G with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system-mismatch amplification mutation assay (ARMS- MAMA) real-time PCR, and serum leptin concentrations were measured with a radioimmunoassay method. Considering the significant linkage disequilibrium observed between the two SNPs, we divided the sample according to the number of GG haplotypes and observed that individuals homozygous for the GG haplotype had higher serum leptin levels in early infancy than the others. Although these preliminary results are based on a limited sample, they suggest that the genetic background seems to play a role in modulating leptin levels in infancy, but changes in leptin levels over infancy and their correlation with obesity need to be further explored. We describe an ARMS-MAMA real-time PCR procedure which could be profitably applied in routine genetic screening.