Oral Microbiota and Immune System Crosstalk: A Translational Research.

BACKGROUND: Oral pathogens may exert the ability to trigger differently the activation of local macrophage immune responses, for instance Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans induce predominantly pro-inflammatory (M1-like phenotypes) responses, while oral commensal microbiota primarily elicits macrophage functions consistent with the anti-inflammatory (M2-like phenotypes). METHODS: In healthy individuals vs. periodontal disease patients' blood samples, the differentiation process from monocyte to M1 and M2 was conducted using two typical growth factors, the granulocyte/macrophage colony stimulating factor (GM-CSF) and the macrophage colony stimulating factor (M-CSF). RESULTS: In contrast with the current literature our outcomes showed a noticeable increase of macrophage polarization from healthy individuals vs. periodontal patients. The biological and clinical significance of these data was discussed. CONCLUSIONS: Our translational findings showed a significant variance between control versus periodontal disease groups in M1 and M2 marker expression within the second group significantly lower skews differentiation of M2-like macrophages towards an M1-like phenotype. Macrophage polarization in periodontal tissue may be responsible for the development and progression of inflammation-induced periodontal tissue damage, including alveolar bone loss, and modulating macrophage function may be a potential strategy for periodontal disease management.

Chronic Periodontitis and Immunity, Towards the Implementation of a Personalized Medicine: A Translational Research on Gene Single Nucleotide Polymorphisms (SNPs) Linked to Chronic Oral Dysbiosis in 96 Caucasian Patients.

Chronic periodontitis (CP) is a complex pathology with a significant impact worldwide causing bone loss. Oral dysbiosis is a highly inflammatory condition associated to a long-term insulting infection and represents an underestimated CP key factor associated with an imbalance of pro-inflammatory and anti-inflammatory gene responses. The presence of a single nucleotide polymorphisms (SNPs) in the promoter region of interleukin 10 (IL-10) gene-1082, -819, and -592 was a possible determinant cause. This translational research aimed to provide outcomes on the role of IL-10 gene expression in bone loss diseases in patients affected by CP. Caucasian patients (n = 96) affected by CP were recruited from the Italian population. The subgingival samples were collected using the Bacterial Periodontal Assessment by Biomolecular Diagnostic® and the characterization of a set of 15 bacterial DNA responsible of periodontitis was performed by real-time multiplex PCR. In addition, two viruses, Epstein-Barr Virus (EBV) and Herpes Simplex Virus 1 (HSV-1), and a pathogenic fungi (Candida albicans) were included as a part of our panel. Our results confirmed an existing association between IL-10 gene polymorphisms and polymorphism of tumor necrosis factor alpha (TNFα), interleukin 1α-ß-RN (IL-1α-ß-RN), collagen type-l alpha (COLIA1), and vitamin D receptor (VDRs) genes in CP. Further studies are needed to improve diagnosis and endorse more effective therapeutic procedures for periodontal disease.

Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.

Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be particularly useful to identify the main respiratory viruses directly from clinical samples, after nucleic acid extraction, and, also, to screen a large number of patients for epidemiological studies.

Molecular markers of influenza B lineages and clades.

Co-circulation of two influenza B virus lineages, B/Yamagata and B/Victoria, has been recognized since the late 1980s. The assessment of the prevalent lineage and the group of viruses in circulation is of importance in order to decide on the vaccine composition and evaluate its efficacy. The molecular characterization of influenza B viruses in circulation has been the aim of this study; this was approached by identifying and locating nucleotide substitutions in the influenza B virus hemagglutinin (HA) and neuraminidase (NA), specific for the lineage and/or clade. By the alignment of 3456 sequences from the influenza GISAID EpiFlu database, a high number of lineage- and group-specific nucleotide positions have been observed in the HA gene, but not in the NA gene. Additionally, an RT-PCR method has been developed, applicable directly to clinical specimens, which amplifies a short HA region that includes a group of unique molecular signatures. Twenty eight influenza B virus-positive respiratory specimens, collected in Tuscany in the seasons 2012-2013 and 2013-2014, were analyzed. The results revealed two clearly distinguishable patterns: one, more frequent, was characterized by all of the nucleotide changes associated with the B/Yamagata lineage (in most cases of Group 2), whereas the other exhibited all of the changes associated with the B/Victoria lineage. It can be concluded that the analysis of this short HA sequence can permit a rapid, highly sensitive determination of influenza B virus lineages and clades.

Reassortment ability of the 2009 pandemic H1N1 influenza virus with circulating human and avian influenza viruses: public health risk implications.

Exploring the reassortment ability of the 2009 pandemic H1N1 (A/H1N1pdm09) influenza virus with other circulating human or avian influenza viruses is the main concern related to the generation of more virulent or new variants having implications for public health. After different coinfection experiments in human A549 cells, by using the A/H1N1pdm09 virus plus one of human seasonal influenza viruses of H1N1 and H3N2 subtype or one of H11, H10, H9, H7 and H1 avian influenza viruses, several reassortant viruses were obtained. Among these, the HA of H1N1 was the main segment of human seasonal influenza virus reassorted in the A/H1N1pdm09 virus backbone. Conversely, HA and each of the three polymerase segments, alone or in combination, of the avian influenza viruses mainly reassorted in the A/H1N1pdm09 virus backbone. Of note, A/H1N1pdm09 viruses that reassorted with HA of H1N1 seasonal human or H11N6 avian viruses or carried different combination of avian origin polymerase segments, exerted a higher replication effectiveness than that of the parental viruses. These results confirm that reassortment of the A/H1N1pdm09 with circulating low pathogenic avian influenza viruses should not be misjudged in the prediction of the next pandemic.

High resolution melting analysis as a tool to detect molecular markers of antiviral resistance in influenza A viruses.

A real-time PCR followed by high resolution melting analysis (HRMA) was developed, for rapid detection of antiviral resistance markers in influenza A viruses, of both H1N1 and H3N2 subtypes. The targets of these assays were the nucleotide substitution G806A (S31N mutation) in the M gene as marker of resistance to adamatanes in influenza viruses A(H3N2), the substitution A356T (E119V mutation) in the N2 gene of influenza viruses A(H3N2) and the substitution C823T (H274Y mutation) in the N1 gene of the pandemic A(H1N1) 2009 virus as markers of oseltamivir resistance. First, the designed primers and the overall protocol of the HRMA were validated using already characterized viral isolates either containing or lacking changes at the tested codons. Then, HRMA was used to search for the marker of oseltamivir resistance in 75 clinical samples, H1N1 2009 positives, analyzed previously by pyrosequencing and Sanger sequencing, and of both adamantane-derivatives and oseltamivir resistance in 57 H3N2 positive clinical samples. The results of HRMA of the H1N1 2009 isolates were in agreement with those obtained by sequencing. As regards the H3N2 isolates, HRMA revealed a widespread resistance to adamantanes with 89.5% nucleotide substitution G806A, while 3% were resistant to oseltamivir (A356T change). HRMA, applied to the detection of markers of resistance to antiviral drugs against influenza A viruses, confirmed to be a procedure flexible, low cost and time-saving, suitable for application to epidemiological surveys and in clinical settings for diagnostic purposes.

Monitoring the susceptibility to oseltamivir of Influenza A(H1N1) 2009 virus by nested-PCR and pyrosequencing during the pandemic and in the season 2010-2011.

For the early detection of the H275Y mutation as a marker of oseltamivir resistance in A(H1N1) pandemic strains, a sensitive and specific pyrosequencing assay was developed. This assay analyses a region 99nts long, encompassing the H275Y site, amplified by a nested PCR. Seventy-five respiratory specimens, obtained from 62 patients during the pandemic and in the 2010-2011 influenza season, in Tuscany, were tested. Resistant strains were demonstrated in 10 patients. In three other patients, resistant and sensitive variants were found. This pyrosequencing assay may be a useful method for monitoring the spread of resistant influenza H1N1 2009 strains.

Human parvovirus PARV4 DNA in tissues from adult individuals: a comparison with human parvovirus B19 (B19V).

BACKGROUND: PARV4 is a new member of the Parvoviridae family not closely related to any of the known human parvoviruses. Viremia seems to be a hallmark of PARV4 infection and viral DNA persistence has been demonstrated in a few tissues. Till now, PARV4 has not been associated with any disease and its prevalence in human population has not been clearly established. This study was aimed to assess the tissue distribution and the ability to persist of PARV4 in comparison to parvovirus B19 (B19V). RESULTS: PARV4 and B19V DNA detection was carried out in various tissues of individuals without suspect of acute viral infection, by a real time PCR and a nested PCR, targeting the ORF2 and the ORF1 respectively. Low amount of PARV4 DNA was found frequently (>40%) in heart and liver of adults individuals, less frequently in lungs and kidneys (23,5 and 18% respectively) and was rare in bone marrow, skin and synovium samples (5,5%, 4% and 5%, respectively). By comparison, B19V DNA sequences were present in the same tissues with a higher frequency (significantly higher in myocardium, skin and bone marrow) except than in liver where the frequency was the same of PARV4 DNA and in plasma samples where B19V frequency was significantly lower than that of PARV4 CONCLUSIONS: The particular tropism of PARV4 for liver and heart, here emerged, suggests to focus further studies on these tissues as possible target for viral replication and on the possible role of PARV4 infection in liver and heart diseases. Neither bone marrow nor kidney seem to be a common target of viral replication.

Human parvovirus B19 (B19V) infection in systemic sclerosis patients.

BACKGROUND: Our previous reports suggested a possible association between parvovirus B19 (B19V) infection and systemic sclerosis (SSc), based on higher prevalence of B19V DNA in SSc patients in respect to controls. METHODS: In the present study, to further evaluate the differences in the pattern of B19 infection in SSc, skin biopsies and bone marrow samples from patients and controls were analysed for B19V DNA detection, genotyping and viral expression. RESULTS: B19V DNA was detected in skin biopsies from 39/49 SSc patients and from 20/28 controls. Bone marrow showed positive in 17/29 SSc patients, 5/21 haematological patients and 0/10 healthy controls. Genotype 1 was more frequent in skin and bone marrow from patients than from controls. Simultaneous persistence of 2 genotypes was detected in SSc skin and bone marrow samples, never in controls. Viral mRNA for capsid protein was detected in the skin of genotype 1-positive patients and not in control skins. CONCLUSION: The results outline some differences in the rate of persistence of B19V DNA, in the simultaneous persistence of 2 genotypes and in the pattern of viral expression among SSc patients and controls.

Tissue persistence of parvovirus B19 genotypes in asymptomatic persons.

Parvovirus B19 (B19V) can persist in immunocompetent symptomatic and non-symptomatic individuals, as demonstrated by the finding of viral DNA in different tissues, in absence of viremia and of anti-B19V IgM. The spread and the nature of this phenomenon have not been clearly determined. In order to investigate the frequency of persistence and the tissue distribution of the three genotypes of B19V, the viral load of the persistent virus and its expression in the affected tissues, 139 tissue samples and 102 sera from 139 asymptomatic individuals have been analyzed by consensus PCRs and genotype specific PCRs for B19V detection and genotyping. Viral load was measured by real time PCR and viral mRNAs were detected by RT-PCR. Altogether, 51% individuals carried B19V DNA, more frequently in solid tissues (65%) than in bone marrow (20%). Genotype 1 was found in 28% tissue samples, genotype 2 in 68% and genotype 3 in 3% only. Viral load ranged from less then 10 copies to 7 x 10(4) copies per 10(6) cells, with the exception of two samples of myocardium with about 10(6) copies per 10(6) cells. mRNA of capsid proteins was present in two bone marrow samples only. In conclusion, in asymptomatic individuals B19V persistence is more common in solid tissues than in bone marrow, and genotype 2 persists more frequently than genotype 1. The results suggest that the virus persists without replicating, at sub-immunogenic levels.

Different behavior of erythrovirus B19 and torquetenovirus in response to a single step of albumin purification.

BACKGROUND: The safety of human serum albumin (HSA) is of special interest with respect to virus transmission because of the wide use of this blood product as a therapeutic agent and also, added to other products, as an excipient or a stabilizer. Conflicting data are reported concerning HSA contamination by small, naked viruses such as the erythrovirus B19 (B19V) and the anellovirus torquetenovirus (TTV). This study has been performed to assess the effect of the HSA purification procedures on the viral contamination. STUDY DESIGN AND METHODS: Known concentrations of B19V and TTV virus were spiked in raw Fraction V, the starting material from fractionated human plasma for HSA purification, which was subsequently submitted to the depth filtration procedure. After spiking, B19V and TTV genome copies were determined by real-time quantitative polymerase chain reaction assays in the mixture at the end of Fraction V dissolution, to determine the virus concentration achieved, in the HSA solution after the filtration step, in the filtered postwashing fluid, and in the supernatant of resuspended Celite. RESULTS: B19V was completely adsorbed by the Celite used as a filter aid in the depth filtration process and was thus undetectable in the resulting HSA-containing fraction. In contrast, in 2 out of 3 experiments, TTV was detected in all samples. CONCLUSION: The different behavior of the two viruses might be a reflection of their different surface charge.