Th1, Th2, Th17 and regulatory T cell pattern in psoriatic patients: modulation of cytokines and gene targets induced by etanercept treatment and correlation with clinical response.

BACKGROUND: Psoriasis is sustained by pro-inflammatory CD4+ T helper cells mainly belonging to the Th1, Th17 and Th22 lineage. OBJECTIVE: To identify whether treatment with the anti-tumour-necrosis-factor antagonist etanercept is able to induce significant modulations in transcription factor and cytokine mRNA gene expressions related to the different T cell immune response polarization (Th1, Th2, Th17 and regulatory T cells, Treg and to correlate them with clinical response. METHODS: The study population included 19 psoriasis patients treated with etanercept and 19 healthy subjects. Blood samples were collected at baseline and every 4 weeks during treatment. Taqman quantitative real-time polymerase chain reaction was applied to analyse the expression of: Stat-4, T-bet, IL-12p35 and IFN-γ (Th1-related); GATA-3, IL-4 (Th2-related); Stat-3, RORγt, IL-23p19 (Th17-related); Foxp3, IL-2 (Treg-related). Flow cytometry was applied to analyse CD4+CD25+(bright)Foxp3+ cells in peripheral blood. RESULTS: Upregulation of Th1 and Th17 and downregulation of Treg subsets was found at baseline. The response to etanercept could be associated with a significant reversal of the Th1/Th17 activation, and a concomitant upregulation of Th2 and Treg subsets. CONCLUSION: Our data may contribute to a better understanding of the mechanisms underlying the achievement of clinical response in psoriasis and could be helpful for the identification of early predictive markers of response.

Detection of the novel variant of influenza virus A/H1N1v in bronchoalveolar lavage of adult hospitalized patients during the 2009/2010 winter season.

AIM: The aim of this study was to report most recent data regarding the occurrence of influenza A virus H1N1v in the lower respiratory tract from a cohort of hospitalized adult patients during the winter season 2009/2010 and investigated the main clinical features and outcomes. METHODS: A total of 130 consecutive BAL specimens (collected from October 2009-March 2010) of 101 patients were retrospectively analyzed for influenza A virus H1N1v positivity using a commercial kit. RESULTS: Overall, 19/130 (14.6%) BAL specimens from 17/101 (16.8%) patients were positive for the novel influenza A H1N1v virus. H1N1v resulted significantly more prevalent in immunocompetent subjects. As regards clinical features, H1N1v resulted more prevalent in respiratory insufficiency or acute respiratory illness. Thirteen patients died during the analytic period; three of them (23.1%) resulted positive to H1N1v but no direct association has been made. CONCLUSION: Our cohort study of influenza A H1N1v detection in BAL from hospitalized adult patients confirms the overall moderate clinical impact of this virus, as reported in most reports worldwide. It remains to be evaluated the role of reassortment with influenza virus strains circulating in the winter season 2010/2011 and its potential pathogenicity.

Prevalence of polyomaviruses BK, JC, SV40, KI, and WU in non-malignant tonsil specimens.

AIM: The recently described polyomaviruses KI and WU have been detected in respiratory samples, stools, tonsils, and blood, particularly in immunocompromised conditions, although little is known about tissue tropism. Herein we investigated the occurrence of KIV and WUV in non-malignant tonsillar specimens by Real-time quantitative PCR; the presence of polyomaviruses BK, JC and SV40-DNA was also evaluated. METHODS: Twenty-nine non-malignant tonsil specimens obtained from children and adults admitted for tonsillectomy were prospectively studied. Real-time quantitative TaqMan PCR for polyomaviruses KI, WU, BK, JC, and SV40 were performed. RESULTS: KI-DNA was positive in 2/29 tonsillar specimens (6.9%), while BK- DNA, JC-DNA, SV-40 DNA, and WU-DNA sequences were not identified. CONCLUSION: Few studies have investigated the prevalence of polyomaviruses in tonsil specimens, with varying results, and data are particularly scant as regards the newly discovered KIV and WUV. Two major questions remain to be definitely answered at this regard: the possibility that human tonsils represent the initial site of infection and/or a latency site and the biological and clinical meaning of KIV and WUV in different contexts and groups of patients, in that it is not clear whether they are simple bystanders or play a role in tonsil disease.

Lower respiratory tract viral infections in hospitalized adult patients.

AIM: The epidemiology of lower respiratory tract (LRT) viral infections in adults is probably underestimated and the high frequency of multiple viral infections complicates the evaluation of the possible role of the single viruses. The aim of this study was to investigate the clinical epidemiology and impact of respiratory viral pathogens, in particular of those singularly detected, in bronchoalveolar lavage (BAL) specimens from hospitalized adult patients. METHODS: A panel for the detection of 16 respiratory viruses was used to prospectively evaluate 324 consecutive specimens obtained from 219 patients over a full-year period. RESULTS: Two-hundred-twenty-one specimens (68.2%) were positive for at least one virus, 119/324 (36.7%) to a single viral agent. The most commonly detected viruses were herpesviruses HHV-7 (26.2%), human cytomegalo-virus (HCMV, 22.2%), HHV-6 (19.8%), EBV (12.7%), enteroviruses and rhinoviruses (both 11.7%), parainfluenza viruses (4.9 %), and metapneumovirus (4.0%). Human cytomegalo-virus was significantly more prevalent as single viral pathogen with a viral load >105 copies/ml associated to pneumonia in solid organ transplant recipients. Other viral pathogens might account for some cases of pneumonia or respiratory insufficiency, although multiple infections were common. CONCLUSIONS: The use of a comprehensive diagnostic panel for respiratory viral infections may be useful to clarify the epidemiology and clinical impact of viral pathogens in hospitalized adult patients. The occurrence of multiple infections is a common finding and results should be interpreted taking into account the clinical context as well as viral load and the biological characteristics of each virus.

Monitoring of human Cytomegalovirus infection by antigenemia and viremia in the first 100 days following renal transplantation and relation to antiviral strategies.

AIM: Human Cytomegalovirus (HCMV) is a relevant pathogen in transplant recipients, particularly in the first three months post-transplantation. The use of antiviral prophylaxis and pre-emptive therapy is able to reduce incidence of HCMV infection and disease. The incidence of HCMV infection and disease in renal transplant recipients in the first 100 days post-transplantation was investigated, in relation with HCMV serological matching and therapeutic management. METHODS: Incidence of HCMV infection in the first 100 days post-transplantation was evaluated by pp65-antigenemia in 165 patients on a total number of 1241 clinical samples. Patients were divided in four groups according to donor/recipient serological matching: D(-)/R(-) (low risk of HCMV disease), D(-)/R+ and D+/R+ (intermediate risk) and D+/R(-) (high risk). Antiviral strategy (prophylaxis in high risk group; pre-emptive therapy in intermediate risk group, no therapy in low risk group) and immunosuppressive protocol were recorded. RESULTS: Incidence of antigenemia-positivity was as follows: 0/3 D(-)/R(-) patients; 59/130 (45.4%) D+/R+; 5/16 (31.3%) D(-)/R+; 4/16 D+/R(-). No significative difference was found between the four groups in terms of incidence of antigenemia-positivity in the first 100 days following transplantation. Antigenemia values >50 pp65-positive/2x10(5) peripheral blood leukocytes (used to start pre-emptive therapy) were present in 18/130 (13.8%) D+/R+; 1/16 (6.2%) D+/R(-); 0/16 D(-)/R+. Viral kinetics in patients with HCMV infection was described. CONCLUSION: No significative difference was found in terms of incidence of HCMV infection in the first 100 days post-transplantation in relation to immunosuppressive protocol and serological matching, suggesting the appropriateness of antiviral strategies and viral monitoring adopted in this setting.

Risk-based control of food-borne pathogens Listeria monocytogenes and Salmonella enterica in the Italian fermented sausages Cacciatore and Felino.

Fermentation is the most important killing step during production of fermented meats to eliminate food-borne pathogens. The objective was to evaluate whether the food-borne pathogens Listeria monocytogenes and Salmonella enterica may survive during the production of two Italian fermented sausages. Sausage batter was inoculated with five strains of L. monocytogenes or S. enterica (ca. 10(5)-10(6) cfu/g) and their kinetic behavior was monitored during production. Both pathogens survived relatively well (in Cacciatore L. monocytogenes and S. enterica inactivation was ca. 0.38±0.23 and 1.10±0.24 log cfu/g, respectively; in Felino was ca. 0.39±0.25 and 1.62±0.38 log cfu/g, respectively) due to the conditions prevailing during production (slow dehydration rate, small reduction of water activity and fermentation temperature mainly below 20 °C during the first 48 h of fermentation). Quantitative analysis of data originating from challenge tests provide critical information on which combinations of the process parameters would potentially lead to better control of the pathogens.

Reproducibility study for the detection of Staphylococcal enterotoxins in dairy products between official Italian national laboratories.

Staphylococcal food poisoning is a common foodborne disease caused by the ingestion of staphylococcal enterotoxins (SEs) produced mainly by enterotoxigenic strains of Staphylococcus aureus. To date, 21 SEs and/or enterotoxin-like types have been identified, several of which represent a potential hazard for consumers. To protect consumer health and to reduce the amount of SE-contaminated food entering the market, European Union legislation regulating food safety requires testing for SEs. The Italian National Reference Laboratory organized a ring trial to test technical and analytical proficiency in the national network of official food laboratories. Twenty-four laboratories took part, and each received and analyzed 24 blind dairy samples. Reproducibility of the results from the laboratories was assessed by the Cohen k index, and accuracy (sensitivity and specificity) was evaluated according to the International Organization for Standardization definition (ISO 16140:2003). Trial results revealed partially satisfactory agreement: 254 of 276 possible paired participants (92%) reached a k value >0.60, which is conventionally recognized as satisfactory. Accuracy was deemed satisfactory; 100% sensitivity and 100% specificity were achieved by 22 and 18 of the 24 laboratories, respectively.

Combined measurement of serum DNA and urine VP1 messenger RNA in monitoring BK virus replication in kidney graft recipients.

Evaluation of BK virus replication is a fundamental tool in the monitoring of renal transplant recipients. Herein, we investigated the role of urine VP1 messenger RNA (mRNA) quantification and combined measurement of serum DNA and urine VP1 mRNA in 428 kidney allograft recipients. BK viremia and viruria were detected in 24 (5.6%) and 54 (12.6%) patients, respectively. A diagnosis of BKV-associated nephropathy (BKVAN) was established in 2 patients, both within the first year posttransplantation. Based on urine VP1 mRNA measurement, BKV replication was observed in 10 (2.1%) patients, 2 of whom displayed BKVAN. Urine VP1 mRNA was detected in all cases in association with viremia except 5 and in all cases with viruria. No difference among VP1 mRNA levels was noted between the 2 BKVAN patients and the highest values in patients without BKVAN. The urine VP1 mRNA result by analysis using the operating characteristics was not superior to viremia, despite the improvement obtained with the combined measurement of viremia (cut-off, 16,000 copies/mL) and urine VP1 mRNA (>10,000 copies/10(3) cells). In conclusion, VP1 mRNA measurements may complement viremia and viruria to monitor BKV replication, although its use is limited by its technical complexity in comparison with DNA detection.

Evaluation and significance of cytomegalovirus-specific cellular immune response in lung transplant recipients.

In lung transplant recipients, cytomegalovirus (CMV) has been associated with direct ie, organ and systemic infection/disease, and indirect effects, including predisposition to develop acute rejection episodes and chronic allograft dysfunction. Cellular immune responses have been demonstrated to play a role in the control of CMV replication. We evaluated CMV-specific cellular responses among lung transplant recipients associated with the onset of organ infection/disease. Cellular responses were evaluated by an Elispot assay of 48 specimens from 24 patients. All samples were evaluated beyond 1 year after transplantation; CMV DNA was concomitantly detected in bronchoalveolar lavage (BAL) and whole blood specimens. Each patient received a combined prolonged antiviral prophylaxis with CMV Ig for 12 months and gancyclovir or valgancyclovir for 3 weeks after postoperative day 21. Nine patients (37.5%) showed transient or persistent CMV nonresponses including donor-recipient negative serologic matching in 2 cases. Positive CMV DNA results were observed in 18/48 BAL specimens (37.5%) from 12 patients (50%). A viral load of >10(4) copies/mL was observed in only 3 cases, 2 of whom were positive also on whole blood. Among these 3 patients, 2 were responders and BAL (as well as whole blood) specimens collected subsequently were negative for CMV DNA; 1 nonresponder patient exhibited a viral load of 426,492 copies/mL BAL (DNAemia, <2,000 copies/mL), developed CMV pneumonia (confirmed by histopathology and immunohistochemistry) and died within 28 days. The prevalence of CMV DNA positivity on BAL did not differ in relation to the immune response; the mean viral load on BAL showed significantly higher results among nonresponders than responders, namely, 1.4 × 10(5) ± 2.4 × 10(5) copies/ml versus 7.9 × 10(3) ± 1.4 × 10(4) (P=.02). Evaluation of CMV-specific cellular immune responses by in vitro immunologic monitoring complements virologic monitoring, helping to identify lung transplant recipients at risk of developing organ infection/disease.

Prevalence and clinical impact of polyomaviruses KI and WU in lung transplant recipients.

The newly discovered polyomaviruses KI and WU (KIV and WUV) were isolated from secretions of patients with respiratory symptoms as well as in blood, spleen, lymphoid tissues, and stools, especially in immunocompromised conditions. The aim of this work was to evaluate the prevalence of KIV and WUV in bronchoalveolar lavage (BAL) from lung transplant recipients. We also examined potential correlations between these viruses and occurrences of pneumonia, acute respiratory insufficiency, or other acute respiratory conditions and acute rejection episodes. Discharge diagnosis was based on the International Classification of Diseases-Italian version 2002, based on the 9th-revision clinical modification. A rejection episode was diagnosed by transbronchial lung biopsy in accordance with the 2007 International Society for Heart and Lung Transplantation Working Formulation. Overall, we analyzed 53 BALs obtained from 24 transplant recipients. Positive polymerase chain reaction results were observed in 6 samples (11.3%) from 6 patients (25%), versus 7 samples (13.2%) from 7 patients (29.2%) for KIV and WUV, respectively. Regarding the diagnosis of pneumonia, the prevalence was 22.2% and 33.3% for KIV and WUV, respectively. In cases of acute respiratory insufficiency or other acute respiratory conditions, 2 out of 9 samples were positive for KIV (22.2%) and 4 out of 9 for WUV (44.4%). An Acute rejection episode (ARE) was diagnosed in 7 instances among 6 lung transplant patients: The corresponding BAL specimens showed positive results for KIV in 3 out of 7 (42.8%) cases with ARE vs 3 out of 46 (6.5%) without an ARE (P < .05), and for WUV in 3 out of 7 (42.8%) vs 4 out of 46 (8.7%) (P < .05), respectively. Although the small number of specimens limits the statistical analysis, our results showed a higher prevalence of WUV compared with KIV. The compromised pulmonary environment in the lung allograft may cause reactivation of these viruses. Their roles in this context need to be further evaluated.