Panagiotis Pantelidis

Molecular Epidemiology of Human Enterovirus 71 in the United Kingdom from 1998 to 2006

ABSTRACT The last decade witnessed a significant increase in epidemic activity of human enterovirus 71 (EV71) in the Western Pacific Region (WPR). In most European countries, this risk is unrecognized despite occasional cases of severe disease and two severe outbreaks in Eastern Europe 30 years ago. In this study we report the first examination of the molecular epidemiology of EV71 in the United Kingdom from 1998 to 2006. Genomic regions encoding the 1D coat protein (VP1) and 3D polymerase (Pol) from 32 EV71 isolates associated with neurological or cutaneous manifestations were sequenced. Phylogenetic analyses of VP1 and 3D Pol sequences identified genotype C as the dominant strain. Several United Kingdom isolates had genetic linkages with predated C1 or C2 strains from Europe and the WPR. Recombination events were not detected between United Kingdom strains. However, a previously published Taiwanese strain was identified as an intergenotypic recombinant. EV71 genotype C appears to have continuous circulation in the United Kingdom from 1998 to 2006 with repeated introductions of new strains replacing previous strains. It is necessary to continuously monitor the molecular evolution and recombination events of EV71.

Serum Interleukin 6 Is Predictive of Early Functional Decline and Mortality in Interstitial Lung Disease Associated with Systemic Sclerosis

Objective. Biomarkers of progression of interstitial lung disease (ILD) are needed to allow early therapeutic intervention in patients with scleroderma-associated disease (SSc-ILD). Methods. A panel of 8 serum cytokines [interleukin 6 (IL-6), IL-8, IL-10, CCL2, CXCL10, vascular endothelial growth factor, fibroblast growth factor 2, and CX3CL1] was assessed by Luminex bead technology in exploratory cohorts of 74 patients with SSc and 58 patients with idiopathic pulmonary fibrosis (IPF). Mortality and significant lung function decline [forced vital capacity (FVC) ≥ 10%; DLCO ≥ 15%] from date of serum collection were evaluated by proportional hazards analysis. Based on these findings, the prognostic value of serum IL-6, evaluated by ELISA, was assessed in a larger test cohort of 212 patients with SSc-ILD. Results. In the exploratory cohort, only serum IL-6 was an independent predictor of DLCO decline in both IPF and SSc-ILD. The IL-6 threshold level most predictive of DLCO decline within a year was 7.67 pg/ml. In the larger test cohort, serum IL-6 > 7.67 pg/ml was predictive of decline in FVC (HR 2.58 ± 0.98, p = 0.01) and in DLCO (HR 3.2 ± 1.7, p = 0.02) within the first year, and predictive of death within the first 30 months (HR 2.69 ± 0.96, p = 0.005). When stratified according to severity (FVC < 70%), serum IL-6 > 7.67 pg/ml was predictive of functional decline or death within the first year in patients with milder disease (OR 3.1, 95% CI 1.4–7.2, p = 0.007), but not in those with severe ILD. Conclusion. In SSc-ILD, serum IL-6 levels appear to be predictive of early disease progression in patients with mild ILD, and could be used to target treatment in this group, if confirmed by prospective studies.

Increased vitronectin and endothelin-1 in the breath condensate of patients with fibrosing lung disease

Background: Non-specific interstitial pneumonia (NSIP) and fibrosing alveolitis associated with systemic sclerosis (FASSc) are diseases of unknown aetiology that are characterised by the accumulation of mononuclear cells, followed by the progressive deposition of collagen within the interstitium and subsequent destruction of lung airspace. Better understanding of mediators involved in fibrosis may be useful for early diagnosis and in clinical monitoring of disease progression. Objective: The aim of this study was to investigate the presence of two profibrotic markers, the vitronectin and the endothelin-1 (ET-1) in the airways of NSIP and FASSc patients. Methods: Ten NSIP (6 males, age 57 ± 2 years) and 15 FASSc (8 males, age 55 ± 4 years) patients were recruited along with 10 normal subjects (4 male, age 52 ± 2 years). Vitronectin and ET-1 concentrations were measured in their breath condensate, using a specific enzyme immunoassay. Results: Higher levels of vitronectin and ET-1 were observed in NSIP and FASSc patients [median 92.8 (91.7–93.9) µg/ml; median 8.3 (7.9–9.3) pg/ml] than in control subjects [median 80.3 (89.3–91.4) µg/ml; p < 0.01; median 5.3 (4.9–5.9) pg/ml, p < 0.0001]. We also found increased concentrations of vitronectin in patients with clinical deterioration compared to those remaining stable and in ex-smokers compared to non-smokers and, increased vitronectin and ET-1 in patients treated with steroids compared to untreated patients. Conclusion: These findings justify further studies of vitronectin and ET-1 levels in exhaled breath condensate, as a means of monitoring activity and predicting progression of pulmonary fibrosis.

Variation in Iron Homeostasis Genes Between Patients With ARDS and Healthy Control Subjects

BACKGROUND Abnormal plasma and lung iron mobilization is associated with the onset and progression of ARDS and is detectable in specific at-risk populations. Patients with ARDS also have pronounced oxidative and nitrosative stress that can be catalyzed and thereby aggravated by the bioavailability of redox active iron. ARDS of pulmonary and extrapulmonary origin may differ pathophysiologically and require different ventilatory strategies. Evidence suggests that genetic predisposition is relevant to the pathogenesis of ARDS. We therefore explored the hypothesis that polymorphisms from a panel of genes encoding iron-metabolizing proteins determine susceptibility to ARDS. METHODS Retrospective case-control study conducted at the adult ICUs of two university hospitals. Patients with ARDS (n = 122) and healthy control subjects (n = 193) were genotyped. Sequence-specific primer polymerase chain reaction was used to genotype selected biallelic single-nucleotide polymorphisms. An audit of the patient database was conducted, and 104 of the 122 ARDS patients were eligible for the final data analysis. RESULTS Preliminary analysis indicated differences between ARDS and healthy control subjects in the incidence of polymorphism of the gene encoding ferritin light chain. Subgroup analysis indicated the prevalence of ferritin light-chain gene -3381GG homozygotes was increased in patients with ARDS of extrapulmonary origin compared to healthy control subjects. Secondly, a common haplotype in the heme oxygenase 2 gene was reduced in patients with ARDS compared to healthy control subjects and was more evident in those with ARDS of direct or pulmonary etiology. CONCLUSIONS These results provide preliminary evidence to suggest a distinction in the genetic background of the subpopulations studied, inferring that the ferritin light-chain gene genotype confers susceptibility to ARDS, while the heme oxygenase 2 haplotype is protective against the onset of the syndrome. Such data support further previous findings that suggest abnormalities in iron handling resulting in redox imbalance are implicated in the pathogenesis of ARDS.

Loss of Th22 cells is associated with increased immune activation and IDO-1 activity in HIV-1 infection.

Background:Immune activation plays a key role in the immunopathogenesis of HIV-1 infection. Microbial translocation, secondary to loss of epithelial integrity and mucosal immune deficiency, is believed to contribute to systemic immune activation. Interleukin 22 maintains intestinal epithelial barrier integrity and stimulates the secretion of antimicrobial peptides that limit bacterial dissemination and intestinal inflammation. Interleukin 22 is secreted by CD4 T-helper (Th)22 cells independently of interleukin 17A and interferon &ggr;. Th22 cells are characterized by the expression of chemokine receptors (CCR)4, CCR6, and CCR10. Methods:We analyzed the frequency of Th22, Th17, Th1, and CD4 T regulatory (Treg) cells, markers of immune activation (expression of CD38 on CD8 T cells, neopterin, soluble CD14), microbial translocation (lipopolysaccharide-binding protein and 16s ribosomal DNA), and indoleamine 2,3-dioxygenase 1 activity in peripheral blood of antiretroviral therapy (ART)-experienced and ART-naive HIV-1–infected patients and healthy controls. Results:We showed a significant reduction in the frequency of Th22 cells in HIV ART-naive patients compared with the healthy controls and HIV ART-experienced patients. We observed a shift away from Th22 and Th17 to Treg cells, which was partially reversed by effective ART. Markers of immune activation negatively correlated with Th22 and Th17 proportions, and with Th22:Treg and Th17:Treg ratios in ART-naive patients. Increased indoleamine 2,3-dioxygenase 1 activity negatively correlated with Th22:Treg and Th17:Treg ratios in the ART-naive group. Conclusions:Loss of Th22 cells and disruption in the balance of Th22 and Treg cells may contribute toward systemic immune activation and mucosal immune deficiency during HIV-1 infection.

Surfactant gene polymorphisms and interstitial lung diseases

Pulmonary surfactant is a complex mixture of phospholipids and proteins, which is present in the alveolar lining fluid and is essential for normal lung function. Alterations in surfactant composition have been reported in several interstitial lung diseases (ILDs). Furthermore, a mutation in the surfactant protein C gene that results in complete absence of the protein has been shown to be associated with familial ILD. The role of surfactant in lung disease is therefore drawing increasing attention following the elucidation of the genetic basis underlying its surface expression and the proof of surfactant abnormalities in ILD.

A Biomarker Panel (Bioscore) Incorporating Monocytic Surface and Soluble TREM-1 Has High Discriminative Value for Ventilator-Associated Pneumonia: A Prospective Observational Study

Introduction Ventilator-associated pneumonia (VAP) increases mortality in critical illness. However, clinical diagnostic uncertainty persists. We hypothesised that measuring cell-surface and soluble inflammatory markers, incorporating Triggering Receptor Expressed by Myeloid cells (TREM)-1, would improve diagnostic accuracy. Methods A single centre prospective observational study, set in a University Hospital medical-surgical intensive Care unit, recruited 91 patients into 3 groups: 27 patients with VAP, 33 ventilated controls without evidence of pulmonary sepsis (non-VAP), and 31 non-ventilated controls (NVC), without clinical infection, attending for bronchoscopy. Paired samples of Bronchiolo-alveolar lavage fluid (BALF) and blood from each subject were analysed for putative biomarkers of infection: Cellular (TREM-1, CD11b and CD62L) and soluble (IL-1β, IL-6, IL-8, sTREM-1, Procalcitonin). Expression of cellular markers on monocytes and neutrophils were measured by flow cytometry. Soluble inflammatory markers were determined by ELISA. A biomarker panel (‘Bioscore’), was constructed, tested and validated, using Fisher’s discriminant function analysis, to assess its value in distinguishing VAP from non VAP. Results The expression of TREM-1 on monocytes (mTREM-1) and neutrophils (nTREM-1) and concentrations of IL-1β, IL-8, and sTREM-1 in BALF were significantly higher in VAP compared with non-VAP and NVC (p<0.001). The BALF/blood mTREM-1 was significantly higher in VAP patients compared to non-VAP and NVC (0.8 v 0.4 v 0.3 p<0.001). A seven marker Bioscore (BALF/blood ratio mTREM-1 and mCD11b, BALF sTREM-1, IL-8 and IL-1β, and serum CRP and IL-6) correctly identified 88.9% of VAP cases and 100% of non-VAP cases. Conclusion A 7-marker bioscore, incorporating cellular and soluble TREM-1, accurately discriminates VAP from non-pulmonary infection.

Amperometric IFN-γ immunosensors with commercially fabricated PCB sensing electrodes.

Lab-on-a-Chip (LoC) technology has the potential to revolutionize medical Point-of-Care diagnostics. Currently, considerable research efforts are focused on innovative production technologies that will make commercial upscaling of lab-on-chip products financially viable. Printed circuit board (PCB) manufacturing techniques have several advantages in this field. In this paper we focus on transferring a complete IFN-γ enzyme-linked immune-sorbent assay (ELISA) onto a commercial PCB electrochemical biosensing platform, We adapted a commercially available ELISA to detect the enzyme product TMB/H2O2 using amperometry, successfully reproducing the colorimetry-obtained ELISA standard curve. The results demonstrate the potential for the integration of these components into an automated, disposable, electronic ELISA Lab-on-PCB diagnostic platform.

An assay system for point-of-care diagnosis of tuberculosis using commercially manufactured PCB technology

Rapid advances in clinical technologies, detection sensitivity and analytical throughput have delivered a significant expansion in our knowledge of prognostic and diagnostic biomarkers in many common infectious diseases, such as Tuberculosis (TB). During the last decade, a significant number of approaches to TB diagnosis have been attempted at Point-of-Care (PoC), exploiting a large variation of techniques and materials. In this work, we describe an electronics-based Enzyme-Linked ImmunoSorbent Assay (eELISA), using a Lab-on-a-Printed Circuit Board (LoPCB) approach, for TB diagnosis based on cytokine detection. The test relies upon an electrochemical (amperometric) assay, comprising a high-precision bioinstrumentation board and amperometric sensors, produced exclusively using standard PCB manufacturing processes. Electrochemical detection uses standard Au and Ag electrodes together with a bespoke, low-power, multichannel, portable data-acquisition system. We demonstrate high-performance assay chemistry performed at microfluidic volumes on Au pads directly at the PCB surface with improved limit of detection (~10 pg/mL) over standard colorimetric ELISA methods. The assay has also been implemented in plasma, showing the utility of the system for medical applications. This work is a significant step towards the development of a low-cost, portable, high-precision diagnostic and monitoring technology, which once combined with appropriate PCB-based microfluidic networks will provide complete LoPCB platforms.

Upregulation of citrullination pathway: From Autoimmune to Idiopathic Lung Fibrosis

BackgroundIncreased protein citrullination and peptidylarginine deiminases (PADIs), which catalyze the citrullination process, are central in Rheumatoid arthritis pathogenesis and probably involved in the initial steps towards autoimmunity. Approximately, 10% of RA patients develop clinically significantly ILD. A possible shared role of protein citrullination in rheumatoid arthritis associated interstitial lung disease (RA-ILD), and idiopathic pulmonary fibrosis (IPF) pathogenesis remains unclear.MethodsWe evaluated PADI2 and PADI4 mRNA expression in bronchoalveolar lavage fluid (BALF) cells of 59 patients with IPF, 27 patients RA-ILD and 10 healthy controls. PADI 2 and 4 expression was analyzed by western blot and immunohistochemistry. Citrullinated protein levels were also quantified.ResultsPADI4 mRNA and protein levels were higher in RA-ILD and IPF than controls. Furthermore, PADI4 mRNA levels showed an increase among smokers in RA-ILD. PADI4 expression was detected in granulocytes and macrophages in all groups, with the strongest cytoplasmic expression observed in granulocytes in RA-ILD and IPF. PADI2 mRNA and immunostaining of BAL cells, were similar in all groups among smokers. Overall, stronger staining was observed in current smokers. Citrullinated peptides were significantly increased in IPF compared to RA-ILD and controls. In RA-ILD, protein citrullination strongly correlated with PADI4 expression and anti-citrullinated protein antibodies (ACPAs).ConclusionsThese results suggest that the citrullination pathway is upregulated in IPF and in RA-ILD.