Stelios Psarras

Etiology of community-acquired pneumonia in hospitalized school-age children: Evidence for high prevalence of viral infections

Abstract Background. Community-acquired pneumonia (CAP) in young children is most commonly associated with viral infections; however, the role of viruses in CAP of school-age children is still inconclusive. Methods. Seventy-five school-age children hospitalized with CAP were prospectively evaluated for the presence of viral and bacterial pathogens. Nasopharyngeal washes were examined by polymerase chain reaction for viruses and atypical bacteria. Antibody assays to detect bacterial pathogens in acute-phase and convalescent-phase serum samples were also performed. Results. A viral infection was identified in 65% of cases. Rhinovirus RNA was detected in 45% of patients; infection with another virus occurred in 31%. The most common bacterial pathogen was Mycoplasma pneumoniae, which was diagnosed in 35% of cases. Chlamydia pneumoniae DNA was not detected in any patient; results of serological tests were positive in only 2 patients (3%). Mixed infections were documented in 35% of patients, and the majority were a viral-bacterial combination. Conclusions. The high prevalence of viral and mixed viral-bacterial infections supports the notion that the presence of a virus, acting either as a direct or an indirect pathogen, may be the rule rather than the exception in the development of CAP in school-age children requiring hospitalization.

Human metapneumovirus as a causative agent of acute bronchiolitis in infants

Abstract Background: Human Metapneumovirus (hMPV), has been recently isolated from children with acute respiratory tract infections (RTIs), including bronchiolitis, and classified in the Pneumovirinae subfamily within the Paramyxoviridae family. Objectives: Since most bronchiolitis studies fail to detect any viral pathogen in part of the samples, we sought for the presence of hMPV in a well characterized bronchiolitis cohort. Study design: Nasal washes were obtained from 56 children admitted to the hospital for acute bronchiolitis. RNA extraction and subsequent RT-PCR were used to detect hMPV, and correlated the presence of the virus with clinical characteristics of the disease. Results and conclusions: PCR revealed the presence of hMPV in 16% of bronchiolitis cases, whereas respiratory syncytial virus (RSV; 67.9%) was the most frequently encountered viral pathogen. hMPV was identified either as a unique viral pathogen or co-existed with RSV, with whom they shared a similar seasonal distribution. There were no differences in disease characteristics, either clinical or laboratory, between bronchiolitis cases where hMPV was present and those caused by RSV or other viral pathogens. These findings suggest that hMPV is a common and important causative agent in infants with bronchiolitis, with clinical characteristics similar to that of RSV.

Mechanisms of rhinovirus-induced asthma

Abstract Several epidemiological studies using sensitive detection methodologies have confirmed that the majority of acute asthma exacerbations follow upper respiratory tract infections – common colds. Most of these colds are due to human rhinoviruses (RVs). RVs are able to reach and replicate in epithelial cells of the lower airways and can activate these cells to produce pro-inflammatory mediators. Under some circumstances, RVs can also become cytotoxic to the epithelium. Atopic asthmatic individuals produce less interferon-γ and more interleukin-10 than normal subjects in response to RV infection. Symptom severity as well as viral shedding after experimental RV infection, is inversely correlated with ‘atopic’ status, expressed as the interferon-γ to interleukin-5 ratio. Expression of co-stimulatory molecules on immune cells is also affected in atopic asthmatics, suggesting an aberrant immune response to RV that may lead to suboptimal viral clearance and viral persistence. Some of the above effects can be reversed in vitro by corticosteroids, second-generation antihistamines or anti-oxidants; however, the optimal strategy for treating acute asthma exacerbations requires further research at both mechanistic and clinical levels.

Regulation of adverse remodelling by osteopontin in a genetic heart failure model

AIMS Desmin, the muscle-specific intermediate filament protein, is a major target in dilated cardiomyopathy and heart failure in humans and mice. The hallmarks of desmin-deficient (des(-/-)) mice pathology include pronounced myocardial degeneration, extended fibrosis, and osteopontin (OPN) overexpression. We sought to identify the molecular and cellular events regulating adverse cardiac remodelling in des(-/-) mice and their potential link to OPN. METHODS AND RESULTS In situ hybridization, histology, and immunostaining demonstrated that inflammatory cells and not cardiomyocytes were the source of OPN. RNA profile comparison revealed that activation of inflammatory pathways, sustained by innate immunity mechanisms, predominated among all changes occurring in degenerating des(-/-) myocardium. The expression of the most highly up-regulated genes (OPN: 226×, galectin-3: 26×, osteoactivin/Gpnmb/DC-HIL: 160× and metalloprotease-12: 98×) was associated with heart infiltrating macrophages. To evaluate the role of OPN, we generated des(-/-)OPN(-/-) mice and compared their cardiac function and remodelling indices with those of des(-/-). Osteopontin promoted cardiac dysfunction in this model since des(-/-)OPN(-/-) mice showed 53% improvement of left ventricular function, paralleled to an up to 44% reduction in fibrosis. The diminished fibrotic response in the absence of OPN could be partly mediated by a dramatic reduction in myocardial galectin-3 levels, associated with an impaired galectin-3 secretion by OPN-deficient infiltrating macrophages. CONCLUSION Cardiomyocyte death due to desmin deficiency leads to inflammation and subsequent overexpression of a series of remodelling modulators. Among them, OPN seems to be a major regulator of des(-/-) adverse myocardial remodelling and it functions at least by potentiating galectin-3 up-regulation and secretion.

Pathogenesis of respiratory syncytial virus bronchiolitis-related wheezing

Respiratory syncytial virus (RSV) is a common cause of virus infection of the human respiratory tract during the first two years of life, with virtually all children experiencing at least one infection within this period. Although this usually leads to mild respiratory illness, some infants develop more severe disease (bronchiolitis, pneumonia, etc.) affecting the lower airways and frequently requiring hospitalisation. There is evidence that bronchiolitis hospitalisations have increased during the last two decades and many of the hospitalised children develop wheezing later in life. The immune response to the virus is probably a major factor in the development or the expression of the pathological phenotype. In particular, a bias towards type-2 cytokine responses seems to be associated with more severe disease, whereas a type-1 response leads to more effective viral clearance and milder illness. Although the virus by itself triggers a type-1 response, a preexisting type-1 deficiency may contribute to the severity of the disease. In that sense, RSV bronchiolitis may serve as a marker, reflecting predisposition of the individual for virus induced wheezing early in life and/or asthma later in life.

Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells.

BackgroundHuman rhinoviruses (RV), the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro.MethodsMonolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry.ResultsRV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation.ConclusionRV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.

Altered expression of calreticulin during the development of fibrosis

Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well‐established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up‐regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up‐regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin‐induced) and heart fibrosis (desmin‐null) were examined. In the lung model, calreticulin expression was up‐regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF‐β, a well known major contributing factor in several fibrotic processes, was found to up‐regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.

Visualizing the course of antigen-specific CD8 and CD4 T cell responses to a growing tumor

Spontaneous tumors frequently express antigens that can be recognized by the immune system but nevertheless manage to evade immune surveillance. To better understand the mechanism of evasion, we followed CD8 and CD4 T cells reacting against a subcutaneously growing tumor, modified to express influenza hemagglutinin (HA) as surrogate tumor antigen. Adoptive transfer of 8,000 antigen‐specific CD8 T cells was sufficient to protect against challenge with 1x106 tumor cells, while larger numbers of T cells rejected established tumors. HA‐specific CD4 T cells could not reject tumors on their own but helped rejection by CD8 T cells. Rejection of the tumor coincided with prolonged survival of expanded antigen‐specific CD8 and CD4 T cells, while a failing anti‐tumor response was accompanied by transient expansion followed by rapid elimination of antigen‐specific T cells. Thus, a highly immunogenic tumor can evade immune surveillance because of an insufficient number of tumor‐specific T cells and antigen overload, resulting in exhaustion of the immune response. In this scenario, adoptive immunotherapy rather than vaccination promises successful treatment.

Differential proliferative response of fetal and adult human skin fibroblasts to transforming growth factor-β

Since pronounced differences exist between the fetal and adult repair processes, we studied the proliferative response of skin fibroblasts from these two stages to transforming growth factor‐β (TGF‐β), a cytokine with a broad range of activities in tissue repair. Here, we present evidence that TGF‐β inhibits fetal human skin fibroblasts, while it is stimulatory for adult ones. This proliferative effect of TGF‐β was found to be concentration‐ dependent, but isoform‐independent. Furthermore, even a transient exposure of the cells to this growth factor was sufficient to exert its stimulatory or inhibitory action. Accordingly, we have studied the immediate responses provoked by TGF‐β in major signaling pathways, and we have found that it induces a rapid activation of the SMAD pathway, i.e., phosphorylation and nuclear translocation of SMAD2, followed by dephosphorylation, most probably due to degradation by the proteasome. However, similar intensity and kinetics of this activation have been observed in both fetal and adult fibroblasts. On the other hand, curcumin, a natural product with wound healing properties that inhibits several intracellular signaling pathways, was found to completely abrogate the inhibitory effect of TGF‐β1 on human fetal skin fibroblasts, without affecting the stimulatory action on fibroblasts from adult donors. In conclusion, there is a major radical in the proliferative response of fetal and adult human skin fibroblasts to TGF‐β, possibly reflecting the different repair strategies followed in these two stages of development.

Synergistic effects of fluticasone propionate and salmeterol on inhibiting rhinovirus-induced epithelial production of remodelling-associated growth factors.

Background Rhinoviruses (RV), the major trigger of acute asthma exacerbations, are able to infect bronchial epithelium and induce production of pro‐inflammatory, but also angiogenic and pro‐fibrotic mediators. Fluticasone propionate (FP) and salmeterol (S) are clinically effective and act synergistically in controlling persistent asthma; however, their effect on virus‐associated asthma is less clear.