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Atrial fibrillation (AF), the most commonly diagnosed arrhythmia, affects a notable percentage of the population and constitutes a major risk factor for thromboembolic events and other heart-related conditions. Fibrosis plays an important role in the onset and perpetuation of AF through structural and electrical remodelling processes. Multiple molecular pathways are involved in atrial substrate modification and the subsequent maintenance of AF. In this review, we aim to recapitulate underlying molecular pathways leading to atrial fibrosis and to indicate existing gaps in the complex interplay of atrial fibrosis and AF.

Intermediate filaments are major components of the cytoskeleton. Desmin and synemin, cytoplasmic intermediate filament proteins and A-type lamins, nuclear intermediate filament proteins, play key roles in skeletal and cardiac muscle. Desmin, encoded by the DES gene (OMIM *125660) and A-type lamins by the LMNA gene (OMIM *150330), have been involved in striated muscle disorders. Diseases include desmin-related myopathy and cardiomyopathy (desminopathy), which can be manifested with dilated, restrictive, hypertrophic, arrhythmogenic, or even left ventricular non-compaction cardiomyopathy, Emery–Dreifuss Muscular Dystrophy (EDMD2 and EDMD3, due to LMNA mutations), LMNA-related congenital Muscular Dystrophy (L-CMD) and LMNA-linked dilated cardiomyopathy with conduction system defects (CMD1A). Recently, mutations in synemin (SYNM gene, OMIM *606087) have been linked to cardiomyopathy. This review will summarize clinical and molecular aspects of desmin-, lamin- and synemin-related striated muscle disorders with focus on LMNA and DES-associated clinical entities and will suggest pathogenetic hypotheses based on the interplay of desmin and lamin A/C. In healthy muscle, such interplay is responsible for the involvement of this network in mechanosignaling, nuclear positioning and mitochondrial homeostasis, while in disease it is disturbed, leading to myocyte death and activation of inflammation and the associated secretome alterations.

Following an insult by both intrinsic and extrinsic pathways, complex cellular, and molecular interactions determine a successful recovery or inadequate repair of damaged tissue. The efficiency of this process is particularly important in the heart, an organ characterized by very limited regenerative and repair capacity in higher adult vertebrates. Cardiac insult is characteristically associated with fibrosis and heart failure, as a result of cardiomyocyte death, myocardial degeneration, and adverse remodeling. Recent evidence implies that resident non-cardiomyocytes, fibroblasts but also macrophages -pillars of the innate immunity- form part of the inflammatory response and decisively affect the repair process following a cardiac insult. Multiple studies in model organisms (mouse, zebrafish) of various developmental stages (adult and neonatal) combined with genetically engineered cell plasticity and differentiation intervention protocols -mainly targeting cardiac fibroblasts or progenitor cells-reveal particular roles of resident and recruited innate immune cells and their secretome in the coordination of cardiac repair. The interplay of innate immune cells with cardiac fibroblasts and cardiomyocytes is emerging as a crucial platform to help our understanding and, importantly, to allow the development of effective interventions sufficient to minimize cardiac damage and dysfunction after injury.

Intermediate filament (IF) proteins are critical regulators in health and disease. The discovery of hundreds of mutations in IF genes and posttranslational modifications has been linked to a plethora of human diseases, including, among others, cardiomyopathies, muscular dystrophies, progeria, blistering diseases of the epidermis, and neurodegenerative diseases. The major IF proteins that have been linked to cardiomyopathies and heart failure are the muscle-specific cytoskeletal IF protein desmin and the nuclear IF protein lamin, as a subgroup of the known desminopathies and laminopathies, respectively. The studies so far, both with healthy and diseased heart, have demonstrated the importance of these IF protein networks in intracellular and intercellular integration of structure and function, mechanotransduction and gene activation, cardiomyocyte differentiation and survival, mitochondrial homeostasis, and regulation of metabolism. The high coordination of all these processes is obviously of great importance for the maintenance of proper, life-lasting, and continuous contraction of this highly organized cardiac striated muscle and consequently a healthy heart. In this review, we will cover most known information on the role of IFs in the above processes and how their deficiency or disruption leads to cardiomyopathy and heart failure.

Backround:Left atrial (LA) enlargement plays an important role in the development of heart failure (HF) and is a robust prognostic factor. Fibrotic processes have also been advocated to evoke HF through finite signalling proteins.Methods:We examined the association of two such proteins, cystatin C (CysC) and galectin-3 (Gal-3), and other clinical, echocardiographic and biochemical parameters with LA volume index (LAVi) in patients with HF with severely impaired left ventricular ejection fraction (LVEF). Severe renal, liver, autoimmune disease and cancer were exclusion criteria.Results:A total of 40 patients with HF (31 men, age 66.6 ± 1.7) with LVEF = 25.4 ± 0.9% were divided into two groups according to the mean LAVi (51.03 ± 2.9 ml/m2) calculated by two-dimensional transthoracic echocardiography. Greater LAVi was positively associated with LV end-diastolic volume (p = 0.017), LV end-systolic volume (p = 0.025), mitral regurgitant volume (MRV) (p = 0.001), right ventricular systolic pressure (RVSP) (p < ...

In inflammatory bowel disease (IBD), compromised restitution of the epithelial barrier contributes to disease severity. Owing to the complexity in the pathogenesis of IBD, a variety of factors have been implicated in its progress. In this study, we report a functional interaction between macroautophagy and Corticotropin Releasing Hormone (Crh) in the gut. For this purpose we used DSS colitis model on Crh −/− or wild-type (wt) with pharmacological inhibition of autophagy. We uncovered sustained basal autophagy in the gut of Crh −/− mice, which persisted over the course of DSS administration. Autophagy inhibition resulted in partial rescue of Crh −/− mice, while it increased the expression of Crh in the wt gut. Similarly, Crh deficiency was associated with sustained activation of base line autophagy. In vitro models of amino acid deprivation- and LPS-induced autophagy confirmed the in vivo findings. Our results indicate a novel role for Crh in the intestinal epithelium that involves regulation of autophagy, while suggesting the complementary action of the two pathways. These data suggest the intriguing possibility that targeting Crh stimulation in the intestine may provide a novel therapeutic approach to support the integrity of the epithelial barrier and to protect from chronic colitis.

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.

A major part of the burden of asthma is caused by acute exacerbations. Exacerbations have been strongly and consistently associated with respiratory infections. Respiratory viruses and bacteria are therefore possible treatment targets. To have a reasonable estimate of the burden of disease induced by such infectious agents on asthmatic patients, it is necessary to understand their nature and be able to identify them in clinical samples by employing accurate and sensitive methodologies. This systematic review summarizes current knowledge and developments in infection epidemiology of acute asthma in children and adults, describing the known impact for each individual agent and highlighting knowledge gaps. Among infectious agents, human rhinoviruses are the most prevalent in regard to asthma exacerbations. The newly identified type-C rhinoviruses may prove to be particularly relevant. Respiratory syncytial virus and metapneumovirus are important in infants, while influenza viruses seem to induce severe exacerbations mostly in adults. Other agents are relatively less or not clearly associated. Mycoplasma and Chlamydophila pneumoniae seem to be involved more with asthma persistence rather than with disease exacerbations. Recent data suggest that common bacteria may also be involved, but this should be confirmed. Although current information is considerable, improvements in detection methodologies, as well as the wide variation in respect to location, time and populations, underline the need for additional studies that should also take into account interacting factors.

BACKGROUND: Human rhinoviruses (HRVs) and house dust mites (HDMs) are among the most common environmental factors able to induce airway inflammation in asthma. Although epidemiological studies suggest that they also synergize in inducing asthma exacerbations, there is no experimental evidence to support this, nor any information on the possible mechanisms involved. OBJECTIVE: To investigate their interaction on the induction of airway epithelial inflammatory responses in vitro. METHODS: BEAS-2B cells were exposed to activated HDM Dermatophagoides pteronyssinus major allergen I (Der p I), HRVs (HRV1b or HRV16) or both in different sequences. IL-8/CXCL8 release, intercellular adhesion molecule (ICAM)-1 surface expression and nuclear factor kappaB (NF-kappaB) translocation were evaluated. Complementary, primary human bronchial epithelial cells (HBECs) exposed to both Der p I and RVs and IL-8, IL-6, IFN-gamma-induced protein (IP)-10/CXCL10, IFN-lambda1/IL-29, regulated upon activation normal T lymphocyte expressed and secreted (RANTES)/CCL5 release were measured. RESULTS: RV and Der p I up-regulated IL-8 release, ICAM-1 expression and NF-kappaB translocation in BEAS-2B cells. Simultaneous exposure to both factors, as well as when cells were initially exposed to HRV and then to Der p I, resulted in further induction of IL-8 in a synergistic manner. Synergism was not observed when cells were initially exposed to Der p I and then to HRV. This was the pattern in ICAM-1 induction although the phenomenon was not synergistic. Concurrent exposure induced an early synergistic NF-kappaB translocation induction, differentiating with time, partly explaining the above observation. In HBECs, both HRV and Der p I induced IL-8, IL-6, IL-29 and IP-10, while RANTES was induced only by HRV. Synergistic induction was observed only in IL-8. CONCLUSION: HRV and enzymatically active Der p I can act synergistically in the induction of bronchial epithelial IL-8 release, when HRV infection precedes or is concurrent with Der p I exposure. Such a synergy may represent an important mechanism in virus-induced asthma exacerbations.

It is known that oxidative stress plays an important role in the pathogenesis of atherosclerosis and that an association exists between osteopontin (OPN) and atherosclerosis.It was proposed that malondialdehyde (MDA), a biomarker of lipid peroxidation and oxidative stress, would be related to plasma OPN levels in patients with coronary artery disease (CAD).Plasma OPN and MDA levels were measured in 71 patients (60 males and 11 females; mean age 61.7 +/- 10 years). Fifty-eight patients had significant CAD (group I) and 13 patients were free of CAD as defined angiographically (group II). Plasma OPN was measured by enzyme-linked immunosorbent assay (ELISA), while MDA was determined spectrophotometrically. Multivariate regression analysis revealed that ln-transformed OPN levels were independently associated with MDA after adjustment for age, hypertension and diabetes mellitus (R(2) = 0.278, p = 0.0004 and beta regression coefficient = 0.252 [standard error = 0.0958], p = 0.011). OPN and MDA levels were higher in patients with diabetes (73.6 +/- 36.2 ng/ml versus 56.1 +/- 30.9 ng/ml, p = 0.02 and 2.5 +/- 0.5 microM versus 2.0 +/- 0.5 microM, p = 0.002, respectively).The association between OPN and MDA levels in patients with CAD suggests an interaction between OPN and oxidative stress. This interaction may play a role in the pathogenesis of atherosclerosis.

Intermediate filament (IF) cytoskeleton comprises the fine-tuning cellular machinery regulating critical homeostatic mechanisms. In skeletal and cardiac muscle, deficiency or disturbance of the IF network leads to severe pathology, particularly in the latter. The three-dimensional scaffold of the muscle-specific IF protein desmin interconnects key features of the cardiac muscle cells, including the Z-disks, intercalated disks, plasma membrane, nucleus, mitochondria, lysosomes, and potentially sarcoplasmic reticulum. This is crucial for the highly organized striated muscle, in which effective energy production and transmission as well as mechanochemical signaling are tightly coordinated among the organelles and the contractile apparatus. The role of desmin and desmin-associated proteins in the biogenesis, trafficking, and organelle function, as well as the development, differentiation, and survival of the cardiac muscle begins to be enlightened, but the precise mechanisms remain elusive. We propose a set of experimental tools that can be used, in vivo and in vitro, to unravel crucial new pathways by which the IF cytoskeleton facilitates proper organelle function, homeostasis, and cytoprotection and further understand how its disturbance and deficiency lead to disease.

Intermediate filament (IF) cytoskeleton comprises the fine-tuning cellular machinery regulating critical homeostatic mechanisms. In skeletal and cardiac muscle, deficiency or disturbance of the IF network leads to severe pathology, particularly in the latter. The three-dimensional scaffold of the muscle-specific IF protein desmin interconnects key features of the cardiac muscle cells, including the Z-disks, intercalated disks, plasma membrane, nucleus, mitochondria, lysosomes, and potentially sarcoplasmic reticulum. This is crucial for the highly organized striated muscle, in which effective energy production and transmission as well as mechanochemical signaling are tightly coordinated among the organelles and the contractile apparatus. The role of desmin and desmin-associated proteins in the biogenesis, trafficking, and organelle function, as well as the development, differentiation, and survival of the cardiac muscle begins to be enlightened, but the precise mechanisms remain elusive. We propose a set of experimental tools that can be used, in vivo and in vitro, to unravel crucial new pathways by which the IF cytoskeleton facilitates proper organelle function, homeostasis, and cytoprotection and further understand how its disturbance and deficiency lead to disease.

Inflammation may contribute to disease progression in arrhythmogenic cardiomyopathy (ACM). However, its role in this process is unresolved. Our goal was to delineate the pathogenic role of the complement system in a new animal model of ACM and in human disease. Using cardiac histology, echocardiography, and electrocardiography, we have demonstrated that the desmin-null mouse (Des-/-) recapitulates most of the pathognomonic features of human ACM. Massive complement activation was observed in the Des-/- myocardium in areas of necrotic cells debris and inflammatory infiltrate. Analysis of C5aR-/-Des-/- double-null animals and a pharmaceutical approach using a C5a inhibitor were used to delineate the pathogenic role of the complement system in the disease progression. Our findings indicate that inhibiting C5aR (CD88) signaling improves cardiac function, histopathology, arrhythmias, and survival after endurance. Containment of the inflammatory reaction at the initiation of cardiac tissue injury (2-3 weeks of age), with consequently reduced myocardial remodeling and the absence of a direct long-lasting detrimental effect of C5a-C5aR signaling on cardiomyocytes, could explain the beneficial action of C5aR ablation in Des-/- cardiomyopathy. We extend the relevance of these findings to human pathophysiology by showing for the first time significant complement activation in the cardiac tissues of patients with ACM, thus suggesting that complement modulation could be a new therapeutic target for ACM.

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. AIM: The aim of this study was to assess the individual and combined effects of FP and S on RV-induced epithelial production of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). METHODS: Bronchial epithelial cells (BEAS-2B) were exposed in vitro to RV and were subsequently treated with FP and S, at physiologically relevant concentrations, alone or in combination. VEGF and FGF-2 were measured in the supernatants of these cultures using ELISA. RESULTS: FP was able to reduce RV-induced VEGF production in a dose-dependent manner. S also induced a smaller reduction; addition of both factors inhibited VEGF synergistically. FGF-2 production was not inhibited by either FP or S alone, but was significantly reduced when both substances were present in the culture. CONCLUSION: This study demonstrates that FP and S may synergistically inhibit the production of angiogenic and/or pro-fibrotic factors that are induced after RV infection of BEAS-2B and are implicated in airway remodelling, suggesting that this combination may represent an important therapeutic option on virus-induced asthma.

Experimental and clinical evidences suggest that oxidants play a role in the pathogenesis of respiratory disorders characterised by chronic airway inflammation such as asthma and chronic obstructive pulmonary disease (COPD). The respiratory system is chronically exposed to environmental pollutants, including oxidants. Exogenous sources of oxidants are particularly relevant to the pathogenesis of COPD, being cigarette smoke an extremely rich source of oxidants. In addition, the inflammatory cells recruited to the airways of patients with asthma and COPD, have an exceptional capacity to produce oxidants. Many decades of research have produced a significant amount of data indicating pro-oxidative molecular mechanisms putatively relevant in the pathogenesis of the oxidative stress which characterises these diseases, both locally and systemically. As a consequence, a drug therapy able to restore the redox imbalance in asthma and COPD would probably exert clinical and functional benefits. Indeed, currently available therapies for asthma and COPD can exert an inhibitory effect on oxidant production in the airways. However, it is unknown whether the efficacy of the treatment is somehow linked to the pharmacological modulation of the oxidant/antioxidant balance. So far, it appears that the potential role of antioxidant compounds in the treatment of asthma and COPD has not been fully explored.

Respiratory syncytial virus (RSV) subtypes A and B are present either simultaneously or alternate during yearly epidemics. It is still not clear whether clinical severity of acute bronchiolitis differs between the two subtypes.Reverse transcription polymerase chain reaction was used to subtype RSV in previously healthy infants hospitalized with RSV bronchiolitis during a winter epidemic. A severity index based on heart rate, respiratory rate, wheezing, difficulty in feeding and oxygen saturation was calculated upon admission.Infants infected with RSV subtype-A were found to have a significantly higher (more severe) clinical score than those infected with RSV-B. There was no statistically significant difference in duration of hospitalization or need of intensive care. Boys and infants younger than 3 months of age were also more severely affected than girls or older infants, respectively.These results support the notion that RSV-A-induced bronchiolitis is more severe than RSV-B-induced one, in agreement with the majority of previously published studies.

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-gamma 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-gamma 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.

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.

Background Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells. Methods To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses β-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of β-galactosidase. Results Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or β-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50. Conclusions The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells. Copyright © 2003 John Wiley & Sons, Ltd.

Using sensitive diagnostic methodologies, epidemiologic studies during the past decade have allowed the identification of human rhinoviruses (RVs), generally recognized as "common cold viruses," as major asthma precipitants. This association was further established by evaluating the impact of RV infection in airway obstruction and inflammation during naturally acquired or experimentally induced RV colds. There is now strong evidence that RVs can infect and propagate not only in the upper but also in the lower airways. Bronchial and pulmonary epithelia infected by RVs are rich sources of inflammatory mediators, which may initiate or augment airway inflammation and obstruction. Furthermore, in an atopic environment, responses to the virus are skewed by and toward an "atopic," Th2-like balance, which may further enhance inflammation and exacerbate asthma.

The present review focuses and comments on the increasing body of evidence correlating respiratory viral infections with asthma onset and exacerbations.Recent data suggest multiple and some time contrasting roles for viral infection in the origin of asthma. These data also indicate that the immune status of the host, including atopy, may interactively contribute to this process, conferring susceptibility or even resistance to the development of asthma in virus-infected individuals. In the presence of asthma, the role of viral infection in triggering exacerbations is clearly established. Chemokine and cytokine responses of the respiratory epithelium, a biased type 1/type 2 cytokine balance, defective costimulation, as well as abnormal neural control have been suggested as possible mechanisms. The importance of concurrent or synergistic effects of allergen exposure is currently under scrutiny.Viruses may initiate and certainly exacerbate asthma. Mild repeated infections early in life could also stimulate type 1 immune responses conferring protection from atopy and asthma. The host's immune status, the type of viral infection and the timing of exposure to various environmental stimuli are probably the key factors in this process. Mechanistic insights deduced from recent work should allow for the development of intervening strategies in the near future.

Fibrosis is a complex and multifactorial process, affecting the structure and compromising the function of several organs. Among those, renal fibrosis is an important pathological change, eventually leading to renal failure. Proteomic analysis of the renal parenchyma in the well-established rat model of unilateral ureteral obstruction (UUO model) suggested that transgelin was up-regulated during the development of fibrosis. Transgelin up-regulation was confirmed both at the protein and at the mRNA level. It was observed that at early stages of fibrosis transgelin was mainly expressed in the interstitial compartment and, more specifically, in cells surrounding the glomeruli. Subsequently, it was confirmed that transgelin expressing cells were activated fibroblasts, based on their extensive co-expression of α-SMA and their complete lack of co-distribution with markers of other cell types (endothelial, epithelial and cells of the immune system). These periglomerular fibroblasts exhibited staining for transgelin mainly cytoplasmic but occasionally nuclear as well. In addition, transgelin expression in periglomerular fibroblasts was absent in renal fibrosis developed in a hypertensive model, compared to the UUO model. Promoter analysis indicated that there are several conserved motifs for transcription factor binding. Among those, Kruppel-like factor 6 was found to be up-regulated in transgelin positive periglomerular activated fibroblasts, suggesting a possible involvement in the mechanism of transgelin up-regulation. These data strongly suggest that transgelin is up-regulated in the obstructive nephropathy and could be used as a novel marker for renal fibrosis in the future.

Background Human rhinoviruses, major precipitants of asthma exacerbations, induce lower airway inflammation and mediate angiogenesis. The purpose of this study was to assess the possibility that rhinoviruses may also contribute to the fibrotic component of airway remodeling. Methods Levels of basic fibroblast growth factor (bFGF) mRNA and protein were measured following rhinovirus infection of bronchial epithelial cells. The profibrotic effect of epithelial products was assessed by DNA synthesis and matrix metalloproteinase activity assays. Moreover, epithelial cells were exposed to supernatants from cultured peripheral blood mononuclear cells, obtained from healthy donors or atopic asthmatic subjects and subsequently infected by rhinovirus and bFGF release was estimated. bFGF was also measured in respiratory secretions from atopic asthmatic patients before and during rhinovirus-induced asthma exacerbations. Results Rhinovirus epithelial infection stimulated mRNA expression and release of bFGF, the latter being positively correlated with cell death under conditions promoting rhinovirus-induced cytotoxicity. Supernatants from infected cultures induced lung fibroblast proliferation, which was inhibited by anti-bFGF antibody, and demonstrated increased matrix metalloproteinase activity. Rhinovirus-mediated bFGF release was significantly higher in an in vitro simulation of atopic asthmatic environment and, importantly, during rhinovirus-associated asthma exacerbations. Conclusions Rhinovirus infection induces bFGF release by airway epithelium, and stimulates stroma cell proliferation contributing to airway remodeling in asthma. Repeated rhinovirus infections may promote asthma persistence, particularly in the context of atopy; prevention of such infections may influence the natural history of asthma.

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-beta, 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.

Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.

Background Human rhinoviruses, major precipitants of asthma exacerbations, infect the lower airway epithelium inducing inflammation. The possibility that viral infection may mediate angiogenesis, thus contributing to airway remodeling, has not been evaluated. Objective To investigate whether epithelial infection with rhinovirus mediates angiogenesis in vitro , evaluate possible modulation by an atopic environment, and confirm angiogenic factor induction after in vivo rhinovirus infection. Methods Bronchial epithelial cells were infected with rhinovirus and levels of vascular endothelial growth factor (VEGF), and angiopoietins were measured. The angiogenic effect of epithelial products was assessed in in vitro models of angiogenesis. PBMCs, obtained from patients with atopic asthma and normal controls, were exposed to rhinovirus; the ability of supernatants from these cultures differentially to affect rhinovirus-mediated epithelial VEGF production was evaluated. VEGF levels were measured in respiratory secretions from patients with asthma, before and during rhinovirus-induced exacerbations. Results Epithelial infection with rhinovirus specifically stimulated mRNA expression and release of VEGF, but not angiopoietins, in a time-dependent and dose-dependent manner. Supernatants from these cultures were able to induce angiogenesis in vitro , significantly inhibited by a neutralizing anti-VEGF antibody. When bronchial cells were exposed to supernatants of rhinovirus-infected mononuclear cells from normal subjects or atopic patients with asthma, VEGF induction was significantly higher under the influence of the atopic environment. VEGF was elevated during rhinovirus-associated asthma exacerbations. Conclusion Rhinovirus infection, a frequent event, induces VEGF production in bronchial epithelial cells and human airways, an effect enhanced in an atopic environment. Rhinovirus-associated, VEGF-mediated angiogenesis may contribute to airway remodeling in asthma.

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-beta (TGF-beta), a cytokine with a broad range of activities in tissue repair. Here, we present evidence that TGF-beta inhibits fetal human skin fibroblasts, while it is stimulatory for adult ones. This proliferative effect of TGF-beta 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-beta 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-beta1 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-beta, possibly reflecting the different repair strategies followed in these two stages of development.

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.

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.

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 1x10(6) 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.

Background Human 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. Methods Monolayers 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. Results RV1b, 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. Conclusion RV-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.

The study of [125I]PDGF-BB binding to normal human embryonic lung fibroblasts, quiescent when cultured at sparsity in the presence of minute concentrations of homologous PDS, reveals approximately 2 x 10(5) binding sites for PDGF per cell; this number significantly increases during prolonged quiescence of the culture. As late as 48 h after down-regulation of PDGF receptors, the cells restore only partially their capacity to bind PDGF, with aged cells (above CPD 45) responding more rapidly and efficiently than younger ones. TGF-beta significantly enhances restoration of PDGF receptors and, in aged cells in particular, its presence results in total receptor recovery within 24 h, suggesting a concerted action of PDGF and TGF-beta regulating the proliferation of human fibroblasts in tissue regeneration.