Andreina Baj

Human Immunodeficiency Virus-1 Tat Induces Hyperproliferation and Dysregulation of Renal Glomerular Epithelial Cells

Human immunodeficiency virus-associated nephropathy (HIVAN) is etiologically related to the viral infection, but the mechanisms of virus-induced renal injury remain undetermined. Peculiar histopathological features of HIVAN are the enhanced proliferation and the loss of differentiation markers of glomerular epithelial cells (podocytes). We found that podocytes were not permissive to HIV-1 replication. In this study we investigated the effects of the HIV-1 regulatory protein Tat on primary cultures and on a continuous line of podocytes. Our results demonstrated that Tat induced hyperproliferation of these cells in a dose-dependent manner. This activity was primarily mediated by the basic domain of the viral protein. Proteoglycans were required for this phenomenon because Tat-induced increase of podocyte growth was significantly impaired by inhibition of proteoglycan synthesis with beta-D-xyloside. In podocyte cultures Tat promoted both the transcription and the release of basic fibroblast growth factor, which contributed to the enhanced cell proliferation. Moreover, Tat deregulated the podocyte phenotype causing down-regulation of maturity markers such as WT-1 and synaptopodin, alteration of cytoarchitecture, and impairment of permselectivity. Together, these results demonstrate that the interaction of extracellular Tat with podocytes can induce alterations that mimic the pathological changes of podocytes detected in HIVAN.

Proliferative activity of extracellular HIV-1 Tat protein in human epithelial cells: expression profile of pathogenetically relevant genes.

BackgroundTat is being tested as a component of HIV vaccines. Tat activity has been mainly investigated on cells of lymphoid/hematopoietic lineages. HIV-1, however, is known to infect many different cells of both solid organs and mucosal surfaces. The activity of two-exon (aa 1–101) and synthetic (aa 1–86) Tat was studied on mammary and amniotic epithelial cells cultured under low serum conditions.Resultssmall concentrations of Tat (100 ng/ml) stimulated cell proliferation. Tat antibodies neutralized the mitogenic Tat activity. Changes of gene expression in Tat-treated cells were evaluated by RT-PCR and gene-array methods. Within 4 hours of treatment, exposure to Tat is followed by up-regulation of some cell cycle-associated genes (transcription factors, cyclin/cdk complexes, genes of apoptotic pathways) and of genes relevant to HIV pathogenesis [chemokine receptors (CXCR4, CCR3), chemotactic cytokines (SDF-1, RANTES, SCYC1, SCYE1), IL6 family cytokines, inflammatory cytokines, factors of the TGF-beta family (TGFb, BMP-1, BMP-2)]. Up-regulation of anti-inflammatory cytokines (IL-10, IL-19, IL-20), a hallmark of other persistent viral infections, was a remarkable feature of Tat-treated epithelial cell lines.Conclusionextracellular Tat is mitogenic for mammary and amniotic epithelial cells and stimulates the expression of genes of pathogenetic interest in HIV infection. These effects may favor virus replication and may facilitate the mother-to-child transmission of virus.

Culture of skeletal myoblasts from human donors aged over 40 years: dynamics of cell growth and expression of differentiation markers.

BackgroundLocal myogenesis, neoangiogenesis and homing of progenitor cells from the bone marrow appear to contribute to repair of the infarcted myocardium. Implantation into heart tissues of autologous skeletal myoblasts has been associated with improved contractile function in animal models and in humans with acute myocardial ischemia. Since heart infarction is most prevalent in individuals of over 40 years of age, we tested whether culture methods available in our laboratory were adequate to obtain sufficient numbers of differentiated skeletal myoblasts from muscle biopsy specimens obtained from patients aged 41 to 91.Methods and resultsNo matter of donor age, differentiated skeletal muscle cells could be produced in vitro in amounts adequate for cellular therapy (≥300 millions). Using desmin as a cytoplasmic marker, about 50% cultured cells were differentiated along myogenic lineages and expressed proteins proper of skeletal muscle (myosin type I and II, actin, actinin, spectrin and dystrophin). Cytogenetic alterations were not detected in cultured muscle cells that had undergone at least 10 population doublings. Molecular methods employed for the screening of persistent viral infections evidenced that HCV failed to replicate in muscle cells cultured from one patient with chronic HCV infection.ConclusionThe proposed culture methods appear to hold promise for aged patients not only in the field of cardiovascular medicine, but also in the urologic and orthopedic fields.

Intrafamilial spread of enterovirus infections at the clinical onset of type 1 diabetes.

At the clinical onset of type 1 diabetes mellitus (T1D), enterovirus (EV) infections are suspected to play a role. EVs in blood are seen as a possible biomarker of T1D. EV infections may occur in temporal and geographic clusters and may spread within families.

Post-poliomyelitis syndrome as a possible viral disease

This review summarizes current concepts on post-polio syndrome (PPS), a condition that may arise in polio survivors after partial or complete functional recovery followed by a prolonged interval of stable neurological function. PPS affects 15-20 million people worldwide. Epidemiological data are reported, together with the pathogenic pathways that possibly lead to the progressive degeneration and loss of neuromuscular motor units. As a consequence of PPS, polio survivors experience new weakness, generalized fatigue, atrophy of previously unaffected muscles, and a physical decline that may culminate in the loss of independent life. Emphasis is given to the possible pathogenic role of persistent poliovirus infection and chronic inflammation. These factors could contribute to the neurological and physical decline in polio survivors. A perspective is then given on novel anti-poliovirus compounds and monoclonal antibodies that have been developed to contribute to the final phases of polio eradication. These agents could also be useful for the treatment or prevention of PPS. Some of these compounds/antibodies are in early clinical development. Finally, current clinical trials for PPS are reported. In this area, the intravenous infusion of normal human immunoglobulins appears both feasible and promising.

Viral Encephalitis: Etiology, Clinical Features, Diagnosis and Management

Viral encephalitis is worldwide spread pathology with high morbidity and mortality. Its incidence is higher in children. Enteroviruses, varicella zoster virus and herpes simplex viruses are the most frequent agents. However, in spite of the use of modern microbiological and radiological methods, an etiological diagnosis is reached in less than 50% of cases, making a careful differential diagnosis with non viral brain diseases imperative. Pathogenesis is elusive and therapy continues to remain supportive in almost all cases, as the only virus-directed treatment is available for herpesvirus-related encephalitis and a role for steroids continues to be debated. Novel and more targeted therapies are eagerly needed.

Poliovirus type 1 infection of murine PRNP-knockout neuronal cells

Transfection of the prion protein gene (Prnp) into prion-deficient mouse cells was shown to reduce the replication of coxsackievirus B3, an enterovirus. Because mice can be susceptible to poliovirus infection by parenteral routes, the authors tested the susceptibility to poliovirus-1 (PV-1) of a panel of murine neuronal cell lines differing in their ability to express Prnp. The investigated cell lines (prionless HpL3.4 cells, HpL3.4 cells transfected with a Prnp vector, HpL3.4 cells transfected with a void vector, wild-type Hw3.5 Prnp+/+ cells) expressed the murine homologue (Tage4) of human poliovirus receptor (CD155/hPVR). PV-1 infection of Prnp−/− HpL3.4 cells resulted in the production of high viral titers, though viral antigens could be detected in only 0.5% to 2% of cells. Wild-type Prnp+/+ cells and prionless cells transfected with the Prnp gene were not permissive to PV-1. Results of viral titration and immunofluorescence were confirmed by conventional polymerase chain reaction (PCR) and quantitative real-time PCR. Exposure to PV-1 had no influence on the gene expression profile of Prnp+/+ cells. In contrast, PV-1 infection was associated with up regulation of several genes in permissive Prnp−/− cell cultures: type I interferon (IFN) genes, IFN-related developmental regulator 1 (IFNRD1), tumor necrosis factor superfamily member 13b (TNFSF13b), interleukin (IL) − 7, granulocyte/macrophage colony-stimulating factors (CSFs), hepatocyte growth factor (HGF), vascular endothelial growth factor-A, transforming growth factors beta1 and beta3 (TGFb1, TGFb3), as well as a variety of bone morphogenetic proteins endowed with neuroprotective activity. Distinction of permissive from nonpermissive neuronal cells on the basis of Prnp expression suggests that prion-deficient mice could represent an extraordinarily sensitive animal model for poliovirus infection.

Virology of the post-polio syndrome

The three poliovirus serotypes (PVs) cause acute paralytic poliomyelitis. Decades after being hit by polio, survivors may develop a condition known as post-polio syndrome (PPS). PPS is characterized by extreme fatigue, progressing muscular weakness and chronic pain. The pathogenesis is unclear and, thus, empirical therapies are employed. PVs are known to be able to persist in infected host cells both in vitro and in vivo. The understanding of PV genomes has made it possible to set up sensitive and specific molecular tests capable of detecting minute amounts of virus in samples from PPS patients. Current data indicate that complete PV genomes (or genomic fragments) remain present, decades after acute paralysis, in the CNS of these patients. Virus persistence is hypothesized to bring about chronic inflammation, immune-mediated injury and decreased expression of neurotrophic factors. Establishing a pathogenetic link between PV persistence and PPS would be extremely relevant to the development of an etiologic...

Colonization of a Central Venous Catheter by the Hyaline Fungus Fusarium solani Species Complex: A Case Report and SEM Imaging

The incidence of opportunistic infections by filamentous fungi is increasing partly due to the widespread use of central venous catheters (CVC), indwelling medical devices, and antineoplastic/immunosuppressive drugs. The case of a 13-year-old boy under treatment for acute lymphoblastic leukemia is presented. The boy was readmitted to the Pediatric Ward for intermittent fever of unknown origin. Results of blood cultures drawn from peripheral venous sites or through the CVC were compared. CVC-derived bottles (but not those from peripheral veins) yielded hyaline fungi that, based on morphology, were identified as belonging to the Fusarium solani species complex. Gene amplification and direct sequencing of the fungal ITS1 rRNA region and the EF-1alpha gene confirmed the isolate as belonging to the Fusarium solani species complex. Portions of the CVC were analyzed by scanning electron microscopy. Fungi mycelia with long protruding hyphae were seen into the lumen. The firm adhesion of the fungal formation to the inner surface of the catheter was evident. In the absence of systemic infection, catheter removal and prophylactic voriconazole therapy were followed by disappearance of febrile events and recovery. Thus, indwelling catheters are prone to contamination by environmental fungi.

Enteroviruses and causality of type 1 diabetes: how close are we?

Organ-specific autoimmunity frequently affects the endocrine system, including pancreatic islets. Type 1 diabetes mellitus (T1D) derives from the autoimmune destruction of insulin-secreting β-cells that is triggered by environmental factors (1, 2). Genetic predisposition accounts for 36–50% of disease susceptibility as demonstrated in monozygotic twin studies (3–5). Approximately 90% of new cases lack a family history of T1D indicating a large contribution of exogenous factors to pathogenesis. A variety of associations with viral infections have been reported for human diabetes including rubella, mumps, and cytomegalovirus. However, among investigated agents, human enteroviruses (HEVs) appear to play a prominent role (1). HEVs are extremely common RNA viruses that spread mainly through the fecal-oral route. Overall, these agents cause millions of new infections per year worldwide (6). The enterovirus genus comprises over 100 antigenically different virus types (7). The single-stranded 7.5 kb RNA enteroviral genome encodes for capsid proteins and other proteins involved in viral infectivity and replication. Capsid proteins are highly variable among HEV species and types. Neutralizing antibodies raised against capsid proteins are highly type specific but may also cross-react with related types (8). So far, there are no means for preventing T1D. Sets of well-defined autoantibodies have a strong predictive value (9, 10), but the costbenefit ratio of periodical determinations appears not to justify screening programs at the population level. At the diagnosis of T1D, a small fraction Oscar Diaz-Hortaa,c, Andreina Baja, Giuseppe Maccaria, Alessandro Salvatonib and Antonio Tonioloa aDepartment of Experimental Medicine, Laboratory of Medical Microbiology, University of Insubria, Varese, Italy; bDepartment of Experimental Medicine, Pediatric Endocrinology Unit, University of Insubria, Varese, Italy; and cDepartment of Immunology and Genetics, National Institute of Endocrinology, Havana, Cuba