IGF-1 stimulated upregulation of cyclin D1 is mediated via STAT5 signaling pathway in neuronal cells

Signal Transducer and Activator of Transcription (STATs) regulate various target genes such as cyclin D1, MYC, and BCL2 in nonneuronal cells which contribute towards progression as well as prevention of apoptosis and are involved in differentiation and cell survival. However, in neuronal cells, the role of STATs in the activation and regulation of these target genes and their signaling pathways are still not well established. In this study, a robust cyclin D1 expression was observed following IGF-1 stimulation in SY5Y cells as well as neurospheres. JAK/STAT pathway was shown to be involved in this upregulation. A detailed promoter analysis revealed that the specific STAT involved was STAT5, which acted as a positive regulatory element for cyclin D1 expression. Overexpression studies confirmed increase in cyclin D1 expression in response to STAT5a and STAT5b constructs when compared to dominant-negative STAT5. siRNA targeting STAT5, diminished the cyclin D1 expression, further confirming that STAT5 specifically regulated cyclin D1 in neuronal cells. Together, these findings shed new light on the mechanism of IGF-1 mediated upregulation of cyclin D1 expression in neural cell lines as well as in neural stem cells via the JAK/STAT5 signaling cascade

From in silico Protein Epitope Density Prediction to Testing Escherichia coli O157:H7 Vaccine Candidates in a Murine Model of Colonization

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a leading cause of foodborne illnesses worldwide and is a common serotype linked to hemorrhagic colitis and an important cause of hemolytic uremic syndrome (HUS). Treatment of EHEC O157:H7 infections is complicated, as antibiotics can exacerbate Shiga toxin (Stx) production and lead to more severe symptoms including HUS. To date, no vaccines have been approved for human use, exposing a void in both treatment and prevention of EHEC O157:H7 infections. Previously, our lab has shown success in identifying novel vaccine candidates via bio- and immunoinformatics approaches, which are capable of reducing bacterial colonization in an in vivo model of intestinal colonization. In this study, we further characterized seventeen of the identified vaccine candidates at the bioinformatics level and evaluated the protective capacity of the top three candidates when administered as DNA vaccines in our murine model of EHEC O157:H7 colonization. Based on further immunoinformatic predictions, these vaccine candidates were expected to induce neutralizing antibodies in a Th2-skewed immunological response. Immunization of BALB/c mice with two of these candidates resulted in reduced bacterial colonization following EHEC O157:H7 challenge. Additionally, immune sera was shown to prevent bacterial adhesion in vitro to Caco-2 cells. Together, this study provides further validation of our immunoinformatic analyses and identifies promising vaccine candidates against EHEC O157:H7

Alternaria-induced release of IL-18 from damaged airway epithelial cells: an NF-κB dependent mechanism of Th2 differentiation?

A series of epidemiologic studies have identified the fungus Alternaria as a major risk factor for asthma. The airway epithelium plays a critical role in the pathogenesis of allergic asthma. These reports suggest that activated airway epithelial cells can produce cytokines such as IL-25, TSLP and IL-33 that induce Th2 phenotype. However the epithelium-derived products that mediate the pro-asthma effects of Alternaria are not well characterized. We hypothesized that exposure of the airway epithelium to Alternaria releasing cytokines that can induce Th2 differentiation.We used ELISA to measure human and mouse cytokines. Alternaria extract (ALT-E) induced rapid release of IL-18, but not IL-4, IL-9, IL-13, IL-25, IL-33, or TSLP from cultured normal human bronchial epithelial cells; and in the BAL fluids of naïve mice after challenge with ALT-E. Both microscopic and FACS indicated that this release was associated with necrosis of epithelial cells. ALT-E induced much greater IL-18 release compared to 19 major outdoor allergens. Culture of naïve CD4 cells with rmIL-18 induced Th2 differentiation in the absence of IL-4 and STAT6, and this effect was abrogated by disrupting NF- κB p50 or with a NEMO binding peptide inhibitor.Rapid and specific release of IL-18 from Alternaria-exposed damaged airway epithelial cells can directly initiate Th2 differentiation of naïve CD4(+) T-cells via a unique NF-κB dependent pathway

Effect of disruption of NF-κB p50 on Th2 differentiation of naïve CD4 T-cells treated with IL-18.

<p>CD4⁺ T-cells from Wild-type (WT) and NF-κB p50^−/− mice were cultured with IL-18 in the differentiation phase, restimulated with con A, and secretion of IL-4 (<b>A</b>), IL-5 (<b>B</b>) and IL-13 (<b>C</b>) was quantified by ELISA. n = 5−7 per group.</p

Rapid release of cytokines from epithelial cells cultured with ALT-E.

<p>(<b>A, B</b>) NHBE cells were cultured with PBS, ALT-E or other allergens for 15 min, and released cytokines were quantified. T. Alder, Tag Alder; S. Ragweed, Short Ragweed (n = 5 wells per treatment group). (<b>C</b>) BALF cytokine levels in naïve mice 60 min after challenge with 20 µg ALT-E or PBS control (n = 4 per group).</p

Induction of epithelial cell necrosis by ALT-E.

<p>(<b>A</b>) A549 cells were cultured with ALT-E for 30 min. Changes in cell morphology were monitored by live cell imaging. (<b>B</b>) Time course of ALT-E-induced IL-18 release from airway epithelial cells. (<b>C</b>) Exposure of cultured A549 epithelial cells to ALT-E induces cell necrosis. (<b>D</b>) ALT-E challenge of mice induces sloughing of trypan blue positive necrotic airway epithelial cells (n = 4 per group). (<b>E</b>) Caspase 1 inhibitor (Cas.1 inh.) reduces IL-18 release from A549 airway epithelial cells cultured with ALT-E.</p

Effect of IL-18 on Th2 differentiation and GATA3 expression in naïve CD4+ T-cells.

<p>(<b>A,B,C</b>). Effect of NEMO peptide on Th2 differentiation. Negatively selected naïve CD3⁺ CD4⁺ T-cells were cultured with plate bound anti-CD3 and anti-CD28 with PBS (PBS), IL-18 100 ng/ml (IL-18), IL-18 100 ng/ml and 10 µM NEMO Binding Domain Binding Peptide (IL-18+NEMO) for the initial culture period. The cells were washed and restimulated, and secretion of IL-4 (<b>A</b>), IL-5 (<b>B</b>) and IFN-γ(<b>C</b>) were quantified. The data was expressed as fold increase compared to cells cultured with PBS. (<b>D,E,F</b>). GATA3 expression in T-cells. Negatively selected naïve CD3⁺ CD4⁺ T-cells were cultured with plate bound anti-CD3 and anti-CD28 in the presence of PBS (<b>D</b>), IL-4 (<b>E</b>) or IL-18 (<b>F</b>) for 7 days. GATA3 expression was measured by flow cytometric analysis.</p