Salima Haque

Interferons α, β, γ each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation

Abstract: Interferon (IFN)‐α is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN‐α, ‐β and ‐γ inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5′ untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full‐length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES‐dependent mechanisms (pCITE‐GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN‐α, ‐β and ‐γ inhibit replication of sub‐genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV‐ and EMCV‐IRES sequences present in the dicistronic HCV sub‐genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES‐mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication.

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating‐wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex‐3 microcarrier beads to form bead aggregates with extensive three‐dimensional growth between beads and on the aggregate surface. HARV and spinner‐flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G1‐phase cells, larger bead aggregates, enhanced development of filopodia and microvilli‐like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T‐flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three‐dimensional growth.

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

BackgroundAlpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines.ResultsHCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b). Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein.ConclusionThis in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

Characterization of esophageal submucosal glands in pig tissue and cultures.

The submucosal glands (SMGs) of the pig esophagus, like the human, secrete mucin and bicarbonate, which help in luminal acid clearance and epithelial protection. The aim of this study was to characterize histochemically the esophageal SMGs and a primary culture obtained from these glands. Tissues and cultures were stained with hematoxylin and eosin, periodic acid Schiff, Alcian blue, lectins, or cytokeratins. In the perfused esophagus, addition of carbachol increased mucin secretion by approximately 2-fold. The results indicate that [1] a method for culturing SMG cells was developed; [2] conventional staining indicates the presence of sulfated, acidic, and neutral mucopolysaccharides in glands and cultures; [3] lectin binding indicates the presence of N-acetyl glucosamine, N-acetyl neuraminic acid, N-acetyl galactosamine, and α-l-fucose in mucous cells and cultures; [4] cytokeratin and lectin staining indicated similarities with Barrett epithelium (columnar metaplasia of the esophagus); and [5] cholinergic agonists enhance mucin secretion and this could play a significant role in esophageal protection.

Activation of interferon-stimulated response element in huh-7 cells replicating hepatitis C virus subgenomic RNA.

Interferon-alpha (IFNα) binds to receptors on the cell surface, which initiate a cascade of signal transduction pathways that leads to transcription of selected genes. This transduction pathway involves binding of transcription factors to a common cis-acting DNA sequence called IFN-stimulated response element (ISRE). To test whether these signaling pathways are functional in hepatitis C virus (HCV)-replicating cells, we studied the regulation of ISRE-mediated transcription of firefly luciferase gene in stable replicon cell lines. A plasmid construct was prepared (pISRELuc) which contains four tandem repeats of 9-27 ISRE sequences positioned directly upstream of the herpes virus 1 thymidine kinase promoter TATA box that drives the expression of firefly luciferase. Regulation of ISRE-mediated expression of firefly luciferase by IFNα was studied by transfecting this clone into Huh-7 cells replicating HCV subgenomic HCV RNA. The significance of ISRE-mediated transcriptional activation was studied in a replicon cell line by pretreatment of cells with actinomycin D, which inhibits cellular DNA-dependent RNA transcription. IFN treatment activates ISRE-mediated expression of luciferase, indicating that this pathway is functional in Huh-7 cells. Activation of ISRE-mediated transcription of luciferase is relatively high in two Huh-7 stable cell lines replicating HCV subgenomic RNA. Inhibition of ISRE-mediated transcription of luciferase by actinomycin D also makes HCV replication totally resistant to IFNα. These in vitro studies suggest that activation of IFN-inducible genes is important in mounting a successful antiviral response against HCV.

Pancreatic polypeptide islet cell tumor: case report and review of the literature.

Pure pancreatic polypeptide-containing tumors (PPomas) are quite rare. Only 20 cases have been described. In this article we report a 75-year-old woman with such an endocrine islet cell tumor. The patient had no specific symptoms that could be ascribed to the tumor. An abdominal CT scan revealed a 3 cm soft tissue mass arising inferiorly from the tail of the pancreas. Local resection by way of a distal pancreatectomy was performed. A well-circumscribed hemorrhagic multiloculated mass, 3.7 cm in greatest dimension, was present in the tail of the pancreas. The patient has remained well and tumor free for the past 22 months. The endocrine characterization of the tumor was achieved by means of immunohistochemical analysis. Staining specific for insulin, ghicagon, somatostatin, and gastrin was negative. In contrash staining of the tumor for pancreatic polypeptide was strongly positive. A number of nonfunctioning islet cell tumors of the pancreas have been described. The lack of fimction has previously been suggested to indicate the lack of secretion of an endocrine product. This report documents that islet cell tumors may function by secreting pancreatic polypeptide but not cause symptoms.

Metastatic pancreatic carcinoma presenting as colon carcinoma.

Determining the origin of poorly differentiated adenocarcinomas remains a challenge for the pathologist. This manuscript reports the use of a panel of specific immunohistochemical stains to determine the primary site of a tumor in the colon. A 45-year-old man had a right hemicolectomy for adenocarcinoma. Immunohistochemical staining documented that the lesion was a metastasis from a primary pancreatic adenocarcinoma—an unusual pattern of spread. The case emphasizes the important use of immunohistochemistry in identifying the primary source of lesions, allowing for appropriate treatment and staging.

Two Unusual Cases of Adult Intussusception

Adult intussusception is very rare. We report 2 unusual cases, a 58-year-old man with a transverse colo-colonic intussusception caused by a malignant sessile polyp that also had an asymptomatic synchronous neoplasm of the kidney, and an 18-year-old female with an ileocecolic intussusception caused by acute appendicitis. This report stresses the point that intussusception in adults may represent an underlying malignancy. The age of the patient and the anatomic location of the intussusception provide significant input as to the etiology and hence the most appropriate surgical procedure.

Langerhans cell histiocytosis: report of a single organ involvement in a child

Langerhans cell histiocytosis is a rare disorder characterized by abnormal proliferation of Langerhans cells that can affect various organ systems. The disease usually presents as a unifocal lytic bone lesion and can affect any age group. Less frequently it presents as a disseminated disease with multisystem involvement. Hepatic manifestation in Langerhans cell histiocytosis is relatively rare and usually presents as a part of a disseminated process. We report a case of Langerhans cell histiocytosis involving only the liver in a 9‐years‐old child.