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Biomedical subjects

M A Gerber

Publications and source records attributed to M A Gerber.

At least 37 records · Page 2Linked to original sources
PubMed1997

Liver-directed gene transfer in non-human primates.

To develop a primate model for liver-directed gene therapy, we studied several gene transfer vehicles and routes in eight rhesus monkeys (Macaca mulatta). For this purpose, we used first-generation, replication-deficient adenoviral vectors carrying the Escherichia coli lacZ gene (Ad.CMVlacZ) or a lacZ-containing plasmid (pCMV beta) with lipofectamine for transfection. The reporter gene construct was infused into either the portal vasculature, common bile duct, or saphenous vein. Adenovirus-mediated gene transfer via the portal vein resulted in expression of lacZ in over 70% of hepatocytes by days 3-7, but was accompanied by acute hepatitis. Adenovirus-mediated gene transfer via the common bile duct resulted in lacZ expression in less than 10% of hepatocytes and was accompanied by portal inflammation. The animals mounted a significant immune response, as demonstrated by adenoviral antigen-induced T-cell proliferation and production of neutralizing anti-adenovirus antibodies and antibodies to E. coli beta-galactosidase (beta-Gal). Activation of the immune response was associated with rapid decrease of the reporter gene by days 13-21. Lipofectamine-mediated gene transfer was inefficient, and no lacZ expression in the liver was detected. To limit the host immune response, 4 animals were immunosuppressed by cyclophosphamide/prednisone and then infused with the Ad.CMVlacZ via the portal vein or the saphenous vein. The monkeys showed sustained expression of lacZ for up to 35 days with no evidence of inflammation. The primates transduced via the saphenous vein showed a level of beta-Gal expression in the liver similar to that of the portal vein-infused animals. In conclusion, adenovirus-mediated gene transfer to non-human primate livers via the portal vein or saphenous vein is efficient, but it results in transient expression and is accompanied by an immune response to both vector and transgene products and acute hepatitis, whereas lipofectamine-mediated transfer is inefficient. Manipulation of the host immune response may expand potential applications of adenoviral vectors for liver-directed gene transfer.

Adenoviridae↗
PubMed1997

Optical immunoassay test for group A beta-hemolytic streptococcal pharyngitis. An office-based, multicenter investigation.

OBJECTIVE: To compare the accuracy of an optical immunoassay (OIA) for the rapid diagnosis of group A streptococcal pharyngitis with blood agar plate (BAP) culture. DESIGN: Blinded comparison with criterion standard. SETTING: A total of 6 private pediatricians' offices, 3 in Connecticut and 3 in Chicago, III. PATIENTS: A total of 2113 consecutive patients with acute pharyngitis, 983 in Connecticut and 1130 in Chicago. MAIN OUTCOME MEASURES: The sensitivities and specificities of OIA and BAP culture (both performed and interpreted in the office) were determined using a research laboratory's interpretation of a combination of BAP culture and Todd-Hewitt broth (THB) culture of transport tube pledget as criterion standard. RESULTS: Among patients in Connecticut, the sensitivities of the OIA and BAP culture were 94% and 89%, respectively (P=.004), while the specificities were 96% and 99%, respectively (P=.001). Among patients in Chicago, the sensitivities of the OIA and BAP culture were 79% and 72%, respectively (P<.001), while the specificities were 89% and 99%, respectively (P<.001). In each of the 6 pediatricians' offices, the OIA was more sensitive than the BAP culture. Combining the data from Connecticut and Chicago, the overall sensitivities of the OIA and BAP culture were 84% and 78%, respectively (P<.001), while the specificities were 93% and 99%, respectively (P<.001). CONCLUSIONS: The results of this comprehensive office-based investigation suggest that with adequately trained personnel, negative OIA test results may not always need to be routinely confirmed with BAP cultures.

Acute Disease↗
PubMed1997

Histopathology of HCV infection.

The basic morphologic features of acute and chronic viral hepatitis C are similar to those of other hepatitides; however, hepatitis C is characterized by the histologic triad of lymphoid aggregates in portal tracts, epithelial damage of small bile ducts and microvesicular and macrovesicular steatosis of hepatocytes. Significant progress has been made in the demonstration of HCV in infected liver tissues by immunohistochemical and in situ hybridization techniques. The new classification of chronic hepatitis, based on etiology, grading (extent of necroinflammatory activity) and staging (extent of fibrosis) has been widely accepted and will lead to a better understanding of the variable course and response to therapy of this enigmatic disease.

DNA, Viral↗
PubMed1997

Diagnosis and management of group A streptococcal pharyngitis: a practice guideline. Infectious Diseases Society of America.

This is the second in a series of practice guidelines commissioned by the Infectious Diseases Society of America through its Practice Guidelines Committee. The purpose of these guidelines is to provide assistance to clinicians when making decisions on treating the conditions specified in each guideline. The targeted providers are pediatricians, family practitioners, and internists. The targeted patients and setting for the acute pharyngitis guideline are pediatric, adolescent, and adult outpatients with a complaint of sore throat. Funding was provided by the IDSA. Panel members represented experts in adult and pediatric infectious diseases. The guidelines are evidence-based. A standard ranking system was used for the strength of the recommendations and the quality of the evidence cited in the literature reviewed. The document has been subjected to external review by peer reviewers as well as by the Practice Guidelines Committee and was approved by the IDSA Council. An executive summary, algorithms, and tables highlight the major recommendations. Indicators of quality will assist in guideline implementation. The guideline will be listed on the IDSA home page at http://www.idsociety.org.

Adolescent↗
PubMed1997

The inhibitory effects of antisense RNA on hepatitis B virus surface antigen synthesis.

Antisense RNA-mediated inhibition of gene expression has the potential for gene therapy of virus infections. We studied the inhibitory effect of antisense RNA directed against the hepatitis B virus (HBV) genome on the expression of the HBV surface antigen (HBsAg). Three prokaryotic antisense RNA expression constructs were produced which expressed antisense RNA complementary to the entire coding region (1.4 kb) and to 1.0 kb and 582 bp of the 5' region of HBsAg mRNA, respectively. In an in vitro translation system, all three antisense RNAs showed concentration-dependent inhibitory effects on translation of HBsAg mRNA. In a coupled in vitro transcription and translation system, concentration-dependent inhibition of HBsAg synthesis was observed for all above mentioned antisense RNAs. Three mammalian antisense RNA expression vectors were then constructed, expressing the same antisense RNAs as used above. Transfection of the vectors into Hep3B cells (an HBsAg secreting cell line) resulted in almost complete blockage of HBsAg production, whereas control vector transfected cells secreted high levels of HBsAg. The inhibitory effect lasted for more than 10 months post-transfection. To examine the possible mechanism of the antisense RNA effect in the cell line, we measured HBV mRNA levels in the transfected cells and found that the mRNA levels in the antisense RNA expressing cells were much lower than those in the control cells. Therefore, in Hep3B cells, the antisense RNAs inhibited HBsAg synthesis, at least partially, through the reduction of HBV mRNA levels.

Gene Expression Regulation, Viral↗
PubMed1997

HCV cDNA transfection to HepG2 cells.

The establishment of an in vitro system for hepatitis C virus (HCV) propagation is essential to characterize virus replication, virus persistence and viral pathogenicity. The aim was to establish HCV replication in HepG2 cells by gene transfer of infectious HCV cDNA. First, several gene transfer methods were evaluated that employed cationic liposomes (lipofectin, lipofectamine. DOTAP), DEAE-dextran and replication-deficient adenovirus for transfection to HepG2 cells using a lacZ reporter gene. Highest transfection efficiency (20%) of cultured HepG2 cells was obtained by the combined use of lipofectamine and adenovirus. This method was used for transfection of HepG2 cells with HCV cDNA in a mammalian expression plasmid (pRC/CMV). The success and efficacy of HCV transfection to HepG2 cells was evaluated by testing for the presence of genomic and replicative (negative) strands of HCV RNA by strand-specific reverse transcription (RT) followed by nested PCR. Expression of structural and non-structural proteins of hepatitis C virus was detected using polyclonal antibodies to core, NS3, NS4 and NS5. Positive-strand HCV RNA was detected by RT-PCR for over 6 weeks in the HCV cDNA-transfected HepG2 cells. Presence of HCV replication in these cells was confirmed by detecting HCV negative-strand RNA by strand-specific RT-PCR and was observed to continue for over 4 weeks. HCV proteins (core, NS3, NS4 and NS5) were detected in the cytoplasm of the transfected cells by immunostaining. In summary, these findings suggest that replication and translation of HCV were achieved for a prolonged time in HepG2 cells after transfection with HCV cDNA and may provide an in vitro system for HCV studies.

DNA, Complementary↗
PubMed1997

Characterization of ductular hepatocytes in primary liver allograft failure.

Liver regeneration after massive hepatic necrosis is characterized by the presence and formation of ductular hepatocytes (DHs). Several studies suggested that these structures might serve as bipotential progenitor cells, as demonstrated by phenotypic features characteristic of both hepatocytes and biliary epithelial cells. In this investigation, 33 liver allograft explants after primary graft failure 1 to 14 days after orthotopic liver transplantation were examined to study the formation and differentiation of DHs. As demonstrated by earlier studies, antibody to CK-19 defines the biliary epithelial cell lineage, whereas HepPar 1 is hepatocyte specific. Antibodies to CK-19, vimentin and alpha-fetoprotein, HepPar 1, and AE-1 were used for immunoperoxidase staining of 33 failed liver allograft specimens with regeneration. DHs were seen along the limiting plates at Day 1, but reactivity to HepPar 1 started only at Day 4 post injury. Vimentin and alpha-fetoprotein were not detectable in the DHs. The DHs reacted with AE-1 and anti-CK-19 starting at Day 1, but reactivity to HepPar 1 started only at Day 4 post injury. DHs are formed along the limiting plates and acquire phenotypic characteristics of bile duct epithelium as early as Day 1 after liver injury and of hepatocytes only at Day 4 after liver injury. We conclude that DHs might represent bipotential progenitor cells in regenerating liver.

Biomarkers↗
PubMed1997

Detection of hepatitis C virus RNA sequences in hepatocellular carcinoma and its precursors by microdissection polymerase chain reaction.

OBJECTIVE: Chronic hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinoma (HCC), but the mechanism of malignant transformation is unknown. To analyze the association of HCV with HCC, we developed a microdissection technique for the detection by polymerase chain reaction of positive (genomic)- and negative (replicative)-strand HCV RNA in histologically confirmed HCC and the surrounding cirrhotic and macroregenerative nodules. MATERIALS AND METHOD: Five HCCs and one macroregenerative nodule and the surrounding cirrhotic liver tissues of all cases were selected for this study. The method entails extraction of RNA from selected areas of formalin-fixed, hematoxylin-stained histologic sections, followed by strand-specific reverse-transcription double polymerase chain reaction and Southern blotting. RESULTS: Positive- and negative-strand HCV RNA sequences were detected in five of six tumors and the surrounding cirrhotic livers. CONCLUSIONS: These results verify the method of polymerase chain reaction detection of HCV RNA from histologically defined, selected lesions. In addition, the findings suggest that HCV RNA persists and replicates in hepatocytes during malignant transformation.

Aged↗
PubMed1997

Transfection of HepG2 cells with infectious hepatitis C virus genome.

Hepatitis C virus (HCV) represents one of the major causes of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) around the world. Our knowledge of the life cycle of HCV, however, is limited. Current studies are hampered by the lack of a reproducible, high-level in vitro replication system of HCV. We sought to establish HCV replication in HepG2 cells by gene transfer of in vitro transcribed HCV RNA. In preliminary experiments, diethylaminoethyl-dextran led to more efficient gene transfer than cationic liposomes (lipofectin, lipofectamine, and DOTAP). Therefore, in subsequent experiments, HepG2 cells were transfected with full-length (9.6-kb) and near-full-length (9.4-kb) HCV RNA using diethylaminoethyl-dextran. Transfection with subgenomic HCV RNA and mock transfection were used as controls. Positive- and negative-strand HCV RNA sequences were detected by reverse transcription polymerase chain reaction (KT-PCR) for 60 days in the infectious HCV RNA transfected HepG2 cells. The presence of negative-strand HCV RNA, presumably representing replicative intermediates, was confirmed by ribonuclease protection assay. The intracellular levels of HCV RNA were measured by quantitative competitive RT-PCR from 10 to 50 days after transfection and were stable over this time period at moderately high levels (10(8) to 10(10) genomes per mg of total RNA). Expression of viral core and nonstructural proteins was detected in the cytoplasm of transfected cells by immunostaining. Virus-like particles measuring 50 to 60 nm in diameter were found by electron microscopy in cytoplasmic vesicles and conditioned media of the cells transfected with infectious HCV RNA but not in cells transfected with truncated HCV RNA. Culture supernatants of infectious HCV RNA transfected HepG2 cells were infectious for Daudi cells for three passages tested. The truncated HCV RNA lacking NS5 and 3' untranslated region (3' UTR) of HCV was replication incompetent. This is the first demonstration of HCV particles in HepG2 cells after transfection with infectious HCV RNA. We conclude that we have established a reproducible HCV replication system in HepG2 cells that can be used to study the life cycle of HCV and to test anti-HCV agents.

Antigens, Viral↗
PubMed1997

A 67-year-old woman with gastrointestinal hemorrhage and chronic hepatitis.

The hepatitis C virus (HCV) is responsible for the majority of cases of non-A, non-B hepatitis. Affected patients are usually asymptomatic when initially infected; however, between 70% and 80% will maintain infection and develop chronic liver disease. Of these patients, 20% to 50% progress to cirrhosis, and up to 15% may develop hepatocellular carcinoma. Thus, many patients have significant liver disease when diagnosed. The following case report describes a patient with cirrhosis secondary to hepatitis C, transferred to our institution to manage complications related to portal hypertension.

Aged↗
PubMed1996

Lyme disease in children in southeastern Connecticut. Pediatric Lyme Disease Study Group.

BACKGROUND: Although the incidence of Lyme disease is highest in children, there are few prospective data on the clinical manifestations and outcomes in children. METHODS: We conducted a prospective, longitudinal, community-based cohort study of children with newly diagnosed Lyme disease in an area of Connecticut in which the disease is highly endemic. We obtained clinical and demographic information and performed serial antibody tests and follow-up evaluations. RESULTS: Over a period of 20 months, 201 consecutive patients were enrolled; their median age was 7 years (range, 1 to 21). The initial clinical manifestations of Lyme disease were a single erythema migrans lesion in 66 percent, multiple erythema migrans lesions in 23 percent, arthritis in 6 percent, facial-nerve palsy in 3 percent, aseptic meningitis in 2 percent, and carditis in 0.5 percent. At presentation, 37 percent of the patients with a single erythema migrans lesion and 89 percent of those with multiple erythema migrans lesions had antibodies against Borrelia burgdorferi. All but 3 of the 201 patients were treated for two to four weeks with conventional antimicrobial therapy, which was administered orally in 96 percent. All had prompt clinical responses. After four weeks, 94 percent were completely asymptomatic (including the two patients whose parents had refused to allow antimicrobial treatment). At follow-up a mean of 25.4 months later, none of the patients had evidence of either chronic or recurrent Lyme disease. Six patients subsequently had a new episode of erythema migrans. CONCLUSIONS: About 90 percent of children with Lyme disease present with erythema migrans, which is an early stage of the disease. The prognosis is excellent for those with early Lyme disease who are treated promptly with conventional courses of antimicrobial agents.

Adolescent↗
PubMed1996

Use of transient CD4 lymphocyte depletion to prolong transgene expression of E1-deleted adenoviral vectors.

E1-deleted adenoviral vectors are increasingly being utilized for in vivo gene transfer. The potential use of these vectors is limited by transient expression of the transgene and a markedly reduced rate of transduction following readministration, presumably due to a host immune response to the vector. We hypothesized that CD4+ lymphocytes are necessary to generate an immune response to these vectors and that administration of a depleting anti-CD4 antibody (GK1.5) might prolong transgene expression in vivo. We found that pretreatment of mice with a single injection (transient depletion) or weekly injections of GK1.5 (persistent depletion), markedly prolonged expression of an adenovirus-encoded tumor necrosis factor (TNF) inhibitor or luciferase gene compared to controls. Moreover, mice treated with GK1.5 showed no antiadenoviral antibody response to repeat administration of the vector and a second adenoviral transgene could be expressed in these animals. However, control mice developed a significant neutralizing antibody response that prevented transgene expression with administration of a second adenovirus. These findings demonstrate that manipulation of the host immune response may expand potential applications of gene transfer utilizing adenoviral vectors.

Adenovirus E1 Proteins↗
PubMed1996

Identification of bipotential progenitor cells in human liver development.

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver.

Adult↗