Shelley A. Grubman

Antibody responses to bacterial toxoids in children infected with human immunodeficiency virus

Infection with human immunodeficiency retrovirus (also known as HTLV III, LAV,. and ARV) can produce a spectrum of immunologic perturbations ranging from no obvious deficit to severe combined acquired immune deficiency. Adults with acquired immune deficiency syndrome are likely to have lymphopenia and severe cell-mediated immunodeficiency, with a dramatic deficiency of helper T cells. Adults also have hypergammaglobulinemia and diminished capacity to produce antibody after primary or booster immunizations? Even asymptomatic HIV-infected adults have defective B-lymphocyte function as measured by a variety of in vitro assays. 2 In chiidren, HIV infection produces a similar spectrum of immunologic perturbations. A recent study of sick children with AIDS has demonstrated blunted antibody responses to bacteriophage phi X174 after prinmry and secondary immunizations. In addition, class switching (IgM to IgG) was generally absent and antibody responses to pneumococcal vaccine and tetanus toxoid were also diminished? We examined 17 children with HIV infection, who had received at least three immunizations with diphtheriatetanus-pertussis vaccine, for the presence of humoral and ceil-mediated immune responses to diphtheria and tetanus toxoids. These children had neither, a history of opportunistic infection nor biopsy-proved lymphocytic interstitial proliferation at the time of study. METHODS

Correction of the cystic fibrosis defect by gene complementation in human intrahepatic biliary epithelial cell lines

BACKGROUND/AIMS Hepatobiliary disease is the second most common cause of mortality in patients with cystic fibrosis (CF). In the liver, only the intrahepatic biliary epithelial (IBE) cells express cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine whether human CF-derived IBE cells can be infected with adenovirus and the CF phenotype complemented. METHODS IBE cells were isolated from 2 patients with CF and immortalized using retrovirus transduction of SV40 large T antigen. Immortalized cells were infected with the adenovirus vector Ad2/CFTR2 and assayed 2-31 days postinfection for cyclic adenosine monophosphate (cAMP)-induced halide efflux. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe 6-methoxy-N-(3-sulfopropyl)-quinolinium. RESULTS CF-derived IBE cell lines express biliary specific markers and express no cAMP-inducible halide efflux. Following infection with the adenovirus vector Ad2/CFTR2, a cAMP-induced halide efflux was observed for 31 days, although the number of responsive cells decreased with time. CONCLUSIONS Human CF-IBE cells can be infected by adenovirus and the defective CFTR complemented. The loss of responsive cells with time could be due to loss of construct and/or a reduced growth of cells that are overexpressing CFTR. These CF-IBE cell lines offer an opportunity to determine the mechanisms responsible for hepatobiliary disease in the patients with CF.

An In Vitro Model of Infection of Human Biliary Epithelial Cells by Cryptosporidium parvum

Cryptosporidium parvum infection in the immunosuppressed host is frequently complicated by biliary tract involvement. The recent production of human biliary epithelial cell lines was exploited to develop an in vitro model of biliary cryptosporidiosis. Infection with C. parvum oocysts was detected by IFA and ELISA and confirmed by transmission electron microscopy. Inoculation of monolayers with 10(4) to 5 X 10(5) oocysts/well resulted in a dose-dependent increase in infection. Time-course experiments showed that the number of parasitic stages was maximal at 18-24 h after inoculation. Infection was significantly enhanced by bile at concentrations of 50 and 100 microg/mL and inhibited by 400 microg/mL paromomycin. Infection of human biliary cells with C. parvum can be consistently achieved and monitored by use of IFA or ELISA. This system will be of use in evaluating mechanisms of C. parvum infection and response to therapeutic agents in biliary cryptosporidiosis.

Purinoceptor P2U identification and function in human intrahepatic biliary epithelial cell lines

The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined. Human intrahepatic biliary cell lines that we have developed were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides. Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ. Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline. The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium. The purinoceptor P2U-subtype was identified based on the potency rank order of ATP-analogues. Halide efflux increases with P2U-purinoceptor stimulation which is consistent with the opening of a Ca(2+)-sensitive Cl- channel. The physiological significance of P2U-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined.

Matrix Metalloproteinase Activity in Human Intrahepatic Biliary Epithelial Cell Lines From Patients With Autosomal Dominant Polycystic Kidney Disease

Hepatic cysts derived from intrahepatic bile ducts are the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD). Cyst enlargement involves cell proliferation, fluid secretion into cysts, and alterations in extracellular matrix. To study hepatic cyst formation, continuous cell lines from human normal intrahepatic biliary epithelium (IBE) and ADPKD liver cyst-derived epithelium (LCDE) were developed. Because matrix degradation and remodeling are important for cyst formation and growth, we investigated matrix modifying enzymes expressed in IBE and LCDE cell lines. Gelatin substrate zymography showed that two matrix degrading activities with characteristics of matrix metalloproteinases are secreted from these cell lines. Western immunoblotting suggests that these activities correspond to the 72 kDa (Gelatinase A) and 92 kDa (Gelatinase B) type IV collagenases. Although the level of Gelatinase A activity is comparable in both IBE and LCDE cell lines, Gelatinase B activity is substantially increased in LCDE lines.

Chapter 14 Tissue-Specific Expression of Genes Encoding the Rat Voltage-Gated Sodium Channel

Publisher Summary This chapter describes the strategy and techniques used to study the expression of the rat sodium channel genes in different excitable tissues. The voltage-gated sodium channel is responsible for the generation of the action potential in nerve and muscle cells. Previous comparative studies of the functional properties of sodium channels have indicated that sodium channels in these two tissue types are very similar. Recent studies have concluded that sodium channels in nerve and muscle differ in significant ways. Investigators have shown that the sodium channels of skeletal muscle differ from the sodium channels in nerve and cardiac muscle in their affinities for the peptide sodium channel blocker, μ-conotoxin. mRNA purified from brain and muscle produces sodium channels that are both functionally and pharmacologically distinguishable in Xenopus oocyte membrane.