Molecular physiology of reverse cholesterol transport.

The primary immunodeficiency disorders reflect abnormalities in the development and maturation of cells of the immune system. These defects result in an increased susceptibility to infection; recurrent pyogenic infections occur with defects of humoral immunity, and opportunistic infections with defects of cell-mediated immunity. These two broad categories of illness correspond roughly to defects in the two principal types of immunocompetent cells, B lymphocytes and T lymphocytes. Defective development of B cells results in abnormalities in humoral immunity, whereas defects in the development of T cells cause problems with cellular immunity. When pathogens are taken up by macrophages or dendritic cells, . . .

The Primary Immunodeficiencies

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue- nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

Epidemiological surveys have identified a strong inverse relationship between the amount in the plasma of high density lipoproteins (HDL), apolipoprotein AI (ApoA-I), the major protein component of HDL, and the risk for atherosclerosis in humans. It is not known if this relationship arises from a direct antiatherogenic effect of these plasma components or if it is the result of other factors also associated with increases in ApoA-I and HDL levels. Because some strains of mice are susceptible to diet-induced formation of preatherosclerotic fatty streak lesions, and because of available techniques for the genetic manipulation of this organism, the murine system offers a unique setting in which to investigate the process of early atherogenesis. To test the hypothesis that induction of a high plasma concentration of ApoA-I and HDL would inhibit this process, we studied the effects of atherogenic diets on transgenic mice expressing high amounts of human ApoA-I. We report that transgenic mice with high plasma ApoA-I and HDL levels were significantly protected from the development of fatty streak lesions.

Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.

Plasma cholesteryl ester transfer protein

In Western societies high density lipoprotein (HDL) levels correlate inversely with the risk for coronary heart disease. The primary protein component of both human and mouse HDL is apolipoprotein A-I (apoAI), which comprises greater than 70% of HDL protein and 30% of HDL mass. Human HDLs include particles of several distinct size subpopulations, whereas HDLs from inbred C57BL/6 mice contain a single population of particles. To study the regulation of apoAI expression and its role in HDL assembly, we created transgenic C57BL/6 mice containing the human apoAI gene. Two independent lines of transgenic mice with approximately twice the normal plasma levels of total apoAI were studied. The level of mouse apoAI is reduced greater than 4-fold in both transgenic lines, comprising only 4% of total plasma apoAI levels in one transgenic line and 13% in the other. We demonstrate that the mechanism responsible for the decrease in mouse apoAI is posttranscriptional. Parallel to the replacement of mouse with human apoAI, the single HDL species normally present in the plasma of C57BL/6 is replaced by two HDL subclasses similar in size to human HDL2b and HDL3a. The changes in murine apolipoprotein levels and HDL subclass size are inherited by all transgenic offspring of the two founder animals. These results suggest a dominant role of apoAI in determining the HDL particle size distribution and a mechanism involving expression of human apoAI transgenes that alters the plasma levels of mouse apoAI.

https://www.pnas.org/content/pnas/88/2/434.full.pdf

Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses.

The inherited deficiency of purine-nucleoside phosphorylase (PNPase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) in humans is associated with a severe deficiency of the T lymphocytes of the immune system. Because of the unsatisfactory nature of previously described model systems, we have selected, cloned, and characterized a mutant mouse T cell lymphoma (S49) completely deficient in PNPase. Of the four substrates of PNPase, only deoxyguanosine at low concentrations is toxic to the PNPase-deficient (NSU-1) cells. 

In order to delineate the biochemical processes necessary for the sensitivity of the NSU-1 cells to deoxyguanosine, we have isolated a series of secondary mutants resistant to deoxyguanosine from the PNPase-deficient line. One of these mutants is defective in its ability to transport deoxyguanosine into the cell. A second type of mutant cannot phosphorylate the deoxyguanosine and is totally deficient in deoxycytidine kinase activity. A third type of mutant (NSU-1-dGuo-L) can both transport and phosphorylate deoxyguanosine and accumulates dGTP. However, unlike its parent, NSU-1-dGuo-L does not become depleted of dCTP and TTP when exposed to exogenous deoxyguanosine. This observation is accounted for by the fact that the reduction of CDP to dCDP by the ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2′-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2′-oxidoreductase, EC 1.17.4.1) of NSU-1-dGuo-L cells is not normally sensitive to feedback inhibition by dGTP. 

Thus, in order to exert its toxicity deoxyguanosine must be transported into the cell, be phosphorylated by deoxycytidine kinase, and be accumulated as dGTP. By inhibiting ribonucleotide reductase, dGTP depletes the cell of dCTP and to some extent TTP, thus preventing the synthesis of DNA, a process necessary for any proliferation-dependent function of T cells.

https://www.pnas.org/content/pnas/76/3/1074.full.pdf

Isolation and characterization of purine-nucleoside phosphorylase-deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: Genetic model for immunodeficiency disease

Evidence is presented that both X chromosomes are genetically active in clonal cultures of undifferentiated female mouse teratocarcinoma stem cells derived from a spontaneous ovarian tumour. As the cells differentiate in vitro one of the X chromosomes becomes inactivated.

X–chromosome inactivation during differentiation of female teratocarcinoma stem cells in vitro

Continuous delivery of factor VIII (FVIII) protein in hemophiliacs by gene therapy will represent a major clinical advance over the current practice of infrequent administration of purified FVIII. Conceptually, retroviral vectors that can permanently insert the FVIII gene into the DNA of the host cell appear the most suitable vehicles for this specific purpose. However, most retroviral vector systems have shown a poor performance in the production of FVIII from primary cells in vitro and in vivo. Here we report the retroviral-mediated gene delivery of a B-domain-deleted human FVIII by using the MFG vector system. This vector permitted efficient transduction of the majority of the primary cells in culture without the use of a selectable marker. High levels of FVIII were produced by various transduced primary cells in vitro. Upon transplantation of primary fibroblasts into mice, therapeutic levels of FVIII in the circulation were obtained for > 1 week. The capacity of primary cells to deliver the FVIII into the circulation was strongly dependent on the site of implantation. These results represent a major step forward in development of gene therapy for treating hemophilia A.

Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice

Alterations in deoxyribonucleotide metabolism in cultured cells with ribonucleotide reductase activities refractory to feedback inhibition by 2'-deoxyadenosine triphosphate.

S M Clift

Papers

Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses.

Isolation and characterization of purine-nucleoside phosphorylase-deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: Genetic model for immunodeficiency disease

X–chromosome inactivation during differentiation of female teratocarcinoma stem cells in vitro

Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice

Alterations in deoxyribonucleotide metabolism in cultured cells with ribonucleotide reductase activities refractory to feedback inhibition by 2'-deoxyadenosine triphosphate.