Uptake of human immunodeficiency virus envelope protein gp120 by human trophoblast in culture.

OBJECTIVE: Our purpose was to determine whether human trophoblast has a cell surface CD4 antigen that will bind to gp120, the envelope protein of human immunodeficiency virus. STUDY DESIGN: Uptake of iodine 125-labeled gp120 by trophoblast in culture was measured. Particular attention was paid to technical details that may have caused the contradictory results reported by previous investigators: the source of the recombinant gp120, the method of radioiodination, and the isolation procedure of trophoblast to ensure elimination of contaminating cells, particularly macrophages. RESULTS: Uptake of transferrin-free iodine 125-labeled gp120 to trophoblast was unaffected by adding a 200 molar excess of gp120, by preincubating gp120 with soluble CD4 to block the CD4 binding sites on gp120 and by preincubation of trophoblast with a blocking antibody to CD4 (OKT4a). In contrast, uptake of gp120 by CD4-positive H9 human lymphocytes was reduced 79% by a 200 molar excess of gp120 and > 50% by a CD4-blocking antibody. CONCLUSIONS: Uptake of gp120 to trophoblast is by a high capacity, CD4-independent mechanism that is probably nonspecific and may be related to the mechanism for binding other circulating glycoproteins in maternal blood.

The effect of zidovudine and 2'3'-dideoxyinosine on human trophoblast in culture.

Trophoblast from term and first trimester placenta, maintained in culture, were exposed to 20 mumoles/l zidovudine or 2'3'dideoxyinosine. Several indices of function were measured and compared to control trophoblast in parallel culture. The results from individual placentas were examined by Student's t-test and cumulative results by ANOVA. Neither zidovudine or 2'3'-dideoxyinosine had statistically significant effects on the function of term trophoblast, following a 48 hr exposure to the drug as indicated by hCG secretion, protein synthesis and glucose consumption. In one of five placentas exposed to zidovudine, progesterone secretion was reduced as compared to its control but remained in the high range. Zidovudine had no significant effect on cultured trophoblast isolated from first trimester placenta even after prolonged exposure to the drug for eleven days. Both term and first trimester trophoblast in culture tolerate prolonged exposure to high concentrations of zidovudine or 2'3'-dideoxyinosine. Human trophoblast in culture provides a safe in vitro model for the screening of drugs intended for use during pregnancy.

Synthesis of alpha 1-antichymotrypsin and alpha 1-antitrypsin by human trophoblast.

alpha 1-Antichymotrypsin (alpha 1-ACHY) and alpha 1-antitrypsin (alpha 1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of alpha 1-AT and alpha 1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize alpha 1-AT and alpha 1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. alpha 1-ACHY and alpha 1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of alpha 1-AT and alpha 1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled alpha 1-AT and alpha 1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35S]-methionine. It is of some interest that the M(r) of the alpha 1-AT and alpha 1-ACHY secreted by trophoblast were 50,000 and 49,000, respectively, compared with 54,000 and 68,000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and secretion of apolipoprotein E by human placenta and choriocarcinoma cell lines.

The synthesis and secretion of apolipoproteins (apos) by cells from a human choriocarcinoma cell line, JAR, were examined by [35S]-methionine labeling followed by immunoprecipitation and SDS/PAGE. Apo E, but not apos A-I, A-IV, or B, was synthesized and secreted. Apo E was also synthesized by fragments of chorionic villi from human placenta and by another choriocarcinoma line, BeWo. Pulse-chase experiments with JAR cells revealed that apoE was initially synthesized as a 33 kDa protein followed by a 34 KDa protein, probably the result of glycosylation. The latter was secreted into the medium where it was detected coincident with a 21/22 kDa doublet, possibly proteolytic fragments of apo E. Approximately 50 per cent of the apo E in the medium was complexed with lipid as indicated by ultracentrifugation at a density of 1.21 g/ml. The amount of apo E produced by JAR was not affected by preincubation with dibutyryl cAMP and theophylline, or by the cholesterol content of the cells. Following perfusion of an isolated lobule of human placenta with [14C]-amino acids, [14C]-apo E was detected by immunoprecipitation of the maternal and fetal perfusates with 88 per cent in the maternal perfusate. These studies suggest that apo E, which promotes receptor-mediated lipoprotein uptake, is secreted by the trophoblast to facilitate uptake of maternal lipoproteins.

Generation and chracterization of variants of mouse hepatoma cells with defects in hepato-specific gene expression. I. Albumin synthesis variants.

Clonal variants of mouse hepatoma cells that either fail to produce albumin (variant 19/2) or show significantly reduced levels (100-fold less) of albumin production (variant 1/c/1) were isolated from the parental line. Hepa la, after a single exposure to N-methyl-N'-nitrosoguanidine (MNNG). Intracellular levels of albumin in both variants were below detection by our assay. Analyses by cDNA-RNA reassociation kinetics indicate that there are approximately 3900 molecules of cytoplasmic albumin mRNA per cell in the parent and less than 10 molecules per cell in both variants. Southern blotting of the Eco RI restriction fragments of cellular DNA from the parent and variants did not indicate any major deletions in the albumin gene DNA sequences. We conclude that in the two variants studied, processes that regulate albumin production via alterations in the level of cytoplasmic albumin mRNA have been affected. Our analyses have also shown that alpha-fetoprotein (AFP) production is lacking in one variant (19/2) and is slightly reduced in the other (1/c/1). Transferrin secretion is lower than the parental line in both variants. Thus multiple nonlethal defects in hepatic gene expression can be obtained in Hepa la cells in culture that will be useful in determining the number and kinds of genes that control the expression of liver-specific loci.