Maurice Panigel

Long-term dual perfusion of isolated human placental lobules with improved oxygenation for infectious diseases research

An improved method for long-term perfusion of the isolated human term placental lobule has been developed to investigate the maternofetal transfer of infectious agents, in particular the human immunodeficiency virus (HIV). The purpose of this paper is to describe those modifications that allow for substantially prolonged perfusions in in a biohazard environment. The method described has been adapted from previous models. The perfusion apparatus has been modified for use within a biohazard hood, and, intravenous bags contain the medium for circulation of perfusates in closed circuits. A Mera Silox-S 0.3 membrane oxygenator delivers more oxygen to the tissue, and, Electromedic Cardioplegia heat exchangers warm the perfusate prior to oxygenation. Viability criteria (glucose consumption, lactate production, de novo production of human placental lactogen (hPL), volume loss, flow, temperature, pressure, oxygen transfer, carbon dioxide production, absence of IgM transfer and light and electron microscopy) demonstrate that the placental tissue remains in a functional state throughout the perfusion. Oxygen and glucose consumption are both stable over time; lactate levels remain constant; and hPL continues to be produced. These significant modifications of the perfusion system have permitted the investigators to increase the duration of perfusion to 48 h while preserving normal metabolic function of ultrastructurally intact tissue as demonstrated by ultra structural observations. This perfusion model device provides biohazard precautions and may be applied to other studies of placental physiology.

In vitro infection of Hofbauer cells with a monocyte-tropic strain of HIV-1

Summary In order to further understand the mechanisms of HIV transmission from mother to child, the susceptibility of Hofbauer cells to the monocyte-tropic strain HIV-1Ba-L was tested in vitro. Hofbauer cells were obtained from normal full term placentae after perfusion of two lobules, following the procedure of Uren et al. (1985). Cells isolated from this procedure were cultured on T-25 flasks with RPMI 20% fetal bovine serum and antibiotics. Characterization of these cells following overnight adherence indicated that 60% of the cells were positive for the macrophage antibody HAM56. After five days 93% of the cells were phagocytic and 85% were nonspecific esterase positive. Immunohistochemical analysis with macrophage specific antibodies indicated that 78% were Hofbauer cells. After 5–6 days in culture, 1×107 cells were inoculated with the monocyte-tropic strain HIVBa-L. Virus production was monitored in culture supernatants at different intervals by a p24 assay. There was a ten or greater fold increase of p24 within 3 weeks of culture. Parallel studies on HIVBa-L infected umbilical cord and adult monocytes showed a similar increase. Infection with the lymphocyte-tropic strain HIV/LAV revealed that Hofbauer cells were more susceptible than adult and cord blood macrophages.

HIV-1 INFECTION OF HUMAN PLACENTAL VILLOUS TISSUE IN VITRO

Summary The role of the placenta in the materno-fetal transmission of HIV-1 infection remains unexplained despite continuing progress. Among the many different experimental models (animal, isolated cells, placental tissue and isolated placental perfusion), we have selected human placental villous tissue culture as the one closest to the in utero condition associated with HIV infectivity and allowing experimentation for an adequate period of time The aim of this study was to investigate infectivity of several HIV-1 strains in human placental explants. Two laboratory strains, Ba-L and IIIB, and VI-5 (isolated from an HIV-positive baby) were utilized. Ba-L and VI-5 were Non-Syncytium-Inducing (NSI), and IIIB was Syncytium-Inducing (SI). Placental explants (first trimester and full term) were incubated for 24 hours with free virus of each strain, then intensively rinsed to remove the inoculum. After an additional five days of culture, explants were examined for infection by detection of p24 in the culture medium and by PCR amplification of viral DNA using two pairs of gag gene primers. Ba-L and VI-5 (NSI) infected both first and third trimester placental tissue. Positive indications of viral infection included increasing p24 production during the third day post-inoculation, which remained sustained for three days; and positive DNA-PCR detection in explants on day 6. As a control for infectivity, p24 increases could be completely abolished using HIV-Ig. Strain IIIB (SI) did not infect placental tissue as determined by lack of p24 release and absence of viral DNA by PCR detection. Free HIV-1 could infect the intact human placental villus and, as observed in vivo , NSI HIV-1 strains appeared more successful in infecting in vitro human placental tissue of first and third trimesters.

Free vitamin B12 and transcobalamin II-vitamin B12 complex uptake by the visceral yolk sac of the sprague-dawley rat : Effect of inhibitors

Exogenous free vitamin B12 or B12 bound to human transcobalamin II (TCII) accumulated in the near-term rat visceral yolk sac. The rates of their uptakes in vitro and in vivo increased rapidly with time then reached a plateau, which supports a saturable transport/binding process as the rate-limiting step for the uptake of free and TCII complexed B12. Both uptakes were significantly decreased by trypan blue, colchicine, and low temperature but not by ouabain. Such inhibition suggests that the absorption of free and bound B12 is via an endocytosis process dependent upon energy but not the magnesium-dependent sodium/potassium-activated ATPase. Thus, the role of the visceral yolk sac in vitamin transfer to the conceptus and the alterations in yolk sac function associated with birth defects and diminished growth can be integrally related.