In Vitro Analysis of Defibrotide’s (DF) Uptake by the Human Cell, as Well as DF’s Spesificity for the Viral Envelope Proteins of HIV-! HIV-3, and of Spesific HIV, with Conformatory Data Regarding the Dose Dependency of DF’s Pharmacological Paleiotropism.

To assess DF’s uptake by the human cell, DF was labeled with a photo-activable analogue of biotin. Cellular uptake started with cell surface receptors to be ultimately localized in the human nucleus.This was confirmed with image analysis utilizing a cold CCD camera.The biologic activity after labelling was considered as being preserved based on prior data with other labeled oligonucleotides.Fig. not shown here clearly displays that nuclear uptake has been in all cases directly proportional to the concentration of DF with biotin The cellular uptake without biotin but with cyanine dye Cy5.18 confirmed the significant enhancement of the uptake with biotin-especially by lymphocytes.To further confirm and explore the above commented dose dependency of the cellular uptake of DF with DF’s therapeutic efficacy against HIV, serial experiments were run, PBMNC were prepared in the known manner, The white cells were then concentrated to a level of 2 million cells per 3 ml RPMI 1640 solution. Two subpopulations of cells were created: Con A stimulated versus unstimulated cell lines. The stimulated cell line showed more enhanced uptake of antibody dye, thereby demonstrating increased celllular expression of viral proteins -even in the face nonspesific immune stimulation (ConA). Subpopulations of unstimulated and stimulated cells were then incubated in the presence of discrete concentrations of DF, each successive assay employing successively higher concentrations of DF. Appropriate controls were run. Cell subpopulations were further stratified into 2 groups of intracellular versus extracellular antibody staining. The former group of cells were fixed with 70% ETOH, washed x2, and suspended in a solution containing 200 ml Hank’s solution, with 2 % FCS (Fetal Calf Serum),0.1% sodium azide, and 5 microliters of Alpha-HIV-Cy5.18. and then incubated for 45 min,to be resuspended finally in 1%paraformaldehyde.Cells reserved for surface staining were washed x2 with monoclonal wash with added 5 ml Alpha-HIV-Cy5.18, as well as 20 microliters surface glycoprotein monoclonal antibody solution of CD3-FITC and CD4-RPE. All prepared cells were analized using a Becton-Dickinson FACS 440 dual laser (argon/krypton)flowcytometry.The expression of HIV proteins was determined on a per cell basis. Flouresence was measured on a logorythmic scale but converted to linear scale for analysis. Mean Linear Flourescence Intensity was accepted to be proportional to the HIV protein expression.. Whether protein expression correlates with cell lysis and/or viability of the virus cannot be determined here.. However decreased expression of viral proteins with increasing concentration of DFand presence of ConA. was verifiable in each case. At .10 mg, 20mg, 30mg DF, respectively.protein expression decreased logorythmically. At 30 mg concentration of DF, however, in both ConA stimulated or unstimulated cell lines, protein expression leveledoff.-further escalation of doses not being pharmacologically effective.3 HIV patients from Phase II will be presented under separate cover. A self -regulating dose range between 50mg/kg----275 mg/kg/d)was also reproducably seen in human patients. And in all instances the spectrum of DF’s pharmacological paleiotropism qualitatively and quantitatively with escalation of doses.

Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule. A new model for parietal cell transformation

The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic ‘tubulovesicles’ as the apical surface of the cell progressively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesicles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimulated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with alpha- and beta-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progressive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; consequently, they cannot have a role in parietal cell transformation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is compatible with the localization of cytoskeletal components in these cells.

Relationship between cytosolic free Ca2+ and Na+-Ca2+ exchange in aortic muscle cells

We examined the relationship between extracellular Na+ ([Na+]o) and cytosolic free Ca2+ ([Ca2+]i) in primary and passaged cultures of aortic muscle cells. Removing [Na+]o increased [Ca2+]i by approximately 10-fold in cells that were Na+ loaded. Decreasing [Na+]o from 140 to 32 mM caused the half-maximal increase in [Ca2+]i. [Ca2+]i exhibited a sigmoidal dependence on [Na+]o. Mg2+, a competitive inhibitor of Na2+-Ca2+ antiport in these cells, antagonized the increase in [Ca2+]i produced by lowering [Na+]o. High K+ decreased the potency of Mg2+ 6.5-fold as previously reported for Na+ gradient-dependent 45Ca2+ influx. In contrast to the Na+-loaded cells, removing [Na+]o caused no detectable change in [Ca2+]i in cells with normal Na+ even though the calculated electrochemical driving force for Na+-Ca2+ exchange was large enough to almost maximally increase [Ca2+]i in the Na+-loaded cells. We conclude that Na+-Ca2+ antiport activity is latent in the unstimulated cell at basal intracellular Na+ and Ca2+.