Synergistic cytotoxic effect of azidothymidine and recombinant interferon alpha on normal human bone marrow progenitor cells

Azidothymidine (AZT) and interferon alpha (IFN-alpha) are among the drugs showing strong in vitro activity against the human immunodeficiency virus type-1 (HIV-1). Each drug, however, has significant toxicity against normal marrow progenitor cells that frequently proves dose-limiting in patients. In this study, AZT and recombinant IFN-alpha 2a (rIFN-alpha 2a) were tested as single agents and in combination against normal myeloid (CFU-GM) and erythroid (BFU- E) colony forming cells in a standard methylcellulose culture assay. The data were analyzed using a quantitative computerized analysis based on the median-effect principle and the isobologram equation as described by Chou and Talalay (Adv Enz Regul 22:27, 1984). The ED90 for BFU-E and CFU-GM inhibition was then compared with previously measured in vivo plasma levels of each drug and the ED90 for the anti-HIV-1 effect in vitro. We demonstrate that (a) the drugs are strongly synergistic in inhibiting marrow progenitor cell growth and that this synergism occurs at drug levels that are within the range of measured plasma levels in phase I clinical trials, (b) BFU-E are more sensitive than CFU-GM to the inhibiting effects of AZT, rIFN-alpha 2a or both drugs in combination, (c) the drug concentrations in combination that synergistically inhibit bone marrow progenitors are much higher than those required to inhibit HIV-1 replication in vitro, and (d) the anti- HIV-1 effect for the combination of AZT and rIFN-alpha 2a was clearly superior to the effect of AZT or rIFN-alpha 2a alone as indicated by the combination index and the dose-reduction index. These data suggest that substantially lower doses of AZT and rIFN-alpha than those currently being tested in clinical trials might not only maintain a strong synergistic anti-HIV-1 effect but might also avoid significant hematologic toxicity.     

Differential effect of Serratia protease on platelet surface glycoproteins Ib and V

The effect of a zinc metalloprotease from Serratia marcescens on platelet surface glycoproteins (GP) Ib and V was analyzed. Increasing protease treatments caused progressive loss of GP Ib with appearance of the major fragment, glycocalicin, in the supernatant solution. No GP V was detected in the supernatant solution, and protease-pretreated platelets had the same capacity as control platelets to release fragment 1 of GP V in response to thrombin. The Serratia protease- pretreated platelets did show the lag before thrombin-induced dense granule secretion, characteristic of platelets modified by pretreatment with other nonstimulating proteases. Treatment with Serratia protease gives the only demonstrated selective loss of GP Ib without apparent effect on GP V. It suggests that GP V (1) does not depend on GP Ib for its association with platelets and (2) is not the substrate for protease modification of platelet function.     

Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.     

Primary aldosteronism: diagnosis, localization, and treatment.

New diagnostic techniques have enhanced the detection of primary aldosteronism. However, the response of blood pressure after operation in unilateral and bilateral adrenal disease is different. We have compared four localizing techniques--adrenal venography, adrenal isotopic scanning, a modified adrenal venous sampling for steroid measurements, and the anomalous postural decrease in plasma aldosterone concentration--in 51 patients with primary aldosteronism, all of whom had undergone operative confirmation. Adrenalectomy resulted in normal blood pressure in 59%, improvement in 25%, and no change in 16%. Correct localization of the lesion was obtained in 47% by the adrenal isotopic scan, in 66% by adrenal venography, and in 91% by the modified adrenal venous hormone technique despite four false-positives. Of the 26 patients with an anomalous postural decrease in plasma aldosterone, 88% had a unilateral lesion.     

Viability of M. leprae under normal and adverse atmospheric conditions

Harvested bacilli from fresh lepromas were subjected to drying at room temperature for varying periods, were exposed to direct sun-rays, UVR, and were then injected into the foot pads of thymectomised irradiated mice for checking the viability. The organisms could survive UVR for 30 minutes, direct sunlight for 2 hours and room temperature for 7 days. Details are presented.     

Lymphohematopoietic progenitors of normal mice

We have identified and characterized the lymphohematopoietic progenitors in the bone marrow of normal mice using a single-step methylcellulose culture assay. Lineage-negative Ly-6A/E (Sca-1)+ progenitors isolated from normal mice were plated in methylcellulose culture containing steel factor (SF), interleukin-7 (IL-7), erythropoietin (Ep), and IL-11. After 16 to 17 days of culture, pre-B- cell-containing multilineage myeloid colonies can be microscopically identified; however, flow-cytometric analysis of individual colonies for B220-positive cells proved superior to in situ microscopic identification of lymphomyeloid colonies. Approximately 10% (6/66) of the mixed colonies without a conspicuous B-cell component had B220- positive cells. The single cell origin of the lymphomyeloid colonies was confirmed by micromanipulation. Although the combination of SF, IL- 7, and Ep was sufficient to support formation of lymphomyeloid colonies, addition of IL-11, granulocyte colony-stimulating factor or IL-12 to the combination of SF, IL-7, and Ep increased the number of lymphomyeloid colonies. IL-1 alpha and IL-3 independently inhibited the expression of the B-lymphoid lineage when added to the combination of SF, IL-7, Ep, and IL-11. Approximately four times more lymphohematopoietic progenitors are present in normal mice than in mice treated with 5-fluorouracil.     

Clonidine suppression test--an evaluation of its diagnostic significance in hypertensive patients

The Clonidine Suppression Test (CST) was performed in 8 patients with Labile hypertension (Group I), 8 patients with mild and moderate Essential hypertension (Group IIa), 8 patients with severe Essential hypertension (Group IIb) and 6 patients with pheochromocytoma (Group III). The mean plasma catecholamine (CA) levels as estimated by a Spectrofluorimetric method were significantly reduced 3-4 hours after administration of clonidine (5 micrograms/kg) by mouth in Group I, IIa & IIb patients. Plasma norepinephrine levels fell from 1.82 +/- SEM 0.35 ng/ml to 1.03 +/- 0.11 ng/ml (p less than 0.05) in Group I, 1.64 +/- 0.36 ng/ml to 0.88 +/- 0.12 ng/ml (p less than 0.025) in Group IIa, 1.23 +/- 0.16 ng/ml to 0.86 +/- 0.12 ng/ml (p less than 0.005) in Group IIb patients. Plasma epinephrine levels fell from 0.35 +/- 0.06 ng/ml to 0.16 +/- 0.03 ng/ml (p less than 0.05) in Group I, 0.34 +/- 0.04 ng/ml to 0.22 +/- 0.03 ng/ml (p less than 0.01) in Group IIa, 0.33 +/- 0.06 ng/ml to 0.18 +/- 0.03 ng/ml (p less than 0.025) in Group IIb patients. The blood pressure and heart rate showed a similar response. By contrast, in patients with pheochromocytoma, the mean plasma CA levels did not show any significant fall, and even rose during the CST, but, when repeated post-operatively, showed normal suppression. No serious side effects were noticed. We conclude that the CST is a safe and reliable test for the diagnosis of pheochromocytoma.(ABSTRACT TRUNCATED AT 250 WORDS)