Search PubMed⌕ Search

Biomedical subjects

R F Levine

Publications and source records attributed to R F Levine.

35 records · Page 2Linked to original sources
PubMed1986

Adhesion, spreading and fragmentation of human megakaryocytes exposed to subendothelial extracellular matrix: a scanning electron microscopy study.

Platelet agonists and subendothelial extracellular matrix (ECM) induce morphological and biochemical changes in animal megakaryocytes, reminiscent of the response of platelets to the same substances. We have examined the behavior of human megakaryocytes exposed for up to 36 hours to the ECM produced by cultured bovine corneal endothelial cells. By phase contrast and scanning electron microscopy these megakaryocytes demonstrated non-reversible adherence and flattening with formation of long filopodia, thus confirming that human megakaryocytes acquire platelet functional capacities. In addition, megakaryocyte fragmentation into prospective platelets was apparently induced by the ECM. Up to 50% of the adherent megakaryocytes underwent spontaneous fragmentation into small particles which individually reacted like platelets on the ECM. The interaction of the megakaryocytes with the ECM was specific since no adherence, flattening or fragmentation occurred upon incubation of the megakaryocytes on regular tissue culture plastic or glutaraldehyde fixed ECM. Thus we have demonstrated platelet like behaviour of human megakaryocytes in response to this physiological basement membrane and a possible role of the subendothelium in platelet production which may occur in vivo as megakaryocytes cross the sinusoid walls and enter the blood stream.

Animals↗
PubMed1985

Megakaryocyte interaction with subendothelial extracellular matrix is associated with adhesion, platelet-like shape change, and thromboxane A2 production.

We have examined the morphological and secretory behavior of rat and guinea pig megakaryocytes exposed for up to 24 hours to extracellular matrix produced by cultured bovine endothelial cells. By phase-contrast microscopy of living cells and in more detail by scanning electron microscopy, the megakaryocytes showed a nonreversible adherence, an extensive formation of filopodia around the periphery like the rays of the sun, and a tendency toward flattening. These filopodia were generally linear with attenuated tips and were larger than, but resembled the filopodia of, rat or guinea pig platelets exposed to this extracellular matrix. In contrast, isolated megakaryocytes on glass or on uncoated plastic surfaces did not show these responses; adherence, in the face of gentle agitation before fixation, was minimal, with rare filopodia and no flattening. Megakaryocytes that interacted with the extracellular matrix produced significant amounts of thromboxane A2, but this did not occur on uncoated surfaces and could not be attributed to other contaminating cells in the megakaryocyte suspensions. The appearance in megakaryocytes of these typical platelet responses indicates that megakaryocytes acquire the functional capabilities of platelets by the synthesis and assembly of platelet substances and organelles. Thromboxane production by megakaryocytes stimulated by the extracellular matrix is a readily quantifiable measure of this capacity.

Animals↗
PubMed1984

Genetics of resistance to the African trypanosomes. III. Variant-specific antibody responses of H-2-compatible resistant and susceptible mice.

Genetically based differences in variant-specific immunity to the African trypanosomes were examined. H-2-compatible inbred mouse strains that differed in relative resistance were infected with Trypanosoma rhodesiense clone LouTat 1. Antibody responses to exposed epitopes of the LouTat 1 variant-specific surface glycoprotein (VSG) were measured. Relatively resistant B10.BR mice (H-2k) made predictable IgM antibody responses to the VSG of LouTat 1 which were associated with clearance of the LouTat 1 variant antigenic type from blood; IgG responses to LouTat 1 surface antigen appeared after clearance occurred, and were lower than peak titers of IgM. Intermediately susceptible CBA mice (H-2k) also made predictable IgM and IgG responses which followed the same pattern as the more resistant strain. Peak titers were lower for both Ig classes, however, and a delayed appearance of antibody was correlated with delayed clearance of LouTat 1. In contrast to B10.BR and CBA mice, the susceptible C3H mice (H-2k) failed to make detectable antibodies to LouTat 1 surface antigen and also failed to control the first peak of parasitemia. The absence of immunity in infected C3H mice was selective for antibody to exposed epitopes of LouTat 1 VSG because antibody was detectable to invariant VSG or internal trypanosome antigens. Also, the C3H strain was shown not to be a genetic nonresponder to LouTat 1 surface antigen because VSG-specific antibodies appeared within 1 wk after trypanocidal chemotherapy. Finally, we demonstrated that the susceptibility of C3H mice was not associated with an inability of the mononuclear phagocyte system to clear the parasites because drug cure, passive transfer of immune serum, or sensitization of trypanosomes with antibody all led to trypanosome clearance from blood by the liver. In summary, we show for the first time that major differences in variant-specific immunity occur in MHC-compatible animals after infection with the African trypanosomes.

Animals↗
PubMed1982

Cloning of African trypanosomes in mice immunosuppressed by cyclophosphamide treatment.

The use of cyclophosphamide-treated mice for purpose of cloning the African trypanosomes was assessed. C3HeB/FeJ mice were injected with 200 mg/kg cyclophosphamide (CY) 24-72 hours prior to infection with a single trypanosome isolated from Trypanosoma rhodesiense infected blood samples. All CY-treated mice exhibited depressed parasite-exposure, which was a period of time sufficient to grow trypanosomes from a single organism to a fulminating parasitemia. Cloning efficiency was routinely 45% in these animals. Thus, our study demonstrates that CT-treated mice are a convenient and efficient vehicle for cloning African trypanosomes. Techniques which facilitate the selection of single trypanosomes from infected blood are also described in this report.

Animals↗
PubMed1982

The significance of megakaryocyte size.

Normal guinea pig and human megakaryocytes in suspension were measured with an optical micrometer. The range of megakaryocyte diameters in both species was from 10 to about 65 micrometer. Approximately 20%-25% of megakaryocytes were smaLler than 20 micrometer in diameter and were mostly missed in past studies. However, virtually the entire population of megakaryocytes was larger than all but a very small percent of the other marrow cells. This size range and the existence of a visual threshold size between the megakaryocytes and nonmegakaryocytes were confirmed by flow cytometric analysis of fresh unfixed cells. On human bone marrow smears there was some flattening of all cell types, but the megakaryocytes were consistently at least minimally greater in size than almost all the nonmegakaryocytes. Normal marrow cells greater than 20 micrometer in diameter were always megakaryocytes. Cells 14-20 micrometer were still noticeably larger than the general marrow population; thus easily found, they could be examined for specific morphological criteria. Size, therefore, is a useful first criterion for the identification of megakaryocytes. The larger sizes of megakaryocytes were related to their greater DNA content per cell (polyploidy) compared to nonmegakaryocytes. The relationship between megakaryocyte size, ploidy, and maturation was examined by the simultaneous measurement for the first time of each of these parameters in the same cell. Maturation was quantitated by the new scheme based on the progressive changes in megakaryocytes nuclear configuration. Within each maturation stage the mean cell volume of guinea pig megakaryocytes doubled with each ploidy doubling. Within each ploidy group, the sizes of megakaryocytes increased with maturation stage. However, maturation and polyploidization appear to be linked; the data showed that 80% of the low ploidy (4N-8N) megakaryocytes were immature and that 95% of the platelet-shedding megakaryocytes were 16N-32N.

Animals↗
PubMed1981

Genetics of resistance to African trypanosomes: role of the H-2 locus in determining resistance to infection with Trypanosoma rhodesiense.

Susceptibility and resistance to Trypanosoma rhodesiense infections in inbred and H-2 congenic strains of mice were studied. Mean survival times and patterns of parasitemia were examined. C3HeB/FeJ mice were highly susceptible; CBA/J, A/J, and BALB/cByJ mice displayed an intermediate level of susceptibility; whereas C57BL/10 mice were highly resistant. H-2 congenic strains with the BL/10 background resembled the BL/10 parental type, thereby suggesting that the major histocompatibility complex does not play a major role in regulating resistance and susceptibility to infection with T. rhodesiense.

Animals↗
PubMed1981

The prickly lettuce agglutinin. II. Immunobiological properties.

The prickly lettuce agglutinin (PLA), extracted from leaves of the plant Lactuca scariole, was tested for immunomodulatory effects on C57BL/6 mice. When administered with antigen in vivo, 50 micrograms PLA caused an enhancement of the primary splenic plaque-forming cell (PFC) response to the helper T cell-dependent antigens sheep red blood cells and trinitrophenyl-keyhole limpet hemocyanin; higher doses of PLA (200 micrograms) resulted in depressed responses to these antigens. Similarly, 50 micrograms PLA enhanced the B cell response to SIII, a helper T cell-independent antigen, but higher doses did not cause significant depression of the SIII-specific PFC response. When PLA (less than 25 micrograms) was coincubated with spleen cells and antigen, sheep red blood cells or SIII, in vitro the primary PFC response was enhanced; higher doses of PLA (greater than or equal to 25 micrograms) depressed cell culture viability and also depressed the resultant PFC responses. In the in vitro studies, stimulatory doses of PLA were effective only when added to cultures before or with antigen. In tests for mitogenic effects of PLA on spleen cells, PLA was found to be nonstimulatory. On the basis of these observations, we suggest that PLA may modulate murine splenic primary B cell responses by providing a stimulatory signal to B cells. The nature of the suppressive effect in vivo is unknown but may be attributable to antigenic competition.

Animals↗
PubMed1980

Isolation and characterization of normal human megakaryocytes.

Human megakaryocytes have been isolated from marrow obtained from ribs removed at thoracotomy. All but one of the patients had normal pre-operative platelet and leucocyte counts. Megakaryocytes averaged 0.37% of all cells in marrow cell suspensions from nine consecutive subjects. A 283-fold purification (to 10.3%) was achieved by a density gradient centrifugation followed by two successive velocity sedimentations at unit gravity. The net yield, 12 800 megakaryocytes per specimen, was sufficient for many kinds of morphological study. Bright-field, phase contrast, and electron microscopy were used to characterize the younger and smaller megakaryocytes. Ploidy analyses were carried out on 100--235 megakaryocytes per specimen; 8N was the predominant ploidy class in isolated megakaryocyte populations from three individuals. The mean megakaryocyte diameter was 24 micrometers in three other specimens and the range was 10--48 micrometers. This data had a normal distribution and overlapped minimally with the size range of all other marrow cells. The presence of a distinct size threshold (at 11.5 micrometers) implied that size alone may be a sufficient objective criterion for identification of human megakaryocytes.

Adult↗
PubMed1980

Origin of platelet-derived growth factor in megakaryocytes in guinea pigs.

Growth factor activity, as determined by the stimulation of [3H]thymidine incorporation into the DNA of quiescent 3T3 cells in culture, was found in lysates of guinea pig platelets and megakaryocytes. Quantitative dilution studies demonstrated that, of the cells present in the guinea pig bone marrow, only the megakaryocyte possessed quantitatively significant growth factor activity. The amount of activity present in one megakaryocyte was equivalent to that present in 1,000-5,000 platelets, a value approximately comparable to the number of platelets shed from a single megakaryocyte. It is suggested that guinea pig platelet-derived growth factor has its origin in the megakaryocyte.

Animals↗
PubMed1977

Culture in vitro of isolated guinea pig megakaryocytes: recovery, survival, morphologic changes, and maturation.

Isolated guinea pig megakaryocytes were maintained in liquid cultures for up to 4 days. Megakaryocytes were incubated in siliconized glass vials in Dulbecco's Modified Eagle Medium with 5%-10% guinea pig serum and 2.3% bovine serum albumin. Cultured megakaryocytes did not adhere to glass vials and were almost entirely recovered by aspiration. No reproduction or cell division of megakaryocytes occurred. A small decline in viability occurred promptly on placing the freshly isolated cells in culture medium and could be attributed to reexposure to calcium. On incubation there was little further cell death. Up to 2 days in culture the megakaryocytes remained morphologically intact and appeared similar to megakaryocytes in situ. Megakaryocytes matured in culture with a loss of cytoplasmic basophilia, an increase in granule content, and progressive changes in nuclear configuration. The most mature megakaryocytes developed pseudopod formation but large-scale platelet liberation was not seen. The ability to culture megakaryocytes in vitro will allow more extensive biochemical and physiologic studies of this cell than previously possible.

Animals↗
PubMed1976

Isolation of intact megakaryocytes from guinea pig femoral marrow. Successful harvest made possible with inhibitions of platelet aggregation; enrichment achieved with a two-step separation technique.

Methods have been devised to harvest megakaryocytes from guinea pig femoral marrow and to isolate them in high yield. When marrow tissue was disaggregated the megakaryocytes underwent degenerative changes characterized by the loss of cytoplasmic granules and alterations in membrane topography, similar to the changes seen in aggregating platelets. These morphologic changes were interpreted to mean that megakaryocytes possessed functional attributes of platelets. The use of agents which inhibit platelt aggregation (0.38% sodium citrate. 10(-3) M adenosine, and 2 x 10(-3) M theophylline) in a medium free of bivalent cations prevented these changes. This solution resulted in both an excellent morphologic preservation and a significantly increased recovery of megakaryocytes from marrow tissue. A two-step purification of the intact megakaryocytes was carried out on the basis of their low density and large size, with equilibrium density gradient centrifugation followed by velocity sedimentation. This sequence gave approximately a 100-fold enrichment of megakaryocytes, significantly better than that achieved with either method alone. These techniques for harvesting and concentrating megakaryocytes make it possible for the first time to study megakaryocytes in vitro.

Adenosine↗
PubMed1976

Tannic acid effect on membrane of cell surface origin in guinea pig megakaryocytes and platelets.

Plasma membranes in isolated guinea pig megakaryocytes and washed platelets are poorly stained with the usual methods used to outline cell membranes. The addition of tannic acid and calcium to the initial fixative is useful to enhance electron density of all surface-derived membrane systems in these cells. The method described here shows that the increased electron denisty of membrane after fixation in the presence of tannic acid occurs both at the cell surface and along the invaginated membrane systems.

Animals↗