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Biomedical subjects

P L Carlson

Publications and source records attributed to P L Carlson.

8 recordsLinked to original sources
PubMed1996

Effects of recombinant human thrombopoietin on megakaryocyte colony formation and megakaryocyte ploidy by human CD34+ cells in a serum-free system.

In serum-free cultures of human CD34 cells, recombinant human thrombopoietin (TPO) induced megakaryocyte colony formation a dose-dependent fashion that was further enhanced by the presence of interleukin-3 (IL-3) and stem cell factor (SCF), but not by IL-6, IL-11 or erythropoietin. TPO gave rise to much smaller colonies and at an earlier time than IL_3, indicating that TPO affects predominantly more mature megakaryocytic progenitors. In liquid cultures. TPO increased the percentage and the absolute number of > or = 8N megakaryocytes, but it did not shift their modal ploidy from 2N. TPO-induced endomitosis was totally inhibited by the presence of or previous exposure of cells to, IL-3 and /or SCF. The mechanism by which TPO overcomes in vivo the negative effects of IL-3 and SCF on megakaryocyte ploidy remains unknown.

Adult↗
PubMed1996

B lymphocytic clonal expansion in rheumatoid arthritis.

OBJECTIVE: To learn whether rheumatoid factor (RF), HLA-DR4, or current therapy influences clonal expansion of B lymphocytes (B cells) in persons with rheumatoid arthritis (RA). METHODS: We measured clonal expansion by analysis of cell surface staining for immunoglobulin light chains. Double staining methods detected a B cell marker (CD19) plus either kappa or lambda on peripheral blood lymphocytes from subjects with RA (n = 26) and controls (n = 26). The difference between frequency histograms of surface kappa and lambda staining was determined by the Kolmogorov-Smirnov statistic D that represents the fraction of clonally expanded B cells. RESULTS: The mean D value in RA was over 50% higher than in the controls [0.225 +/- SD 0.155 versus 0.144 +/- 0.025 (p < 0.0001)]. Ten subjects with RA had values exceeding +2 SD for controls (p = 0.0007). Mean D correlated with RF titer (Spearman's rank correlation coefficient rSp + 0.53, p = 0.006). All 10 high D values were found in the RA subgroups with positive serum tests for RF and with the HLA-DR4 positive genotype. The channel of maximal difference between kappa and lambda staining was higher in the RA group than in controls, showing that clonal expansion was most marked among brightly staining cells. Patients with RA currently receiving low dose methotrexate (MTX) tended to have higher D values than those not receiving MTX (mean 0.29 versus 0.18, respectively, p < 0.025). The RA group currently receiving MTX had a higher frequency of abnormal D values (7 of 11 versus 3 of 15 not currently receiving MTX, p = 0.03). This probably reflects preferential use of MTX for severely affected individuals. Confirmatory studies to detect clonal immunoglobulin gene rearrangements were attempted in selected individuals with high D values, but none was demonstrated in total leukocytic or B cell enriched fractions. CONCLUSION: Findings consistent with B cell clonal expansion occur in about 40% of persons with RA, particularly in the subgroups with positive serum tests for RF and with the HLA-DR4 genotype. However, the clonal expansion level must be below the sensitivity of confirmatory methods.

Adult↗
PubMed1994

Replication and endoreplication in developing megakaryocytes in vitro.

To study the relation in time between replication and endoreplication and the relation between appearance of platelet-specific proteins and endoreplication in maturing megakaryocytes, peripheral blood mononuclear cells highly enriched in hematopoietic progenitors were cultured in liquid cultures and plasma clots in the presence of either interleukin-3 (IL-3) and stem cell factor (SCF) or medium conditioned by blood mononuclear cells stimulated by phytohemagglutinin (PHA). In plasma clots, megakaryocytic (MK) colonies appeared first on day 5 and reached a maximum by day 8, whereas the number of cells per colony increased until day 10, indicating that there was a single wave of MK colony formation. In liquid cultures, the first immunologically recognizable megakaryocytes appeared on day 5 and expressed GPIIb/IIIa and thrombospondin only, but all other platelet-specific protein markers appeared within 24 hours. Replating cells from liquid medium into plasma clots showed that 92 +/- 8% of day 6 GPIIb/IIIa-positive cells are capable of replicating. Their replicative potential decreased with age, however, so that between days 6 and 11, a linear correlation was noted between the logarithm of the percentage of megakaryocytes with replicative capacity and their age in culture. Replication ceased completely after day 10. In the presence of IL-3, polyploid megakaryocytes appeared at the same time that GPIIb/IIIa was expressed, and the megakaryocyte distribution into ploidy classes remained unchanged until day 20. In the presence of PHA-leukocyte conditioned medium (PHA-LCM), ploidy of megakaryocytes was shifted toward higher classes after day 6, and the process of endoreplication was completed by day 10. No changes in ploidy distribution were noted between days 10 and 20. These findings indicate that in the cohort of megakaryocytes derived from colony-forming units-megakaryocyte (CFU-MK), endoreplication can occur at an early stage of development, proceeds synchronously with replication, and is completed before the megakaryocytes exhaust their replicative potential.

Adult↗
PubMed1991

Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.

The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.

Agglutination↗
PubMed1990

Effects of gold sodium thiomalate and tenidap sodium (CP-66,248-2) on a model of macrophage differentiation using HL-60 cells.

We studied the effects of gold sodium thiomalate (GST) and a new antirheumatic drug, tenidap sodium ([Z]-5-chloro-2,3-dihydro-3-[hydroxy-2-thienylmethylene]-2-oxo-1H- indole-1-carboxamide, sodium salt), previously known as CP-66,248-2, in a model system of macrophage differentiation using a myelomonocytic cell line. HL-60 cells can be stimulated by vitamin D3 to differentiate along a monocytic pathway. Monocytic HL-60 cells express CD14 (Leu-M3), a macrophage surface marker, and develop the capacity to produce the second complement component (C2) in response to stimulation with cytokines such as gamma-interferon. The effects of GST and tenidap sodium were compared with the effects of dexamethasone and a variety of nonsteroidal antiinflammatory drugs in this model system. We found that GST inhibited the capacity of HL-60 cells to produce C2 but did not inhibit the expression of CD14. Tenidap sodium inhibited C2 production as well as CD14 expression, and it partially reversed the decrease in 3H-thymidine incorporation by HL-60 cells, which accompanies monocytic differentiation. At concentrations that inhibited C2 production by HL-60 cells, tenidap sodium did not inhibit C2 production by monocytes. Neither dexamethasone nor the other nonsteroidal antiinflammatory drugs tested possessed these activities. Thus, both GST and tenidap inhibit markers of monocytic differentiation in HL-60 cells, and this activity may relate to their antirheumatic activities.

Cell Differentiation↗
PubMed1989

Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma.

IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.

Antibodies↗
PubMed1987

Effects of gold sodium thiomalate on interferon stimulation of C2 synthesis and HLA-DR expression by human monocytes.

Gamma-interferon (gamma-IFN) is a T cell-derived lymphokine that has potent macrophage-activating properties. It increases Fc receptor density, increases the formation and release of reactive oxygen intermediates, increases the synthesis and release of complement cascade proteins, especially C2 and factor B, and increases class II (HLA-DR) antigen expression. These effects may play a role in the potentiation of inflammation in rheumatoid arthritis. We examined the possibility that gold sodium thiomalate (GST), an effective treatment for rheumatoid arthritis, would inhibit gamma-IFN-mediated stimulation of monocyte/macrophages. GST in concentrations attainable in vivo was shown to inhibit both spontaneous and gamma-IFN-stimulated C2 production up to 50%. GST inhibition could be only partially overcome with increasing concentrations of gamma-IFN. In addition, GST inhibited gamma-IFN-stimulated HLA-DR expression at the highest concentrations tested (20-50 micrograms/ml). GST alone in low concentrations (0.1-5 micrograms/ml) was found to increase HLA-DR antigen expression as quantitated by several methods, including flow cytometry, cell surface enzyme-linked immunosorbent assay, and Western blotting. This GST-stimulated increase in HLA-DR antigen expression paralleled an increased ability of monocytes to present antigen. The mechanism by which low concentrations of GST stimulate HLA-DR antigen expression is unclear, but was shown by 35S-methionine cell labeling not to involve increased HLA-DR protein synthesis.

Cell Separation↗