Stimulated response of peripheral lymphocytes may distinguish cyclosporine effect in renal transplant recipients receiving a cyclosporine+rapamycin regimen.

BACKGROUND: Clinically, cyclosporine (CSA, Neoral) is titrated to concentrations, and not to pharmacological effect. METHODS: Intracellular interleukin- (IL) 2 was measured in phorbol myristic acid-ionomycin-stimulated peripheral lymphocytes by flow cytometry, after isolation from 14 renal transplant recipients receiving CSA+prednisone, and double-blind rapamycin (rapamycin:placebo=4:1). RESULTS: The proportion (%) of CD4+IL-2+ lymphocytes corresponding to CSA levels (mean+/-SD ng/ml) measured preoperatively (TO=O), and on postoperative day 8, before (356+/-63), and 2 hr after the morning dose (Cmax=1567+/-669), decreased from 39+/-16 to 15+/-8 and 3+/-1.6, respectively. Reciprocally, unresponsive lymphocytes (%CD4+IL-2-) increased with increasing CSA levels and predicted an EC50 of 249 ng/ml (CSA concentration at which CD4+IL-2- cells increased by 50% over baseline) in an Emax pharmacodynamic model. CONCLUSIONS: Clinically, the pharmacological effect of CSA is quantifiable, and lies in the upper end of the predicted range. In our Neoral-treated sample population, Cmax was associated with the least variable "cyclosporine effect." Such information could potentially individualize immunosuppression, and lead to rational dosing strategies.

Specificity of anti-lymphocyte antibodies in sera from patients with AIDS-related complex (ARC) and healthy homosexuals.

The presence and specificity of anti-lymphocyte antibodies (ALA) was investigated in sera from male homosexuals with AIDS-Related Complex (ARC) as well as healthy homosexuals. Individuals in the healthy homosexual group had no detectable antibodies to human immunodeficiency virus (HIV). Antibodies reactive with normal peripheral blood mononuclear cells were detected by Western blot analysis in sera from both groups of homosexuals. Of those individuals whose sera contained ALA, 71% of ARC patients and 83% of healthy homosexuals had antibodies recognizing a 73 kilodalton (kD) molecule. ALA present in ARC sera reacted with CD3+, CD4+ and CD8+ lymphocytes while little reactivity with B cells was observed. Our results indicate that ALA appear in homosexuals prior to HIV infection and are reactive primarily with T lymphocytes. A 73 kD structure associated with the T cell membrane is frequently the target for these antibodies.

Monoclonal antibodies to human osteosarcoma-associated antigen(s).

Monoclonal antibodies (MoAbs) against human osteosarcoma cells were obtained by the production and cloning of hybrids resulting from the fusion of mouse myeloma cells P3 X 63Ag8.653 with spleen cells from partially purified, osteosarcoma-associated antigen (OSAA)-immunized BALB/c mice. OSAAs were isolated from the spent culture medium of a human osteosarcoma cell line (TE-85). Five hybrid clones were established and designated as OSA1, OSA2, OSA3, OSA4, and OSA5. OSA1 and OSA2 had similar activity. All 5 MoAbs reacted strongly with most osteosarcoma cell lines and with all osteosarcoma tissues tested but not with 10 tumor cell lines and 2 tumor tissues from other cancers. OSA3, OSA4, and OSA5 cross-reacted with a fibrosarcoma cell line, a colon cell line, and fibrosarcoma, respectively, as well as with a melanoma cell line. None of the MoAbs were reactive with activated normal human peripheral blood mononuclear cells (PBMC). Immunoprecipitation of membrane protein isolated from LM cells and TE-85 cells with the MoAbs OSA1 and OSA2 conjugated with Staphylococcus aureus yielded a molecule with molecular weight of approximately 92,000. No detectable membrane protein was precipitated when 125I-labeled membrane protein from pooled activated human PBMC and tumor cells of other histologic types were used in the immunoprecipitation.

Flow cytometric analysis of bursal, thymic, and splenic cells from normal and cyclophosphamide-treated embryos.

Cell flow cytometry was used to assess the life cycle of embryonic lymphocytes. Cells were prepared for flow cytometry by fixation for 30 min in a final concentration of 70% ethyl alcohol, treated with ribonuclease (50 to 70 Kunitz/ml) and incubated for 30 min. in propidium iodide. Our data demonstrated that bursal lymphocytes from 20-day embryos (DE) exhibited significantly fewer cells in pre-DNA (deoxyribonucleic acid) and more cells in DNA synthesis (S) than thymic cells from 20 DE, and thymic lymphocytes from 16 DE expressed a more active S than bursal cells. Lymphocytes were separated from granulocytes by a Ficoll-hypaque double density gradient. The DNA cycle of bursal lymphocytes was more easily disrupted with cyclophosphamide (Cy) than the DNA cycle of thymic lymphocytes. However, unfractionated splenic cells from embryos treated with Cy revealed a greater percentage of cells in S and post-DNA synthesis and mitosis than splenic cells from control embryos. Because the splenic cells of Cy-treated embryos were predominantly immature granulocytes, it was concluded that Cy stimulated myelopoiesis in the spleen.

Cell flow cytometry of fixed and unfixed bursal and thymic cells.

Analysis in a cell flow cytometer (Ortho Spectrum III, Ortho Inst.) of single cell suspensions from the bursa and thymus of 20-day embryos revealed two distinct cell clusters. The two clusters were less apparent after fixation of the cells in paraformaldehyde and assumed a comet-like appearance at 30 min fixation in ethyl alcohol (EA). The G (postmitotic), S [deoxyribonucleic acid (DNA) synthesis], and G2/M (premitotic and mitotic) phases of the life cycle were visualized in two cell flow cytometers (Ortho Spectrum III and FACS IV, Bectin Dickinson) after treating the cells with EA, ribonuclease (RNase), and propidium iodide (PI, a fluorescent dye). Bursal cell suspensions exposed to the EA-RNase-PI protocol and stored for 3 weeks in phosphate-buffered saline showed minor changes in the G1 coefficient of variation, G1, and S percentages, but marked changes in these parameters occurred after 6 weeks of storage. Thymic cells treated in a similar fashion could not be maintained for 3 weeks. Bursal and thymic cells may remain in the EA for one day and possibly as long as 7 days prior to preparing them for DNA life cycle analysis. Paraformaldehyde was not a satisfactory cell fixative for assessing a cell's DNA life cycle.

The use of Ficoll-Hypaque double density gradients in the separation of avian granulocytes from other cell types for the purpose of cell flow cytometric analysis.

Avian peripheral blood and embryonic spleen cells were prepared for cell flow cytometry. The Ortho Spectrum III was the flow cytometer used in these experiments. The major objectives were to identify the location of lymphocytes and granulocytes in the cytogram displayed by flow cytometry, to develop a technique which would allow the collection of granulocytes relatively free of other cell types and to characterize the cell cycle within these cell populations. The cytogram of fresh avian cells developed in the Ortho Spectrum III revealed three characteristic cell clusters. Peripheral blood or embryonic spleen cells were separated on a Ficoll-Hypaque double density gradients into two distinct layers and a pellet. Light microscopic examination revealed the top layer of cells to be primarily lymphocytes while the middle layer of cells was granulocytes. Presentation of the cells from these layers to the Ortho Spectrum III revealed that granulocytes made up Cluster 3 while lymphocytes were included in the other clusters. The Ortho Spectrum III was employed to determine the presence of G1 (pre-DNA synthesis), S (DNA synthesis), and G2/M (post-DNA synthesis and mitosis) phases of cells in Clusters 1 and 2 and Cluster 3. While all the cells from peripheral blood were in G1, the embryonic spleen revealed cells in G1, S and G2/M in both Clusters 1 and 2 and Cluster 3.

Immune complexes and antinuclear, antinucleolar, and anticentromere antibodies in scleroderma.

Forty-one patients with various forms of systemic sclerosis (scleroderma) and positive antinuclear antibodies of nucleolar (ten patients), speckled (eleven patients), or centromere pattern (twenty patients) were selected for study of immune complexes by the radioisotope labeled Clq binding and the radioisotope labeled protein A binding methods. The presence of immune complexes was found by the Clq binding assay in sixteen patients (39%) and by a protein A binding assay in eight patients (20%). Overall, 46% of patients (19/41) had immune complexes. A lower incidence of organ involvement and fewer positive results in the screening of serum immune complexes were observed in patients with centromere antibody (35%) than in patients with nucleolar (60%) or speckled pattern (55%). Patients with immune complexes had higher frequencies of kidney, heart, and muscle involvement and digital ulceration than did patients with no detectable immune complexes, but the differences were not statistically significant. Diffuse skin involvement was not related to the presence of immune complexes.

Effect of prostaglandins on mouse erythrocyte rosette-forming human lymphocytes.

Prostaglandins (PGs) E2 and F2 alpha, preincubated with human lymphocytes for short periods of time inhibit mouse erythrocyte rosette formation. The presence of calcium ions does not influence this effect, which is dose-dependent and relatively temperature-independent. These observations indicate the PG treatment of lymphocytes may be useful to distinguish a subclass of IgM-bearing mouse erythrocyte rosette-forming B lymphocytes, which is sensible to the modulating effect of Pgs.

Interaction between prostaglandins and human T lymphocytes: effect of PGE2 on E-receptor expression.

E-rosette formation is not modified by preincubation of lymphocytes with prostaglandins (PGs) E1, F1 alpha, and F2 alpha. On the contrary, short preincubation with PGE2 affects active rosette-forming cells (Ea-RFC) and, only slightly, total RFC. This effect appears to be dose-dependent and relatively temperature-independent; it does not require calcium ions. Incubation with a fraction enriched in late RFC showed that PGE2 does not affect late rosette formation. It is postulated that PGE2 may redistribute surface receptors for sheep erythrocytes on T lymphocyte membranes. Thus, sensitivity to PGE2 may be considered another difference between early and late RFC.

Antiserums for immunofluorescent enumeration of human T lymphocytes utilizing fluoresceinated staphylococcal protein A.

Five lots (100 ml or more) of heterologous antiserums specific for human T lymphocytes were prepared using human or Rhesus monkey thymocytes as immunogens. After appropriate adsorptions, these antiserums reacted by immunofluorescence with 68% of human peripheral blood mononuclear cells and 98% of human thymocytes, with E-rosette--positive cells but not with EAC-rosette--positive cells or five human B-lymphoblastoid-cell lines. Blocking experiments showed that Rhesus monkey thymocytes share thymic antigenic determinant(s) with humans. E-rosette receptors modulated independently from T-cell heteroantigens. Non-E--rosetting neoplastic T cells were identified in several patients with lymphoproliferative malignancies. Applying both the E-rosette assay and the anti-T-cell serum provides a better method of defining the biologic properties of normal and neoplastic T lymphocytes. Standardization of immunofluorescent conjugates for human T- or B-cell enumeration is simplified if large lots of well-characterized antiserums are available.

Rosette formation and inhibition in cervical dysplasia and carcinoma in situ.

Total and early rosettes and rosette inhibition were measured in patients with cervical dysplasia and carcinoma in situ. Both total and early rosettes were significantly depressed in patients with carcinoma in situ; early rosettes were also significantly lower than were controls in women with severe dysplasia. Rosette inhibition titers were increased in most patients with moderate dysplasia and in all patients with either severe dysplasia or carcinoma in situ. Thus, the Rosette inhivition test may be useful in detecting, in a precancerous state, patients at risk for cancer.

Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity.

A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H(2)O(2)-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H(2)O(2) were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H(2)O(2); F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H(2)O(2) production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H(2)O(2) in human granulocytes.