Biomedical subjects
K Rhyner
Publications and source records attributed to K Rhyner.
[Headache and vision disorders].
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[Shoulder-thorax pain].
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CSF-responsive bone marrow cells in liquid culture: characterization and screening applications.
In a previous study, colony-stimulating factor (CSF) activity assayed in colony culture correlated closely with 3HTdR uptake by human marrow cells depleted of adherent cells. To use this assay for screening media for CSF and immunotoxins for marrow toxicity, cells growing in liquid culture were compared to conventional granulocyte/macrophage (CFU-gm) colony assays. CSF dose-response relationships for liquid and colony-forming assays were nearly identical. 3HTdR uptake by nonadherent marrow cells was CSF dose-related, and there was a linear relationship between number of cells cultured and 3HTdR uptake. Ricin cytotoxicity curves for liquid cultures and CFU-gm were identical on day 7 but showed some disparity with day 14 cultures. Results with all cultures showed 3HTdR uptake to be most closely correlated with CFU-gm colony, rather than cluster, growth. Myeloid cell differentiation in liquid culture was similar to colony cultures, producing mixtures of granulocytes, macrophages and eosinophils. By combining cell and differential counts, production of various myeloid cells could be quantitated. Cytotoxicity of anti-Ia for CFU-gm and liquid culture cells was compared and the majority of both cell populations expressed Ia-like antigens. Simultaneous staining for surface antigens and DNA content was used to characterize proliferating marrow cells, and the vast majority of cells expressed myeloid markers. Transferrin receptors were displayed by cells in S/G2/M and appeared after CSF stimulation on G0/G1 cells. We conclude liquid cultures can be used to screen conditioned media for human CSF and to screen for cytotoxicity to normal myeloid precursor cells. Behavior of CSF-responsive cells in liquid culture appears most closely related to that of CFU-gm colony-forming cells, and characterization of CSF-stimulated cells allows quantitative as well as qualitative estimates of myeloid cell production.
Induction of colony-stimulating factor response in myeloid leukaemia cell lines.
Untreated, late passage HL60 promyelocytic and KG1 myeloblastic leukaemia cells did not increase proliferation with placenta or Mo T cell conditioned medium containing colony-stimulating factor (CSF) nor with partially purified, recombinant granulocyte/macrophage (GM)-CSF. However, after induction with DMSO or 1,25-dihydroxy-vitamin D3, HL60 cells showed dose-dependent increases in proliferation with crude and purified CSFs. CSF responses and macrophage differentiation were induced in KG1 cells by treatment with tetradecanoylphorbol-acetate (TPA). When cells were exposed to inducing agents for varying periods, washed and exposed to CSF, proliferative responses were related to time of exposure. Cells exposed for 1-4 d showed post-induction CSF-induced proliferation, but cells induced for 5-6 d were inhibited by CSF. Induction of CSF response appeared linked to differentiation, since KG1 cells differentiated with TPA and developed CSF-induced proliferative responses, but showed no differentiation or CSF induced proliferation after treatment with vitamin D3. When HL60 cells were continuously exposed to DMSO or vitamin D3, overall cell production was increased by placenta conditioned medium, but cultures still became senescent and died after several weeks. Cells continuously cultured with DMSO were predominantly macrophages, indicating lineages of DMSO-induced differentiation were modified by continuous culture or the presence of CSF. After treatment with chemical inducers, proliferation of myeloid leukaemia lines is stimulated by CSF, providing a model for post-deterministic regulation of normal and malignant myeloid cell production.
Toxoplasmosis in hairy-cell leukaemia.
Toxoplasma serology was performed in 28 patients with hairy-cell leukaemia and was positive in eight patients (29%). In two patients (7%) reactivated toxoplasmosis was proven by either isolation of Toxoplasma gondii or by significant antibody titre rise with generation of specific IgM-antibodies. In four patients (14%), a clinical diagnosis of active toxoplasmosis was based on signs and symptoms, serologic tests, and response to specific treatment. The high proportion of patients in which active toxoplasmosis was proven or probable (six; 21%) may be related to the presence of severe monocytopenia. In patients with hairy-cell leukaemia developing fever of unknown origin and myositis, toxoplasma serology should be performed, particularly because treatment of active toxoplasmosis usually is successful.
[Chronic myeloid leukemia: multivariant analysis of initial findings regarding its effect on mean life expectancy].
Clinical signs, complete blood counts and bone marrow differential counts at the time of diagnosis were investigated in 75 consecutive patients with chronic myelogenous leukemia in a study of prognostic parameters. All patients were followed until death (62 patients) or for a mean follow-up time of 69 months (13 patients). Clinical signs and hematological values were analyzed by univariate and multivariate statistical methods. The only parameter relevant to mean survival time in multivariate analysis is the percentage of bone marrow myeloblasts at diagnosis. Patients with less than 5% myeloblasts at diagnosis had a mean survival time of 48 months compared with 16 months for those with greater than or equal to 5% (p less than 0.01).
[Polyostotic eosinophilic granuloma with pituitary insufficiency].
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Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells.
Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin-free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation.
Role of transferrin, Fe, and transferrin receptors in myeloid leukemia cell growth. Studies with an antitransferrin receptor monoclonal antibody.
In previous studies, antitransferrin receptor antibody 42/6 inhibited growth of normal granulocyte/macrophage progenitors and some malignant myeloid cells. In these studies, leukemia cell lines cultured without serum and fresh leukemia cells were used to investigate the roles of Fe, transferrin receptors, and transferrin in leukemia cell growth, and mechanisms of 42/6 inhibition and resistance. HL60 and KG-1 leukemia cells grown in serum-free medium were inhibited by 42/6. In contrast to results in fetal calf serum (FCS), soluble Fe (ferric nitriloacetate) reversed 42/6 growth inhibition of serum-free HL60 cells. When HL60 cells were adapted for growth in serum-free, transferrin-free medium, they became refractory to 42/6 growth inhibition. By using radiolabeled transferrin and 42/6, HL60 cells cultured in FCS and transferrin displayed similar quantities of transferrin receptors (29,000-30,000/cell) and similar Kd's (3.8-4.9 X 10(-9) M). Cells grown in transferrin-free medium showed a similar Kd (3.1 X 10(-9) M), but fewer transferrin binding sites (5,000/cell). Transferrin-independent cells contained a log higher concentration of intracellular ferritin. For both FCS and serum-free HL60 cells, calculated affinities for 42/6 were lower (5.7-10.0 X 10(-9) M), but the number of binding sites was three- to fourfold higher. To investigate further the relationship between receptor display and antibody inhibition in proliferating normal and malignant myeloid cells, simultaneous immunofluorescence was used to determine the cell cycle status of transferrin receptor-positive cells. Malignant cells in S + G2/M displayed approximately 50% of the amount of transferrin receptors detected in normal dividing colony-stimulating factor-stimulated marrow cells. Receptor display by dividing cells from two patients with acute nonlymphocytic leukemia was variable. When HL60 cells were exposed to dimethyl sulfoxide, transferrin receptor display decreased, and 42/6 growth inhibition was abrogated or greatly diminished. The presence of 42/6 did not prevent dimethyl sulfoxide-induced HL60 differentiation in serum-containing or serum-free cultures. We conclude that human leukemia cells require Fe for growth and that 42/6 inhibits transferrin-dependent cells by Fe deprivation. Some dividing normal and differentiating malignant cells display reduced transferrin receptors, and can also escape antibody inhibition. The increased ferritin levels and decreased transferrin receptors in transferrin-independent HL60 cells confirm the inverse relationship between cell ferritin content and transferrin receptor display. These studies indicate a critical role for Fe in leukemia cell growth and possible roles in cellular differentiation.
Morphologic alterations of blood cells in the impedance aggregometer.
The scanning (SEM) and transmission (TEM) electron microscopic appearance of blood cells was studied in correlation with the aggregometer tracing recorded after activation of whole blood samples by collagen or ADP. Early morphologic alterations of platelets characterized by the formation of marginal pseudopods and bulbous protrusions were not indicated by the aggregometer. The initial increase in impedance was caused by the attachment of platelets displaying typical shape change morphology at the surface of the electrode wires joint with collagenous fibrils in collagen activated specimens. During further increase in impedance, aggregates were detectable in the blood suspension and at the electrode, the number and size of which increased up to the maximal extension of the aggregometer tracing. Using low doses of ADP (2-3 microM), dissociation of aggregates in the blood suspension was detectable by SEM, which was not recorded by the aggregometer tracing indicating further limitation of the impedance aggregometer. In collagen activated samples, platelet aggregates were covered by PMN and monocytes that in TEM displayed distinct phagocytosis of platelet fragments and fibrin masses. In ADP specimens, activation of leukocytes was only rarely detectable. The detection of mixed aggregates may be important for further employment of the impedance aggregometer in the diagnosis of hematologic diseases.
Preclinical studies on the use of selective antibody-ricin conjugates in autologous bone marrow transplantation.
Whole-ricin immunoconjugates were synthesized with the pan-T cell antibodies T101 and 3A1 and assayed in the presence of 0.1 mol/L lactose. Their toxicity for cell lines, peripheral blood T lymphocytes, and normal bone marrow progenitors was compared with that of whole ricin. In the presence of 0.1 mol/L lactose, normal cells and cell lines exhibited the following sensitivities to ricin: 8392 (human malignant B cell line) less than E rosette-positive lymphocytes less than bone marrow progenitors less than 8402 (human T ALL) less than CEM (human T ALL). Ricin sensitivities correlated with ricin binding as determined by immunofluorescence. In the presence of lactose, peripheral blood T cells were resistant to 0.1 nmol/L ricin, but a similar concentration of T101-ricin inhibited normal and malignant T colony formation by greater than 98%. 3A1-ricin was slightly less effective. At a conjugate concentration of 0.1 nmol/L, bone marrow progenitor colony formation was inhibited by 30% or less; T101-positive cells were at least tenfold more sensitive than normal progenitors. When mixtures of 10% CEM cells and marrow cells were incubated with T101-ricin, 95% of CEM colonies were killed, and 96% of marrow granulocyte/ macrophage progenitors survived. Some free ricin was released from immunotoxin-treated cells, producing minimal inhibition of protein synthesis or cell growth. We conclude that (a) normal blood cells and malignant cell lines exhibit varying degrees of ricin sensitivity in the presence of lactose; (b) T101-ricin is at least tenfold more toxic to T lymphocytes than to bone marrow progenitor cells and is effective in mixtures of normal and malignant cells; and (c) treatment of infiltrated marrow with anti-T cell immunotoxins should safely remove target T cells without excessively damaging normal progenitors or producing excessive free ricin. Anti-T cell, whole-ricin immunotoxins merit trials for autologous transplantation.
Decreased thiosulfate sulfur transferase (rhodanese) in Leber's hereditary optic atrophy.
In mammals the major portion of cyanide is converted to thiocyanate by the liver enzyme thiosulfate sulfur transferase (TST) (rhodanese). We have found a much reduced activity of this enzyme in liver biopsies from two affected males of a family with Leber's hereditary optic atrophy and in two isolated cases of the same disease, (compared to liver biopsies from controls or liver samples obtained at autopsy). In one of the patients we studied the effect of a 3-day thiosulfate infusion. The urinary excretion of thiocyanate which was low prior to the infusion was raised during the thiosulfate treatment; in a healthy control person the same thiosulfate infusion did not alter the thiocyanate excretion rate. This suggests that cyanide detoxification which is suboptimal in patients with Leber's disease may be increased by thiosulfate infusion.
[Divergence between the pharmacologic and clinical effects of inhibitors of prostaglandin synthesis].
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[Temperature-dependent spontaneous platelet aggregation in polycythemia vera and primary thrombocythemia: a diagnostic criterion].
In twenty patients suffering from myeloproliferative syndromes (nine with polycythaemia vera, 11 with primary thrombocythaemia) platelet aggregation was tested following incubation of blood samples at 4 degrees C, room temperature, 30 degrees C and 37 degrees C. A spontaneous platelet aggregation following incubation at room temperature took place in 65% of patients with primary thrombocythaemia. At the two higher temperatures, positive results were seen in 72% and 92% of cases, respectively. Seven of nine patients (78%) showed a pathological spontaneous platelet aggregation at room temperature. Pathological spontaneous platelet aggregation at 30 degrees C occurred in eight of nine (89%) cases. The spontaneous platelet aggregation test is a simple method to diagnose primary thrombocythaemia and asymptomatic polycythaemia vera, the sensitivity of which increases considerably following incubation of blood samples at two and three different temperatures.
[Are blood platelets released in the lungs?].
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Thiamphenicol induced bone marrow suppression as a therapy of myeloproliferative diseases.
Sixteen patients, suffering from myeloproliferative diseases (9 polycythaemia vera, 7 primary thrombocythaemia) and 20 control subjects were treated for 14 days with 1 g of thiamphenicol per day. The effect of 40 treatment cycles was studied. The regimen resulted in lowering the haemoglobin values by 4.8% in controls (p greater than or equal to 0.001) and patients (p greater than or equal to 0.01); the reticulocyte count dropped 43% in the patient group (p greater than or equal to 0.01) and 32% in the controls (p greater than or equal to 0.05); the thrombocyte count was decreased 42% (p greater than or equal to 0.0001) vs 29% (p greater than or equal to 0.0001) in the control group. The administration of 1 g/day of thiamphenicol for a 14 day period reduced the myelopoietic activity by 40%. The decrease of all values was statistically significant. After discontinuation of thiamphenicol therapy the haematological parameters returned to the initial values within 1-2 months. The myelodepressant activity of thiamphenicol may therefore be applied to the therapy of the myeloproliferative diseases in order to reduce the risk of spontaneous haemorrhages and thrombosis.
Surface morphology of human platelets during in vitro aggregation.
The alterations of surface morphology of human platelets during the course of the aggregometer tracing were studied by scanning electron microscopy (SEM). Prior to activation the platelet rich plasma was pre-incubated at 37 degrees C for at least 30 min in order to obtain a sufficient number of discoid platelets. Immediately following the addition of collagen or ADP platelets showed slender pseudopods which thereafter were replaced by bulbous protrusions. The decrease in percent light transmission (%T) in the aggregometer tracing was characterized in SEM by a significant enlargement of bulbous protrusions and by the transformation of platelet shape from disc to sphere. During the increase in %T platelets forming primary aggregates displayed spiny pseudopods at their surface indicating that, during in vitro aggregation induced by collagen or ADP, two generations of pseudopods are formed. Using a low dose of ADP, the return of aggregometer tracing to the base line was regularly accompanied by dissociation of primary aggregates, but platelets remained spheroid and displayed pseudopods for a long time. Our study indicates that the course of aggregometer tracing is closely associated to the surface morphology of platelets. Single morphological changes, however, are not reflected by the aggregometer method.