Lymphoproliferative disease of "LAK cell" precursor large granular lymphocytes in association with celiac disease.

We have investigated a case of lymphoproliferative disease of large granular lymphocytes (LDGL) occurring in association with celiac disease, anemia, neutropenia, and carcinomas of the endometrium, breast, and skin. The large granular lymphocyte (LGL) proliferation was monoclonal, T cell in origin, with T cell receptor beta-chain gene rearrangement, and a CD3+, CD8+, CD16+/- phenotype. In spite of the high frequency of LGL, natural killer (NK) cell activity was absent. Stimulation with interleukin-2 in vitro, however, resulted in high lymphokine-activated killer (LAK) cell activity against NK-resistant targets. The T-cell nature of the LAK precursor cells is in contrast to the majority seen in normal peripheral blood. Therapeutic trials of cyclosporin A, low-dose cyclophosphamide, and levamisole were unsuccessful in reducing transfusion requirements. This case is unique in the association of LDGL with celiac disease. It is also unique in that the patient had been followed for several years prior to the onset of the LDGL. The case extends the list of lymphoproliferative disorders documented to be associated with celiac disease and, conversely, adds to our knowledge of lymphoproliferative disorder of LGL and its "dysimmune" manifestations.

Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells.

A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtitre plate spectrophotometer. In the present study, optimal conditions for chemosensitivity testing of human leukemia samples were determined, and the relative chemosensitivity of five patient samples was tested.

The Family Physician's Role in Managing Chronic Leukemia.

This article provides a brief update on the clinical approach to chronic myelogenous leukemia and chronic lymphocytic leukemia, based on advances in pathbiology and the effect of new concepts on treatment policies. These disorders were selected because family physicians take most responsibility for the day-to-day management of these most common forms of chronic leukemias.

Toxicity of agar for neutrophilic colonies.

The soft agar culture system is widely used to study in vitro regulation of granulopoiesis. This report is presented to illustrate how agar itself affects the kinetics of colony growth and thus, the results obtained with the assay. The main finding was that agar caused cells to lyse as they matured. Death appeared to occur at the myelocyte/metamyelocyte transition. Thus colonies did not develop a non-growth fraction of metamyelocyte, bands and segmented forms. Colonies grew in a multiphasic fashion owing to the maturation/differentiation process. Growth was characterized by an initial logarithmic phase, followed by a phase of non-logarithmic growth in which the size of colonies depended on the relative rates of cell production and loss. When clonogenic cells exhausted their self-renewal capacity, colonies entered a decay phase, during which they decreased in size and disappeared. In pulsing experiments the onset of decay phase was delayed if the addition of growth factor was delayed. Conversely, the onset of the decay phase was premature if mitosis was arrested with vincristine. During the decay phase the proportion of cells with damaged membranes (permeable to propidium iodide) remained constant. It was concluded that data accumulated during the decay phase cannot be interpreted reliably. This point should be considered in designing experiments and interpreting the results with this assay system.

Effects of lactoferrin on human granulopoiesis in vitro.

It has been proposed that Lactoferrin plays a negative feedback role in regulating granulopoiesis through its ability to suppress Colony-Stimulating Factor production. However, an analysis of experimental data, based on cell kinetic principles, does not support this contention. The basic point is that the concentration of Colony-Stimulating Factor determines the rate of clonal amplification, and mean clonal size. The latter is a reliable index with which to quantify the level of stimulation in a system. With this index it was found that Lactoferrin: did not suppress the de novo production of colony factor by blood mononuclear leukocytes affected the rate of cell proliferation both in the absence and presence of preformed Colony-Stimulating Factor. It was also found that Lactoferrin influenced myelocyte progression through the cell cycle. These findings suggest that the action of Lactoferrin is more complex than was previously believed and its relevance as a regulator of granulopoiesis is unclear.

Kinetic analysis of mechanisms regulating granulopoiesis in cultured human bone marrow.

In the past, in vitro perturbations of granulocytic progenitor cells have been measured by comparing the number of colonies that have grown to a threshold size in a given time. In our hands this method has proven to be both insensitive and unreliable. In this paper kinetic evidence is presented that the number of colonies, however defined, is subject to the influence of unpredictable independent variables which include the age distribution of the clonogenic cells, the proportion of them which are dormant, and the factors which promote their recruitment, their expansion into colonies and the rate at which colonies die. Results are improved by an early analysis of total clonal number (TC) as a measure of recruitment and mean clonal size (MCS) as a measure of clonal expansion rate.

The generation of colony stimulating activity by lectin stimulated human peripheral blood mononuclear leucocytes.

In order to clarify the role of T-lymphocytes and monocytes in the regulation of granulopoiesis, we have examined the production of colony stimulating activity by human peripheral blood mononuclear cells in response to phytohemagglutinin. T-cells prepared by adherence and sheep erythrocyte rosettes synthesized appreciable quantities of colony stimulating activity, but after further depletion using a monoclonal antibody against monocytes, this synthesis was much reduced, and DNA synthesis abolished. It was possible to restore DNA synthesis by adding back small numbers of monocytes, without restoring the production of colony stimulating activity. Similarly, depleting monocytes from unfractionated mononuclear leucocytes with monoclonal antibody caused a marked reduction in the amount of colony stimulating activity generated. In contrast, depleting T-cells from whole mononuclear cells, or from a sheep cell rosette negative fraction, had little effect on colony stimulating activity production. Mononuclear leucocytes were also depleted of helper and suppressor T-cell subsets. Colony stimulating activity production following these procedures was highly variable, and no clear pattern emerged. We postulate that monocytes are the major source of the colony stimulating activity measured by our assay. We have failed to establish a clear role for T-lymphocytes in either producing or regulating colony stimulating activity, although the difficulties inherent in depletion experiments do not allow such a role to be excluded.

Kinetic analysis of cloning patterns by human marrow cells in leukemia.

In leukemia and preleukemic disorders the progeny of a single cell proliferate and ultimately come to occupy the hemopoietic system. In the process normal stem cells are suppressed and in time may become extinct. This implies that neoplastic clones have a biological advantage. In this paper evidence is presented that the cloning of granulocytic colony forming cells in the clonal hemopathies is influenced by cell products that regulate cloning of normal colony forming cells. We have attempted to develop an approach to the study of clone-clone interactions in order to determine at what level(s) the battle between clones is fought. Future studies on relative responsiveness might help in understanding the mechanisms by which normal hemopoiesis is suppressed during the evolution of leukemia and re-established during remission induction.

A simple method for analysis of DNA histograms in human myeloid cells perturbed by stimulators of DNA synthesis.

In the past, phase fractionation has been used as a determinate of the level of stimulation of cells perturbed by stimulators and inhibitors of DNA synthesis. This method has, in our hands, proven to be insensitive and unreliable with human myeloid cells. A new method of analysis is described in this paper which involves utilization of the magnitude of the slope of a line fit to the mid-portion of S phase as an index of the level of stimulation of cultured human myeloid cells. This method is fast, reliable and simple to implement on an inexpensive microcomputer.

Lithium and hematopoiesis.

Some of lithium's effects on blood cell formation suggest that the element may be of value in treating hematologic disorders. Lithium enhances granulopoiesis and thereby induces neutrophilia. Two possible mechanisms of action are suggested: a direct action on the pluripotent stem cells, or an inhibition of the suppressor cells (thymus-dependent lymphocytes) that limit hematopoiesis. Lithium also inhibits erythropoiesis. Although most studies use concentrations at or above pharmacologic levels there is evidence that lithium plays a role in normal cell metabolism.

Factors influencing in vitro production of colony-stimulating factor by mononuclear leukocytes from humans.

The purpose of this study was to identify factors that influence the production of colony-stimulating factor by leukocytes of humans. The use of nonadherent light-density bone marrow cells is semisolid agar cultures to assay the concentrations of colony-stimulating factor in the supernatant of monocyte and mononuclear leukocyte cultures made it possible to distinguish between colony-stimulating factor, which stimulates colony-forming cells directly, and monocyte-dependent stimulating activity, which acts indirectly, by increasing the monocyte production of colony-stimulating factor. Colony-stimulating factor was not detectable in the cytosol of monocytes; that detected in culture must, therefore, have been newly synthesized. Synthesis was enhanced independently by heat-inactivated human serum and by semipurified serum fractions enriched with monocyte-dependent stimulating activity. The kinetics of the production of colony-stimulating factor in the presence and absence of monocyte-dependent stimulating activity indicated that the latter facilitated monocyte production of the former. Factors released from neutrophils were shown to reduce the production of colony-stimulating factor and thr proliferation of colony-forming cells and thus may provide a feedback control mechanism limiting the proliferation of neutrophils.

Action of inhibitor released from neutrophils and leukemic blast cells.

The concept that polymorphonuclear leukocytes, or neutrophils, play a role in feedback control of granulopoiesis has been supported by the finding in bone marrow culture studies that mature neutrophils inhibited formation of granulocytic colonies. The study described in this paper was done to investigate the mechanisms involved. With the use of a modified assay it was found that mature neutrophils released factors that reduced the proliferation of colony-forming cells in cultures stimulated by cell-free colony-stimulating factor. In myeloproliferative and myelodysplastic disorders the amount of inhibitor released by the neutrophils varied greatly. Leukemic blast cells also released inhibitor, and in some cases the amount released per cell was greater than the amount released from normal mature neutrophils. The inhibitory factors released from the neutrophils differed from those previously described in the literature in terms of mode of action and apparent molecular size.

Microvascular closure in malignant histiocytosis.

Two patients with malignant histiocytosis were found to have capillary occlusion by aggregates of neoplastic histiocytes, in skeletal muscle in one, and in renal glomeruli in the other. One patient had clinical evidence of similar occlusions in the arterioles and capillaries of the ocular fundi. Occlusion of small vessels by tumour cells may explain the confusion of both patients.

Effect of lithium on granulopoiesis in culture.

Lithium carbonate therapy is associated with polymorphonuclear leukocytosis. In vitro studies have shown that lithium ions stimulate formation of granulocytic colonies. In a study undertaken to determine how lithium acts, colony-forming cells uncontaminated by monocytes (which elaborate colony-stimulating factor [CSF] in vitro) were obtained by means of a two-step cell separation procedure. The effects of lithium on colony formation were then studied in (a) cultures stimulated by humoral CSF, (b) cultures in which monocytes were relied upon to synthesize CSF de novo and (c) unstimulated cultures. Lithium enhanced the action of CSF but did not stimulate colony formation in the absence of CSF. In monocyte-stimulated cultures, colony formation increased with lithium concentrations up to 1 mmol/L but this increase paralleled that in CSF-stimulated cultures and therefore was not due to increased CSF production by monocytes. At higher concentrations of lithium, colony formation decreased in the monocyte-stimulated cultures but increased in the CSF-stimulated cultures. A lithium concentration of 4 mmol/L gave the greatest enhancing effect on colony formation in CSF-stimulated cultures and a concentration greater than 1 mmol/L inhibited de novo synthesis of CSF by monocytes.

In vitro regulation of granulopoiesis in human leukemia: application of an assay for colony-inhibiting cells.

We describe two assays to detect the action of colony-inhibiting cells. In the first assay, we used a simple density separation technique to remove dense neutrophils (PMN) from suspensions of blood and of bone marrow cells prior to culture in semisolid agar. Conditions were arranged to ensure that control suspensions of unseparated cells and test suspensions of buoyant mononuclear cells differed only in their content of neutrophils. The control and test suspensions contained equal numbers of mononuclear cells (and granulocyte precursors). Colony (and cluster) formation was invariably enhanced in neutrophil-depleted cultures of normal cells. In the second assay, dense PMN, treated by an adherence separation procedure, were recovered, and the non-adherent dense PMN were added back to PMN-depleted cultures. A reproducible dose-related decrease in colony (and cluster) formation to basal levels resulted. The inhibitory effect was identical when the PMN were added directly to the culture (overlayer) or to the underlayer. In PMN-depleted cultures obtained from patients with leukemia and other hemopoietic disorders, neither colony nor cluster formation was enhanced, and sometimes it was reduced. When we compared the effect of adding patient and normal non-adherent PMN to target cultures of normal and patient PMN-depleted cells, some leukemic PMN were noninhibitory. Our results suggest that abnormalities of cellular interactions in vitro detected in the first assay may have more than one explanation, as shown when they are subjected to the closer scrutiny possible with the second assay.