Studies on the pathogenesis of an immune defect in multiple myeloma.

The reduced capacity of patients with multiple myeloma to respond to antigen challenge is well recognized. Response to antigen involves antigen recognition, cell proliferation, and synthesis and secretion of antibody. This study examines this sequence of events in peripheral blood lymphocytes from untreated and treated patients with myeloma, from individuals with benign monoclonal gammopathy, and from normal healthy donors. Antigen-binding capacity was assessed by testing the ability of lymphocytes to bind radio-labeled pneumococcal polysaccharide, tetanus toxoid, or diptheria toxin. The in vitro proliferative response to these antigens as well as to pokeweed mitogen and streptokinase-streptodornase was evaluated. The secretion of immunoglobulin in response to pneumococcal polysaccharide, tetanus toxoid, and pokeweed mitogen by 2-4 x 10(6) lymphocytes in 7-day cultures was determined. The effects of coculture of myeloma peripheral blood lymphocytes and normal peripheral blood lymphocytes on immunoglobulin production and mixed leukocyte reactions were explored. All myeloma patients had normal numbers (3-8/5,000 cells) of antigen-binding cells. However, most showed a diminished antigen-induced blast transformation as measured by uptake of [(125)I]5-iodo-2'-deoxyuridine in culture. Immunoglobulin production in response to specific antigen in myeloma lymphocytes was 30-80% less than in normal lymphocytes. Immunoglobulin synthesis and mixed leukocyte responses by normal peripheral blood lymphocytes could be suppressed by myeloma lymphocytes. Multiple suppressor populations were present. Thus, the immune defect in myeloma is beyond the antigen recognition step and involves both the proliferation of antigen-sensitive cells and immunoglobulin production. Further suppressive effects are imposed on normal cells, implying defects in immunoregulation in this disease.

Multiple myeloma: an immunologic profile. II. Bone marrow studies.

Bone marrow mononuclear cell populations were studied in 35 patients without myeloma, 39 patients with multiple myeloma, and 15 patients with benign monoclonal gammopathy. Bone marrow mononuclear cell receptors, responses to mitogens or allogeneic stimuli, and suppressive effects on in vitro peripheral blood lymphocyte (PBL) function were studied. In bone marrow cell populations from patients with untreated multiple myeloma, the percent of complement receptor-bearing cells and the pokeweed mitogen- and concanavalin A-stimulated responses were significantly greater than were those in bone marrow cell populations from patients without myeloma. Sheep red blood cell receptor-bearing cells were significantly greater in marrow populations from treated multiple myeloma patients compared to those from untreated multiple myeloma patients. Sheep red blood cell receptor-bearing cells from the bone marrow of multiple myeloma patients suppressed responses of the multiple myeloma patients' PBL's to autologous mitomycin C-treated bone marrow plasma cells and to allogeneic stimuli in one-way mixed leukocyte culture. Complement receptor-bearing cells suppressed the response to pokeweed mitogen. The presence of lymphocytes in the marrow compartment that are capable of suppressing the response of myeloma patients' PBL's to plasma cell antigens may be significant in the pathogenesis of multiple myeloma.

Establishment and characterization of a CD5 positive, immunosuppressive human myeloma cell line.

A continuously growing, in vitro cell line with plasma cell characteristics has been established from the bone marrow of a patient with multiple myeloma. Surface marker characterization of the cells revealed a combination of markers normally associated with different developmental stages in the B cell lineage. The cell secretes immunoglobulin at a relatively low rate. The cell also expresses CD5 and secretes a factor which suppresses in vitro mitogen and antigen induced immunoglobulin synthesis by normal PBL.

Factors affecting the in vitro evolution of a myeloma cell line.

A continuously growing plasma cell line has been established from the bone marrow of a multiple myeloma patient. Initial growth of the cells was dependent on the presence of bone marrow stromal cells. Following initial outgrowth the cells were maintained by transfer onto non-autocthonous bone marrow stromal cultures. Following approximately one year of continuous growth, a subline was derived which could be grown independently of feeder cells. These stromal-cell-independent myeloma cells nevertheless retained dependence for a growth factor present in stromal-cell-conditioned media. The relevant factor in the conditioned media was determined to be interleukin-6 (IL-6). The cells also ultimately became independent of the conditioned media. These latter cells were shown to contain mRNA for IL-6 and eventually began to secrete IL-6. This cell line has thus progressed from complete dependence on stromal cells to IL-6-dependent growth in the absence of stromal cells to complete self sufficient growth. This in vitro progression may reflect an in vivo pattern of myeloma development.

RB status as a determinant of response to UCN-01 in non-small cell lung carcinoma.

7-Hydroxystaurosporine (UCN-01), a protein kinase inhibitor in clinical development, demonstrates potent antineoplastic activity. To determine whether specific genetic abnormalities would modulate the response to UCN-01, a model of human non-small cell lung carcinoma (NSCLC) cell lines with differential abnormalities of p16CDKN2, RB, and p53 was used for these studies. Cell growth was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and cell cycling was studied using flow cytometric analysis of DNA content. Changes in protein levels and phosphorylation were assessed by Western blotting. In cell lines expressing wild-type RB (A549 and Calul), UCN-01 treatment resulted in dose-dependent growth inhibition, arrest of cells in G1, and a reduction of cells in S phase. p16CDKN2-null cells showed similar growth inhibition to normal fetal lung fibroblasts. UCN-01-induced growth arrest was accompanied by induction of p21CDKN1 and a shift of Rb to the hypophosphorylated state in both p53 wild-type and mutant cell lines. In contrast, UCN-01 treatment of the RB-null cell line H596 resulted in less growth inhibition. To test the role of RB in response to UCN-01, effects of treatment were examined in two human isogenic models of RB expression: the bladder cancer cell line 5637 (RB-null) and the prostate cancer cell line DU-145 (RB-mutant). In the Rb-expressing 5637 subline (RB5), UCN-01 treatment resulted in Rb hypophosphorylation and an accumulation in G1 in contrast to the parent line. Similarly, the wild-type Rb-expressing DU-145 sublines (DU1.1 and B5) showed increased G1 arrest compared with the parent cells. We conclude that UCN-01-induced G1 arrest can occur in cells null for p53 and p16CDKN2, and that RB status influences the ability of UCN-01 to induce a G1 arrest. These data suggest that the molecular profile of cell cycle regulating genes in individual tumors may predict responsiveness and provide insight into optimal therapeutic application of this new antineoplastic agent.

Cocoa inhibits platelet activation and function.

BACKGROUND: Epidemiologic studies have shown inverse associations between dietary polyphenols and mortality from coronary heart disease. However, the basis for this protective association is uncertain. Food polyphenols reportedly have antioxidant properties and decrease platelet function in vitro. OBJECTIVE: This study sought to evaluate whether consumption of a polyphenol-rich cocoa beverage modulates human platelet activation and primary hemostasis. DESIGN: Peripheral blood was obtained from 30 healthy subjects before and 2 and 6 h after ingestion of a cocoa beverage (n = 10), a caffeine-containing control beverage (n = 10), or water (n = 10). Platelet activation was measured in terms of expression of activation-dependent platelet antigens and platelet microparticle formation by using fluorescent-labeled monoclonal antibodies and flow cytometry. Primary platelet-related hemostasis was measured with a platelet function analyzer. RESULTS: Ex vivo epinephrine- or ADP-stimulated expression of the fibrinogen-binding conformation of glycoprotein IIb-IIIa was lower 2 and 6 h after consumption of cocoa than before consumption. Cocoa consumption also decreased ADP-stimulated P-selectin expression. In contrast, epinephrine-induced platelet glycoprotein IIb-IIIa expression increased after consumption of the caffeine-containing beverage but not after water consumption. Platelet microparticle formation decreased 2 and 6 h after cocoa consumption but increased after caffeine and water consumption. Primary hemostasis in response to epinephrine in vitro was inhibited 6 h after cocoa consumption. The caffeine-containing beverage inhibited ADP-induced primary hemostasis 2 and 6 h after consumption. CONCLUSIONS: Cocoa consumption suppressed ADP- or epinephrine-stimulated platelet activation and platelet microparticle formation. Cocoa consumption had an aspirin-like effect on primary hemostasis.

Marshall-Smith syndrome: two case reports and a review of pulmonary manifestations.

Two patients with the Marshall-Smith syndrome are described. Both had significant and fatal respiratory distress attributable to this condition. Congenital, functional, and acquired abnormalities of the respiratory tract are described in nine of the 11 case reports in the literature and are characteristic of this syndrome as well as a primary cause of failure to thrive and death in these patients. Unusual immunologic findings in one of our two patients are the first to be reported in the Marshall-Smith syndrome. Quantitation of immune function in other patients with this condition will be helpful in determining the significance of these results. It is hoped that the etiology of the syndrome will be discovered as more cases are recognized and reported by pediatricians caring for infants with failure to thrive, advanced bone age, and chronic respiratory symptomatology.

Donor cell induced CD69 expression and intracellular IL-2 and IL-4 production by peripheral blood lymphocytes isolated from kidney transplant recipients.

Flow cytometry assays, which measure CD69 activation and intracellular cytokine production, have been used to measure peripheral blood lymphocyte (PBL) responses to in vitro antigen exposure. In the present study, we show that, in healthy individuals and immunosuppressed kidney transplant recipients, CD69 expression and intracellular cytokine production by peripheral blood T cells compare favorably to thymidine uptake as a measure of PBL response to alloantigen in mixed leukocyte culture (MLC). Heparinized whole blood from 23 healthy individuals was incubated for 24-48 h with 3rd party allogeneic monocytes; blood from twelve kidney transplant recipients was incubated with monocytes from their kidney donor and with monocytes from unrelated individuals. The percentage of T cells expressing surface CD69 or intracellular IL-2 or IL-4 was determined by 3-color flow cytometry. We identified 5 donor-specific response patterns in our kidney transplant group. One transplant recipient was hyporesponsive; his cells did not express CD69 or produce IL-2 in response to either donor or 3rd party allogeneic cells. All other transplant recipients expressed CD69 and IL-2 in response to 3rd party allogeneic cells. Two had no response to donor cells (donor-specific hyporesponsiveness), three had donor-specific anergy (CD69 expression without cytokine production in response to donor cells), five had a donor-specific Thl response (CD69 expression and IL-2 production in response to donor cells), and one had a donor-specific Th2 response (CD69 expression and IL-4 but not IL-2 production in response to donor cells). Rapid measures of donor-specific hyporesponsiveness such as CD69 activation antigen expression and intracellular cytokine production may prove valuable in monitoring lymphocyte function and aid in the long-term management of kidney transplant recipients.

Platelet-erythrocyte adhesion in sickle cell disease.

BACKGROUND: The abnormal adherence of sickle red blood cells (sRBC) to other cell types likely contributes to vaso-occlusion. Increased numbers of platelet-erythrocyte aggregates (PEA) and platelet activation have been described in sickle cell disease. The present study was undertaken to determine the contribution, if any, of the extracellular matrix protein thrombospondin to the adhesion of sRBC and platelets. METHODS: Platelet activation and PEA were measured using fluorescent-labeled monoclonal antibodies and flow cytometry. Platelet red-cell adhesion was measured by a gravity sedimentation assay. Erythrocyte-bound thrombospondin (TSP) was determined by enzyme-linked immunoabsorbant assay (ELISA). RESULTS: Our studies demonstrate significant platelet activation and adhesion of sRBC to platelets in sickle cell disease. Thrombospondin was detected on sRBC. There was variable inhibition of sRBC-platelet adhesion by antibodies to CD36 (thrombospondin receptor) and antibodies to thrombospondin. CONCLUSIONS: Thrombospondin on sRBC may mediate, at least in part, sRBC-platelet adhesion in sickle cell disease. The study of heterotypic cell-cell interactions is important in understanding the pathogenesis of vaso-occlusion in sickle cell disease.

Prolonged circulation of activated platelets following plasmapheresis.

The extent and duration of in vivo platelet activation were determined in 12 volunteer donors undergoing automated plasmapheresis. Expression of P-selectin, activated GpIIb/IIIa, and platelet microparticle formation were measured by flow cytometry on peripheral blood samples obtained immediately before and after plasmapheresis and at 24 hour intervals thereafter for up to 3 days. Although no adverse effects were noted in any donor, immediately after apheresis 3-87% of circulating platelets expressed P-selectin; by 48 hours, 0.5-50% expressed P-selectin; and by 72 hours, all donors studied had fewer than 5% P-selectin expression on circulating platelets. Results were similar for the expression of the activated conformation of GpIIb/IIIa. There was a positive correlation with in vitro P-selectin expression in response to ADP in the pre-apheresis sample and the number of platelet microparticles detected in the donor following plasmapheresis. In addition, the percent expression of P-selectin and activated GpIIb/IIIa in response to ADP was reproducible in each individual studied on five separate occasions (CV < or = 8%). Platelets activated during plasmapheresis using an automated device may circulate for at least 48 hours, and pre-plasmapheresis response of platelets to the agonist ADP correlated with platelet microparticle formation post-plasmapheresis.

CD5 positive immunoregulatory B cell subsets.

B-chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease often expressed as a clonal expansion of CD5+ B cells. We report the characterization of CD5+ B cells from two unique B-CLL patients. Cells from patient 1 coexpressed CD5 (leu-1), CD19 (Leu-12), CD20 (B1), and HLA-DR; they were CD10 (J5), CD21 (B2), CD22 (Leu-14), CD25 (IL2-R1), PCA-1, surface, and cytoplasmic Ig negative. They suppressed normal peripheral blood lymphocyte (PBL) pokeweed mitogen (PWM) -stimulated immunoglobulin (Ig) synthesis greater than 80%. Cells from patient 2 were CD5 (Leu-1), CD19 (Leu-12), CD20 (B1), CD21 (B2), CD22 (Leu-14), HLA-DR, IgM, and kappa positive. They were negative for CD10 (J5), CD25 (IL2-R1), and PCA-1. These cells did not suppress normal PBL PWM-stimulated Ig synthesis but produced a monoclonal IgM kappa protein with rheumatoid factor-like activity. These observations suggest that there are different CD5+ B cell subsets, one immunosuppressive and the other autoreactive.

Plasma cell antigens in human multiple myeloma.

Tumor-specific antigens capable of eliciting a response from autologous lymphocytes have been described in mouse plasmacytoma systems. This paper presents evidence of similar antigens in human myeloma. Myeloma plasma cells isolated from the bone marrow of 27 patients stimulated autologous and allogeneic peripheral blood lymphocytes (PBL) from myeloma patients in mixed leukocyte culture. Plasma cells isolated fromt the bone marrow of normal patients or patients with benign plasmacytosis failed to stimulate PBL in mixed leukocyte culture. Similar results were found in passive cytotoxicity assays with the use of chinken red blood cells (CRBC) coated with 3M KC1 plasma cell extracts from myeloma patients. PBL from myeloma patients caused 30 to 80 percent chromium-51 release from tanned chromium-labeled plasma cell extract-coated CRBC targets, whereas PBL from normal patients or patients with benign plasmacytosis caused only 10 to 25 percent chromium-51 release. This study indicates the presence of material resembling tumor-specific antigens on human myeloma plasma cells. Immune response to suce antigens is elicited in autologous and allogeneic myeloma patients.

Effects of serial plasmapheresis on serum IgA levels in IgA-deficient blood donors with IgA-suppressor T cells.

Seventeen IgA-deficient blood donors, without antibodies to IgA, underwent plasmapheresis four to eight consecutive times at intervals of 8 weeks or less to provide fresh-frozen plasma for patients with anti-IgA. Blood samples, drawn for analysis no more than 1 hour before plasmapheresis and again at the conclusion of each procedure, were analyzed for lymphocyte subpopulations and serum IgA levels. Five lymphocyte subpopulations, including natural killer cells, the suppressor-inducer CD4 subset, the suppressor-precursor CD8 subset, non-major histocompatibility complex (MHC)-restricted cytotoxic T cells, and CD5+ B cells, were all decreased significantly after plasmapheresis (p less than 0.05). In a subgroup of IgA-deficient donors with excessive IgA-suppressor T-cell activity, serum IgA increased to levels exceeding 0.05 g per L following the fourth consecutive plasmapheresis procedure. Serum IgA levels did not similarly increase in IgA-deficient donors without excessive IgA-suppressor T-cell activity or in controls without IgA deficiency. Our study shows the potential, in a subpopulation of IgA-deficient donors who undergo frequent plasmapheresis, for a transient increase in serum IgA to a level no longer considered IgA deficient.

Removal by white cell-reduction filters of activated platelets expressing CD62.

White cell (WBC)-reduction filters that remove more than 99 percent of the WBCs from platelet concentrates are rapidly being introduced into routine use. Using activation-dependent monoclonal antibodies and flow cytometry, platelet activation was evaluated before and after WBC reduction in 10 platelet concentrates prepared manually from whole blood obtained from five male and five female regular volunteer blood donors. In general no significant increases were found in platelet activation markers after WBC reduction using filters. However, if platelets were activated during preparation, increased numbers of platelets were found expressing the activation marker CD62, and this correlated with the decrease in the platelet count after WBC reduction. These observations may explain increased platelet loss following WBC reduction in some platelet components.

Multiple myeloma: an immunologic profile. Cytotoxic and suppressive effects of the EA rosette-forming cell.

Lymphoid cells bearing receptors for human IgG anti-Rh antibody but not otherwise identifiable as T or B cells were isolated from the peripheral blood of human multiple myeloma patients. These cells do not demonstrate surface immunoglobulin, do not secrete immunoglobulin in culture after stimulation with pokeweed mitogen, and do not possess receptors for the third component of complement. They do not form rosettes with sheep erythrocytes, and do not respond to known T cell mitogens. These cells are not phagocytic and do not possess demonstrable nonspecific esterase. Thus, they must be considered at present, members of the "third population" lymphoid cells. These cells are active in the cytotoxic response by myeloma peripheral blood lymphocytes to myeloma plasma cells accounting for approximately 60% of such activity. They are also responsible for part of the suppression of pokeweed mitogen-stimulated immunoglobulin production, and at equivalent suppressor to target cell ratios are more active than immunoregulatory monocytes. Thus, these cells play an important, not previously widely appreciated role in the pathophysiology of the human myeloma patient.

In vitro cytotoxic response to human myeloma plasma cells by peripheral blood leukocytes from patients with multiple myeloma and benign monoclonal gammopathy.

Peripheral blood leukocytes (PBL) from myeloma patients were studied for their capacity to lyse plasma cells from myeloma patients, benign monoclonal gammopathy (BMG) patients, and nonneoplastic disease patients. Plasma cells were isolated from bone marrow, labeled with 51Cr, and cultured with PBL isolated from patients with myeloma, BMG, or nonneoplastic disease, as well as normal individuals. PBL from patients with multiple myeloma demonstrated responses to autologous or allogeneic myeloma plasma cells. Optimum conditions for cytotoxic response included a responder-to-stimulator ratio of 1:1 and an effector-to-target ratio of 20:1. PBL from normal individuals or patients with BMG failed to demonstrate this response. However, PBL from BMG patients, but not normal individuals, could be induced to kill myeloma plasma cells (but not nonmyeloma plasma cells) by simultaneous stimulation with allogeneic lymphocytes and myeloma plasma cells.