Mechanisms of lymphocyte activation: the role of suppressor cells in the proliferative responses of chronic lymphatic leukemia lymphocytes.

Binding of (125)I-leukoagglutinin (LPHA) to lymphocyte membrane receptors at equilibrium generated similar curvilinear Scatchard plots in 20 patients with bursa-derived (B)-cell-type chronic lymphatic leukemia (CLL) and 15 controls. If biphasic plots are assumed, the two linear components show markedly diminished receptor capacity (15 and 137 ng/10(6) lymphocytes) in CLL as compared to controls (60 and 668 ng). In contrast, affinity was similar in patients (1.0 x 10(8) M(-1) and 2.1 x 10(6) M(-1)) and controls (1.8 x 10(8) M(-1) and 1.5 x 10(6) M(-1)). Highly purified B cells from patients and controls generated binding data comparable to that obtained from the mixed lymphocyte (ML) suspensions from which they originated. Maximal DNA synthesis of highly purified, normal, thymus-derived (T) and B cells in response to LPHA stimulation was comparable to that of ML (mitotic index [MI] 19.9, 20.1, and 23.4, respectively), though B-cell responses were slightly delayed. In CLL the markedly decreased and delayed DNA synthesis by ML (MI 2.3), and by highly purified T (MI 1.6) and B (MI 1.9) cells seemed out of proportion to their decreased receptor capacity for LPHA. The impaired mitogenic responses of leukemic cells from five patients were not enhanced when cocultured with normal lymphocytes. In contrast, cells from eight patients inhibited cocultured normal lymphocyte responses to LPHA by 94.3%. Sera from these patients and supernates from their cultured cells did not mediate this suppressor effect. These observations indicate that the decreased DNA synthesis observed in CLL is not an attribute of B cells and does not represent the expected response of a few residual normal T lymphocytes, but rather reflects impaired responses by all CLL cells. The defect does not relate to the density or function of membrane receptors for LPHA, to the presence of inhibitors in these patients' sera, or to depletion of helper T cells. Our data strongly suggest that one mechanism for the immunoincompetence observed in CLL reflects excessive suppressor-cell activity.

Quantitation of immunocompetence in Hodgkin's disease.

In vitro cellular immunocompetence was investigated on 35 patients with Hodgkin's disease by studying their in vitro lymphocyte responsiveness to full range stimulation achieved by a spectrum of phytohemagglutinin concentrations. When compared to the normal lymphocyte profile elicited from 35 control subjects, the Hodgkin's patterns of response enabled the identification of a quantifiable lymphocyte defect present in most patients regardless of their clinical status. Increasing severity of this defect was found with progression of the disease and was most pronounced in patients with skin anergy and absolute lymphopenia. The marked abnormality observed in patients restudied after intensive therapy returned towards normal in patients achieving a long lasting, unmaintained complete remission. The data suggest the early presence of an intrinsic functional lymphocyte defect, increasing severity of which may lead to progressive immunoincompetence, reflected to vitro by imparied lymphocyte responsiveness and in vivo by skin anergy and ultimately lymphopenia.

Binding interaction studies of selected receptor subpopulations after partial cross-linking receptor-ligand complexes with a photoactivated heterobifunctional reagent.

Certain hormonal and nonhormonal binding systems such as the leukoagglutinin-lymphocyte model exhibit complex receptor-ligand interactions that result in nonlinear Scatchard plots. Such plots are interpreted as indicating either homogeneous negatively interacting binding sites or heterogeneous sites with different and fixed affinity. We assessed the validity of these interpretations in our system by conjugating the ligand to a photoactivated heterobifunctional agent and cross-linking the conjugate to a subset of receptors before studying the binding interactions of non-cross-linked sites. Conjugation did not qualitatively or quantitatively affect the binding properties of the ligand. Cross-linking was specific, efficient, and stable and had no effect on irrelevant surface receptors. Cross-linking of only 3% of the total receptors resulted in 50% decreased ligand binding to high affinity sites consistent with a calculated inactivation of 85% and 2% of high and low affinity sites, respectively. Such preferential inactivation of high affinity sites in an unequivocal demonstration of binding sites heterogeneity in this system and shows a clear rejection of the homogeneous cooperation model.

Nutritional requirements of human malignant (leukemic) cell lines: implications for adjuvant therapy.

Metabolic requirements of malignant cell lines derived from patients with chronic myelogenous and acute lymphoblastic leukemias were compared to those of proliferating normal cells (mitogen-stimulated human lymphocytes) and circulating blasts from acute myeloblastic and acute lymphoblastic leukemias. Requirements were judged by degree of amino acid (AA) utilization in short-term cultures and assessed by the effect of selective AA deprivation on cell growth. Cell growth was measured by DNA synthesis and growth rate analysis. Six AAs (serine, threonine, methionine, valine, phenylalanine, and lysine) were appreciably utilized (52-87%) by IM-9, CEM, MOLT-4, and K-562 cells, but little or no utilization of these or any other AAs were noted in HSB cells, in leukemic blasts, or in mitogen-stimulated normal lymphocytes in short-term culture. Omission of lysine from culture media greatly inhibited cell growth (DNA synthesis by 91%), and cell density (by 83%) of IM-9 cells. However, omission of lysine, valine, serine, threonine, methionine, or phenylalanine had less of an effect on CEM and MOLT-4 cell lines. These observations demonstrate that under the conditions used the IM-9 cell line is uniquely dependent on extracellular lysine levels in contrast to the other cell lines studied. This suggests that human malignancies other than acute lymphoblastic leukemia which exhibits an obligate dependence on extracellular asparagine might be manageable by enzymatic degradation in vivo or by dietary restriction of indispensable AAs.

Chronic lymphocytic leukemia: an updated review.

PURPOSE: To review recent advances in the pathogenesis, biology, diagnosis, and management of chronic lymphocytic leukemia (CLL). DESIGN: A literature search restricted to English-language articles, abstracts, book chapters, and reports published between 1975 and 1993 was conducted both electronically using MEDLINE and CANCERLIT and manually using the bibliographies of the electronically retrieved data base. Of approximately 1,000 publications identified for analysis, 233 were selected as representative of important advances in CLL. RESULTS: The last 10 years have witnessed renewed interest in the biology and treatment of CLL, a disease long viewed by clinicians as following an indolent course and perceived by investigators as uninspiring. This interest, based on advances in understanding the lineage and biology of CLL and on the advent of new and more efficacious chemotherapeutic agents, has led to improvements in patient survival and, for the first time, to complete remissions (CRs) in large subsets of patients. As we learn to use these agents better, especially in combination chemotherapy, alternative therapeutic modalities, are being vigorously pursued, including high-dose ablative chemotherapy with allogeneic or autologous bone marrow transplantation (BMT) rescue and the use of powerful tumor-cell target-specific immunoreagents. These therapeutic advances, coupled with the recent availability of molecular probes to ascertain objectively minimal remnant disease posttreatment, provide a basis for developing and assessing ever more efficacious and potentially curative treatment strategies for the management of this disease. CONCLUSION: For the first time since the original 1924 monograph by Minot and Isaacs, the cure of subsets of patients with CLL appears not to be an unreasonable goal.

Drug-induced liver injury. In vitro demonstration of hypersensitivity to both phenytoin and phenobarbital.

Fever, lymphadenopathy, exfoliative dermatitis, and evidence of drug-induced liver injury developed in a 16-year-old girl three weeks after beginning therapy with phenytoin and phenobarbital. This clinical syndrome can be caused by either of these structurally related drugs but has been more frequently attributed to phenytoin. In vitro studies disclosed marked reactivity of this patient's lymphocytes to concentrations of both drugs, which encompassed their measured serum levels. The demonstration of dual reactivity raises concerns about continuing administration of phenobarbital during an apparent phenytoin-induced reaction. Whether this potential risk is greater than the risk of stopping all anticonvulsant medications in a patient with a seizure disorder is not known and remains to be established.

Quantitation of erythropoietin stimulatory activity using [3H]thymidine uptake by K562 cells.

A microassay for erythropoietin (Ep) activity in serum using [3H]thymidine uptake by K562 cells is presented. The method is similar to that of Krystal except that cells of the K562 human pluripotent leukemia cell line replace spleen cells from phenylhydrazine-treated anemic mice. Response to the hormone by K562 cells and spleen cells was colinear. Using the Krystal bioassay, 14 young hemoglobin S homozygotes had Ep activity levels of 17.9-113.8 mU/ml serum, whereas the new method with K562 cells gave a range of 19.2-115.3 mU/ml. The correlation coefficient between the two sets of data (r) was 0.999 (p less than 0.001). With the modified technique we have assayed 34 sickle cell patients, whose sera ranged from 19.2 to 1400 mU of Ep/ml with corresponding hemoglobin concentrations of 10.7 g % to 3.0 g %. Values for normal subjects were 22.1 +/- 2.1 mU/ml (n = 7). The stimulation of [3H]thymidine uptake is significantly inhibited by an anti-Ep antiserum. The assay permits quantification of stimulatory activities in a large number of samples with relative ease and is also suitable to explore the interactions of erythropoietic factors with their appropriate receptors on stem cells.

A simple technique for the rapid enrichment of class and subclass hybridoma switch variants. A 1000-fold enrichment in half the time, for half the cost.

Switching parental hybrids in vitro to downstream switch variant clones producing more desirable monoclonal antibodies (MoAbs) requires either labor intensive and time consuming subcloning techniques, or fluorescence activated sorting of the desired clones. We tested the hypothesis that enrichment of downstream switch variant clones might be achieved by selective lysis of upstream hybridoma cells followed by expansion of the enriched downstream clone. Using a parental hybridoma with surface and secretory IgM, we attempted to enrich downstream switch variant clones producing class (IgG) and subclass (IgG1 or IgG2a) MoAbs. Enrichment of downstream IgG, IgG1 and IgG2a MoAb-producing switch variants was achieved by single or repeated antibody-dependent, complement-mediated lysis of the upstream IgM-bearing parental hybridoma cells followed by limited subcloning. Two exposures of parental hybridoma cells to lysis followed by plating at 100 cells/well enriched the frequency of switch variants up to 1235-fold, enabling the development of IgG1 or IgG2a-producing subclones exhibiting high yield antibody production. Using this protocol, production time and costs were reduced by > 50% when compared to the standard technique. This novel technique for the rapid isolation and expansion of switch variant clones should be ideal for most laboratories, particularly those without access to cell sorting capabilities.

Immunologically diagnosed malignancy in Sjögren's pseudolymphoma.

Studies of lymphocyte markers in a patient with Sjögren's syndrome who exhibited histologically benign lymphoproliferation in the lung revealed a malignant cell clone. T and B cells were quantitated according to their ability to form spontaneous rosettes with sheep erythrocytes and to fluoresce with fluorescein-conjugated antiserums, respectively. Circulating lymphocytes were 66 percent T cells (N = 58 +/- 2 per cent) and 14 percent B cells (N = 22+/- 1 percent), the latter exhibiting normal polyclonal distribution of membrane immunoglobulins. However, lymphocyte suspensions obtained from fresh lymph node and from biopsy specimens from a lymphoid lung nodule revealed 95 percent and 88 percent B cells, with 1 percent and 2 percent T cells, respectively. Moreover, when cryostat-frozen sections from both tissues were reacted with each of the heavy and light chain-specific antiserums, most cells demonstrated the presence of intracytoplasmic mu kappa immunoglobulin exclusively. Twenty-two months later, a clinically and histologically classic lymphoma developed. Repeat marker studies performed on cells freshly isolated and on frozen sections from the histologically malignant lymph node revealed persistence of the monoclonal marker on most cells.

CD20 and CD5 expression in B-chronic lymphocytic leukemia.

In order to quantitate a previously noted decrease in CD20 fluorescence intensity (FI) on B-CLL lymphocytes, binding capacities [BC x 10(3) +/- 1SD = number of antibodies bound per cell] were calculated. The mean (N = 5) BC x 10(3) +/- 1SD of CD20 reagents for normal B-PBL and B-CLL lymphocytes confirmed this observation. B-PBL and B-CLL were 56 +/- 11 and 61 +/- 14, and 19 +/- 15 and 18 +/- 16, respectively, for Leu 16 and B1. Although adequate compensation standards for the determination of CD5 and CD20 coexpression are not available, qualitatively, the density of CD5 on both normal B-PBL and B-CLL is less compared to the expression of CD5 by normal T cells. CD5 expression on B-CLL seems to be linked to the lower levels of CD20, whereas CD5 expression may appear to be absent on CLL lymphocytes expressing normal levels of CD20. Levels of CD20 in B-CLL suggest involvement of one or two genes (alleles) whose decreased expression may be linked to CD5 expression.

Radiation-induced clastogenic plasma factors.

Ionizing irradiation induces chromosomal aberrations in directly exposed cells and is known to have mutagenic and carcinogenic potential for the exposed host. Under controlled conditions, we examined whether such clastogenic effects of irradiation might be due in part to radiation-induced plasma factors. Irradiated cells and sera from CF-Nelson rats were used at 15 min, and 1, 7, 14, and 56-70 days after total body irradiation (250 R, n = 67 or 400 R, n = 39). Control rats (n = 44) served as donors of nonirradiated sera and cells. In addition, sera from six rats were irradiated (250 R or 400 R) in vitro. On the average, 298 metaphases from six rats were studied at each time-point. Cytogenetic abnormalities observed included chromatid- and chromosome-type lesions and hyperdiploidy. The frequency of abnormalities was comparable at both radiation doses. Nonirradiated cells exposed in vitro to irradiated serum (15 min postirradiation) exhibited a 36- to 48-fold increment in hyperdiploidy (p = 0.0001) and a 2.- to 2.2-fold rise in chromatid gaps and breaks (p less than 0.01), but none of the chromosome-type aberrations seen in cells exposed to radiation. The clastogenic activity of irradiated plasma persisted in circulation for the 10-wk duration of the study and was not abrogated by dilution with nonirradiated serum. Serum irradiated in vitro was not clastogenic. This study shows that irradiation of rats results in the prompt appearance of clastogenic activity in their plasma. This activity is not due to radiation-induced depletion of protective factors nor to chemical-physical changes of normal plasma components, but results from circulating factors released by irradiated cells.

Monoclonal antibody production to human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2.

Monoclonal antibodies against human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.

Hodgkin's disease: basing treatment decisions on prognostic factors.

The purpose of this study was to review the current status of risk factor assessment in Hodgkin's disease (HD) clinically useful for managing this disease. Regarding database retrieval and selection a literature search restricted to English-language articles, abstracts, book chapters and reports published between 1980 and 1993 was conducted both electronically using MEDLINE and CANCERLIT and manually using the bibliographies of the retrieved database. Out of approximately 500 publications identified for analysis, 34 were selected as illustrative. Results showed that most patients with Hodgkin's disease are curable at the outset with standard radio- or chemo-therapy, depending on stage and other risk factors. However, up to 40% of patients will either fail at initial induction, or experience early or late relapses. Risk factors analysis at these various times provide a solid base for selecting the therapy best suited to optimize outcome for each individual patient. Patients with truly refractory disease pose a serious challenge to clinicians and are best managed in specialized centers conducting controlled clinical trials. In conclusion it appears that although approximately 75% of newly diagnosed patients with HD can expect long-term, disease-free survival, refractory patients exhibit a dismal survival. Improving their outcome will require innovative approaches.

The chronic lymphocytic leukemia antigen (cCLLa) as immunotherapy target: pharmacokinetics and biodistribution of two divalent, ricin-based immunotoxins in xenografted athymic mice.

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is potentially suitable for targeted immunotherapy given its restriction to clonal CLL cells and lack of expression by normal lymphocytes. In order to assess the pharmacokinetics and biodistribution of two potent anti-cCLLa immunotoxins (ITs) were examined in the mouse model. The IgG fraction of anti-cCLLa monoclonal antibody CLL2m was conjugated with 125I-labeled intact (RTA) or deglycosylated (dgA) ricin chain A, injected intravenously into athymic mice engrafted with cCLLa-expressing human tumors, and monitored over 120 hours. Blood concentrations of CLL2m/125I-RTA and CLL2m/125I-dgA were best fit to biexponential equations but the latter exhibited a lower alphaT1/2 and betaT1/2 (4.1 and 102 min vs 5.9 and 126 min), a smaller volume of distribution (5.1 g vs 9.7 g), and a lower blood clearance (2.2 g/hr vs 4.6 g/hr). Both ITs exhibited preferential tumor uptake that followed distinct kinetics: rising tumor uptake for 2 hrs post-injection (while tissue uptake decreased), reaching tumor/non-tumoral tissue uptake ratios up to 16.9; and slower dissociation rates of tumor- vs tissue-bound ITs (>45% vs <20% remaining tissue-bound 6 hrs post-injection, respectively). Non-specific liver uptake was not prominent for either IT. In vivo IT deconjugation reached 50% approximately 12 hours pos-injection. The pharmacokinetics and biodistribution data in the mouse model suggest that ricin-based anti-cCLLa ITs are suitable for use in human trials.

The chronic lymphocytic leukemia antigen (cCLLa) as immunotherapy target: assessment of LD50 and MTD of four ricin-based anti-cCLLa immunotoxins (ITs) in Balb/c mice.

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is a promising immunotherapy target given its disease-restricted expression, its highest prevalence among CLL surface antigens, and its lack of expression by normal T- and B-lymphocytes. The objectives of this study were to assess the 50% lethal dose (LD50) and the maximum tolerated dose (MTD) in Balb/c mice of four anti-cCLLa immunotoxins (ITs) derived from the intact monoclonal antibody (MoAb) or its Fab fraction, each conjugated to either ricin chain-A (RTA) or its deglycosylated derivative (dgA). The IgG fraction of anti-cCLLa monoclonal antibody CLL2m and its Fab fraction were conjugated to RTA or dgA to generate four ITs: IgG/RTA, IgG/dgA, Fab/RTA and Fab/dgA. Progressive concentrations of each IT (ranging between 2.60 mg/kg and 100.00 mg/kg) were injected intravenously into groups of 5 mice each. After injection, mice were monitored daily for 10 days for survival. Observed mortality data in each group were matched to those in Weil's tables for estimating LD50 (mg/kg) from the moving average interpolation method. Estimated LD50 (in mg/kg) were: IgG/RTA, 13.33; Fab/RTA, 25.53; IgG/dgA, 55.33; Fab/dgA, 55.33. Their respective MTD (mg/kg), defined as the highest dose level survived by all mice, were 8.78, 13.17, 29.63 and 29.63. Depending on the animal-to-human extrapolation method used, the calculated LD50 and MTD in humans ranged from 1.2 mg/kg and 0.8 mg/kg (IgG/RTA), to 55.6 mg/kg and 36.9 mg/kg (IgG/dgA and Fab/dgA), respectively. The following conclusions are drawn. 1. Antibody valence exerted little influence on either the LD50 or the MTD; 2. The LD50 to MTD ratios were approximately 2:1; 3. dgA-derived ITs were approximately one half as toxic as their RTA-derived counterparts; and 4. Extrapolation of LD50 and MTD mouse data to humans resulted in dose levels comparable to or exceeding those reported in most IT human trials. These data suggest the suitability of anti-cCLLa ITs for clinical immunotherapy trials.