An interlaboratory study of a candidate reference method for platelet counting.

A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio. A multicenter comparison was performed to determine whether the method met the necessary criteria and was precise enough to be recommended as a new reference method. Each laboratory analyzed serial dilutions of normal specimens, stabilized material, and at least 60 patient specimens with a range of platelet counts from 1 to 400 x 10(3)/microL (1-400 x 10(9)/L). Pooled analysis of the serial dilutions showed that RBC-platelet and RBC-RBC coincidence events became negligible at sufficiently high dilutions (i.e., > 1:1,000). All laboratories demonstrated excellent intra-assay and acceptable interlaboratory precision. Two antibodies (CD61 and CD41) were used for identifying platelets and individually gave acceptable results, but in a minority of samples, staining differences were observed. The optimum method thus uses a double-labeling procedure with a final dilution factor of 1:1,000. The study demonstrated that this method meets the criteria for a reference platelet count.

Automated quantitation of hemoglobin-based blood substitutes in whole blood samples.

It is necessary to develop methods for accurate monitoring of cell-free hemoglobin in circulation. Routine monitoring of circulating cell-free hemoglobin will be useful for evaluating the efficacy of blood substitute administration andfor determining the clearance rates of the blood substitute from circulation. In addition, discriminating between cell-free hemoglobin and cell-associated hemoglobin will enable accurate determination of RBC indices, mean cell hemoglobin and mean corpuscular hemoglobin concentration, in individuals receiving hemoglobin-based blood substitutes. As colorimetric methods used by hematology analyzers to quantitate the hemoglobin value of a blood sample cannot distinguish between cell-associated and cell-free hemoglobin, it is currently not feasible to quantitate the levels of hemoglobin substitutes in circulation. The advent of a technology that measures volume and hemoglobin concentration of individual RBCs provides an alternative strategy for quantitating the cell-associated hemoglobin in a blood sample. We document that the combined use of cell-based and colorimetric hemoglobin measurements provides accurate discrimination between cell-associated and cell-free hemoglobin over a wide range of hemoglobin levels. This strategy should enable rapid and accurate monitoring of the levels of cell-free hemoglobin substitutes in the circulation of recipients of these blood substitutes.

Improved platelet counting using two-dimensional laser light scatter.

Clinical management of platelet disorders depends on accurate platelet counts. We evaluated a new analytic approach for platelet counting based on improved platelet discrimination. Current automated counting methods provide accurate platelet counts for most samples but often are unable to discriminate platelets accurately from nonplatelet particles such as microcytic RBCs, RBC fragments, and cellular debris that may falsely elevate platelet counts. The new approach measures 2 light-scatter angles of platelets and nonplatelet particles as they pass through a laser beam. The volume and refractive index of each platelet and particle are derived from the light-scatter measurements using the Mie scattering theory. Together, these 2 measurements provide improved platelet discrimination compared with 1-dimensional methods. With its improved discrimination, 2-dimensional platelet analysis provides more accurate platelet counts in samples containing interfering particles and may contribute to more effective clinical management of patients with platelet disorders.

[Near infrared spectroscopy: a noninvasive optical method for monitoring cerebral oxygenation and hemodynamics]. / Spektroskopia w bliskiej podczerwieni--nieinwazyjna metoda optyczna monitorowania oksygenacji i hemodynamiki mózgu.

The Near Infrared Spectroscopy (NIRS) has been established as a non-invasive method for monitoring the oxygenation state of human brain at the bedside. This possible by observing spectral changes in the tissue caused by presence of oxygenated hemoglobin, deoxygenated hemoglobin and cytochrome aa3. In this paper the technical data of the NIRS instrument and its clinical application particularly in investigation of cerebral oxygenation and hemodynamic has been described.

Modification of cytokine production by piroxicam.

OBJECTIVE: Nonsteroidal antiinflammatory drugs have been thought to act by inhibiting the enzyme, cyclooxygenase (prostaglandin H synthetase). We sought to demonstrate additional biologic actions of this class of drugs including effects on cytokine production. METHODS: We administered the nonsteroidal antiinflammatory drug piroxicam 20 mg to 6 healthy volunteers daily for 7 days. Before and for one week after drug administration, concentrations of interleukin 1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha (TNF alpha) and interferon-gamma (IFN-gamma), produced by anti-CD3 stimulated peripheral blood mononuclear cells, were measured. RESULTS: Piroxicam treatment resulted in elevation of levels of IL-2, depression of IL-1, IL-6, TNF alpha and IFN-gamma, and no consistent effect on IL-4. CONCLUSION: Piroxicam modulates production of various cytokines in a complex fashion when administered to healthy individuals.

[Electrophysiologic methods in differential diagnosis of syncope]. / Róznicowa diagnostyka elektrofizjologiczna stanów o symptomatologii synkopalnej.

The purpose of the study was the assessment of the diagnostic possibilities and usefulness of 24-hour cassette EEG/ECG recording in the differential diagnosis of short attacks of consciousness disturbances and/or syncopal states, and demonstration of interrelations between arrhythmias and seizures of cerebral origin. 24-EEG/ECG was obtained in 71 patients, including 34 with cardiological history who had above disturbances. Recording of 24-EEG/ECG made possible establishing of correct diagnosis in 15 cases (21.1%): in 1 case it was sinus arrest, in 14--epilepsy. In another 5 cases (7%) arrhythmia or conduction disturbances were found which could have been the case of their attacks, they all were patients with cardiological history. The analysis of EEG/ECG records of 47 epileptic seizures in 16 patients showed that in 11 of them (69%) the seizures were associated with sinus tachycardia without other arrhythmias. These results demonstrated the complex cause-effect relationship of cardiocerebral disorders in the aetiology of these states, and the usefulness of 24-EEG/ECG in their detection.

Immunosuppression by glucocorticoids: inhibition of production of multiple lymphokines by in vivo administration of dexamethasone.

Glucocorticoids are extremely potent immunosuppressive agents, capable of directly affecting the function of lymphocytes. We studied the effect of in vivo dexamethasone (DEX) administration on anti-CD3-induced lymphocyte proliferation and lymphokine production in mice. To characterize the kinetics and dose responsiveness of lymphocytes to DEX, splenocytes from BALB/c mice that had received a single dose of DEX in vivo were cultured in vitro with suboptimal and optimal concentrations of anti-CD3 monoclonal antibody. Cell proliferation in response to suboptimal concentrations of anti-CD3 was decreased by DEX doses of > or = 30 mg/kg; much higher doses (> or = 200 mg/kg) were required to inhibit cell proliferation in response to optimal anti-CD3 stimulation. Inhibition of suboptimal anti-CD3-stimulated proliferation was evident within 4 hr after DEX administration, was maintained for at least 24 hr, and was no longer evident at 7 and 14 days. Lymphokine secretion induced by optimal doses of anti-CD3 in vitro was differentially affected by in vivo DEX treatment. IL-1 alpha, IL-4, IL-6, IL-10, and IFN-gamma levels were decreased by treatment with low doses of DEX (30 mg/kg), whereas higher doses were required to inhibit production of IL-2, IL-3, and TNF. GM-CSF (granulocyte-macrophage-colony stimulating factor) was least susceptible to DEX inhibition. Low-dose (30 mg/kg) DEX treatment significantly reduced anti-CD3-stimulated production of most lymphokines tested at 4 and 12 hr; by 24 hr the levels of most lymphokines had begun to return to control values. Hence, our data indicate that administration of a single dose of DEX (30 mg/kg for 4 hr) results in significant suppression of lymphokine production and cell proliferation that precedes any significant cell loss and can be used as a reversible model of immunosuppression.

[Diagnostic value of 24-hour simultaneous EEG and ECG monitoring of patients with heart diseases and atypical consciousness disorders]. / Znaczenie diagnostyczne calodobowego jednoczasowego monitorowania EEG i ECG u osób z chorobami serca i nietypowymi zaburzeniami swiadomosci.

In 24 patients with diagnostically not clear, short, recurrent episodes of consciousness disturbances and heart diseases and/or a history of arrhythmia simultaneous 24-hour recording was done of eeg and ecg. In the differential diagnosis epilepsy was considered, especially since in most cases routine eeg records demonstrated slight episodic changes. During 24-hour recording in 8 cases typical episodes of consciousness disturbances developed but in none of them these episodes were associated with arrhythmia which ruled out their cardiogenic origin. In 2 cases EEG recording served for establishing the diagnosis of partial complex seizures, 2 patients had hyperventilation syncope, one had TIA, in the remaining 3 cases absence of eeg and ecg changes during these episodes and coexistence of anxiety neurosis suggested functional origin. So the combined 24-hour eeg+ecg recording made possible establishing of diagnosis in 1/3 of these patients, enabling adequate treatment to be instituted.

[2 cases of Sneddon syndrome]. / Dwa przypadki zespolu Sneddona.

Two cases of Sneddon syndrome (S.s.) in a 33 and 53-year-old women who developed arterial hypertension, cerebral ischaemic signs and who have livedo reticularis or livedo racemosa, are reported. The authors describe clinical, radiological and biological features of this rare disease, as well as diagnostic investigations including the measurements of the antiphospholipid antibodies (APA). The possible role of APA in the pathogenesis of S.s. is discussed.

Hybridoma-derived human suppressor factors: inhibition of growth of tumor cell lines and effect on cytotoxic cells.

With the objective of developing human T-T cell hybrids producing B-cell growth factor, we fused concanavalin A-activated T lymphocytes with cells of the Jurkat T cell line. The hybrids were selected on the basis of their ability to form colonies in soft agar, whereas the parent Jurkat T cell line did not. T-T cell hybrids were HLA-typed, screened by functional tests, and recloned by limiting dilution. In addition to obtaining B-cell growth factor-producing hybrids, we also obtained certain other T-T cell hybrids (as determined by HLA-typing) producing suppressor factors inhibiting proliferative responses and antibody production by human lymphocytes. Subsequently, a suppressor factor with similar inhibitory properties was identified in supernatants of the Jurkat T cell line. However, the Jurkat factor exhibited different biochemical and functional properties than the hybridoma-derived suppressor factors. Using two-parameter cell cycle analysis and the metachromatic fluorochrome acridine orange, we found that the hybridoma-derived 160 and 169 suppressor factors arrested phytohemagglutinin-induced proliferative of peripheral blood mononuclear cells in the G0/G1 phase of the cell cycle, whereas the Jurkat suppressor factor arrested proliferation in the S phase. Incubation of peripheral blood mononuclear cells with the 160, 169, or Jurkat suppressor factors for 24 hr at 37 degrees C, followed by washing, did not alter their cell cycle progression (or RNA content) in response to stimulation with phytohemagglutinin. The hybridoma-derived 160 and 169 suppressor factors and the Jurkat factor inhibited the growth but not the viability of cells from the following human tumor cell lines: A673 sarcoma cell line, SK-LC-6 and SK-LC-14 lung cell lines, SB, Raji, and Daudi lymphoblastoid cell lines, and FARR malignant melanoma cell line. In contrast, it did not affect the growth of murine L1210 cells and FS-4 normal human diploid fibroblasts. The hybridoma-derived 160 suppressor factor was selected to investigate its effect on cell-mediated cytotoxicity. The 160 suppressor factor did not inhibit natural killer cytotoxicity or its augmentation by interferon alpha or interleukin 2 or the generation of lymphokine-activated killer cells. However, this factor partially inhibited the generation of specific T cell-mediated cytotoxicity.

Phase I evaluation of a combination of monoclonal antibody R24 and interleukin 2 in patients with metastatic melanoma.

A combination of recombinant human interleukin 2 (rhIL-2) and mouse monoclonal antibody R24 (recognizing the ganglioside GD3) was evaluated in patients with metastatic melanoma in a phase I trial. rhIL-2 was given at a constant daily dose of 1 x 10(6) units/m2 i.v. over 6 h on days 1-5 and 8-12. R24 was given on days 8-12 at four dose levels (1, 3, 8, and 12 mg/m2 daily). Twenty patients were evaluable for toxicity and response, five at each dose level. The toxicity of the combination was not overlapping and generally mild. There was a rebound peripheral blood T-lymphocytosis at the end of treatment increasing with the dose of R24. The median lymphocyte count on day 12 of treatment was 3108 +/- 554/ml in patients treated at R24 doses of 8 and 12 mg/m2 versus 2239 +/- 672/ml at doses of 1 and 3 mg/m2. This evidence and other data suggested that R24 enhanced IL-2-mediated T-cell activation in vivo. Two patients demonstrated increases in R24-mediated antibody-dependent cellular cytotoxicity for GD3-expressing cells during treatment. rhIL-2 appeared to accelerate the development of human anti-mouse antibody; three patients developed human anti-mouse antibody by the fifth day of R24 treatment, earlier than observed in prior studies using R24 alone and one patient during the first week of rhIL-2 alone, prior to R24 treatment. One patient had a partial response in soft tissue sites lasting 6 months and two patients had minor responses. This clinical trial extends the previous observation that R24 enhances lymphocyte proliferation in vitro.

Caffeine increases sensitivity of DNA to denaturation in chromatin of L1210 cells.

Exposure of exponentially growing L1210 cells to 5 mM and higher concentrations of caffeine perturbs their progression through the cell cycle and results in increased sensitivity of DNA in situ to denaturation. The latter is detected by the increased metachromatic stainability of DNA with acridine orange (AO) and sensitivity to S1 nuclease, measured by flow cytometry. Decreased DNA stability is generally characteristic of chromatin condensation and in untreated cells is observed in mitosis or quiescence (G0). The caffeine-induced decrease in DNA stability affects the interphase cells regardless of their position in the cycle and the changes are stochastic, concentration- and time-dependent. Populations of cells responding to caffeine are very heterogenous with respect to the degree of destabilization of DNA; sensitivity of DNA to denaturation of the maximally affected cells is similar to that of untreated cells in mitosis. The present method allows one to quantitatively express effects of caffeine on nuclear chromatin in individual cells of large cell populations and may be employed in studies correlating chromatin changes induced by this agent with its effects in modulation of cell sensitivity to radiation or antitumour drugs.

DNA in situ sensitivity to denaturation as a marker of human breast tumors.

DNA content and in situ sensitivity to denaturation were analyzed by flow cytometry of individual cell nuclei isolated from 40 breast carcinomas, nine fibroadenomas, and 14 samples of normal breast tissue. The extent of DNA denaturation induced by acid was expressed as alpha t, which represents the fraction of DNA staining metachromatically red with the fluorochrome acridine orange. In all cases of normal breast tissue DNA was very sensitive to denaturation and the frequency distribution of alpha t values was unimodal with over 90% of cells having alpha t above 0.6. All fibroadenomas were diploid; four had unimodal alpha t as in normal tissue and five had a bimodal distribution with an additional peak below 0.6. Twenty-seven adenocarcinomas (67%) had a DNA index above 1.0; of these 24 had bimodal alpha t distributions. Among 13 diploid carcinomas 10 had bimodal alpha t distributions. Statistically significant differences were observed in alpha t distributions of normal versus tumor breast tissue (P less than 0.005). In normal tissue and in all tumors a predominant proportion of cells with S and G2 + M DNA content were characterized by DNA resistant to denaturation (alpha t below 0.6). Of interest, the diploid cells from aneuploid tumors which may represent reactive host cells often displayed bimodal distributions of alpha t. These results may be interpreted in light of earlier studies demonstrating increased resistance of DNA to denaturation in diffuse chromatin of proliferating and/or transcriptionally active cells, and greater sensitivity to denaturation of DNA in condensed chromatin of quiescent cells. Thus, the presence of the second peaks representing cells with low alpha t values in breast tumors may indicate a high proportion of proliferating cells, whereas high alpha t populations may represent quiescent and differentiating (condensed chromatin) or dying (pycnotic nuclei) cells. It is likely that the low alpha t diploid cells detected in aneuploid tumors may represent the reactive (transcriptionally active and/or proliferating) infiltrating host cells (i.e., lymphocytes, monocytes) whose presence may also be of prognostic value. The data suggest that a DNA denaturability assay may be useful to characterize tumor and infiltrating host cell populations.

[A minor epidemic of pertussis cough in Brno]. / Mensí epidemie pertusoidního kasle v Brne.

In the course of six months the authors investigated 32 children aged 1-32 months with pertussoid cough. This number included one child where pertussis was confirmed in a 25-month-old properly vaccinated child and parapertussis in an 8-month-old incompletely vaccinated infant. In the remaining children by cultivation or serological examination a different aetiology was found (RS virus, adenovirus, parainfluenza 2, M. pneumoniae, H. influenzae, Branhamella catarrhalis).

Effect of swainsonine on stimulation and cell cycle progression of human lymphocytes.

The indolizidine alkaloid swainsonine (SW) is an inhibitor of lysosomal alpha-mannosidase reported to have antimetastatic activity in animal models. The cells grown in its presence develop truncated (hybrid) surface oligosaccharides that may alter their functional properties dependent on interactions of various ligands with membrane receptors. In the present study we observe that SW enhances stimulation of human lymphocytes induced by suboptimal concentration of concanavalin A. The enhancement is manifested by an increased proportion of cells undergoing transition from G0 (G1Q) to G1 and progressing through the cell cycle (S + G2 + M). In contrast, SW suppresses stimulation of lymphocytes by phytohemagglutinin, and the degree of suppression is greater when measured by the number of cells progressing through the cell cycle (S + G2 + M) than by the proportion of cells entering G1 phase. The suppression remains evident even when SW is added 12 h after phytohemagglutinin, suggesting that SW modifies membrane receptors that develop in G1 and are necessary for cell entrance to S phase. The modification of receptors by SW thus up-regulates stimulation by concanavalin A and down-regulates stimulation by phytohemagglutinin. SW has no effect on lymphocyte stimulation induced by OKT3 monoclonal antibody or on the progression of cells from three leukemic cell lines, HL-60, L1210, and MOLT-4, through the cell cycle. The present data are compatible with the possibility that the reported suppression of the growth of metastatic mouse tumors by SW may be due to the immunomodulatory properties of this alkaloid.

Human suppressor factors constitutively produced by T-T cell hybridomas: functional and biochemical characterization.

We have recently developed a new method (Hybridoma 6:589, 1987) for the generation of human T-T cell hybrids. This method is based on a new selection procedure that involves cloning the hybrids in soft agar, screening by HLA-typing or appropriate functional tests and recloning by limiting dilution. T-T cell hybrids were separated from the parent line on the basis of their ability to form colonies in soft agar, whereas the parent lymphoblastoid T cell lines did not. HAT medium was not used in our selection procedure. Using this method, we have succeeded in developing human T-T cell hybrids (as determined by HLA-typing) constitutively producing B cell growth factor (BCGF) (Hybridoma 6:589, 1987) or suppressor factors. These hybrids were obtained by fusing MLC or Con A T cell blasts with cells from the Molt 4 or Jurkat lymphoblastoid T cell lines. T-T cell hybridomas, derived by fusing Con A-stimulated lymphocytes with cells from the Jurkat T cell line, produced suppressor factors inhibiting: (1) proliferative response in vitro of human peripheral blood mononuclear leukocytes to mitogens and to allogeneic cells in mixed lymphocyte culture; and (2) immunoglobulin synthesis and secretion by mononuclear leukocytes in the PWM-induced differentiation system in vitro. A suppressor factor with these inhibitory properties was also identified in supernatants of the Jurkat T cell line. These suppressor factors were ammonium sulphate precipitable, pH 2 labile, non-dialyzable and they were inactivated by treatment at 56 degrees C for 30 minutes. They exhibited a molecular weight in the range of 50,000-70,000, as determined by gel filtration, and were not gamma or alpha interferon or lymphotoxin/TNF. They did not lyse human lymphoblastoid tumor cell lines nor did they affect the viability and cell numbers of human mononuclear cells even after prolonged incubation (88 hr). They appeared to be cytostatic rather than cytotoxic molecules. The Jurkat suppressor factor is different from those produced by the hybrids on the basis of: (a) different isoelectric points; and (b) the ability of the Jurkat factor to arrest proliferation to PHA of human mononuclear cells in the S phase, whereas the 160 and 169 factors arrest proliferation at the G1 phase of the cell cycle. Certain of these suppressor factors (produced by the hybrids 153, 160, 170, and the Jurkat T cell line) also inhibited proliferative responses of mouse lymphocytes in vitro. In contrast, suppressor factors produced by the 169 and 77 hybrids did not inhibit any murine responses.

Hybridoma-derived human suppressor factors: differential effects on mouse lymphocytes.

We have recently identified a family of suppressor factors produced by certain human T-T cell hybridomas that we developed (references 1 and 2) and by the Jurkat T cell line. These suppressor factors significantly inhibited proliferative responses to mitogens and allogeneic cells in mixed lymphocyte culture and antibody production by human peripheral blood mononuclear cells. We investigated and report here the effect of these suppressor factors on certain in vitro murine immune responses. Suppressor factors produced by certain of these hybrids, such as 153, 160, 170 and by the Jurkat T-cell line were able to inhibit: (1) proliferative responses to mitogens of mouse thymocytes and splenocytes; (2) proliferative responses of mouse splenocytes to allogeneic cells in mixed lymphocyte cultures; (3) primary in vitro antibody responses of mouse spleen lymphocytes to sheep erythrocytes; (4) primary in vitro antibody responses of mouse spleen lymphocytes to a T-cell independent antigen (TNP-Ficoll). Inhibition of murine immune responses in vitro by these suppressor factors was regular and reproducible and it was observed in a large number of experiments. In contrast, suppressor factors produced by the 169 and by the 77(38F3) hybrids did not suppress the murine immune responses. The basis for these differences are not known at the present. The ability of human suppressor factors to inhibit effectively mouse immune responses provides an additional opportunity for the characterization of the properties of these factors in vivo using mouse models of human disease.

Induction of suppressor cells to T- and B-cell proliferative responses and immunoglobulin production by monoclonal antibodies recognizing the CD3 T-cell differentiation antigen.

Monoclonal antibodies (mAb's) recognizing the CD3 T-cell differentiation antigen induced the generation of suppressor cells. These cells inhibited (1) proliferative responses of human peripheral blood mononuclear cells (PBMC) to PHA and allogeneic cells in mixed leukocyte culture; (2) proliferative responses of purified E-rosette-negative cells to Staphylococcus aureus Cowans I; and (3) de novo immunoglobulin synthesis and secretion in the pokeweed mitogen (PWM)-induced differentiation system. Monoclonal antibodies recognizing other T-cell differentiation antigens (anti-Leu 2a, anti-Leu 3a, and anti-Leu 5) did not induce the generation of suppressor cells, even at very high antibody concentrations. Statistically significant differences were not observed in the ability of the OKT3 and anti-Leu 4 mAb's to induce suppressor cells. Monocytes were not required for the generation of anti-CD3-induced suppressor cells. F(ab')2 fragments of the OKT3 mAb's were equally effective when compared with intact antibody molecules in inducing suppressor cells, although they did not induce proliferative responses. Proliferation was not required for the induction of suppressor cells. Irradiation (2500 rad) of PBMC before incubation with the anti-CD3 mAb did not affect the generation of suppressor cells. Furthermore, anti-CD3-induced suppressor cells were radioresistant. Addition of recombinant IL-2 to the cultures of responding cells and suppressor cells did not reverse the suppression. In vitro treatment of anti-CD3-induced suppressor cells with either the OKT4 mAb plus complement or the OKT8 mAb plus complement partially decreased the suppression of proliferative responses of PBMC to PHA or allogeneic cells in mixed lymphocytes culture. However, treatment with both OKT4 and OKT8 mAb's plus complement or the OKT11 mAb plus complement completely abolished the suppression. These results suggest that the suppressor cells are of the T11+T4+T8- and T11+T4-T8+ phenotypes. In other experiments, T4+T8- and T8+T4- cells were isolated from PBMC treated for 48 hr with anti-CD3 mAbs. Both these two populations significantly inhibited proliferative responses of autologous PBMC to PHA and de novo immunoglobulin synthesis and secretion by mixtures of purified T4 and B cells from normal donors, in the PWM-induced differentiation system. These results demonstrate that anti-CD3-induced suppressor cells are of the T4 or T8 phenotype. Treatment of purified T4+T8- and T8+T4- cells with anti-CD3 mAb's resulted in the generation of suppressor cells, suggesting that the precursors of the anti-CD3-induced suppressor cells can belong to either of these two populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Leukaemic B cells from patients with chronic lymphocytic leukaemia suppress immunoglobulin production by lymphocytes from normal donors.

We observed that highly purified E-rosette-negative largely leukaemic B cells from 9 out of 15 patients with chronic lymphocytic leukaemia (CLL) significantly suppressed immunoglobulin production by mixtures of T4 and B cells from normal donors in the presence of pokeweed mitogen (PWM). This suppression by leukaemic B cells was concentration-dependent. Addition of equal numbers of B cells from normal donors to the mixtures of normal T4 and B cells increased, or had no effect on the production of IgM, IgA, and IgG. Treatment of purified largely leukaemic B cells from patients with CLL with either the anti-B1 or anti-Leu 1 monoclonal antibody plus complement abolished their ability to suppress immunoglobulin production. In contrast, treatment with either the anti-Leu 5 or the OKM1 monoclonal antibody plus complement had no effect on the suppression. These results suggest that leukaemic B cells from certain patients with CLL may exhibit, or can be induced to exhibit, immunosuppressive properties.

Defective helper function of purified T4 cells and excessive suppressor activity of purified T8 cells in patients with B-cell chronic lymphocytic leukemia. T4 suppressor effector cells are present in certain patients.

We investigated helper and suppressor functions to B-cell responses and T-T cell interactions of purified T4 and T8 cells from 20 untreated patients with B-cell chronic lymphocytic leukemia (CLL). Appropriate mixtures of purified T4 or T8 cells from patients with CLL were cultured with purified B cells or T4 and B cells from normal donors for 7 days with pokeweed mitogen (PWM). IgM, IgA, and IgG produced were determined in the supernatants of these cultures by a heavy chain-specific enzyme-linked immunosorbent assay (ELISA) and compared to those obtained by the corresponding mixtures of T4, T8, and B cells from normal donors. Purified T4 cells from 14 of 20 patients with CLL exhibited defective helper function (P less than .001) to immunoglobulin (Ig) production by purified B cells from normal donors. Purified T4 cells from 6 of these 14 patients were able to suppress significantly (P less than .001) and in a concentration-dependent manner Ig production by mixtures of T4 and B cells from normal donors, in the absence of T8 cells. These suppressor effector T4 cells from certain patients were partially radiosensitive. Purified T8 cells from 8 of 20 patients with CLL exhibited excessive suppressor activity. These cells significantly suppressed (P less than .001), Ig production by mixtures of T4 and B cells from normal donors to a degree significantly higher (P less than .005) than that observed by equal numbers of T8 cells from normal donors. This inhibition was dependent on the numbers of the T8 CLL cells added to the cultures. Excessive suppressor activity by T8 CLL cells was at least in part radiosensitive in four of eight patients. These results demonstrate a wide range of immunoregulatory T-cell abnormalities in patients with CLL. Naturally occurring T4 suppressor effector cells, directly inhibiting Ig production by mixtures of T4 and B cells, in the absence of T8 cells, are present in certain patients with CLL.