Leukemia-associated antigens detected by heterologous antisera.

An antiserum was produced by immunization of rabbits with the membrane fraction of a lymphoblastoid cell line, RPMI 4265. This antiserum reacted against leukemia-associated antigens on immature blast cells of 24 patients with acute leukemia (13 myeloblastic, 11 lymphoblastic). No reactivity was observed against morphologically normal blood mononuclear cells from patients in remission, cells from normal control subjects and patients with unrelated disorders, phytohemagglutinin-induced lymphoblasts, or normal bone marrow cells. Reactivity against leukemia cells was not reduced by absorption with fetal tissues. These findings were consistent with the presence of tumor-associated antigens on leukemia cells. The antigens were detectable neither during hematologic remission nor on cells from patients with unrelated diseases.

Recognition by human and rabbit sera of common antigens to leukemia blast cells, peripheral blood B-lymphocytes, and monocytes.

A human serum (obtained from a multiparous and multiple-transfused patient with chronic myelogenous leukemia) and a rabbit antiserum (obtained by immunization with papain extracts from a B-lymphoblastoid cell line) showed reactivity against antigenic specificities (different from HLA) expressed on peripheral blood B-lymphocytes, unmarked lymphocytes, and monocytes. These antigenic determinants were expressed on myeloblasts and lymphoblasts from patients with acute leukemia (during the active phase of their disease) and on B-lymphoblastoid cell lines and lymphocytes from patients with chronic lymphocytic leukemia. Purified peripheral blood T-lymphocytes, mitogen (phytohemagglutinin)-activated T-lymphocytes, and lymphoblasts (with T-cell characteristics) obtained from patients with acute lymphoblastic leukemia or established lymphoblastoid cell lines lacked these antigenic specificities. Absorption experiments indicate that the antigen(s) detected on normal mononuclear cell populations, leukemia cells, and B-lymphoblastoid cell lines were either identical or highly cross-reactive.

Antigens shared by leukemic blast cell and lymphoblastoid cell lines detected by lymphocyte-dependent antibody.

Lymphocyte-dependent antibodies (LDA's) directed against antigenic determinants present on lymphoblastoid cell lines as well as human leukemia blast cells were demonstrated in heterologous antisera obtained by immunizing rabbits with a membrane fraction from RPMI-4265 (a lymphoblastoid cell line derived from a patient with chronic myelogenous leukemia). LDA was present at high titers against B-lymphoblastoid, myelomonocytic, and stem cell lines. The T-lymphoblastoid cell line MOLT-4, however, did not react. LDA was demonstrated against acute myelogenous as well as lymphoblastic leukemia cells. The reactivity was not directed against phytohemagglutinin-induced blastoid antigens, fetal antigens, or fetal calf serum. Absorptions with lymphoblastoid cell lines removed all LDA reactivity. Similar results were obtained by absorbing the rabbit antiserum with acute lymphoblastic and/or acute myelogeneous leukemia cells. These findings indicate the presence of cross-reactive antigens between lymphoblastoid cell lines and leukemia cells. Furthermore, cross-reactivity between acute lymphoblastic and acute myelogenous leukemia cells was demonstrated.

A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells.

The progressive rise of mature CD5+ B lymphocytes, despite the low proportion of proliferating cells, has led to the notion that B cell chronic lymphocytic leukemia (B-CLL) is primarily related to defective apoptosis. The microenvironment likely plays a prominent role because the malignant cells progressively accumulate in vivo, whereas they rapidly undergo spontaneous apoptosis when cultured in vitro. To assess microenvironment-mediated survival signals, B-CLL cells were cultured with a murine fibroblast cell line, Ltk-, with and without an agonistic antibody to CD40. Spontaneous apoptosis was associated with the loss of Akt and NF-kappaB activities. Interactions with fibroblasts sustained a basal level of Akt and NF-kappaB activities, which was dependent on phosphatidylinositol-3 kinase (PI3K). Constitutive activity of the PI3K pathway in B-CLL cells when cultured with fibroblasts prevented the downregulation of the prosurvival Bcl-2 family protein Bcl-xL and the caspase inhibitor proteins FLIPL and XIAP, and consequently caspase-3 activation and apoptosis. CD40 crosslinking in B-CLL cells did not further prevent murine fibroblasts-mediated apoptosis but induced cell proliferation, which was associated with an increase of Akt and NF-kappaB activation compared with cells cultured with fibroblasts alone. The PI3K pathway seems to play a pivotal role in B-CLL cell survival and growth.

Induction of apoptosis by 2-chlorodeoxyadenosine in B cell chronic lymphocytic leukemia.

We investigated whether 2-chlorodexoyadenosine could induce apoptosis in B cell chronic lymphocytic leukemia (B-CLL) cells in vitro using clinically achievable drug doses, measuring apoptosis ratio by flow cytometry. B cells were isolated from previously untreated patients and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with 2-chlorodeoxyadenosine at different concentrations, for 24 and 48 h. Distribution of cellular DNA content and quantitative analysis of apoptosis were determined by standard propidium iodide staining and flow cytometry. Spontaneous apoptosis occurred in B-CLL cells incubated in vitro in the absence of drug, but the level of apoptosis was greater in cells treated with 2-chlorodeoxyadenosine after the second day of culture. The present in vitro study of B-CLL cells from previously untreated patients suggests this chemotherapeutic agent activates a program of cell death by apoptosis using a drug dose equivalent to the physiological concentration used in patients in vivo. These data reveal an interesting possibility in the 2-chlorodeoxyadenosine treatment of untreated patients by neoplastic B cell apoptosis induction.

Flow-cytometric assessment of lymphocyte cytokine production in tuberculosis.

We assessed by flow-cytometry the Th1/Th2 profiles in peripheral blood lymphocytes (PBL) from patients with active tuberculosis (TB), before and after antituberculous therapy, and from healthy tuberculin-positive and -negative reactors. PBL from patients showed a reduced potential for Th1-cytokine (notably IFN- gamma) production after culture with a policlonal stimulus. When these PBL from patients were cultured with a M. tuberculosis (MTB)-specific antigen such as PPD (10 microg/ml), there was no detectable production of Th1 cytokines. Only the Th2 cytokine IL10 was detected in PBL from patients but not from controls. However, at the site of the tuberculosis disease, T lymphocytes from bronchoalveolar lavage, after culture with PPD, produced IFN- gamma. After completion of tuberculosis therapy, PBL did not produce IL10. These data indicate that the immunosuppression observed in PBL during active tuberculosis infection may be related to IL10 production, and to the compartmentalization of the antigen-Th1 response to sites of active MTB infection.

Studies of lymphocyte-dependent antibodies to leukemia-associated antigens using frozen stored leukemia target cells.

The ability of frozen stored leukemia blast cells to participate in a lymphocyte-dependent antibody assay (LDA) is demonstrated. Frozen stored acute myelogenous and lymphocytic blast cells retained antigenicity and high viability (larger than or equal to 80%). High titer LDA reactivity against frozen stored leukemic blasts was demonstrated with heterologous (rabbit) and homologous (leukemic patient) antisera. Similarly, we demonstrated the ability of frozen stored peripheral blood mononuclear cells to mediate effectively the destruction of antibody-coated leukemia blast cells stored frozen in liquid nitrogen before use. These findings will facilitate the study of LDA and antibody-dependent cellular cytotoxicity in human leukemia patients using a completely autogeneic system.

Impaired natural killer cytotoxicity in peripheral blood mononuclear cells in Graves' disease.

We studied the natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) in patients with Graves' disease (GD). Peripheral blood mononuclear cells from 20 untreated hyperthyroid patients with GD showed a significantly reduced NK activity against 51 Cr-labeled K562 cells (33.9 +/- 15.9%), while in 32 euthyroid patients under antithyroid drug therapy. NK activity was similar to that of controls (46.9 +/- 17.3 and 49.9 +/- 20.2%, respectively). Furthermore, normalization of thyroid function with antithyroid drugs was associated with a significant increase and normalization of NK activity during the follow-up of nine GD patients (from 29.2 +/- 17.9 to 48.1 +/- 16.5%). This phenomenon could not be ascribed to a defective number of NK cells because the amounts of CD56+ and CD16+ cells in PBMC from both hyperthyroid and euthyroid GD patients were within normal ranges. Natural killer activity of PBMC from patients with toxic multinodular goiter was similar to that of normal controls (45 +/- 12.8 to 49.9 +/- 20%). No correlation was found between natural killer activity and serum levels of free thyroxine, TSH-inhibitory immunoglobulins, thyroidal antibodies to thryoglobulin and thyroidal microsomal antigen, dose or duration of antithyroid drug therapy. Natural killer activity from both controls and GD patients was enhanced in vitro by addition of recombinant interleukin 2 (IL-2), reaching control levels in hyperthyroid patients. These abnormalities were not associated with a defective IL-2 production by T cells, nor with a decreased IL-2R expression. We conclude that in untreated Graves' disease there is a decrease in NK cell activity in PBMC, probably dependent on the autoimmune process.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of intracellular IL-2, TNF-alpha, and IFN-gamma by T cells in B-CLL.

BACKGROUND: Recent evidence indicates that the slowly expanding population of CD5(+) B cells that characterizes B-cell chronic lymphocytic leukemia (B-CLL) could be related to defects in the response to cytokine produced by T cells that regulate apoptosis. We studied the intracellular expressions of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in T-helper 1 cells (Th1 response) of B-CLL. METHODS: Peripheral blood mononuclear cells from 21 healthy individuals and purified T cells from 21 early-stage and 15 late-stage B-CLL patients were activated with phorbol myristate acetate and ionomycin. The Th1 cytoplasmic cytokines were evaluated in CD4(+) and CD8(+) T cells by flow cytometry. RESULTS: The percentages of CD4(+) and CD8(+) T cells positive for IL-2 were significantly lower in B-CLL patients than in healthy individuals (P = 0.030 and 0.049, respectively). No significant differences in TNF-alpha or IFN-gamma intracellular expressions were found between patients and healthy individuals. TNF-alpha- and IFN-gamma-expressing CD8 T cells were disease stage dependent, being significantly higher in late-stage patients (P < 0.001 for both cytokines). CONCLUSIONS: Our present observations suggested that Th1 cytokines may be of major importance in the pathogenesis of B-CLL.

Selective cytotoxic effect of tamoxifen on epithelial and fibroblastic cells transformed by different oncogenes.

To study the correlation between oncogenes and the cytotoxic effect of tamoxifen in estrogen negative (ER) cells, we transformed mouse keratinocyte and fibroblastic cell lines with several oncogenes and studied cell viability, thymidine incorporation and PKC levels. We show that v-myc and v-H-ras oncogenes increase sensitivity in both cell types and that Neu and mutant p53 also increase sensitivity to tamoxifen, more significantly in the epithelial cells. Conversely, transformation with adenovirus E1a oncogene induces resistance to tamoxifen in both cell types. These results indicate that tamoxifen may be effective in different kinds of malignant cells depending on the oncogenic alterations present in the cells.

Natural killer cell activity in laryngeal carcinoma.

We studied the relationship of natural killer cell activity from peripheral blood mononuclear cells with the clinical and pathologic stage of disease in 23 male patients with previously untreated carcinoma of the larynx and 22 healthy male control subjects. Levels of natural killer cell activity against K-562 target cells were similar in control subjects and patients, regardless of stage, tumor size, and clinical cervical adenopathies. Natural killer cell activity, however, was significantly decreased in patients with pathologic cervical lymph node involvement. The number of natural killer cells, as estimated by CD16 and CD56 monoclonal antibodies, was similar in all groups of subjects. We conclude that in patients with laryngeal carcinoma, there is a correlation between deficient natural killer cell activity and nodal metastases, which may represent a prognostic indicator in these patients.

Prognostic significance of natural killer cell activity in patients with laryngeal carcinoma.

BACKGROUND: For laryngeal carcinoma, the present TNM clinical staging system does not seem completely satisfactory as a guide for providing a prognosis for survival. We believe that natural killer cell activity would probably have a role in a more reliable system. Therefore, we analyzed the disease outcome with previously untreated epidermoid carcinoma of the larynx, evaluating classic clinical and pathologic factors, as well as natural killer cell activity in peripheral blood samples of the patients. OBJECTIVES: To determine the level of natural killer cell activity in patients with laryngeal carcinoma and to analyze the prognostic value of this finding when associated with other clinical and pathologic variables. DESIGN: Prospective cohort study of surveillance of laryngeal cancer. Mean follow-up of 42.8 months. SETTINGS: Tertiary care referral center and ambulatory and hospitalized care. PARTICIPANTS: We compared 81 men (mean age, 62.4 years; range, 35-89 years) with laryngeal carcinoma with 44 healthy men serving as control subjects (mean age, 57.6 years; range, 37-82 years). RESULTS: Natural killer cell activity was significantly reduced in patients who had died of cancer-related causes in comparison with tumor-free survivors. Overall actuarial survival differed significantly in histologically assessed nodal involvement and low natural killer cell activity. Use of the Cox proportional hazards regression model showed that the factors that seem to have a prognostic effect on survival are histologically determined nodal involvement and low natural killer cell activity. CONCLUSIONS: These results support the prognostic significance of the determination of pretreatment natural killer cell activity in peripheral blood samples from patients with laryngeal carcinoma and suggest that assessment of adding such a determination to the current tumor staging system is advisable.

Deficiency of naive T cells in patients with sudden deafness.

BACKGROUND: Although there are a number of reports concerned with the role of immunity in the sudden onset of progressive sensorineural hearing loss, there are few references dealing with the involvement of immune-mediated mechanisms in sudden deafness. OBJECTIVES: To study the phenotype of peripheral blood lymphocytes in a group of patients with sudden deafness by use of 3-color flow cytometry. DESIGN: The study was carried out prior to the start of steroid therapy. Fourteen patients underwent a follow-up study once steroid therapy had been completed. Prospective analysis, case-control. SETTING: Tertiary case referral center, ambulatory and hospitalized care. PATIENTS: Twenty-two patients (13 men and 9 women; mean age, 45.3 years) were compared with 14 healthy control subjects (9 men and 5 women; mean age, 36 years). Patients were divided in 2 groups according to their response to steroid therapy. RESULTS: Decreased numbers of both CD4+ helper cells (38.4% vs 45.5%; P = .04) and CD8+ cytotoxic cells (17.5% vs 22.3%; P = .02) were observed in patients and compared with those in the control subjects, as well as reduced numbers of CD4+CD45RA+ cells (14.4% vs 29.3%; P = .01) and CD8+CD45RA+ naive cells (18.2% vs 25.4%; P = .04). In the group of patients with a good response to steroid therapy (group 1), a tendency toward normalization of the CD4+ (pretreatment, 38.6%; posttreatment, 44.6%), CD4+CD45RA+ (pretreatment, 15.2%; posttreatment, 21.7%), and CD4+CD45RO+ (pretreatment, 21.1%; posttreatment, 18.2%) cell counts was observed, with a slight decrease in the CD8+ population (pretreatment, 18%; posttreatment, 15.7%). However, in patients with a poorer response (group 2), while there were increases in the CD4+ (pretreatment, 38%, posttreatment, 50%) and CD4+CD45RA+ (pretreatment, 12.8%; posttreatment, 16.7%) cell counts after steroid therapy, there was a significant increment in the CD4+CD45RO+ memory cell count (pretreatment, 14.1%; posttreatment, 28.5%) and low CD8+CD45RA+ counts (pretreatment, 14.6%; posttreatment, 15.5%). No differences were observed in the numbers of B or natural killer cells or in the presence of activation antigens CD25 and HLA-DR when pretreatment and posttreatment levels were compared. CONCLUSION: These results demonstrate significant abnormalities in the subpopulations of lymphocytes in patients with sudden hearing loss, suggesting the existence of immune-mediated responses in the inner ear as possible etiopathogenic factors in this entity.

Budd-Chiari syndrome due to obstruction of the inferior vena cava. A report of two cases.

Two cases of Budd-Chiari syndrome caused by membranous occlusion of the proximal portion of the inferior vena cava are presented. Both were treated surgically, using the modified technique of Kimura in one case, and a resection with direct vision and extracorporeal circulation in the other. Both patients progressed well. We discuss the etiology and pathogenesis of the syndrome, the diagnostic means that can be used, and the importance of early treatment.

Functional and phenotypic analysis of T-lymphocytes in laryngeal carcinoma.

We studied the functional response and phenotypic characterization of peripheral blood T cells and their correlation with the clinical stage of disease in 29 males with previously untreated carcinoma of the larynx and 24 healthy male controls. Peripheral blood T cells, phenotypically CD2+ CD3+, were significantly decreased in the patients relative to the controls. Patients with advanced locoregional extension (T4 and N1, 2, 3) also showed a diminution of the CD4+ subpopulation of T cells. DNA synthesis by purified T cells showed similar blastogenic responses in patients and controls; the interleukin-2 production of phytohemagglutinin stimulated lymphocytes was also normal. We conclude that in patients with laryngeal carcinoma there is a phenotypic alteration of the T cells that is variable according to tumor stage, without functional alterations in blastogenic capacity or IL-2 production.

Natural killer activity in patients with breast cancer.

The clinical significance of the natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) was analyzed in 83 breast cancer patients and 24 healthy control women. Similar levels of NK cytotoxic activity against K-562 target cells were found in PBMC from either untreated or surgically treated patients with local or disseminated breast cancer and from normal controls. However, a transitory and significant decrease (p less than 0.05) of the NK activity of PBMC from breast cancer patients was found during chemotherapy. But, according to quantitative flow cytometry analysis, similar percentages of phenotypically defined NK cells (CD16+, CD11b+, HNK-1+) were found in PBMC from patients, whether prior to or during chemotherapy, and healthy controls. Our results demonstrate that in breast cancer patients, the percentage of NK cells present in PBMC and their lytic activity are independent of the clinical and pathological stage of the disease.

[Correlation between 99m Tc-tetrofosmin uptake and P-glycoprotein expression in non-small-cell lung cancer]. / Correlación entre la captación de 99m Tc-tetrofosmín y la expresión de P-glicoproteína en cáncer de pulmón no microcítico.

UNLABELLED: We used thoracic SPECT to study the 99mTc-tetrofosmin (TF) uptake in patients with non-small-cell-lung-carcinoma (NSCLC). The results were compared with the percentage of P-glycoprotein (Pgp) found in flow cytometric analysis (FC) of samples of surgically-resected tumor tissue. MATERIAL AND METHODS: A total of 21 patients with NSCLC were studied by means 99mTC-TF and thoracic SPECT. Image analysis included the determination of the TF uptake rate in the lung mass with respect to that of healthy tissue of the contralateral lung. These rates were compared with the percentage of Pgp expression according to FC. FC analysis was also carried out in 16 samples of healthy lung tissue obtained from the patients. RESULTS: In healthy lung tissue, the mean Pgp expression according to FC was 4.58 +/- 1.87%. The cutoff value used to differentiate between Pgp positive and Pgp negative tumors was considered to be the mean plus two standard deviations (8.32). The Pgp-positive tumors (> 8.32%) presented significantly lower uptake levels (1.28 +/- 0.39) than the Pgp-negative lesions (1.66 +/- 0.33) (p = 0.029). CONCLUSIONS: There is a inverse correlation between the Pgp expression as determined by FC analysis and 99mTc-TF in NSCLC. Thus, this radiopharmaceutical provides rapid and non-invasive information on Pgp expression in these lesions.

[Study of pulmonary lesions with (99m)Tc-Tetrafosmin and chest spect. Determination of uptake related factors, diagnostic value and prognosis]. / Estudio de las lesiones pulmonares con(99M)Tc-Tetrofosmin y SPECT de tórax. Determinación de los factores que intervienen en la captación, valor diagnóstico y pronóstico.

One hundred fifteen patients with 119 pulmonary lesions in which malignancy was suspected underwent a SPECT study with 99mTc-Tetrofosmin (TTF) to assess the possible factors involved in the uptake of the radiopharmaceutical. The TTF uptake rate in the lung tumor with respect to that of healthy tissue (TTF index) was evaluated in terms of: benignity and malignancy, histological type, stage, cell differentiation, size, necrosis, survival and the influence of P-glycoprotein (Pgp), detected by immunohistochemistry, on the TTF uptake. The mean TTF index in the 18 benign lesions studied was 1.01 0.05, while that of the 101 malignant lesions was 1.59 0.45 (p < 0.001), with a positive predictive value of 97.7% and a negative predictive value of 50%. The comparison of the histological types, degree of cell differentiation, necrosis and stage revealed no statistically significant differences. With respect to size, those tumors measuring > 3 cm showed greater uptake than smaller lesions. In patients with non-small cell lung cancer, a positive relationship was observed between the TTF index and survival, a circumstance that did not occur in patients with small cell lung cancer. In the cases in which the presence of Pgp was assessed, there was an inverse relationship between the TTF ratio and Pgp expression. In conclusion, thoracic SPECT with 99mTc-TTF has a high positive predictive value for the presence of lung cancer, although a negative study does not rule out the existence of the disease. The reason for this is the inverse relationship between 99mTc-TTF uptake and the density of Pgp expression.

[Preliminary study by flow cytometry of the cell cycle and DNA quantification in cytokeratin-positive cells in tumors of the head and neck]. / Estudio preliminar del ciclo celular y cuantificación del ADN de células citokeratina positivas en tumores de cabeza y cuello mediante citometría de flujo.

DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumor tissue of 27 epithelial head and neck carcinomas. Epithelial cells were labeled with a fluorescein-isothiocyanate-conjugated cytokeratin antibody to study the possible influence of contaminating stromal and inflammatory cells on the results of cell-cycle analysis of tumor cells. The patient sample included 26 men and 1 women with a mean age of 60 years. Without cytokeratin gating, 11/27 tumors (40.74%) were aneuploid. After selecting the cytokeratin population, 10 more aneuploid tumors were found that had not been detected when considering the total population. Therefore, aneuploid tumors increased from 40.74% to 77.74%. The remaining 6/27 (22.26%) tumors were diploid. In the tumors that were either aneuploid without cytokeratin gating or diploid, the S-phase and G2M phase were significantly higher after cytokeratin staining, specially in diploid tumors (24.2% versus 10% and 6.8% versus 3.2%, respectively, p < 0.01). Therefore, in head and neck tumors cytokeratin staining optimizes both the determination of DNA ploidy and cell-cycle analysis, which is advantageous for tumor staging and prognosis assessment in these patients.

[Study of spontaneous cytotoxic activity in laryngeal carcinoma: prognostic value]. / Estudio de la actividad citotóxica espontánea en el carcinoma de laringe: valor pronóstico.

The status of natural killer (NK) cell activity in peripheral blood, based on number and functional state, was studied in relation to the clinical and histopathologic stage of 52 patients with laryngeal carcinoma and in 23 healthy controls. The number of NK cells, estimated using CD16 and CD56 monoclonal antibodies, was similar in patients and controls and showed no relation to tumor size and nodal involvement. NK cell function did not show significant differences in spontaneous cytotoxic activity either overall or in relation to tumor size and the presence of palpable lymph nodes. However, cytotoxic activity was significantly lower in patients who had histologically confirmed nodal involvement. NK activity under 36% (the percentage of specific lysis at an effector:target dilution of 50:1) was suggested the probable presence of nodal metastases and was a highly sensitive and specific test. In patients with laryngeal carcinoma, NK-cell cytotoxic activity may be an independent prognostic parameter for evaluating cervical lymph node involvement.