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.

The dinucleotide repeat polymorphism in the 3'UTR of the CD154 gene has a functional role on protein expression and is associated with systemic lupus erythematosus.

OBJECTIVE: To investigate the association of the (CA)n dinucleotide repeat in the 3' untranslated region (3'UTR) of the CD154 gene with systemic lupus erythematosus (SLE), and its functional role in protein expression. METHODS: The allelic and genotypic distributions of the polymorphism were compared in 80 patients with SLE and 80 controls. A complete clinical and analytical database was recorded in each patient in order to correlate the clinical manifestations in SLE with different alleles. To investigate the functional role of the polymorphism, the CD154 protein expression on activated lymphocytes from healthy homozygous controls was evaluated by flow cytometry. RESULTS: The 24 CA allele was the most represented in controls (p = 0.029), whereas the alleles containing >24 CA repeats were found in patients (p = 0.0043). Furthermore, when only homozygous women were considered, most controls carried two 24 CA alleles (p = 0.041), whereas most patients carried two alleles containing >24 CA repeats (p = 0.032). Also, patients carrying at least one 24 CA allele had less neurological involvement (p = 0.034), and carriers of at least one allele with fewer than 24 CA repeats presented more livedo reticularis (p = 0.006) and anti-Sm (p = 0.01) and anti-RNP (p = 0.038) autoantibodies. CD154 maximum expression in activated lymphocytes from all controls was reached after 54 hours, but it was more prolonged in controls carrying two alleles with >24 CA repeats (p = 0.0068). CONCLUSION: The CD154 3'UTR microsatellite is associated with SLE, and the most represented alleles in patients were accompanied by a more prolonged protein expression in activated lymphocytes from controls.

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.

[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.

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.

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

Se ha realizado el estudio mediante SPECT y 99mTc-Tetrofosmin (TTF) en 115 pacientes con 119 lesiones pulmonares sospechosas de malignidad para valorar los posibles factores que intervienen en la captación del radiofármaco. Los índices de captación del tumor respecto al tejido sano (índice TTF) se evaluaron respecto a: benignidad y malignidad, tipo histológico, estadio, diferenciación celular, tamaño, necrosis, supervivencia y la influencia de la P-glicoproteína (Pgp) en la captación de TTF, detectada mediante inmunohistoquímica. En las 18 lesiones benignas estudiadas la media del índice TTF fue 1,01 ñ 0,05 mientras que en las 101 malignas 1,59 ñ 0,45 (p 3cm presentaron mayor captación que los inferiores. En los pacientes con cáncer de pulmón de célula no pequeña se detectó una relación positiva entre el índice TTF y la supervivencia, no así en los pacientes con cáncer de pulmón de célula pequeña. En los tumores en los que se determinó la presencia de Pgp existía una relación inversa entre los índices de TTF y la expresión de Pgp. En suma, el estudio mediante SPECT torácico y 99mTc-TTF tiene un alto valor predictivo positivo para la presencia de cáncer de pulmón, aunque un estudio negativo no excluye su existencia. Esto es debido a que existe una relación inversa entre la captación de 99mTc-TTF y la densidad de expresión de la Pgp (AU)

CD154 polymorphism in Spanish populations. Differences in the allelic distribution between Canary islanders and Peninsulars.

The CD154 gene contains a dinucleotide repeat (CA)n in the 3' untranslated region. Allelic distribution in Spanish populations from two areas with different genetic background, the Canary Islands and Peninsula, are described. Seven alleles with different allelic distribution between the two groups, were found. This represents a highly polymorphic marker, useful for genetic studies on a critical molecule in immunity.

Modulation of apoptosis by cytokines in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the slow and progressive accumulation of monoclonal apparently mature, CD5(+) B lymphocytes. The majority of circulating cells appear to be nondividing, and it has been suggested that a prolonged life span is mainly responsible for the accumulation of the leukemic cells. However, spontaneous programmed cell death by apoptosis occurs when B chronic lymphocytic leukemia cells are cultured in vitro. This may be because of the lack of an unidentified essential cytokine present in vivo. Thus, we investigate interleukin-2 (IL-2), IL-4, IL-6 and IL-10 in vitro effects on apoptosis of B cells from 32 previously untreated patients with B-CLL in initial clinical stages. B cells were isolated from peripheral blood, and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with the different cytokines, 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 cytokines. Our results indicate that both IL-2 and IL-4, but not IL-6, inhibit in vitro apoptosis in a large percentage of B-CLL patients. IL-10 increases in vitro apoptotic cell number in stage 0 patients, but not in stage I and II. These data support the hypothesis that IL-2 or IL-4, may be cell survival factors in vivo and that IL-10 might be a candidate for immune therapy of early B-CLL.

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.

[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.

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.

Physiological differences between professional and elite road cyclists.

The purpose of this study was to compare the physiological responses of professional and elite road cyclists during an incremental cycle ergometer test. Twenty-five elite cyclists (EC; 23+/-1 yr) and 25 professional cyclists (PC; 25+/-2yr) performed a ramp protocol (increases of 25 W x min(-1)) during which the following parameters were measured: oxygen consumption (VO2), pulmonary ventilation (VE), ventilatory equivalents for oxygen and carbon dioxide (VE x VO2(-1) and VE x VCO2(-1), respectively), respiratory exchange ratio (RER), ventilatory thresholds 1 and 2 (VT1 and VT2, respectively), blood lactate, and electromyographic activity (EMG) of the vastus lateralis. Significant differences existed between the two groups mainly at submaximal intensities, since both VT1 and VT2 occurred at a higher exercise intensity (p<0.001) in PC than in EC (VT2: 80.4+/-6.6 vs 87.0+/- 5.9% VO2max in EC and PC, respectively). Lactate levels showed a similar response in both groups at low-to-moderate intensities (< 300 W), and thereafter blood lactate was significantly higher in EC. Finally, the "electromyographic threshold" (EMGT) occurred at a significantly higher intensity (p < 0.05) in PC when compared to EC (64.7+/-14.2 vs 56.0+/-14.9% VO2max, respectively). It was concluded that, in comparison with EC, PC exhibit some remarkable physiological characteristics such as a high VT2, an important reliance on fat metabolism even at high power outputs, and several neuromuscular adaptations.

[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.

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.

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.

Natural killer cell proliferation and renal disease: a functional and phenotypic study.

We describe a 57-year-old woman who presented with a constitutional syndrome, glomerulonephritis, and lymphocytosis. The phenotypic study, using flow cytometry, showed an expansion of natural killer (NK) cells (CD2+, CD3-, CD16+, CD56+, and CD7+). We performed a functional study of peripheral blood mononuclear cells (PBMCs) and of purified CD16+ cells (NK cells) and CD3+ cells (normal T cells). The expanded NK cell population, CD16+, did not proliferate with phytohemagglutinin (PHA) or anti-CD3 but showed a dose-dependent proliferation with recombinant interleukin-2 (rIL-2) and also proliferated with phorbol dibutyrate. This population showed very strong NK and lymphokine-activated killer cell (LAK) activities. The patient's symptoms resolved spontaneously without treatment. Three years later, however, there is still abnormal renal function, and the expansion of NK cells persists, although with no indication of malignancy. We review the features of the different large granular lymphocyte proliferations and their seldom described relationship with renal disease.

[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.