Comparative analysis of the pathological events involved in immune and non-immune TRALI models.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is characterized by leukocyte transmigration and alveolar capillary leakage shortly after transfusion. TRALI pathogenesis has not been fully elucidated. In some cases, the infusion of alloantibodies (immune model), whereas in others the combination of neutrophil priming by proinflammatory molecules with the subsequent infusion of biological response modifiers (BRMs) in the hemocomponent (non-immune model) have been implicated. Our aim was to compare the pathological events involved in TRALI induced by antibodies or BRMs using murine models. MATERIALS AND METHODS: In the immune model, human HNA-2(+) neutrophils were incubated in vitro with a monoclonal antibody (anti-CD177, clone 7D8) directed against the HNA-2 antigen and injected i.v. in NOD/SCID mice. In the non-immune model, BALB/c mice were treated with low doses of lipopolysaccharide (LPS) followed by platelet-activating factor (PAF) infusion 2 h later. Forty minutes after PAF administration, or 6 h after neutrophil injection, lungs were isolated and histological analysis, determination of a variety of cytokines and chemokines including keratinocyte-derived chemokine (KC), MIP-2, the interleukins IL-1ß, IL-6, IL-8 as well as TNFα, cell influx and alveolar capillary leakage were performed. RESULTS: In both models, characteristic histological findings of TRALI and an increase in KC and MIP-2 levels were detected. In contrast to the immune model, in the non-immune model, there was a dramatic increase in IL-1ß and TNFα. However, capillary leakage was only detected if PAF was administrated. CONCLUSIONS: Regardless of the triggering event(s), KC, MIP-2 and integrins participate in TRALI pathogenesis, whereas PAF is essential for capillary leakage when two events are involved.

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells.

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 microM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 microM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 microM, respectively. The critical micellar concentration (CMC) of ODPC was 200 microM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (H) variation of 7.3 kcal mol(-1). The presence of 25 microM ODPC decreased T(c) and H to 39.3 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 microM destabilized the liposomes (36.3 degrees C, 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.

Transcriptional changes in U343 MG-a glioblastoma cell line exposed to ionizing radiation.

Glioblastoma multiforme (GBM) is a highly invasive and radioresistant brain tumor. Aiming to study how glioma cells respond to gamma-rays in terms of biological processes involved in cellular responses, we performed experiments at cellular context and gene expression analysis in U343-MG-a GBM cells irradiated with 1 Gy and collected at 6 h post-irradiation. The survival rate was approximately 61% for 1 Gy and was completely reduced at 16 Gy. By performing the microarray technique, 859 cDNA clones were analyzed. The Significance Analysis of Microarray algorithm indicated 196 significant expressed genes (false discovery rate (FDR) = 0.42%): 67 down-regulated and 97 up-regulated genes, which belong to several classes: metabolism, adhesion/cytoskeleton, signal transduction, cell cycle/apoptosis, membrane transport, DNA repair/DNA damage signaling, transcription factor, intracellular signaling, and RNA processing. Differential expression patterns of five selected genes (HSPA9B, INPP5A, PIP5K1A, FANCG, and TPP2) observed by the microarray analysis were further confirmed by the quantitative real time RT-PCR method, which demonstrated an up-regulation status of those genes. These results indicate a broad spectrum of biological processes (which may reflect the radio-resistance of U343 cells) that were altered in irradiated glioma cells, so as to guarantee cell survival.

Telomere length analysis in monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia Binet A.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic clinical entity characterized by the proliferation of monoclonal B cells not meeting the diagnosis criteria for chronic lymphocytic leukemia (CLL). MBL may precede the development of CLL, but the molecular mechanisms responsible for disease progression and evolution are not completely known. Telomeres are usually short in CLL and their attrition may contribute to disease evolution. Here, we determined the telomere lengths of CD5+CD19+ cells in MBL, CLL, and healthy volunteers. Twenty-one CLL patients, 11 subjects with high-count MBL, and 6 with low-count MBL were enrolled. Two hundred and sixty-one healthy volunteers aged 0 to 88 years were studied as controls. After diagnosis confirmation, a flow cytometry CD19+CD5+-based cell sorting was performed for the study groups. Telomere length was determined by qPCR. Telomere length was similar in the 3 study groups but shorter in these groups compared to normal age-matched subjects that had been enrolled in a previous study from our group. These findings suggest that telomere shortening is an early event in CLL leukemogenesis.

Adhesion molecule profiles of B-cell non-Hodgkin's lymphomas in the leukemic phase.

We evaluated the expression of 10 adhesion molecules on peripheral blood tumor cells of 17 patients with chronic lymphocytic leukemia, 17 with mantle-cell lymphoma, and 13 with nodal or splenic marginal B-cell lymphoma, all in the leukemic phase and before the beginning of any therapy. The diagnosis of B-cell non-Hodgkin's lymphomas was based on cytological, histological, immunophenotypic, and molecular biology methods. The mean fluorescence intensity of the adhesion molecules in tumor cells was measured by flow cytometry of CD19-positive cells and differed amongst the types of lymphomas. Comparison of chronic lymphocytic leukemia and mantle-cell lymphoma showed that the former presented a higher expression of CD11c and CD49c, and a lower expression of CD11b and CD49d adhesion molecules. Comparison of chronic lymphocytic leukemia and marginal B-cell lymphoma showed that the former presented a higher expression of CD49c and a lower expression of CD11a, CD11b, CD18, CD49d, CD29, and CD54. Finally, comparison of mantle-cell lymphoma and marginal B-cell lymphoma showed that marginal B-cell lymphoma had a higher expression of CD11a, CD11c, CD18, CD29, and CD54. Thus, the CD49c/CD49d pair consistently demonstrated a distinct pattern of expression in chronic lymphocytic leukemia compared with mantle-cell lymphoma and marginal B-cell lymphoma, which could be helpful for the differential diagnosis. Moreover, the distinct profiles of adhesion molecules in these diseases may be responsible for their different capacities to invade the blood stream.

Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model.

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor alpha (RARalpha) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARalpha fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARalpha develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARalpha TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 +/- 16.68, 10.83 +/- 8.11, 7.4 +/- 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARalpha TM present a specific immunophenotype.

CD10 and CD19 fluorescence intensity of B-cell precursors in normal and leukemic bone marrow. Clinical characterization of CD10(+strong) and CD10(+weak) common acute lymphoblastic leukemia.

In order to assess the age-related changes in CD10 and CD19 fluorescence intensity (FI) the present study analyzed by flow cytometry 56 sternal biopsies from 'normal' infants, children and adults undergoing cardiac surgery. The CD10(+weak) subset was predominant in all age groups, representing approximately 50% of the bone marrow (BM) lymphoid cells in children younger than 4 years. Both CD10+ subsets significantly decreased with age but their ratio did not differ significantly. Moreover, the intensity of CD10 and CD19 fluorescence in the strong and weak subsets did not vary with age. The CD19 intensity was significantly higher in CD10(+weak) than in CD10(+strong) cells. In addition, we classified as CD10(+weak) or CD10(+strong) the leukemic cells from BM aspirates of 117 patients with common acute lymphoblastic leukemia (cALL) (78 children and 39 adults). A higher frequency of cases expressing the CD19+ CD10(+strong) phenotype was observed both in children and adults. Children of the CD10(+weak) group tended to be older than those of the CD10(+strong) group (median = 7 vs. 4 years, P = 0.07), and presented a significantly higher frequency of splenomegaly (93.7 vs. 55%, P = 0.04), which was massive in about 60% of these cases. Among adults, a significantly higher frequency of cases expressing the CD10(+weak) phenotype was observed in females. No other clinical or biological difference was detected between the two groups either for children or adults. Concerning the treatment outcome, we did not observe significant differences in complete remission rate (CRR) or in disease free survival (DFS) among the 32 children and 28 adults analyzed. Finally, we compared the CD10 and CD19 intensity in normal and leukemic BM. Overexpression of either or both antigens in leukemic cells was observed in 42.4% of the cALL cases. In these cases, using cut off values of 110 afu for the CD10 FI and of 100 afu for the CD19 FI, the detection of leukemic cells was possible at levels of 0.2% based on CD10 analysis, of 0.6% based on CD19, and 0.02% when both antigens were overexpressed. In conclusion, we demonstrated that the heterogeneity of CD10 and CD19 fluorescence intensity is of no clinical relevance in cALL, although its study may be helpful for the diagnosis and the detection of minimal residual disease.

Characterization of acute lymphoblastic leukemia subtypes in Brazilian patients.

The distribution of the acute lymphoblastic leukemia (ALL) subsets in 225 consecutive Brazilian patients was determined by an immunophenotypic study with an extensive panel of monoclonal antibodies. All subsets were detected and their relative frequencies were similar to those described in developed countries, except for the B-mature subset which had a higher frequency, especially in adults. Associated myeloid markers were expressed by 11% of the ALL and CD10 by 15.9% of T-ALL cases. Besides, the incidence rates determined for the region of Ribeirão Preto showed that the overall incidence of ALL was 12.5 cases/10(6) people years (PY) (5 cases/10(6) PY in non-Whites versus 14 cases/10(6) PY in Whites); the incidence of childhood ALL was 25.5 cases/10(6) PY (8.1 versus 29.8 cases/10(6) PY in non-Whites and Whites, respectively) and the incidence of ALL in adults was 6.2 cases/10(6) PY (5.5 versus 6.1 cases/10(6) Py in non-Whites and Whites, respectively). The significantly lower incidence rate of ALL in non-White children was associated with a selective deficit of the common subtype and a lack of the typical age peak of incidence in this group. The ALL features demonstrated here in Brazilian non-White children resemble those described in the American non-Whites before the seventies and those in British and American Whites at the beginning of the century.

Neutrophil chemotaxis in sickle cell anaemia, sickle cell beta zero thalassaemia, and after splenectomy.

Neutrophil chemotaxis was evaluated in 28 patients with sickle cell anaemia, 10 patient with sickle cell beta zero thalassaemia, 25 patients who had undergone splenectomy, and 38 controls. The mean distance migrated by patients' neutrophils was not significantly different from that of neutrophils from controls. Although several immunological variables have been reported to be changed after loss of splenic function, we were unable to show a defect in neutrophil chemotaxis that could account for the increased susceptibility to infection.

Circulating immune complexes after splenectomy.

Circulating immune complexes were evaluated in 25 patients (age range 10 to 46 years) who had undergone splenectomy for non-malignant conditions by studying a polyethylene glycol insoluble serum fraction. Although the extent of binding to Clq was within normal limits, these patients had increased concentrations of factor B in the immune complex serum fraction. These findings indicate that an unusual type of circulating immune complex may be detected after splenectomy, suggesting a possible role for the spleen in the removal of circulating immune complexes.

Neutrophil function in megaloblastic anemia.

Chemotaxis, nitroblue tetrazolium (NBT) reduction and the presence of Fc gamma receptors on surface membranes were measured in the peripheral blood neutrophils of 20 patients with megaloblastic anemia (15 with folate deficiency and 5 with pernicious anemia). The percent of NBT- or Fc gamma-positive neutrophils was the same for patients and normal controls. In contrast, chemotaxis was decreased approximately 20% (P less than 0.05) in megaloblastic anemia and when anemia was corrected in 3 cases, chemotaxis increased to levels within the normal range. In contrast to what one would expect on the basis of these laboratory findings, there are no reports of increased susceptibility to infections in these patients.

T-cell subsets in patients with aplastic anemia.

Abnormalities of lymphocyte subpopulations have been described in patients with aplastic anemia. In the present report we extend these studies by measuring T cell subsets identified by the presence of Fc receptors for IgM (T mu-lymphocyte) and IgG (T gamma-lymphocyte) in 22 patients and in 48 normal controls. The absolute number of T mu and T gamma lymphocytes was normal in the majority of cases. The percentages of T mu cells was increased in 4 cases and decreased in 2; T gamma cells were increased in 6 patients. The levels of serum immunoglobulins did not correlate with the T mu/T gamma ratio. The pathogenesis of aplastic anemia is discussed in terms of these immunological abnormalities.

Age-associated changes of memory (CD45RO+) and naive (CD45R+) T cells.

1. The total number of lymphocytes and the percentage of CD45RO+ (putative memory T cell) and CD45R+ (putative naive T cell) were determined in 15 cord blood samples, 66 healthy children ranging in age from 1 to 18 years, 16 adults (23-59 years) and 16 aged individuals (60-96 years). 2. The total number of lymphocytes decreased with age and reached the adult range in children aged 11-14 years. CD45R+ T cells were the predominant cell type in cord blood and decreased with age up to the adult group, while the percentage of CD45RO+ T cells was low in cord blood and increased with age. No significant difference was observed between the adult and the aged groups for either lymphocyte subset. 3. These data support the view that CD45RO+ and CD45R+ T-cell subsets represent maturational stages of T cells.

Cell-mediated immunity in Brazilian hemophilia patients.

The purpose of this study was to correlate the immunological features of healthy hemophiliac A patients treated with commercial cryoprecipitates with those who received factor VIII concentrates associated or not with cryoprecipitates. The absolute number of total lymphocytes, T3, T4 and B lymphocytes did not differ for either group of patients or the controls. The number of T8 lymphocytes was higher in the group treated only with cryoprecipitates than for the controls. The T4/T8 lymphocyte ratios for both groups of patients were significantly lower than in the controls. This was due to a decrease in the percentage of T4 and increase of T8 lymphocytes. K cell activity was lower in both groups of patients than the controls. These results indicate that both cryoprecipitates and commercial factor VIII concentrate replacements had similar effects on the development of lymphocyte abnormalities.

Circulating immune complexes in sickle cell-beta zero thalassemia.

A serum fraction from patients with sickle cell-beta zero thalassemia prepared by treatment with polyethyleneglycol showed increased amounts of C1q-precipitable immune complexes, i.e., 216 micrograms/dl (range, 141-266 micrograms/dl) vs 181 micrograms/dl (range, 152-228 micrograms/dl) for controls (P less than 0.05), as well as increased amounts of protein. Levels of IgG, IgA, IgM, C3, C4 and factor B in the same fraction were within the normal range.

Immunophenotype of normal and leukemic bone marrow B-precursors in a Brazilian population. A comparative analysis by quantitative fluorescence cytometry.

The distinction between normal and leukemic bone marrow (BM) B-precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia (ALL). In order to evaluate the potential use of quantitative fluorescence cytometry (QFC) for this distinction, we studied 21 normal individuals and 40 patients with CD10+ ALL. We characterized the age-related changes of the CD10, CD19, TdT, CD34 and CD79a densities in normal and leukemic BM. Compared to normal adults, the B-precursors from normal children expressed significantly lower values of CD34-specific antibody binding capacity (SABC) (median value of 86.6 vs 160.2 arbitrary units (a.u.) in children and adults, respectively). No significant age-related difference was observed in the expression of the other markers in the normal BM, or in any of the markers in the leukemic BM. Based on the literature, we set the cut-off value for the normal CD10 expression at 45 x 10(3) a.u. for both age groups. For the remaining markers we established the cut-off values based on the minimum-maximum values in the normal BM in each age group. The expression of CD10 was higher than the cut-off in 30 ALL cases and in 18 of them there was a concomitant aberrant expression of other markers. In 9 of the 10 CD10+ ALL with normal CD10 SABC values, the expression of at least one other marker was aberrant. In conclusion, the distinction between normal and leukemic cells by QFC was possible in 38/40 CD10+ ALL cases.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes.

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

Antibody-dependent cellular cytotoxicity in some lymphoreticular diseases.

Antibody-dependent cellular cytotoxicity mediated by K cells against chicken erythrocytes was measured in 113 patients with malignant lymphoreticular disorders and compared with 230 controls. The results were expressed as the specific cytotoxicity of a fixed number of cells and also by cytotoxic capacity, which measures the number of cytolytic units in 1 ml of blood. The values for cytotoxic capacity were normal in the group of untreated patients with non-Hodgkin's lymphomas, multiple myeloma or chronic lymphocytic leukemia and in most of the patients with Hodgkin's disease or acute lymphoblastic leukemia. However, decreased specific cytotoxicity was observed in these same lymphoid leukemia patients, which may be due to dilution of effector cells. The effect of chemotherapy in reducing K-cell activity is more evident in patients with multiple myeloma, followed by patients with Hodgkin's disease, and finally by patients with non-Hodgkin's lymphomas. No case of K-cell neoplastic disease was observed in this series.

Lymphocyte subpopulations in patients with advanced breast cancer submitted to neoadjuvant chemotherapy.

AIMS AND BACKGROUND: There is an enhanced immune response in patients with breast cancer after the use of chemotherapy. The objective of this study was therefore to investigate alterations in the number of peripheral lymphocytes in patients with breast cancer after neoadjuvant chemotherapy (NC) and the relationship with prognosis. METHODS: Thirty women were analyzed. Their UICC staging was IIb (only T3N0 included) and III (N3 not included). Sample analysis was performed using flow cytometry before the first cycle and 18 to 21 days after the last cycle of NC. The lymphocyte subsets studied were: T (CD3, CD4, CD8), B (CD19, CD23), natural killer (NK) (CD56, CD16), and interleukin-2 (CD25). CD3, CD56, CD8, and CD16 lymphocytes were analyzed with double marking. After x = 3.8 +/- 1.3 cycles of 5-fluorouracil, epirubicin and cyclophosphamide (FEC), 16 patients showed a complete or partial response (group 1). After three cycles 14 showed no response or tumor progression (group 2). A control group of healthy women was used for pretreatment analysis. RESULTS: Before NC there was a significant increase in B lymphocytes and NK cells in comparison to the control group. After NC there was a significant percentage increase in CD3, CD4, CD8, CD25 and CD3+CD56+ cells and a decrease in CD19, CD23, CD56, CD16 and CD16+CD8+ cells. There was a significant fall in the absolute number of CD4, CD19, CD23, CD56, CD16 and CD16+CD8+ lymphocytes and an increase in CD3+CD56+ lymphocytes. Before NC the ratio CD4/CD8 in group 1 was 2.25 +/- 0.5 and in group 2 it was 1.79 +/- 0.5 (P <0.05). CONCLUSIONS: Patients with advanced breast cancer showed increases in B and NK lymphocytes. Neoadjuvant chemotherapy (FEC) caused an increase in CD3+CD56+ and a decrease in B lymphocytes. Patients with an increased CD4/CD8 ratio have a better chance of responding to neoadjuvant chemotherapy.

Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease.

The absolute numbers of total leukocytes, lymphocytes. T cells, helper/inducer, suppressor/cytotoxic and B cells were decreased in the peripheral blood of patients with chronic Chagas' disease. Since antilymphocyte antibodies were present only in a minority of patients they probably cannot account for the abnormalities in lymphocyte subsets. Patient neutrophils stimulated with endotoxin-treated autologous plasma showed depressed chemotactic activity and this seems to be an intrinsic cellular defect rather than plasma inhibition. Random migration of neutrophils was normal. Reduction of nitroblue tetrazolium by endotoxin-stimulated neutrophils was also decreased. These findings further document the presence of immunosuppression in human Chagas' disease. They may be relevant to autoimmunity, defense against microorganisms and against tumor cells at least in a subset of patients with more severe abnormalities.