Decreased chemiluminescence in leukocyte adhesion deficiency presenting with recurrent sepsis, amoebiasis and Candida albicans urinary tract infection.

Leukocyte adhesion deficiency (LAD) is a rare disorder of cellular immunity, generally due to various mutations producing reduced or altered expression of membrane integrins. The authors report a case of LAD due to integrins expression imbalance. LAD was suspected after recurrent sepsis, fungal infection and amoebiasis with persistent leukocytosis. Neutrophils were studied with chemiluminescence showing decreased functional activity: up to now, this seems the first chemiluminescence study of neutrophil function and the first report of amoebiasis at the onset in LAD.

Phosphatidylserine exposure in platelet concentrates during the storage period: differences between the platelets collected with different cell separators.

BACKGROUND AND OBJECTIVES: Platelet alterations occur during the production and storage of platelet concentrates, the so called "storage lesion". We studied the platelet alterations during the storage period in apheresis concentrates, employing flow cytometry for phosphatidylserine (PS) detection on platelets during the five days of storage. MATERIAL AND METHODS: Twenty-seven single donor platelet concentrates harvested with the Cobe Trima, Baxter Amicus, or Haemonetics MCS+ were analyzed for PS exposure by flow cytometry on the day of production (day 1) and on days 3 and 5 of storage. Furthermore PS expression was analyzed in platelet donors' blood samples withdrawn before plateletpheresis. RESULTS: PS expression on platelets gave the following median values: in blood donors before apheresis it was 1.12% (0.13-1.78) in platelets concentrates on the first day (2 h after apheresis) 2.06% (0.66-15.2), the third day 6.57% (1.98-51.13) and the fifth day 23.04% (3.86-80.23). All differences between median values of PS expression in blood samples before apheresis, and platelets concentrates on days 1, 3 and 5 of storage, are statistically significant. The expression of PS in platelet concentrates was analyzed in relation to the blood cell separator used for the collection procedure and showed the following results: on day 1 the median values of PS in platelet concentrates collected with the three different blood cell separators, Trima, Cobe and MCS, did not show statistically significant differences. On day 3, the platelets concentrates collected with the Trima and with the MCS showed differences that were statistically significant. Those were respectively 10.59% (4.56-51.13) and 3.53% (1.98-12.61), p = 0.005. The PS expression in platelet concentrates collected with the Trima and MCS showed differences that are also statistically significant on day 5 at respectively 32.4% (9.61-80.23) and 8.57% (3.86-48.42), p = 0.005. CONCLUSIONS: PS exposure in platelet concentrates on days 3 and 5 rise to levels that could compromise the quality of the platelet units. Improvements in standardized platelet quality controls, and in platelet collection systems are required to reduce the storage lesions in platelets concentrates.

CD34+/CD105+ cells are enriched in primitive circulating progenitors residing in the G0 phase of the cell cycle and contain all bone marrow and cord blood CD34+/CD38low/- precursors.

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte-macrophage colony-forming units (GM-CFU) and burst-forming units - erythroid (BFU-E) were retained in the CD34+/CD105- fraction, whereas rare GM-CFU/BFU-E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti-TGF-beta1 antibody demonstrated CD34+/CD105+ cells capable of colony-forming activity without any significant effect on CD34+/CD105- cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105- cells. CD34+/CD105+ cells had a significantly higher long-term culture-initiating cell (LTC-IC) frequency than CD34+/CD105- cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki-67- cells whereas 82% of CD34+/CD105- were DNA 2n G1Ki-67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105- contained a small CD25+ subset. Three-colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/- primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.

Bezafibrate as differentiating factor of human myeloid leukemia cells.

Bezafibrate belongs to the class of fibric acid derivatives usually used as antihyperlipidemia agents. From the biochemical point of view, these drugs show intriguing properties which leads one to think they may promote a differentiation process in tumour cells. This new pharmacological activity of fibrates could partially depend on the induction of an oxidative stress. To test this hypothesis, the effect of bezafibrate, as well as of clofibric acid and gemfibrozil, on growth, functional and cytochemical characteristics of human leukaemia-derived cell lines HL-60, U-937 and K-562 has been studied in some details. The results show that bezafibrate, gemfibrozil and clofibric acid, do induce differentiation in human myeloid leukaemia cell lines as indicated by several differentiation markers. Moreover fibrates, in dose dependent manner, significantly alter the cell cycle distributions, mainly leading to G0/G1 phase increment and G2/M phase reduction. The differentiating activity of fibrates could have significant implications both for the pharmacotoxicological profile of this class of compounds and for the pathophysiology of neoplastic disease.

Large volume leukapheresis for collecting hemopoietic progenitors: role of CD 34+ precount in predicting successful collection.

In this work we evaluated the efficacy of stem cell collection with Large Volume Procedures. (LVP), and analysed the importance of the CD34+ cell precount in promoting the collection of a sufficient number of CD34+ cells for transplantation, using the Univariate Logistic Regression analysis. Eighty-nine leukapheresis were performed in 49 patients with hematological malignancies and solid tumors, mobilized with chemotherapy plus Granulocyte Colony Stimulating Factor (G-CSF). For each procedure 15.8 liters of blood were processed. The median value of Nucleated Cells (NC) and CD34+ cells precount was respectively 8.29 x 10(9)/ml (range 1.13/45.4) and 43.08 x 103/ml (range 1.06/795.2). Results show the capability of LVP to collect large quantities of hemopoietic progenitors with a median CD34+ cell total yield of 215.02 x 10(6) (range 5.03/2210). The yields per patients' body weight were: CD34+ cells 3.23 x 10(6)/kg (range 0.081/41.58). The regression analysis between blood cell precounts and collection yields gave the following correlations: the CD34+ cell precount correlates with CD34+ yield (r = 0.78 p < 0.00) and with CD34+ cell yield/kg (r = 0.76 p < 0.00). The number of CD34+ cells processed correlated with the number of CD34+ cells collected/kg (r = 0.83 p < 0.000). To investigate the importance of CD 34+ cell precount in promoting CD34+ cell yields > or =2.5 x 10(6)/kg we performed a Univariate Logistic Regression analysis that showed in our patients a probability of collecting > or =2.5 x 10(6) CD34+/kg that rose from 0.6 to 0.95 for CD 34+ precounts that oscillated from 30 to 40 x 10(3) CD34+ cells/ml, respectively. The Univariate Logistic Regression gave a probability of collecting > or =2.5 x 10(6) CD34+ cells/kg that oscillated between 0.64/0.98 for values of CD34+ cells processed from 6 x 10(6)/kg to 8 x 10(6)/kg, p < 0.000. Sixty-three percent of patients reached the target dose of 2.5 x 10(6) CD34+ cells/kg with only one LVP. Until now 12 patients have been transplanted and all have had a prompt and complete lasting recovery. These results confirm the efficacy of LVP in harvesting hemopoietic progenitors and their ability in reconstituting hemopoiesis of transplanted patients, enabling the estimation of CD34+ precounts and CD34+ cells processed values, highly predictive for the collection of > or =2.5 x 10(6) CD34+ cells/kg. Furthermore, the Logistic Model suggests that the best strategy to plan a successful CD34+ cell collection procedure is to identify for each patient the amount of CD34+ cells/kg to be processed rather than the fixed processing of 3/5 blood volumes in all patients.

Induction of CD69 antigen on normal CD4+ and CD8+ lymphocyte subsets and its relationship with the phenotype of responding T-cells.

We evaluated phenotype and apoptotic status of normal CD4+CD69+ and CD8+CD69+ peripheral blood T-lymphocytes after short-term challenge with escalating concentrations of phytohemagglutinin (PHA). The frequency of CD69-coexpressing CD4+ and CD8+ T-cells and CD69 staining intensity increased following T-cell mitogenic stimulation; these changes were proportional to PHA concentration in culture medium. A considerable fraction of lymphocytes underwent blast transformation, displaying increased forward and side scatter signals. Interestingly enough, PHA-responsive T-cells exhibited a predominantly CD25negCD38negTCRalphabetapos phenotype; APO-1/Fas antigen (CD95) could be detected on a minority of activated CD69+ T-cells. A considerable proportion of CD69+ lymphocytes expressed intracellular perforin; in addition, an average 16+/-6% CD69+ T-lymphocytes were apoptotic after 4 h of stimulation, as evaluated by 7-amino-actinomycin-D staining and by annexin-V binding. CD69+ activated lymphocytes comprise phenotypically heterogeneous cell subpopulations potentially devoted to diverse immunological functions, i.e., proliferation, apoptosis, or cell cytotoxicity; moreover, our findings indicate that CD69 expression is proportional to the intensity of the activating stimulus and that the capacity to upregulate CD69 antigen following short-term mitogenic challenge may be restricted to unactivated CD38negCD25negTCRalphabetapos T-lymphocytes.

Expression of thrombospondin receptor (CD36) in B-cell chronic lymphocytic leukemia as an indicator of tumor cell dissemination.

BACKGROUND AND OBJECTIVE: The expression of CD36 antigen has not been conclusively associated with human B-lymphocytes although CD36 was recently detected in a human B-cell angiotropic lymphoma where it might be involved in lymphoblast-endothelial cell adhesion. We investigated the expression of CD36 in B-cell chronic lymphocytic leukemia (CLL) by multiparameter flow cytometry; results were correlated with clinical features. DESIGN AND METHODS: CD36 expression was evaluated on peripheral blood and bone marrow samples from 24 patients affected by CD5+ B-CLL. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation, were labeled with fluorochrome-conjugated monoclonal antibodies under standard experimental conditions and were analyzed by flow cytometry. CD36 expression was quantified both in terms of frequency of CD19+CD36+ cells and of mean fluorescence intensity (MFI-R) of CD36+ cell populations. The intensity of CD36 expression was arbitrarily classified as weak (MFI-R ranging from 3 to 6; score 0), moderate (MFI-R ranging from 6 to 9; score 1), intermediate (MFI-R ranging from 9 to 11; score 2) or strong (MFI-R ranging from 11 to 17; score 3). RESULTS: CD36 could be detected on 3% (range 2-5) of normal CD19+ B-lymphocytes and on 45% (range 30-75) of neoplastic CD19+ B-cells. When CLL patients were stratified according to CD36 staining intensity, higher hemoglobin levels (Hb) were recorded in patients assigned to score 0 (Hb = 14.3 g/dL; range 13.9-15.1) compared to patients scoring 1-2 (Hb = 11.2; range 10.3-12.2) or 3 (Hb = 9.8; range 9.6-11.6; p=0.0053). Similarly, higher platelet counts (Plt) were found in patients scoring 0 (Plt = 282x10(3)/microL; range 244-319), compared to patients with intermediate (Plt = 175x10(3)/microL; range 144-238) and high scores (Plt = 149x10(3)/microL; range 103-230; p=0.044); lymphocyte count (Ly) was significantly higher in patients assigned to score 3-4 (Ly = 23.3x10(3)/microL, range 13-30) compared to score 0-2 (Ly = 9.8x10(3)/microL, range 8.5-10.8; p=0.045). CLL patients expressing CD36 at intermediate-to-strong intensity (MFI-R = 14, range 9-16) were more frequently assigned to Rai stages III-IV than stages I-II (CD36 MFI-R = 9, range 6.5-11; p=0.005) and stage 0 (CD36 MFI-R = 6, range 4-7.3; p<0.001). Interestingly, bone marrow diffuse histology was strongly associated with higher CD36 expression (MFI-R = 8.7; range 4.7-13.9) compared to non-diffuse patterns of bone marrow infiltration (MFI-R = 6.7; range 5.2-9.3; p=0.0019). In multivariate regression analysis, CD36 staining intensity significantly and independently correlated with diffuse BM histology (p=0.033). INTERPRETATION AND CONCLUSIONS: The present report provides the first evidence of CD36 expression on CD19+ B-cells from CLL; the correlations with clinical parameters strongly support the view that CD36 might favor tumor cell spreading. Whether high CD36 expression levels on CLL CD19+ B-cells identify an aggressive disease subset remains to be further confirmed in larger series of patients.

Immunophenotypic profile of peripheral blood eosinophils in acute graft-vs.-host disease.

We used a flow cytometry technique, the "FOG" method (formaldehyde and octylglucopyranoside), to investigate the expression of activation antigens, i.e., CD4, CD23, CD25, HLA-DR, and the EG2 epitope of eosinophilic cationic protein, on peripheral blood eosinophils (PBEs) in leukemic patients who had developed acute graft-vs.-host disease (aGVHD) with eosinophilia after allogeneic bone marrow transplantation (alloBMT) or leukocyte buffy coat infusion. A comparative analysis was performed in transplanted patients not affected by aGVHD and in other conditions commonly associated with peripheral eosinophilia, i.e., interleukin (IL)-2 immunotherapy and allergy. CD25, recognizing the p55 subunit of IL-2 receptor, was detected in all patients with aGVHD except two who, at the onset of eosinophilia, were already receiving methylprednisolone intravenously. The specificity of our findings is confirmed by the absence of reactivity with anti-CD25 mAb in PBEs from transplanted patients not affected by aGVHD. Interestingly, the expression of CD25 progressively declined after steroid therapy. CD25 was also expressed after rhIL-2 administration, probably reflecting analogous mechanisms of eosinophil activation. No aGVHD or rhIL-2-treated patient showed reactivity with anti-CD4, CD23, or HLA-DR. CD25 and CD23 antigens were detected in 29% and 36% of allergic patients only. The accessibility of the EG2 epitope was significantly enhanced in all study groups compared with controls. In vitro activation of normal eosinophils with phorbol 12-myristate 13-acetate upregulated CD9 and EG2 expression but failed to induce the CD25 antigen, suggesting that selective activating stimuli may be required. The functional significance of in vivo CD25 expression and the role of activated PBEs in the development of cellular and cytokine-mediated tissue destructive processes in aGVHD remain to be clarified.

Cytotoxicity of paclitaxel and docetaxel in human neuroblastoma cell lines.

Taxanes are an important new class of anticancer agents that inhibit cell division by the unique mechanism of increasing the rate of microtubule assembly and preventing microtubule depolymerisation. Using the colony inhibition assay, we compared the cytotoxicity of paclitaxel and docetaxel in three human neuroblastoma (NB) cell lines, SH-SY5Y, BE(2)M17 and CHP100. Different exposure times (3, 6, 12, 24, 48 and 72 h) and different concentrations ranging from 0.1 nM to 10 microM were tested. Both paclitaxel and docetaxel show antineoplastic activity in human NB cell lines. Taxanes' antitumour activity varied among the different cell lines, CHP100 being the most sensitive and SH-SY5Y the least sensitive. Paclitaxel cytotoxicity appears schedule-dependent, with marked cell kill observed only for exposures of 24 h or longer. Docetaxel cytotoxicity was dependent upon prolonged exposure only in the SH-SY5Y cell line, while an exposure time of 3-6 h resulted in exponential cell kill in the other two cell lines. Docetaxel was more cytotoxic than paclitaxel with a mean ratio of (paclitaxel/docetaxel) IC50 values ranging from 2 to 11. For both taxanes, we observed good correlation between cytotoxic effect and percentage of cells blocked in G2/M phase. A cytotoxic effect occurred at concentrations comparable with those achieved in the plasma of patients treated with these agents in initial clinical trials. The full potential of prolonged infusion or repeated daily administrations of taxanes should be explored in clinical studies, and responses to taxanes in neuroblastoma should be assessed in paediatric phase II studies.

Further investigations on the expression of HLA-DR, CD33 and CD13 surface antigens in purified bone marrow and peripheral blood CD34+ haematopoietic progenitor cells.

We evaluated the HLA-DR, CD33 and CD13 antigen expression on CD34+ haematopoietic progenitor cells (HPC) isolated from the bone marrow (BM) and peripheral blood (PB) of normal donors. The majority of both BM and PB CD34+ HPC expressed CD13 and HLA-DR. The coexpression of CD34 and CD33 was found in a minor CD34+ subset. After 7 d of culture in the presence of interleukin-3 and granulocyte-macrophage colony-stimulating factor, CD33 expression was detected in about 50% of HPC. At this point CD34 antigen expression was lost and CD13 and HLA-DR expression was partially lost. After 14 d of culture, the majority of HPC were CD33+. HPC maintained the capacity to generate colony forming unit granulocyte-macrophage but they lost the capacity to generate burst forming unit-erythroid. A correlation was found between the percentage of CD34+/HLA-DR+ cells and the total number of colony forming cells in unfractionated samples from BM and PB of patients with malignancies. These studies demonstrate that, in normal conditions, only a minor subset of CD34+ cells coexpress CD33 antigen either in BM or in PB and CD33 antigen is a lineage marker which is coexpressed with HLA-DR and CD13 on a progenitor committed to the granulocytic-macrophagic lineage.

Lymphocyte subset counting by immunoperoxidase using an automated routine hematologic analyzer.

Using monoclonal antibodies against CD2, CD4, CD8 and CD19 antigens and an automated biotin-avidin immunoperoxidase technique on whole blood samples, we evaluated the technical performance and clinical usefulness of lymphocyte subset counting by the routine hematology analyzer Technicon H*1. Statistical evaluation demonstrated excellent precision and very good correlation with the immunofluorimetric flow cytometer Ortho Spectrum III. Correlation between manual immunofluorescence at the microscope and the H*1 method was much poorer, owing to the high intrinsic imprecision of the manual method. Reference ranges obtained with the H*1 immunoperoxidase method in 44 healthy subjects closely matched those obtained with the Spectrum III. In 46 subjects with or at risk for HIV infection, we found with the H*1 method a significant decrease in CD4+ cells and in the CD4+/CD8+ cell ratio, which was progressively more marked in HIV- negative patients with lymphadenopathic syndrome, AIDS-related complex, and in patients with full-blown AIDS.