Implementing a routine flow cytometry assay for nucleated red blood cell counts in cord blood units.

INTRODUCTION: As required by standards organizations, Héma-Québec Cord Blood Bank performs enumeration of nucleated red blood cells (NRBCs) in cord blood units (CBUs). This study presents the validation and implementation approaches developed to transfer the routine NRBC enumeration from the manual blood film method to a flow cytometric assay. METHODS: The flow cytometry method was adapted from Tsuji (Cytometry, 37, 1999, 291). This assay was validated to assess the specificity, detection limit, repeatability, and reproducibility of the method, including interoperator and interlaboratory testing. Finally, postimplementation follow-up and adjustments were performed for CBU over a 7-month period. RESULTS: Blood film and flow cytometry NRBC enumerations showed a strong correlation (n = 40; Pearson's r correlation = 0.90). Validation was successful as exemplified by the correlation in interlaboratory testing (n = 30; r = 0.98). During implementation, our routine laboratory analyses revealed that CBU with low NRBC content (≤2%), representing 26% of all CBU tested, resulted in 15% of repeated reading and/or staining and was the principal source of nonconformity. Small adjustments in the standard operating procedures (SOPs), including a fixed 200-event setting in the NRBC gate for the second reading of the replicates, have completely solved this issue. CONCLUSION: Flow cytometric NRBC enumerations, now implemented in Héma-Québec Public Cord Blood Bank, is an improvement in the efficiency of our operations by integrating the count for NRBC into our flow cytometry platform.

The experience of pain and anxiety in rectal cancer patients during high-dose-rate brachytherapy.

BACKGROUND: Pain and anxiety have been reported as primary concerns for patients with head-and-neck, gynecologic, and prostate cancers undergoing high dose rate (hdr) brachytherapy. However, almost no research has been published on the degree to which these symptoms are experienced by rectal cancer patients undergoing hdr brachytherapy. We conducted a pilot study examining the experiences of rectal cancer patients during hdr brachytherapy, specifically the intensity and trajectory of their anxiety and pain. METHODS: Rectal cancer patients (n = 25) who received hdr brachytherapy treatment at a hospital in Montreal, Quebec, completed verbal analog scales for pain and anxiety at 4 time points over 4 treatment days. RESULTS: On all 4 days, a subset of patients reported moderate-to-severe anxiety before applicator insertion. Pain increased significantly from the time patients were lying on the table to immediately after insertion of the applicator (p < 0.001). Insertion of the applicator appears to be the most painful part of the procedure, and although anxiety declined to below baseline after applicator removal, pain remained somewhat elevated. Some patients required conscious sedation; however, reports of moderate-to-severe pain were more frequent from patients who received pain medications than from patients who did not receive such medication (p < 0.05). CONCLUSIONS: Most patients with rectal cancer tolerated hdr rectal brachytherapy well, although the procedure is stressful and painful for some. Insertion of the applicator was found to be the point of maximal pain, and medication was not always completely successful at alleviating the pain, suggesting that additional psychosocial interventions might be needed, with particular emphasis on the time of applicator insertion.

c-Src tyrosine kinase co-associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes.

c-Src is the normal human cellular protein homologue of the viral oncogene v-src. c-Src activity was reported recently to increase in CD40-activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c-Src in this process, we investigated the effects of c-Src over-expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c-Src (c-Src/dominant-positive) or a dominant-negative c-Src (c-Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c-Src/dominant-positive-infected cells. In contrast, over-expression of c-Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c-Src may compete with endogenous c-Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c-Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c-Src and STAT5b co-associate during B lymphocyte proliferation. These results confirm an important role for c-Src in the expansion of normal human B lymphocytes in vitro, in which c-Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.

Screening for depressive symptoms in patients with unresectable lung cancer.

INTRODUCTION: Early identification of psychological distress and depression is important to optimise the quality of life in patients with advanced non-small cell lung cancer (NSCLC). The prevalence of depression may vary, depending on the time since diagnosis of cancer, results of the treatment and the prognosis. The purpose of this study was to compare the efficacy of a self-administered screening tool (Hospital Anxiety and Depression Scale (HADS)) with a health professional administered tool (Montgomery-Asberg Depression Rating Scale (MADRS)) and to explore the variability of major affective symptoms in patients with unresectable lung cancer during the initial 7-8 weeks of chemotherapy treatment for their illness. MATERIAL AND METHODS: Patients with newly diagnosed unresectable lung cancer were screened on four occasions for anxiety and depressive symptoms simultaneously using the self-rated HADS and the MADRS administered by a psycho-oncologist or a trained research associate. The first assessment was done within 1 week of diagnosis and was repeated on 3 occasions during the initial 2 cycles of chemotherapy. RESULTS: Forty-nine patients, aged 38-82 years (median age 63 years) were enrolled. All patients had advanced NSCLC (stages 3A, 3B and 4) and 61% (30 patients) had an ECOG performance status (PS) of 1 or greater. The point prevalence of depression measured by an interviewer using the MADRS at visits 1-4 was 49%, 51%, 47%, and 41%, respectively. The point prevalence of self-reported depression (HADS) was significantly (p < 0.001) lower at each assessment point (18%, 20%, 6%, 12%) compared to health professional detected depression (MADRS). Although MADRS and HADS showed very strong (Pearson's correlation = 0.8) and significant (p < 0.001) correlation, the concordance rate in identifying the same cases of depression was only 54%. CLINICAL IMPLICATION AND CONCLUSION: The prevalence of depression among advanced lung cancer patients is high and varies very little during the first 2 cycles of chemotherapy. Among a variety of tools available for the screening of depression, a semi-structured interview is more effective at identifying clinically significant depression than a self-administered questionnaire.

Telomere-independent reduction of human B lymphocyte: proliferation during long-term culture.

Telomeres and telomerase, the telomere lengthening enzyme, have been shown to play a central role in the long-term ability of cells to proliferate and maintain viability. In opposition to transformed cells, normal somatic cells express a low level of telomerase, which results in the gradual shortening of their telomeres after each division and in cell senescence once a critical telomere length is reached. We have tested the hypothesis that shortening of telomeres could limit the expansion of normal human B lymphocytes maintained in long-term culture using a CD40/CD154 system. Measurement of temolerase activity in cell lysates showed a rapid up-regulation of telomerase following the initiation of the culture that was dependent on the CD40 signaling. The high level of telomerase activity and the corresponding long telomere structures remained constant for the 35 day culture period in which a gradual reduction of the cell expansion rate is observed. We conclude that the gradual in vitro senescence of cultured B cells does not correlate with a corresponding loss of telomerase activity and of telomere length. Rather the phenomenon may be related to an intrinsic property of the proliferating B cells to differentiate into Ig-secreting cells.

Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

CD5+ B cell-dependent regulation of the murine T-cell independent immune response against the human blood group A antigen.

The CD5+B lymphocyte (B1a) population is known to be involved in most immune responses to microorganism TI antigens. Moreover, xid mice deficient for immune responses against TI-2 antigens are known to lack the B1a population, suggesting a role for B1a cells in TI-2 immune responses. We previously established that the oligosaccharide human blood group A antigen stimulated murine TI-2 immune responses. In this work, we show that the frequency of anti-A-secreting hybridomas was higher in mice with larger splenic B1a populations and that in vivo anti-CD5 treatment reduced anti-A immune response without affecting the response against TD RBC antigens. A similar effect was observed by in vitro anti-CD5 treatment of splenocytes. The in vivo anti-CD5 treatment also interfered with the immunization-dependent increase in splenocyte numbers. These results are in agreement with an important role for the B-cell CD5 receptor in the regulation of TI-2 immune responses possibly mediated by its interaction with the CD72 ligand.

Induction of LFA-1 independent human B cell proliferation and differentiation by binding of CD40 with its ligand.

Engagement of CD40 on resting B cells in the presence of IL-4 triggers B cell proliferation, differentiation and homotypic adhesion. This study was designed to investigate the role of LFA-1/ICAM-1 interactions in homotypic adhesion and proliferation of CD40-activated human B lymphocytes. Freshly isolated B cells were cultured in vitro in the presence of IL-4 and of L cells expressing CD40L, the CD40 ligand. The addition to the culture medium of LFA-1 and ICAM-1 antibodies inhibited homotypic B lymphocyte adhesion. However, these antibodies failed to affect B lymphocyte proliferation and antibody production. These results indicate that aggregation and proliferation are independent events although both induced by CD40 activation.

Hypocalcemia decreases the early and late responses to epidermal growth factor in rat hepatocytes.

Extreme variations in extracellular Ca2+ concentrations ([Ca2+]e) modify the signaling generated by many hormones and growth factors. However, the influence of physiological changes in [Ca2+]e on the response to hepatic mitogens remains largely unknown. To study the influence of [Ca2+]e on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca2+]e similar to those observed in normocalcemia or hypocalcemia. To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca2+]e prevailing in vivo. Intracellular Ca2+ concentrations ([Ca2+]i) and DNA synthesis were evaluated after increasing doses of EGF. [Ca2+]e strongly influenced the [Ca2+]i response to EGF with significantly smaller [Ca2+]i increases in hepatocytes of normal rats kept in low [Ca2+]e compared with those kept in normal [Ca2+]e. In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca2+]e. In normal [Ca2+]e, the EGF-induced increases in [Ca2+]i were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca2+]e significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca2+]e. Furthermore, the blunted responses in [Ca2+]i mobilization and DNA synthesis associated with hypocalcemia could not be overcome by increasing concentrations of EGF nor by normalization of [Ca2+]e in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Type 2 T-cell-independent murine immune response to the human AB0 blood group antigens.

The human AB0 blood group determinants are simple carbohydrate structures which are widely distributed in nature. Much work has been done on the structure of the A and B antigens but little is known on the regulation of anti-A and anti-B immune responses. To develop a model system, we have characterized the AB0 immunity of normal Balb/c mice and found a significant level of serum natural anti-A but almost no anti-B. This finding and the known IgM predominance among immune anti-A produced in the Balb/c mouse indicate that the AB0 immunity of this mouse strain is comparable to the one of human blood group B individuals. Following immunization with human red blood cells, similar levels of anti-A were produced in normal and athymic Balb/c mice showing that the anti-A response is T cell independent. Furthermore, no anti-A or anti-B antibodies were produced in CBA/xid mice indicating a type 2 T-cell-independent immune response. These results may contribute to a better understanding of human AB0 responses and establish the mouse as a suitable model to study the immunobiology of AB0 antigens.

Efficient preparation of human monoclonal antibody-secreting heterohybridomas using peripheral B lymphocytes cultured in the CD40 system.

The use of peripheral B lymphocytes in the successful preparation of human monoclonal antibodies by hybridoma technology is highly dependent on lymphocyte activation procedures. We studied the ability of peripheral human B lymphocytes cultured in vitro and activated through their CD40 antigen (CD40 system) (Banchereau et al., 1991) to form antibody-secreting heterohybridomas after fusion with murine X63Ag8.653 myeloma cells. The frequency of antibody-secreting heterohybridomas formation was greatly increased (15 times) by culture of B cells in the CD40 system. The CD40 system offers many advantages over other procedures of B lymphocyte activation representing a significant technological advance in the preparation of human monoclonal antibodies by standard hybridoma technology.

A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus.

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.

Negative effect of multiple antigen injections on the yield of murine monoclonal antibodies obtained by hybridoma technology.

One of the critical steps in the preparation of monoclonal antibodies is the obtainment, by in vivo immunization and boost, of the maximal number of antigen-activated B lymphocytes. In hybridoma laboratories, the common procedure is to immunize a group of mice with several antigen injections and use the mouse showing the highest serum antibody titre for the fusion experiment. The observation that the use of mice hyperimmunized with human red blood cells failed to yield a high number of monoclonal antibodies, led us to study the effect of multiple antigen injection prior to the fusion experiment. The results obtained showed that the maximal yields of monoclonal antibodies were obtained using mice that had received only one or two antigen injections while the mice immunized with three antigen injections consistently yielded at least a three-fold reduction in the number of monoclonal antibodies. The negative effect could not be reversed by prolonged resting of the animals and suggests the induction of a tolerance/suppression state which can prevent the final activation step. These results point out to the importance of avoiding the hyperimmunization of mice for the preparation of a high number of monoclonal antibodies by standard hybridoma technology.

Intracellular xylitol-phosphate hydrolysis and efflux of xylitol in Streptococcus sobrinus.

The parental strain Streptococcus sobrinus (Streptococcus mutans ATCC 27352), which is known to transport, phosphorylate and accumulate xylitol intracellularly as nonmetabolizable xylitol-phosphate (xylitol-sensitive (XS) strain) and its xylitol-resistant (XR) spontaneous mutant were used to further investigate the inhibitory action of xylitol on oral streptococci. Fructose-grown XR cells did not accumulate xylitol-phosphate, indicating that the inducible fructose PTS is incapable of transporting the pentitol. The intracellularly accumulated pentitol-phosphate by the XS cells did not prevent the subsequent uptake and degradation of glucose or fructose, despite a drop in the PEP pool and a 50% inhibition of the glucose but not the fructose catabolism. Intracellular dephosphorylation of the pentitol-phosphate and release of xylitol in the extracellular medium resulted in a rapid decrease of the intracellular level of this nonmetabolizable product. A Mg(++)- or Mn(++)-independent sugar-phosphate hydrolysing activity capable of splitting xylitol-phosphate was demonstrated in both XS and XR strains. Preincubation in the presence of N1-ethylmaleimide (NEM) and xylitol or NEM and fructose resulted in the subsequent inhibition of both xylitol uptake and efflux. The efflux kinetic at various temperatures is compatible with a facilitated diffusion by the phosphotransferase system EIIfru without, however, excluding the existence of an additional exit route, but it excludes a simple diffusion exit process. The results are consistent with the existence of a xylitol futile cycle contributing to the growth inhibition of S. sobrinus by the pentitol without excluding a toxic effect of xylitol-phosphate. Discrepancies in the literature on the action of xylitol on S. mutans could be explained in the light of the evidence presented.

Acute T-cell leukemia/lymphoma mimicking Hodgkin's disease with secondary HTLV I seroconversion.

The authors observed a pleiomorphic lymphoma mimicking Hodgkin's lymphoma in a French Guyana black woman lacking antibodies for human T-cell lymphoma/leukemia virus type I (HTLV I). After two courses of chemotherapy with either mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) or doxorubicin, bleomycin, vincaleukoblastine, and dacarbazine (ABVD), a typical acute T-cell leukemia/lymphoma developed with HTLV I seroconversion. Specific HTLV I DNA sequences were detected using the polymerase chain reaction (PCR) on a lymph node biopsy obtained before chemotherapy. The mechanisms of the seroconversion are discussed.

Preparation and Purification of Xylitol-5-Phosphate from a Cell Extract of Lactobacillus casei Cl-16.

A simple procedure which yields pure xylitol-5-phosphate is described. A cell extract of Lactobacillus casei Cl-16 from a 6-liter culture was used to synthesize up to 70 mg of xylitol-5-phosphate overnight from xylitol and phosphoenolpyruvate via a xylitol phosphoenolpyruvate:phosphotransferase system with a 53% yield. Centrifugation, filtration, precipitation as a barium salt, and ion-exchange batch chromatography permitted recovery of nearly 90% of the phosphorylated product synthesized. Thin-layer chromatography and enzymatic analysis indicated a purity level of more than 99%. The method was used to synthesize [U-C]xylitol-5-phosphate, and it is suitable for the synthesis of many other nonmetabolizable sugar phosphates.

Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture.

The membrane-bound, sugar-specific enzyme II (EII) component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Streptococcus mutans Ingbritt is repressed by growth on glucose under various conditions in continuous culture. Compared with optimal PTS conditions (i.e., glucose limitation, dilution rate [D] of 0.1 h-1, and pH 7.0), EII activity for glucose (EIIGlc) and mannose (EIIMan) in cells grown at a D of 0.4 h-1 and pH 5.5 with the same glucose concentration was reduced 24- to 27-fold. EII activity with methyl alpha-glucoside and 2-deoxyglucose was reduced 6- and 26-fold, respectively. Growth with excess glucose (i.e., nitrogen limitation) resulted in 26- to 88-fold repression of EII activity with these substrates. The above conditions of low pH, high dilution rate, and excess glucose also repressed EII activity for fructose (EIIFru), but to a lesser extent (two- to fivefold). Conversely, growth of S. mutans DR0001 at a D of 0.2 h-1 and pH 5.5 resulted in increased EIIGlc and EIIMan activity. Unlike the EII component, the HPr concentration in S. mutans Ingbritt varied only twofold (5.5 to 11.4 nmol/mg of protein) despite growth at pH 5.5 with limiting and excess glucose. The HPr concentrations in S. mutans DR0001 and the glucose-PTS-defective mutant DR0001/6 were similar. In a companion study, the soluble components of the PTS (i.e., HPr, EI, and EIIILac) in Streptococcus sobrinus grown on limiting lactose in a chemostat were not influenced significantly by growth at various pHs (7.0 and 5.0) and growth rates (D of 0.1, 0.54, and 0.8 h-1). However, growth on lactose resulted in repression of both EIIGlc and EIIFru, confirming earlier results with batch-grown cells. Thus, the glucose-PTS in some strains of S. mutans is regulated at the level of EII synthesis by certain environmental conditions.

Immunobiology of the oral mucosa in the mouse.

The incidence of immunoglobulin (Ig)-synthesizing cells, Thy 1-positive cells and macrophages in the murine oral mucosa was investigated. Immunofluorescence studies of frozen tissue sections showed that IgA-, IgM- and IgG-containing cells and Thy 1-bearing cells were closely associated with the minor salivary glands. A quantitative analysis was then undertaken using single cell suspensions of the tissue. After mechanical disruption or enzymatic digestion of the mucosa, lymphoid cells were recovered almost exclusively from the mucosa of the posterior soft palate where we observed a dense accumulation of minor salivary glands. Thy 1-bearing cells were found at a higher frequency (25% of recovered cells) than membrane Ig-positive B lymphocytes (6-7%) in these suspensions. Cytoplasmic Ig+ cells accounted for about 6% of recovered cells, whereas plaque-forming cells (Ig-secreting cells) occurred at the same frequency as in the spleen (0.1%). Plasma cells of the IgA and IgM isotypes predominated over IgG-secreting cells (A:M:G ratio = 1:1:0.2); this distribution did not directly correlate with the isotype distribution of salivary Igs (A:M:G ratio = 1:0.003:0.07). In addition, about 10-14% of the cells in our preparations were esterase-positive mononuclear cells. Present data indicate that the murine oral mucosa contains both effector and regulatory cells required for the development and expression of local antibody responses.