Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients

Recent studies have shown that normal human T cells contain a high- molecular-weight (mol wt) protein exhibiting B cell growth factor (BCGF) activity. Other studies have shown that virally transformed human B cells also secrete a high-mol-wt BCGF-like molecule in vitro. We have studied neoplastic B cells from patients with untreated hairy cell leukemia (HCL) to ascertain whether such cytoplasmic BCGF activity is present in the tumor cells. Studies on HCL cells from four patients indicated that BCGF-like activity was in fact present in the cytosolic extracts when tested on autochthonous HCL cells as well as on normal BCGF-dependent human B cell lines. Chromatographic analysis indicated that the BCGF activity from HCL cells was similar in mol wt as well as function to the normal T cell-derived cytosolic BCGF activity. These studies suggest that HCL cells contain and, in some cases, secrete a high-mol-wt growth factor that can be autostimulatory and appears to resemble a similar growth factor molecule found in normal human T cells.     

Enhancement of in vitro beta-thalassemic and normal hematopoiesis by a noncytotoxic monoclonal antibody, 9.1C3: evidence for negative regulation of hematopoiesis by monocytes and natural killer cells

The enhancement of in vitro human hematopoiesis by the addition of a noncytotoxic monoclonal antibody, 9.1C3, is described. Enhancement of all aspects of in vitro hematopoiesis was observed on addition of 9.1C3 antibody to cultures of mononuclear cells from normal bone marrow, cord blood, and peripheral blood from beta-thalassemia major patients. In cultures with no exogenous colony-stimulating factor (CSF), the addition of 9.1C3 resulted in a two- to eightfold increase in nonerythroid colony formation. Similarly, for cultures maximally stimulated with CSF, the addition of 9.1C3 antibody resulted in a one- to fourfold increase in colony formation. These effects were abrogated by the removal of either adherent, Leu-M3+ or Leu-7+ cells. Colony- forming cells were shown to be present among the 9.1C3-negative cells when mononuclear cells were sorted by flow cytometry. Media conditioned in the presence of 9.1C3 and mononuclear cells were able to enhance colony formation in vitro for normal nonadherent bone marrow cells beyond that achieved with supramaximal amounts of human placental- conditioned medium and erythropoietin. The data suggest that natural killer cells interact with monocytes to exert a negative regulatory control on in vitro granulopoiesis and erythropoiesis. Consequently, the number of progenitor and multipotential cells in cultures of unfractionated cell populations may be greatly underestimated.     

Predictors and outcomes of treatment in hip hemiarthroplasty dislocation

Introduction

Dislocation following hip hemiarthroplasty (HHA), its incidence, predictors, treatment outcomes and mortality were investigated in a single centre series.

Methods

The prospectively collected data on neck of femur fracture admissions compiled over 11 years were reviewed. Place of residence, place of fall, past medical history, intraoperative factors (grade of surgeon, delay in surgery, type of implant and operative time), postoperative complications and mortality were compared between patients who suffered a dislocation and those who did not. In the dislocation group, the mean number of dislocations, reduction method, type and fate of implant, and mortality were investigated.

Results

Prospective data on 8,631 admissions were collected; 41% of these were managed with a HHA. The dislocation rate was 0.76%. A delay in surgery of >24 hours was associated with a fourfold increase in the dislocation risk. The majority (81%) of dislocations occurred in the first six weeks and closed manipulation was the definitive treatment in only 23% of the cases. The mortality rate was not increased following HHA dislocation.

Conclusions

The delay in surgery was the most important predictor of HHA dislocation. Closed reduction was associated with a high failure rate. While an initial attempt at closed reduction for a first dislocation is recommended, for redislocators, we recommend early exploration/revision as an alternative to repeat manipulations.     

The clonal proliferation in vitro of enriched populations of human promyelocytes and myelocytes

The proliferative capacity of normal human promyelocytes and myelocytes was demonstrated and characterized on the basis of clonal proliferation in agar. An enriched population of normal human promyelocytes and myelocytes was obtained from bone marrow using the monoclonal antibody WEM G11 and the fluorescence-activated cell sorter (FACS). In cultures stimulated by placental-conditioned medium, these cells generated peak total clone numbers between days 3 and 5 of culture. Clones disappeared rapidly thereafter. These clones were mainly of subcolony size at day 7, although some colonies were generated by this population. The clones were primarily neutrophilic in type. These cells had a plating efficiency of up to 50%, and clonal proliferation was dependent on stimulation by colony-stimulating factor (CSF).     

In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E

A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein- deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.     

Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony- stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.     

Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.     

Isolation and characterization of growth factor(s) from a human B-cell lymphoma

We demonstrate that human neoplastic B cells (Br cells) contain a cytoplasmic protein of molecular mass 60 Kd that exhibits B-cell growth factor (BCGF) activity on growth factor-dependent long-term human B cells as well as on autochthonous tumor cells. This 60-Kd protein is recognized by antibodies against a similar intracellular 60-Kd protein derived from normal human lymphocytes. These results demonstrate that the two proteins share epitope homology. Microculture bioassays indicate that neoplastic and normal 60-Kd proteins are capable of driving neoplastic B cells through S-phase. Western immunoblot analysis indicates that neoplastic B cells secrete 60- as well as 14-Kd protein. Immunoaffinity-purified proteins secreted by Br cells exhibit BCGF activity in anti-IgM or dextran sulfate-preactivated human B cells. In addition, a double-antibody immunofluorescence staining technique was used to demonstrate that Br cells express cell surface receptors for BCGF molecule(s). These studies provide support for the autocrine growth model for neoplastic human B cells and suggest that the autocrine growth factor derived from such tumor cells is similar if not identical to normal BCGF molecules.     

STUDY OF GLOMERULAR FUNCTIONS IN NEONATES

Glomerular function of neonates (25 full term and equal number of preterm neonates) at birth, on day seven and day fourteen were estimated by endogenous Creatinine clearance (CCr). The Preterm were divided into three groups viz. Group I (Gestation age (GA) 30-32 weeks), Group II (GA 33-34 weeks) and Group III (GA 35-36 weeks). Group IV consisted of 25 term neonates. Serum creatinine (in mg/dl) in all the groups of preterm ranged from 0.92 ± 0.153 to 1.204 ± 0.154 and in term neonate from 0.562 ± 0.175 to 1.148 ± 0.247 showing that the levels were inversely proportional to the period of gestation. Besides the Serum Creatinine levels in all groups of Preterm as well as term neonates were found to fall significantly (p < 0.001) during the first and second week. Glomerular filtration rate (GFR) in Group I were 16.603 ± 2.519, 19.786 ± 2.078 and 23.720 ± 2320 on day one, seven and fourteen respectively showing progressive improvement during the first two weeks. The GFRs were also found to be directly proportional to the GA. In addition the levels of GFR were found increasing significantly during the second week compared to that on day seven in all the groups of preterm neonates. GFR''s in Group I & II on all the three occasions were significantly lower (p < 0.001) than those of term counterparts, however the GFR on the first day in Group III neonates was lower than group IV, the difference was insignificant (p > 0.05). The increase in GFR in Group I on the three occasions was linear but insignificant (p > 0.05). The rise was more rapid & significant (p < 0.001) during the second week in Groups II & III. On the contrary the rate of improvement of GFR in full terms was quite rapid during the first week and gradual over the second week of life.KEY WORDS: GFR preterm & term neonates, Serum creatinine     

Pentoxifylline inhibits integrin-mediated adherence of interleukin-2- activated human peripheral blood lymphocytes to human umbilical vein endothelial cells, matrix components, and cultured tumor cells

Peripheral blood lymphocytes (PBLs) cultured in the presence of recombinant human interleukin-2 (rhIL-2) develop a natural killer (NK) cell phenotype (CD16+, CD56+, CD3-) and are referred to as lymphokine- activated killer cells (LAK). In developing the LAK phenotype, enhanced adherence to matrix components and endothelial cells have been described. In this report we investigated the functional behavior of adhesion receptors in rhIL-2-activated PBLs by in vitro adhesion assay and by flow cytometry. Compared to PBLs, IL-2-activated PBLs had increased integrin-mediated adherence to: (1) fibronectin (FN), (2) human umbilical vein endothelial (HUVE) cells, and (3) cultured melanoma and pancreatic tumor cell lines. This increase in adherence was mediated by increased surface expression of members of the beta 1 and beta 2 integrin subfamilies, as determined by flow cytometric analysis. No induction of an activation-dependent beta 1 (CD29) epitope was detected. We also investigated the effects of the methylxanthine derivative pentoxifylline (PTX) on PBLs and rhIL-2-activated PBL adhesion. PBLs co-cultivated in the presence of rhIL-2 (1,000 U/mL) and PTX exhibited reduced adherence to FN, HUVE and cultured tumor cell lines. This inhibition by PTX was concentration- and time-dependent. The increased expression of integrins induced by rhIL-2 was only in part inhibited by PTX, suggesting that PTX induced a subpopulation of integrins that are expressed but functionally inactive.