Biomedical subjects

H Bass

Publications and source records attributed to H Bass.

At least 19 recordsLinked to original sources

PubMed2004

Provision of Epstein-Barr virus-transformed B-cell lines in a routine tissue typing laboratory: practicalities and applications.

A bank of Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BCLs) has been established in this laboratory over the last 7 years. Novel, rare and unusual HLA phenotypes were highlighted during routine clinical testing and during typing of volunteer haematopoietic stem cell donors for the Welsh Bone Marrow Donor Registry. The BCLs are routinely used as laboratory reagents, as a source of DNA for reference purposes and for research work. Consenting donors are tested for hepatitis B surface antigens (HBsAg), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) before their B-lymphocytes are transformed. Strict bio-identity checks are performed before and after transformation and after each BCL expansion. Each BCL is tested regularly for mycoplasma contamination. A total of 230 blood samples were transformed. One hundred and fifty-nine sterile samples produced 157 BCLs (98.7% success), while 71 non-sterile samples produced 50 BCLs (70.4% success), giving an overall success rate of 90.0%. Fifteen of the transformation failures have since been repeated successfully. Factors contributing to the high success rate and reasons for the 23 failed transformations are discussed. The successful development and use of pools of BCLs for HLA antibody screening by flow cytometry are described. Whilst certain training and health and safety issues require close attention, it is clear that EBV transformation of B lymphocytes and/or the use of BCLs is feasible in a routine tissue typing laboratory and that BCLs are a valuable resource. Careful adherence to the methods and procedures detailed here should virtually guarantee successful transformation (98.7%) from good quality, sterile whole blood samples.

B-Lymphocytes↗

PubMed1997

Evaluation of the Short Parkinson's Evaluation Scale: a new friendly scale for the evaluation of Parkinson's disease in clinical drug trials.

The extensive use of the Unified Parkinson's Disease Rating Scale (UPDRS) has revealed low interrater reliability in some items and redundancy in others. In view of these shortcomings, we have structured a new scale that includes a zero-to three-point scale for each item in the evaluation of PD. The mental axis includes memory, thought disorders, and depression. Activities of daily living (ADL) includes eight items: speech, eating, feeding, dressing, hygiene, handwriting, walking, and turning in bed. The motor examination includes eight items: speech, tremor, rest and posture, rigidity, finger tapping, arising from chair, gait, and postural stability. Complications of therapy were also included: dyskinesias, dystonia, motor fluctuations, and freezing episodes, collected by history. In addition, a global scoring for motor fluctuations that should complement the Hoehn and Yahr Scale was incorporated. In this report, we present a statistical analysis of the ADL, motor evaluation, and complications of therapy sections. Concerning the interrater reliability mean, Kendall's W values were >0.9 for most of the items in the Short Parkinson's Evaluation Scale (SPES). Kendall's W <0.8 (motor evaluation) was found for two items of the SPES and nine items of the UPDRS. The mean interrater reliability for both scales across all seven centers (seven Kendall's W for seven centers) (Mann-Whitney test) showed no statistical differences between the scales. Spearman's correlations between items of both scales were significant. Factor analysis of the SPES and UPDRS data revealed a four-factor solution that explained approximately 60% of the data. All participating centers found the SPES easier to apply and quicker to complete, when compared with the UPDRS. The results obtained strongly favor the introduction of SPES for clinical practice.

Activities of Daily Living↗

PubMed1995

Lipopolysaccharide regulation of lipoprotein lipase expression in murine macrophages.

The enzyme lipoprotein lipase is expressed in a number of cell types and plays a central role in lipid metabolism. Multiple factors regulate its expression in a tissue-specific manner. In murine macrophages, lipopolysaccharide inhibits lipoprotein lipase enzyme activity. The current work examines this process in the established J774 macrophage line and primary peritoneal macrophages from endotoxin-sensitive (C3HeB/Fej) and endotoxin-resistant (C3H/Hej) murine strains. Lipopolysaccharide inhibition of macrophage lipoprotein lipase occurred at the enzyme and mRNA levels in a time- and concentration-dependent manner. Cells from endotoxin-resistant animals maintained their expression of lipoprotein lipase following treatment with lipopolysaccharide. Results of gel retention assays showed that lipopolysaccharide treatment of the J774 macrophages altered the level of nuclear proteins recognizing and binding the lipoprotein lipase promoter DNA. Nuclear extracts from resting J774 cells contained proteins which bound specifically to the octamer motif and to the CAAT box within the lipoprotein lipase promoter. Exposure of the J774 cells to lipopolysaccharide for 16 h increased the level of protein-octamer DNA complexes. Similar responses were obtained in endotoxin-sensitive, but not endotoxin-resistant, primary macrophages following in vitro treatment with lipopolysaccharide. This finding suggests that transcriptional events may contribute to the lipopolysaccharide regulation of macrophage lipoprotein lipase expression.

Animals↗

PubMed1994

Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein.

The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone-induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor alpha protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.

Adipocytes↗

PubMed1993

Effects of albumin and apolipoprotein C-II on the acyl-chain specificity of lipoprotein lipase catalysis.

In this study we used monoacid triacylglycerols of various acyl-chain lengths as substrates for probing the active-site structure and substrate specificity of lipoprotein lipase (LPL). An unexpected finding was that the albumin ligand binding site is accessible not only to long-chain fatty acids for its recognized functional role as a fatty acid acceptor, but also to short- and medium-chain monoacid triacylglycerol substrates. The observed striking inhibitory effect (99%) of albumin on the LPL-catalyzed hydrolysis of trihexanoylglycerol is probably the result of the high affinity interaction of albumin with this substrate. Spectrophotometric analyses indicated that there is one high affinity binding site per albumin molecule (apparent KD = 1.8 +/- 0.9 microM) for the interaction with trihexanoylglycerol. Despite LPL acyl-chain specificity being obscured by the substrate binding effect of albumin, a systematic study of the lipolysis reaction under various assay conditions demonstrated that tributyroylglycerol represents the best substrate for LPL, and the preferential order of LPL catalysis for both the basal and apoC-II-activated activities is: C4 > C6 > C8 > C10 > C12 > C18:1. In some assay conditions, the presence of albumin affects the above-mentioned order, which can be attributed to substrate binding by albumin, rather than an alteration in the specificity of LPL. The synergistic effect of apoC-II and albumin resulted in the preferential activation of LPL for the hydrolysis of long-chain triacylglycerols. Even with optimal assay conditions for the hydrolysis of long chain triacylglycerols, there is still a preferential reactivity of LPL with short- and medium-chain triacylglycerols.

Animals↗

PubMed1992

Adipogenesis in a myeloid supporting bone marrow stromal cell line.

The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long-term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow stromal cell lines is heterogeneous, they share a common mechanism of adipogenesis.

Adipose Tissue↗

PubMed1992

Different lymphoid cell populations produce varied levels of neopterin, beta 2-microglobulin and soluble IL-2 receptor when stimulated with IL-2, interferon-gamma or tumour necrosis factor-alpha.

Immune activation is central to many immune disorders. Clinical investigations have shown that immune activation can be quantified by measurements of soluble immune activation products in serum. Most in vitro studies of these immune activation products have focused on single products. In this study the specific cell sources and the major lymphokines inducing multiple activation products were investigated. In vitro addition of interferon-gamma (IFN-gamma) or IL-2 stimulated peripheral blood mononuclear cells to produce neopterin, beta 2-microglobulin (beta 2-M) and soluble IL-2 receptor (sIL-2R). These two lymphokines can act independently, because neutralizing antibodies to one of the lymphokines did not block the inducing activity of the other. Tumour necrosis factor-alpha (TNF-alpha) was also investigated and shown to be a less powerful inducer than IL-2 or INF-gamma. Separated lymphoid subpopulations responded differently to specific lymphokines. Monocytes produced only neopterin and only in response to INF-gamma. T cells released beta 2-M and sIL-2R in response to IL-2. B cells, however, were capable of producing all three immune activation products. Neopterin production in B cells was induced by either INF-gamma of IL-2, indicating that B cells have additional mechanisms for responding to lymphokines. To investigate whether these in vitro findings also occur in vivo, sera from patients who had received either rIL-2 or INF-gamma treatment were tested. INF-gamma administration led to substantial increases in serum neopterin but only a moderate beta 2-M increase and no increase in the serum sIL-2R levels. rIL-2 administration caused a substantial increase of all three serum immune activation products, consistent with our in vitro findings. The results confirm that increased serum levels of soluble immune activation products are indicators of increased cytokine production by lymphocytes and monocytes and also that B cells can be a prominent source of immune activation products.

B-Lymphocytes↗

PubMed1992

Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C-III.

In this study we have examined effects of synthetic polypeptide fragments of apoC-III on the kinetic properties of lipoprotein lipase (LPL) activity. Based on the loss of 79% of LPL-inhibitory activity after CNBr cleavage at the N-terminal portion of apoC-III and a systematic search for synthetic peptides with LPL-inhibitory activity spanning the apoC-III sequence, we concluded that the N-terminal domain is the most important in the modulation of LPL activity. In addition, there are multiple attachment sites in apoC-III for its interaction with LPL and these sites reside in the hydrophilic sequences of apoC-III. Probably for this reason the intact apo-CIII exhibited higher inhibitory potential than its peptide components. Based on the deduced inhibition constants derived for the synthetic apoC-III1-79 we concluded that apoC-III is likely to exhibit a physiological role in regulating LPL activity since the derived dissociation constants for the LPL-apoC-III interaction are within the physiological concentration range of plasma apoC-III. In addition, as the synthetic apoC-III1-79 lacks the carbohydrate moiety, we also concluded that the presence of the oligosaccharide in native apoC-III is not essential for its inhibitory activity on LPL. The fact that the I50 (concentration for inhibition of LPL at 50% activity) decreases for apoC-III-1 when assayed in the presence of apoC-II indicated that the activator actually caused an increased affinity between LPL and apoC-III and demonstrated that apoC-III does not compete for the activator site of apoC-II.

Amino Acid Sequence↗

PubMed1991

Thymic irradiation inhibits the rapid recovery of TH1 but not TH2-like functions of CD4+ T cells after total lymphoid irradiation.

Four to six weeks after total lymphoid irradiation (TLI), there is a selective deficit in the CD4+ T cells which secrete IL-2, proliferate in the MLR, and induce GVHD (Th1-like functions). A similar deficit in CD4+ T cells which secrete IL-4 and help antibody responses (Th2-like functions) is not observed. In the present study, shielding of the thymus with lead during TLI increased the Th1-like functions of CD4+ cells. Mice without thymus shields showed a marked selective reduction in the medullary stromal cells identified with the monoclonal antibody, MD1, and the severe reduction was prevented with thymus shields. Thus, shielding the thymus prevents the depletion of thymic medullary stromal cells and allows for a rapid recovery of Th1-like functions in the mouse spleen after TLI. Th2-like functions recover rapidly after TLI whether or not the thymus is irradiated.

Animals↗

PubMed1990

Natural killer lines and clones with apparent antigen specificity.

Fresh CD3-, CD16+ lymphocytes that adhered to selected allogeneic lymphoblastoid cell lines (LCL) were cultured with LCL in the presence of IL-2-containing medium. The resulting lines as well as clones derived from these lines expressed CD16 and/or CD56, but lacked detectable CD3 or TCR-alpha/beta or TCR-gamma/delta complexes on the cell surface. Northern blot analysis failed to detect CD3 epsilon or TCR-beta transcripts, but revealed the presence of a TCR-gamma chain transcript in one of these lines. In addition to displaying potent cytolytic activity against K562 erythroleukemia cells (a classical NK target), the vast majority of these lines and clones lysed their specific stimulator LCL to a significantly greater extent than irrelevant LCL. This selective killing was inhibited by the addition of cold stimulator LCL or K562 cells, or anti-LFA 1 mAbs, but not by irrelevant LCL or mAbs to CD3, class I or class II MHC antigens. These results indicate that some CD3- lymphocytes, phenotypically indistinguishable from NK cells, can recognize and lyse allogeneic targets in a specific manner.

Antibodies, Monoclonal↗

PubMed1990

Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of Th1 and Th2 cells.

Many long term mouse Th clones express either the type 1 or type 2 Th cell (Th1 or Th2) cytokine secretion phenotype. In this report we present two lines of evidence for the existence of additional Th differentiation states. Lectin-stimulated spleen cells secreted moderate levels of IL-2 compared with long term Th1 clones, whereas the levels of other cytokines were more than 100-fold lower than those produced by either Th1 or Th2 clones. This suggests that many spleen cells produce substantial amounts of IL-2 but little or no IL-4, IL-5, IFN-gamma, IL-3, and granulocyte/macrophage-CSF. In contrast to long term Th clones, many short term alloreactive clones displayed cytokine secretion phenotypes intermediate between the Th1 and Th2 patterns. The proportion of recognizable Th1 and Th2 clones at early times in culture was greatly increased by immunization of the mice from which the responder and stimulator cells were derived; Brucella abortus immunization resulted in the isolation of exclusively Th1 clones, whereas infection with Nippostrongylus brasiliensis resulted in a strong trend toward the isolation of Th2 clones. The immunization of mice from which responder cells were derived strongly affected the type of Th clone obtained, whereas the source of stimulator cells had much less effect, suggesting that the commitment of Th cells to the Th1 or Th2 phenotypes occurred mainly in vivo. A model for the possible relationships of the various Th cells is presented.

Animals↗

PubMed1990

Deficits in T helper cells after total lymphoid irradiation (TLI): reduced IL-2 secretion and normal IL-2 receptor expression in the mixed leukocyte reaction (MLR).

Spleen cells from BALB/c mice treated with total lymphoid irradiation (TLI) and from normal, unirradiated mice were compared in the mixed leukocyte reaction (MLR). Although the percentage of CD4+ cells in the spleen was close to normal, 4 to 6 weeks after TLI, the MLR of unfractionated spleen cells from irradiated mice was more than 10-fold lower than controls. A similar reduction was observed when purified CD4+ cells were used as responders in the MLR. Secretion of IL-2 by cells from irradiated mice was also about 10-fold lower than controls. However, the percentage of CD4+ and CD8+ cells which expressed IL-2 surface receptors during the MLR was similar using spleen cells from irradiated and control mice. Addition of an exogenous source of IL-2 restored the proliferative capacity of the irradiated cells and suggests that the lack of IL-2 secretion is the likely explanation of the marked deficit in the MLR of CD4+ spleen cells after TLI.

Animals↗

PubMed1989

Evidence for mouse Th1- and Th2-like helper T cells in vivo. Selective reduction of Th1-like cells after total lymphoid irradiation.

Purified CD4+ BALB/c spleen T cells obtained 4-6 wk after total lymphoid irradiation (TLI) helped normal syngeneic B cells to produce a vigorous antibody response to TNP keyhole limpet hemocyanin in adoptive cell transfer experiments. However, the same cells failed to transfer delayed-type hypersensitivity to the adoptive hosts as measured by a foot pad swelling assay. In addition, purified CD4+ cells from TLI-treated mice were unable to induce graft vs. host disease in lethally irradiated allogeneic C57BL/Ka recipient mice. In response to mitogen stimulation, unfractionated spleen cells obtained from TLI mice secreted normal levels of IL-4 and IL-5, but markedly reduced levels of IL-2 and INF-gamma. A total of 229 CD4+ clones from spleen cells of both normal and TLI-treated mice were established, and the cytokine secretion pattern from each clone was analyzed. The results demonstrate that the ratio of Th1- and Th2-like clones in the spleens of normal BALB/c mice is 1:0.6, whereas the ratio in TLI mice is approximately 1:7. These results suggest that Th2-like cells recover rapidly (at approximately 4-6 wk) after TLI treatment and account for the early return of antibody helper activity and secretion of IL-4 and IL-5, but Th1-like cells recover more slowly (in approximately 3 mo) after irradiation, and this accounts for the deficit in cell-mediated immunity and the reduced amount of IL-2 and IFN-gamma secretion.

Animals↗

PubMed1985

Microwave-thawed plasma for cryoprecipitate production.

A microwave oven has been used to obtain rapid and controlled thawing of frozen plasma packs for cryoprecipitate production. The resulting factor VIII yields were higher than the average of 77.8 IU (in a volume of 17.2 ml) obtained by the slow overnight thaw procedures in routine use. Assays on 10 individual packs showed mean results of 142 +/- 24.5 IU factor VIII procoagulant activity, 188.6 +/- 51.68 IU factor VIII-related antigen, 0.152 +/- 0.05 g fibrinogen and 218.9 +/- 66.5 IU fibronectin in a volume of 23.2 +/- 6.6 ml. The results of this preliminary study indicate that microwave thawing of plasma is worthy of further investigation and should be reconsidered for routine production of cryoprecipitate and high yield fibronectin.

Antigens↗

PubMed1985