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Biomedical subjects

B Walker

Publications and source records attributed to B Walker.

At least 73 records · Page 4Linked to original sources
PubMed1999

Protease-activated receptor-2 involvement in hypotension in normal and endotoxemic rats in vivo.

BACKGROUND: The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. METHODS AND RESULTS: Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin-SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription-polymerase chain reaction analysis. CONCLUSIONS: Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.

Animals↗
PubMed1999

Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy.

Combination therapy for HIV-1 infection can reduce plasma virus to undetectable levels, indicating that prolonged treatment might eradicate the infection. However, HIV-1 can persist in a latent form in resting CD4+ T cells. We measured the decay rate of this latent reservoir in 34 treated adults whose plasma virus levels were undetectable. The mean half-life of the latent reservoir was very long (43.9 months). If the latent reservoir consists of only 1 x 10(5) cells, eradication could take as long as 60 years. Thus, latent infection of resting CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy.

Adult↗
PubMed1999

Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin.

We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP(18-27), and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml(-1)). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth.

Bombesin↗
PubMed1999

Preliminary assessment of whole-blood, red-cell and platelet- leucodepleting filters for possible induction of prion release by leucocyte fragmentation during room temperature processing.

Universal leucodepletion is being introduced in the U.K. to reduce a theoretical risk of Creutzfeldt-Jakob disease (CJD) transmission. If CJD infectivity is associated with leucocytes, any cell fragmentation associated with filtration could reduce the potential benefit. Four types each of whole blood, red cell and platelet leucodepletion filters were assessed after holding of blood units for at least 4 h at 22 degrees C. In all cases the mean residual leucocyte content was <1 000 000 per unit, with only two individual filtered whole blood units having a leucocyte content exceeding this. Evidence of leucocyte fragmentation during filtration was sought but not found by assay of soluble elastase, beta-thromboglobulin and normal prion protein, as well as by isotopic labelling of leucocyte external membrane. These preliminary studies indicate that it was possible to prepare leucodepleted blood components by filtration at room temperature, and that this appeared not to be associated with overt cell fragmentation. Definitive demonstration that fragmentation does not occur requires the development of improved general (non-specific) assays for cell membrane fragments.

Blood Component Removal↗
PubMed1999

Treatment of human immunodeficiency virus infection with hydroxyurea, didanosine, and a protease inhibitor before seroconversion is associated with normalized immune parameters and limited viral reservoir.

Current treatments for human immunodeficiency virus (HIV) require uninterrupted drug administration because they are unable to reconstitute the immune response and do not affect the viral reservoir. Ten patients were treated during acute HIV infection before complete Western blot (WB) seroconversion with the combination of hydroxyurea, didanosine, and indinavir. This treatment was associated with the normalization of some immune parameters and functions. No loss of naive CD4 T lymphocytes was observed, and recovery of up to 35% of naive CD8 T lymphocytes occurred in several weeks. A vigorous HIV-specific T helper response (stimulation index >8) was observed in 7 of 8 patients treated before complete WB seroconversion but in only 1 of 5 controls treated after seroconversion. In addition, a limited latent viral reservoir (<0.02-0.5 infectious units/106 cells) was documented in quiescent peripheral blood lymphocytes after treatment initiated before complete WB seroconversion.

Anti-HIV Agents↗
PubMed1999

Gene organisation determines evolution of function in the chicken MHC.

Some years ago, we used our data for class I genes, proteins and peptide-binding specificities to develop the hypothesis that the chicken B-F/B-L region represents a "minimal essential MHC". In this view, the B locus contains the classical (highly expressed and polymorphic) class I alpha and class II beta multigene families, which are reduced to one or two members, with many other genes moved away or deleted from the chicken genome altogether. We found that a single dominantly expressed class I gene determines the immune response to certain infectious pathogens, due to peptide-binding specificity and cell-surface expression level. This stands in stark contrast to well-studied mammals like humans and mice, in which every haplotype is more-or-less responsive to every pathogen and vaccine, presumably due to the multigene family of MHC molecules present. In order to approach the basis for a single dominantly expressed class I molecule, we have sequenced a portion of the B complex and examined the location and polymorphism of the class I (B-F) alpha, TAP and class II (B-L) beta genes. The region is remarkably compact and simple, with many of the genes expected from the MHC of mammals absent, including LMP, class II alpha and DO genes as well as most class III region genes. However, unexpected genes were present, including tapasin and putative natural killer receptor genes. The region is also organised differently from mammals, with the TAPs in between the class I genes, the tapasin gene in between the class II (B-L) beta genes, and the C4 gene outside of the class I alpha and class II beta genes. The close proximity of TAP and class I alpha genes leads to the possibility of co-evolution, which can drive the use of a single dominantly expressed class I molecule with peptide-binding specificity like the TAP molecule. There is also a single dominantly expressed class II beta gene, but the reason for this is not yet clear. Finally, the presence of the C4 gene outside of the classical class I alpha and class II beta genes suggests the possibility that this organisation was ancestral, although a number of models of organisation and evolution are still possible, given the presence of the Rfp-Y region with non-classical class I alpha and class II beta genes as well as the presence of multigene families of B-G and rRNA genes.

Animals↗
PubMed1999

T cell reactivity to human insulinoma cell line (CM) antigens in patients with type 1 diabetes.

Autoimmune (type 1) diabetes mellitus results from a progressive destruction of insulin secreting beta cells operated by T lymphocytes in pancreatic islets. Circulating autoreactive T cells to specific beta cell antigens are detected in patients with type 1 diabetes. To date, several beta cell autoantigens have been identified in this disease (GAD, IA-2, 38kD secretory protein, insulin, ICA69 etc.), however, it is possible that also other unidentified self molecules contribute to trigger beta cell autoimmunity. In this study we used the human insulinoma cell line CM as source of beta cell antigens to detect reactive T lymphocytes in patients with type 1 diabetes mellitus. This cell line has been previously shown to express a number of recognized beta cell antigens. Since the expression of several beta cell antigens is affected by glucose stimulation we tested two preparations of CM cells cultured under different conditions containing low (0.8 mM) and high glucose concentration (11 mM). T cell proliferation was measured using cells from 32 patients with type 1 diabetes (19 of recent onset and 13 at 3 to 22 months from diagnosis) and 27 age-matched control subjects. A significant increase in T cell proliferation to CM cells grown in high glucose conditions (11 mM) (p < 0.05) was found in type 1 diabetic patients compared to controls. No significant differences were observed when using CM cells cultured at the low glucose concentration. Furthermore, the response to both extracts of CM cells was independent of disease duration (p = 0.6 for both CM cells cultured at 0.8 and 11 mM glucose). These data indicate that T cell reactivity to homogenates of CM cells is detectable in patients with type 1 diabetes and suggest that this human insulinoma cell line is an interesting potential source of beta cell material for immunological studies of autoimmune diabetes.

Adolescent↗
PubMed1999

A novel, simple method of functional spleen volume calculation by liver-spleen scan.

UNLABELLED: Spleen enlargement is commonly associated with portal hypertension from cirrhosis and may cause thrombocytopenia. Thus, accurate assessment of spleen size may be helpful in the clinical evaluation. Spleen length is not a precise estimate of spleen size because of the variation in spleen configuration, and spleen volumes measured by edging techniques can be tedious. We present a new method of measuring the functional spleen volume by liver-spleen scan (LSSs), validation experiments and some clinical data. METHODS: The method involves measurement of the total spleen counts by SPECT and dividing by a representative voxel concentration on a single frame to obtain the organ volume. Validation included phantom studies and clinical evaluation in 443 consecutive patients, including 216 with histologic assessments of chronic liver disease (CLD) and 11 healthy volunteers. RESULTS: A calibration factor determined from phantoms was used to convert the calculated volume (CV) to the "true" volume (V): V = CV (0.956) - 66.5 (r = 0.9991; P < 0.001). The volume calculations were validated in a second group of phantoms (r= 0.981; P < 0.0001). Spleen volumes were expressed as volume (cm3) and as volume per pound ideal body weight (IBW) (cm3/lb) (the conversion factor to convert cm3/lb IBW to cm3/kg IBW is 2.2). Clinical studies of reproducibility included demonstration of a significant (P < 0.0001) linear correlation between volumes calculated from repeat LSSs within 9 mo of the initial LSS in 11 healthy volunteers and 32 patients with CLD: y = 1.02x - 25; r = 0.968. The correlation with spleen volumes from autopsy or splenectomy was significant: y = 0.766x + 57; r = 0.845; P < 0.001. The normal spleen volume in 11 patients was 201 +/- 77 cm3 and 1.43 +/- 0.68 cm3/lb IBW (upper limits of normal: 335 cm3 or 2.5 cm3/lb IBW). In 443 consecutive LSSs over 15 mo, half of the patients had spleen volumes above the upper limits of healthy volunteers, and CLD was present in 90.9% of these patients. In 216 patients with histologically proven liver disease, a progressive increase in the percentage of spleen volumes above the upper limits of normal was noted from no fibrosis (10%) to mild to moderate fibrosis (36.7%) to early cirrhosis (52%) to advanced liver disease (75%). The correlation of spleen volume with platelet count was excellent (r = 0.7635; P < 0.005). CONCLUSION: This novel spleen volume measurement detects serious liver disease and correlates with splenic hyperfunction.

Chronic Disease↗
PubMed1999

Choosing the right computer system.

We are living in a world where virtually any information you desire can be acquired in a matter of moments with the click of a mouse. The computer is a ubiquitous fixture in elementary schools, universities, small companies, large companies, and homes. Many dental offices have incorporated computers as an integral part of their management systems. However, the role of the computer is expanding in the dental office as new hardware and software advancements emerge. The growing popularity of digital radiography and photography is making the possibility of a completely digital patient record more desirable. The trend for expanding the role of dental office computer systems is reflected in the increased number of companies that offer computer packages. The purchase of one of these new systems represents a significant commitment on the part of the dentist and staff. Not only do the systems have a substantial price tag, but they require a great deal of time and effort to become fully integrated into the daily office routine. To help the reader gain some clarity on the blur of new hardware and software available, I have enlisted the help of three recognized authorities on the subject of office organization and computer systems. This article is not intended to provide a ranking of features and shortcomings of specific products that are available, but rather to present a process by which the reader might be able to make better choices when selecting or upgrading a computer system.

Appointments and Schedules↗
PubMed1998

Mechanisms of protection induced by attenuated simian immunodeficiency virus. II. Lymphocyte depletion does not abrogate protection.

To determine the role that cellular immune responses play in the protection conferred by vaccination with attenuated SIVmac32H (pC8), we have attempted to deplete macaques of their CD8+ cells prior to challenge with wild-type SIVmac32H (pJ5). In two of four pC8-infected macaques, N109 and N112, a transient partial depletion of CD8+ cells by antibody treatment was achieved. On the day of challenge peripheral CD2+CD4-CD8+ cell counts were reduced by 92 and 95%, respectively, in animals N109 and N112 and their lymph nodes revealed a 46 and 58% reduction, respectively, in CD2+CD4-CD8+ cells. Two other pC8-immunized macaques, N110 and N111, treated in the same way, did not show significant depletion of CD8+ cells. None of these four pC8-immunized animals became infected when challenged with 50 MID50 of pJ5. Treatment of a further four pC8-infected and protected macaques and two naive control animals with Campath-1H antibody successfully depleted peripheral CD3+ cell counts by >99% in all treated animals. Campath-1H depletion resulted in enhanced, longer lasting lymphoid depletion. Yet subsequent challenge with 20 MID50 of pJ5 still failed to infect the pC8-immunized animals. All eight of the naive controls, including two Campath-1H-treated animals, became infected following challenge. In summary, partial depletion of circulating CD8+ cells or total lymphocytes prior to challenge failed to abrogate the protection conferred by vaccination with pC8.

Animals↗
PubMed1998

A sequence within the cytoplasmic tail of GpIIb independently activates platelet aggregation and thromboxane synthesis.

All integrin alpha subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989-995, of the platelet integrin subunit glycoprotein GpIIb (alphaIIb), palmitoyl-KVGFFKR (Ppep; 10 microM), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A2 (TXA2) synthesis (EC50 = 45 +/- 5 microM), secretion of alpha-granules detected as enhanced surface expression of P-selectin (EC50 = 52 +/- 8 microM), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC50 = 3.7 +/- 1 microM). The TXA2 receptor antagonist, SQ29548, PGE1, and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991-994) as the critical residues in all peptide-mediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA2 synthesis and secretion.

Acetates↗
PubMed1998

The synthesis and utilization of 2,4-dinitrophenyl-labeled irreversible peptidyl diazomethyl ketone inhibitors.

Biotinylated diazomethyl ketones have been much used to detect cysteine proteases on Western blots, but high background may occur due to the presence of endogenously biotinylated proteins. In order to overcome this problem, we have examined the potential utility of a peptidyl diazomethyl ketone labeled with a 2,4-dinitrophenyl (DNP) group as a potential inhibitor and disclosing agent of cathepsin B/L-like proteases. This DNP-labeled peptide, DNP-Ahx-Gly-Phe-Ala-CHN2, was used to detect a 30-kDa cathepsin L-like protease, produced by Fasciola hepatica, on Western blots with high sensitivity and relatively low background staining. The DNP-labeled diazomethyl ketone was also found to be a good inhibitor for bovine cathepsin B, human cathepsin L, and a novel F. hepatica cathepsin L-like protease, when assayed fluorimetrically, displaying second-order rate constants (ki/Ki) of 7.93 x 10(2), 2.16 x 10(2), and 3.73 x 10(2) M-1 s-1, respectively.

2,4-Dinitrophenol↗
PubMed1998

Synthesis and proteinase inhibitory properties of diphenyl phosphonate analogues of aspartic and glutamic acids.

The synthesis of diphenyl phosphonate analogues of aspartic and glutamic acid, and their inhibitory activity against S. aureus V8 protease and granzyme B, is described. The study has revealed difficulties with protecting group compatibility in the synthesis of these analogues. Two analogues, Acetyl. AspP (OPh)2 and Acetyl.GluP (OPh)2 were found to function as irreversible inactivators of V8 proteinase, yet exhibit no activity against granzyme B.

Aspartic Acid↗