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Biomedical subjects

B F Gibbs

Publications and source records attributed to B F Gibbs.

At least 55 records · Page 3Linked to original sources
PubMed1997

The effect of recombinant stem cell factor on human skin and lung mast cells and basophil leukocytes.

Recombinant human stem cell factor (rhSCF) induced histamine release from human skin and lung mast cells but had no effect on human basophil leukocytes. More importantly, rhSCF enhanced the release of histamine in response to IgE-crosslinking of human mast cells. This potentiation was observed at rhSCF concentrations which induced histamine release, and also at lower concentrations of the ligand which by themselves produced no effect. Very limited potentiation was observed with human basophil leukocytes. The enhancing effect of SCF was unique to IgE-dependent stimulation and when SCF was incubated with the neurotransmitter substance P and the calcium ionophore A23187, no augmentation of histamine release was observed with any of the cell types tested. These findings suggest that endogenous SCF may contribute to the regulation of mast cell function and may hence play a role in diverse allergic and inflammatory reactions.

Antibodies, Anti-Idiotypic↗
PubMed1997

A three-step procedure for the purification of human basophils from buffy coat blood.

OBJECTIVE AND DESIGN: We report a method for basophil purification from buffy coats, which avoids positive selection of the cells and gives rise to good purity, yield and functional integrity of the cells. SUBJECTS: Buffy coat blood (concentrated leukocyte fraction derived from 450 ml venipuncture donations) obtained from healthy blood donors (n = 51). METHODS: Basophils were enriched by a three-step process starting with Ficoll density centrifugation (1.6 +/- 0.1% basophil purity) followed by counter current centrifugal elutriation (17.7 +/- 1.4% basophil purity). The final stage involved negative selection using Dynal immunomagnetic beads directed against CD2, CD14, CD16 and CD19 positive cell contaminants. Functional integrity of which was assessed by comparing the anti-IgE or calcium ionophore A23187 induced histamine release from basophils obtained from each enrichment step. Furthermore, basophil morphology was investigated using light and electron microscopy. RESULTS: The final mean basophil purity of 67.3 +/- 1.4% with a yield of 3.5 +/- 0.5 x 10(6) basophils and a recovery of 21.8 +/- 2.4% was achieved. Net histamine release from basophils stimulated with optimal concentrations of anti-human IgE was 39.1 +/- 6.5% after Ficoll centrifugation, 41.6 +/- 7.7% following elutriation and 35.7 +/- 6.8% from the final purified fraction. Additionally, basophils enriched with our method showed intact morphology by electron microscopy and were functionally active to non-immunological stimulation. CONCLUSIONS: These results compare favourably with previous studies, which have often required the use of positive selection via the Fc epsilon RI receptor, which may result in cell degranulation, or cell sorting, which cannot be applied to large cell numbers. Our method provides a reproducible technique for basophil enrichment when large numbers of functionally intact basophils are required.

Antigen-Antibody Reactions↗
PubMed1996

Simple and rapid high-performance liquid chromatographic method for the determination of aspartame and its metabolites in foods.

A method for the determination of aspartame (N-L-alpha-aspartyl-L-phenylalanine methyl ester) and its metabolites, applicable on a routine quality assurance basis, is described. Liquid samples (diet Coke, 7-Up, Pepsi, etc.) were injected directly onto a mini-cartridge reversed-phase column on a high-performance liquid chromatographic system, whereas solid samples (Equal, hot chocolate powder, pudding, etc.) were extracted with water. Optimising chromatographic conditions resulted in resolved components of interest within 12 min. The by-products were confirmed by mass spectrometry. Although the method was developed on a two-pump HPLC system fitted with a diode-array detector, it is straightforward and can be transformed to the simplest HPLC configuration. Using a single-piston pump (with damper), a fixed-wavelength detector and a recorder/integrator, the degradation of products can be monitored as they decompose. The results obtained were in harmony with previously reported tedious methods. The method is simple, rapid, quantitative and does not involve complex, hazardous or toxic chemistry.

Aspartame↗
PubMed1996

Human basophils release interleukin-4 after stimulation with Schistosoma mansoni egg antigen.

The elevated interleukin (IL)-4 and IgE production in Schistosoma mansoni infection seems to be induced essentially by the egg stage of the parasite. The underlying mechanism, however, is not known. Since basophils from human peripheral blood can produce IL-4, we asked, whether soluble S. mansoni egg antigens (SEA) would trigger basophils to release IL-4. Basophils from healthy human donors (n = 32) without prior history of schistosomiasis were incubated with SEA in the presence of IL-3. In all donors, IL-4 was produced at different concentrations. The IL-4 production was dependent on the dose of SEA, was correlated with the purity of the basophil preparation, and the IL-4 concentration in the culture supernatant was maximal 5 h after stimulation with SEA. In addition to its IL-4-stimulatory effect, SEA triggered basophils to degranulate, thereby releasing histamine and sulfidoleukotrienes. Stripping of receptor-bound IgE from basophils inhibited both SEA- and anti-IgE-induced, but not ionomycin-induced IL-4 production. Moreover, resensitization of stripped basophils with stripping supernatants or human serum restored SEA-induced IL-4 production. This suggests that IgE is involved in the mechanism of IL-4 induction by SEA. Since IL-4 is induced in basophils from nonexposed donors, basophils may play a role as an early source of IL-4 in S. mansoni infection.

Animals↗
PubMed1996

Purified human peripheral blood basophils release interleukin-13 and preformed interleukin-4 following immunological activation.

Recent studies have shown that human basophils, like mast cells, generate interleukin (IL)-4 following immunological activation and may thus participate in late-phase allergic and inflammatory processes. Here, we report the capacity of human basophils to release IL-13 within 24 h following stimulation with anti-IgE. Additionally, in 14 out of 31 experiments, we observed that basophils rapidly release performed IL-4 within 5-10 min, as well as newly generated IL-4, which was released 4 h following stimulation of the cells with anti-IgE. In contrast to the biphasic release of IL-4 from the cells, no preformed IL-13 was detected at earlier times (5-30 min). Preformed IL-4 and IL-4 and IL-13 generated de novo were also released after stimulation of the cells with IL-3; an enhanced production of these cytokines was observed using a combination of IL-3 and anti-IgE. We conclude from these data that, by releasing performed IL-4 and IL-4 and IL-13 generated de novo, human basophils may be centrally involved in the orchestration of allergic inflammation by providing a trigger to IL-4-mediated T helper 2 lymphocyte activation, B cell IgE switching, and increased vascular adhesion molecule expression.

Antibodies, Anti-Idiotypic↗
PubMed1996

Effects of basophil-priming and stimulating cytokines on histamine release from isolated human skin mast cells.

Cell priming and stimulation of different cytokines (which include chemokines and growth factors) are typical features of human basophils. Recently, it has been shown that the macrophage chemotactic protein-1 (MCP-1), RANTES and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are potent direct secretagogues for human basophils and that interleukin-3 (IL-3), IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are priming factors for subsequent potentiation of mediator release from basophils induced by different stimuli. This observation may be clinically important for the activation and recruitment of inflammatory cells in different immune responses of the skin (e.g. late-phase reactions). The aim of the present study was to investigate whether cytokines and chemokines are also capable of priming or stimulating isolated human skin mast cells (SMC). SMC were either stimulated directly with the cytokines alone or preincubated with these factors for 10 min before being activated with suboptimal concentrations of anti-IgE, A23187 or substance P. IL-3, IL-5, GM-CSF, platelet factor-4 (PF-4), IL-8, MCP-1 and MIP-1 alpha (each at concentrations of 1 ng/ml to 1 microgram/ml, log steps) did not significantly modulate histamine release from SMC induced by the three different secretagogues. RANTES exhibited a weak but significant potentiating effect on IgE-mediated activation. Stem cell factor (SCF) as a positive control was able to prime mast cell histamine release strongly. In addition, PF-4, MCP-1, RANTES and MIP-1 alpha were incapable of inducing direct histamine release from SMC. In experiments with isolated human peripheral basophils, however, we observed potent Fc epsilon RI-mediated priming effects evoked through IL-3, IL-5 and GM-CSF. We conclude that SMC derived from healthy donors are not targets of (immuno)modulatory factors that prime or stimulate basophils.

Basophils↗
PubMed1996

Heterogeneity of human skin mast cells and human basophils. I. Pharmacological experiments with activators and inhibitors of protein kinase C.

Skin mast cells and basophilic leukocytes are known as key elements of acute and subacute IgE-mediated immune responses of the skin. The present paper investigated pharmacological aspects of signal transduction pathways of both cell types using activators and inhibitors of protein kinase C (PKC). The nonselective inhibitor K252a suppressed Fc epsilon RI-mediated histamine release from basophils and skin mast cells dose-dependently with IC50 values of 0.01 and 0.28 mumol/l. However, preincubation of both cell populations with kinase inhibitors showing in vitro selectivity for PKC (Ro 31-7549, calphostin C, GF 109203X) revealed a distinct modulation of cell response: IgE-mediated mediator release was inhibited only in skin mast cells, whereas in experiments with basophils a concentration-dependent potentiation of exocytosis was observed. Further evidence for heterogenous biochemical signals following activation of both cell types derived from studies with the phorbol ester TPA. With respect to acute and late-phase IgE-mediated skin reactions, we suggest that distinct signal transduction mechanisms at the level of PKC (isozymes) in basophils and skin mast cells might reflect their functional heterogeneity.

Animals↗
PubMed1995

Proparathyroid hormone is preferentially cleaved to parathyroid hormone by the prohormone convertase furin. A mass spectrometric study.

Parathyroid hormone (PTH), an 84-amino acid peptide, is the major regulator of blood calcium homeostasis. Its mRNA, in addition to encoding the mature peptide, also encodes a "pre" sequence of 25 amino acids and a basic "pro" hexapeptide. To assess which of the subtilisin-like prohormone convertases can process proPTH to PTH we coinfected cells with a vaccinia virus construct expressing human preproPTH and vaccinia virus constructs expressing furin, PC1 or PC2. BSC-40 cells, having a constitutive secretory pathway, and GH4C1 cells, having a regulated secretory pathway, were used. PTH biosynthetic products in cell extracts and media were purified by high performance liquid chromatography, identified by radioimmunoassay, and unambiguously defined as either proPTH or PTH by ion-spray mass spectrometry. In both cell types, furin was the most effective in processing proPTH to PTH. In all cases only PTH was released into the medium. In addition, partially purified furin and PC1 were tested for their ability to appropriately cleave a tridecapeptide spanning the prohormone cleavage site found in proPTH. Here too furin was much more effective at cleaving at the correct site. Northern blot analysis and in situ hybridization showed that furin and preproPTH mRNA are co-expressed in the parathyroid, whereas PC1, PC2, and PC5 are not and PACE4 is expressed only at very low levels. Taken together these studies strongly suggest that furin is the enzyme responsible for the physiological processing of proPTH to PTH.

Amino Acid Sequence↗
PubMed1995

Small peptide mimics of nerve growth factor bind TrkA receptors and affect biological responses.

Small monomeric cyclic analogs that mimic the beta-turn regions of nerve growth factor (NGF) were designed and synthesized. Potent competitive antagonists were derived from the NGF beta-turn C-D, which inhibited [125I] NGF binding to TrkA receptors and specifically inhibited optimal NGF-mediated neurite outgrowth in PC12 cells. The cyclic beta-turn A'-A" analog also inhibited NGF binding to TrkA receptors but with lower potency. These data indicate that beta-turns C-D and A'-A" are critical for TrkA binding and may confer neurotrophin receptor specificity. Furthermore, structural requirements for binding are absolute, because unconstrained analogs derived from the same regions had no effect. Compounds that mimic NGF will be useful in deciphering the interactions of NGF and its receptors and in rational drug design.

Amino Acid Sequence↗
PubMed1995

Cost-effective carotid endarterectomy in community practice.

The purpose of this study was to compare hospital charges for carotid endarterectomy on a surgeon-specific basis. The cost of carotid endarterectomy is influenced by preoperative evaluation, operating time, use of the intensive care unit, length of hospital stay, and surgical results. Length of stay and average hospital charges for 18 doctors performing 344 carotid endarterectomies at three hospitals were analyzed. Outcome data were also reviewed. The results demonstrated a wide variation in hospital charges among surgeons. Surgeons using the most cost-effective measures achieved comparable or superior outcomes. In an era of managed care, severe cost constraints mandate that surgeons perform similar studies in their own communities so that cost-effective clinical pathways can be developed. With the use of appropriate guidelines, carotid endarterectomy can be performed at relatively low cost without sacrificing quality.

California↗
PubMed1994

Design of potent bivalent thrombin inhibitors based on hirudin sequence: incorporation of nonsubstrate-type active site inhibitors.

Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a K(i) value of 2.2 x 10(-14) M. It consists of an active site blocking moiety, hirudin1-48, a fibrinogen-recognition exo-site binding moiety, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, hirudin1-48, was replaced by small nonsubstrate-type active site inhibitors of thrombin, e.g., dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by omega-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudin55-65 was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human alpha-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues. The use of dansyl-Arg-(D-pipecolic acid) improved the affinity (K(i)) of the inhibitor 10-100-fold (down to 1.70 x 10(-11) M) compared to that of the similar compounds having D-Phe-Pro-Arg as their substrate-type inhibitor moiety (K(i) = 10(-9)-10(-10) M). The linker connected to P2 residue eliminated the scissile peptide bond. The inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1- or 2-naphthalenesulfonyl group for in vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Purification of antimicrobial peptides from an extract of the skin of Xenopus laevis using heparin-affinity HPLC: characterization by ion-spray mass spectrometry.

A simple scheme was developed for the rapid purification of antimicrobial peptides from the skin of Xenopus laevis. An extract of the frog skin was prepared using an acidic medium designed to maximize the solubilization of low-molecular-weight peptides. This extract was subjected to an enrichment procedure using C18 Sep Pak cartridges to yield a salt-free fraction, devoid of high-molecular-weight proteins. This fraction was in turn subjected to heparin affinity high-performance liquid chromatography on a Shodex AF-Pak column. All the antibacterial activity bound to the column and could be eluted using a linear gradient of increasing sodium chloride concentration. Antibacterial activity emerged from the column in fractions corresponding to a sodium chloride concentration of 0.45 M. Reversed-phase high-performance liquid chromatography resolved this material into a series of compounds which could be readily characterized using a combination of amino acid analysis and ion-spray mass spectrometry. Each peptide was found to be antimicrobial and each was positively identified as belonging to a family of amphipathic helix-forming peptides characterized by other investigators. Listed in their order of elution from the reversed-phase column the peptides were magainin 2, magainin 1, peptide-glycine-leucine amide, xenopsin precursor fragment, levitide precursor fragment, and a mixture of fragments derived from the caerulein precursor. These peptides owe their antimicrobial properties to a predeliction to forming amphipathic alpha-helical structures when associated with lipid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Design of a linker for trivalent thrombin inhibitors: interaction of the main chain of the linker with thrombin.

N alpha-Acetyl[D-Phe45,Arg47]hirudin45-65 (P53) is a bivalent thrombin inhibitor (Ki = 5.6 nM) that consists of an active site inhibitor segment, [N alpha-acetyl-(dF)PRP]; a fibrinogen recognition exo site inhibitor segment, hirudin55-65 (DFEEIPEEYLQ-OH); and a linker, hirudin49-54 (QSHNDG), connecting these inhibitor segments (DiMaio et al., 1990). The structure-function relationships of the linker were studied using a combination of various omega-amino acids, which modified the length of the linker as well as the number and the locations of peptide bonds. Linkers with 14-18 atoms (counting only the atoms contributing to the length of the linker) showed a competitive inhibition with Ki = 1.7-3.4 nM. The potency of the inhibitors with 12-13-atom linkers was sensitive to the chemical structure of the linker. The high-potency inhibitors showed a competitive inhibition, while the low-potency inhibitors showed a hyperbolic inhibition. Among them, an inhibitor with a 13-atom linker showed the highest potency (Ki = 0.51 nM, an 11-fold improvement from that of P53 above), indicating that this is an optimal linker length. Since linkers with 6-10 atoms failed to bridge the active site and exo site inhibitor segments, a minimum of 11 atoms was required to bridge them, even though the potency of the inhibitor with an 11-atom linker was weak (Ki = 26 nM). Molecular dynamics simulation of the inhibitors with 13-atom linkers suggested that some linkers serve as a functional domain with the amide bond of the linker interacting with thrombin through hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Surgical treatment of aneurysm of the persistent sciatic artery.

Persistent sciatic artery (PSA) is a rare congenital malformation that is complicated by aneurysm formation in more than 25% of the reported cases. Two cases of aneurysm of the PSA are presented. Twenty-six aneurysms of the PSA, including our two cases, have been reported in the English literature in the last three decades. Early surgical treatment is warranted to decrease the 25% amputation rate associated with thromboembolic complications. The posterior, transgluteal repair of this aneurysm affords excellent exposure and avoids a long bypass graft, multiple incisions, and staged procedures. Magnetic resonance imaging may be helpful in preoperative evaluation of the feasibility of proximal, extrapelvic vascular control.

Aged↗