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M J Raftery

Publications and source records attributed to M J Raftery.

At least 19 recordsLinked to original sources
PubMed2001

Novel intra- and inter-molecular sulfinamide bonds in S100A8 produced by hypochlorite oxidation.

Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Murine S100A8 (A8) is a major cytoplasmic protein in neutrophils and is secreted by macrophages in response to inflammatory stimuli. After incubation with reagent HOCl for 10 min, approximately 85% of A8 was converted to 4 oxidation products, with electrospay ionization mass spectrometry masses of m/z 10354, 10388, 10354 +/- 1, and 20707 +/- 3. All were resistant to reduction by dithiothreitol. Initial formation of a reactive Cys sulfenic acid intermediate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) to HOCl-treated A8 to form stable adducts. Matrix-assisted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in the full-length proteins. Both Met(36/73) were converted to Met(36/73) sulfoxides. An additional product with an unusual mass addition of m/z 14 (+/-0.2) was identified and corresponded to the addition of oxygen to Cys(41), conjugation to various epsilon-amines of Lys(6), Lys(34/35), or Lys(87) with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide cross-links. Specific fragmentations identified in matrix-assisted laser desorption-post source decay spectra and low energy collisional-induced dissociation tandem mass spectroscopy spectra of sulfinamide-containing digest peptides confirmed Lys(34/35) to Cys(41) sulfinamide bonds. HOCl oxidation of mutants lacking Cys(41) (Ala(41)S100A8) or specific Lys residues (e.g. Lys(34/35), Ala(34/35)S100A8) did not form sulfinamide cross-links. HOCl generated by myeloperoxidase and H(2)O(2) and by phorbol 12-myristate 13-acetate-activated neutrophils also formed these products(.) In contrast to the disulfide-linked dimer, oxidized monomer retained normal chemotactic activity for neutrophils. Sulfinamide bond formation represents a novel oxidative cross-linking process between thiols and amines and may be a general consequence of HOCl protein oxidation in inflammation not identified previously. Similar modifications in other proteins could potentially regulate normal and pathological processes during aging, atherogenesis, fibrosis, and neurogenerative diseases.

Amino Acid Sequence↗
PubMed2001

Targeting the function of mature dendritic cells by human cytomegalovirus: a multilayered viral defense strategy.

Human cytomegalovirus (HCMV) can suppress and evade the immune system. We have identified as a mechanism the ability of HCMV to infect dendritic cells (DC), which initiate the antiviral immune response. HCMV-infected DC show enhanced expression of costimulatory molecules. In contrast, MHC molecules are partially downregulated, leading to a reduced antigen-presenting capacity. Moreover, the apoptosis-inducing ligands CD95L (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) are upregulated, thereby enabling HCMV-infected DC to delete activated T lymphocytes. This additional layer of viral defense is complemented by nondeletional mechanisms, which suppress surviving T cells. Thus, infection of DC allows the virus to blunt the antiviral T cell response by a multilayered defense strategy and could play a pivotal role in HCMV-triggered immunosuppression.

Antigen Presentation↗
PubMed2001

Recurrence of Wegener's granulomatosis 13 years after renal transplantation.

Wegener's granulomatosis (WG) can cause renal failure, requiring long-term renal replacement therapy. Renal transplantation in patients with WG is successful, but the risk for recurrence of the disease necessitates continued vigilance. We report a patient that originally presented with acute renal failure secondary to a pauci-immune focal necrotizing crescentic glomerulonephritis. Subsequent nasal involvement and serologic tests for antineutrophil cytoplasmic antibodies suggested a diagnosis of WG.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2001

An unexpected cause of muscle pain in diabetes.

Diabetic muscle infarction is a rare condition which may present to a rheumatologist. It was first reported in 1965. Two illustrative cases are described here and the mechanisms of pathogenesis discussed. Analysis of the published data, results of the muscle biopsies, and a technetium-99m sestamibi scan suggest that the condition, which occurs against a background of diabetic microangiopathy, can be triggered by an ischaemic event and causes extensive muscle necrosis through hypoxia-reperfusion injury and compartment syndrome.

Biopsy↗
PubMed2001

Proinflammatory properties of the human S100 protein S100A12.

S100 proteins represent a new class of chemoattractants. Here we extend earlier evidence for the proinflammatory properties of human S100A12. A12 induced migration of monocytoid cells, with optimal activity at 10(-10) M and potency of >10(-9) M C5a. Neutrophils were poorly responsive, and lymphocyte migration was not affected. Actin polymerization in monocytoid cells was accompanied by a sustained [Ca(2+)]i flux of a magnitude comparable with C5a. A12 elicited a transient infiltration of neutrophils (4-8 h) and more delayed recruitment of monocytes (8-24 h) in vivo. A12 (approximately 70 nM) was present in synovial fluid (SF) from rheumatoid arthritis patients, and synovium contained A12-positive neutrophils in the sublining and interstitial region, often surrounding the perivasculature but rarely in the synovial lining layer, although some macrophages were positive. The A12 gene was transiently up-regulated in monocytes by tumor necrosis factor alpha (6 h); induction by lipopolysaccharide (LPS) was sustained (12-48 h). A12 may contribute to leukocyte migration in chronic inflammatory responses.

Actins↗
PubMed2000

Cytomegalovirus infection of vascular cells induces expression of pro-inflammatory adhesion molecules by paracrine action of secreted interleukin-1beta.

BACKGROUND: Infection with human cytomegalovirus (HCMV) has been associated with vascular disease processes such as vascular allograft rejection, transplantation vasculopathy, restenosis after angioplasty, and native atherosclerosis. To elucidate underlying pathomechanisms, the effect of acute HCMV infection on the expression of pro-inflammatory adhesion molecules on human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (hvSMC) was examined. METHODS AND RESULTS: Cells were infected in vitro with clinical strains of HCMV and the resulting changes in adhesion molecule expression were quantified by histology and flow cytometric analysis. On HUVEC, surface expression of vascular cell adhesion molecule-1 and E-selectin was induced de novo on HCMV infection and intercellular adhesion molecule-1 expression was increased by >200%. On hvSMC, intercellular adhesion molecule-1 surface expression induced de novo, although vascular cell adhesion molecule-1 and E-selectin were not changed. Expression of major histocompatibility complex (MHC) class II, lymphocyte-function associated antigen 3 (LFA-3; CD58), and CD40 was not altered by HCMV infection in either cell type. In partially infected cultures, up-regulation of surface molecules also occurred on noninfected cells, suggesting a paracrine mechanism via a soluble factor. Expression of surface molecules could be enhanced in noninfected HUVEC and hvSMC by incubation with virus-free conditioned supernatant from HCMV-infected cells or by coincubation in transwells with infected cells. The responsible agent could be identified as IL- interleukin- (IL) 1beta by detection of de novo secretion of IL-1beta by HCMV-infected cells and by prevention of adhesion molecule up-regulation after addition of an IL-1-converting enzyme inhibitor or IL-1 receptor antagonist. Surface molecule up-regulation could be suppressed by UV inactivation of virus, but not by treatment of cell cultures with inhibitors of viral replication (ganciclovir). CONCLUSION: We propose that HCMV infection induces IL-1beta release and subsequent up-regulation of pro-inflammatory adhesion molecules on noninfected neighboring cells through a paracrine mechanism. This may lead to local potentiation of the inflammatory effects of HCMV infection, not amenable to current therapeutic antiviral strategies.

Cell Adhesion Molecules↗
PubMed2000

Safety and tolerability of a multidose formulation of epoetin beta in dialysis patients. Collaborative Study Group.

AIM: Previous studies in healthy volunteers and renal patients have demonstrated the favorable tolerability of a new multidose formulation of epoetin beta. The aim of this open, multicenter study was to further assess the safety, tolerability and efficacy of this formulation ofepoetin beta in patients with end-stage renal disease (ESRD). MATERIALS AND METHODS: 375 adult patients receiving maintenance epoetin therapy for renal anemia were switched to the multidose formulation of epoetin beta for 12 weeks, using the same dosage and route of administration. RESULTS: Adverse events were experienced by 123 patients (33%), most commonly hypertension (5.6%) and hypotension (4.5%). Few patients (2%) were prematurely withdrawn because of tolerability concerns. No clinically relevant changes in blood pressure or laboratory variables were observed. Compared with baseline, hemoglobin and hematocrit values remained essentially unchanged during treatment with this new formulation of epoetin beta. No changes in iron metabolism parameters were apparent, and nearly all patients (94%) did not require blood transfusions during the study. CONCLUSION: The results of this study indicate that the multidose formulation of epoetin beta is safe and well tolerated in patients with ESRD. Moreover, switching patients to this new formulation of epoetin beta does not compromise therapeutic efficacy.

Aged↗
PubMed1999

Herpes simplex virus type 1 infection of activated cytotoxic T cells: Induction of fratricide as a mechanism of viral immune evasion.

Herpes simplex virus type 1 (HSV1), a large DNA-containing virus, is endemic in all human populations investigated. After infection of mucocutaneous surfaces, HSV1 establishes a latent infection in nerve cells. Recently, it was demonstrated that HSV1 can also infect activated T lymphocytes. However, the consequences of T cell infection for viral pathogenesis and immunity are unknown. We have observed that in contrast to the situation in human fibroblasts, in human T cell lines antigen presentation by major histocompatibility complex class I molecules is not blocked after HSV1 infection. Moreover, HSV1 infection of T cells results in rapid elimination of antiviral T cells by fratricide. To dissect the underlying molecular events, we used a transgenic mouse model of HSV1 infection to demonstrate that CD95 (Apo-1, Fas)-triggered apoptosis is essential for HSV1-induced fratricide, whereas tumor necrosis factor (TNF) also contributes to this phenomenon but to a lesser extent. By contrast, neither TRAIL (TNF-related apoptosis-inducing ligand) nor perforin were involved. Finally, we defined two mechanisms associated with HSV1-associated fratricide of antiviral T cells: (a) T cell receptor-mediated upregulation of CD95 ligand and (b) a viral "competence-to-die" signal that renders activated T lymphocytes susceptible to CD95 signaling. We propose that induction of fratricide is an important immune evasion mechanism of HSV1, helping the virus to persist in the host organism throughout its lifetime.

Animals↗
PubMed1999

Oxidation regulates the inflammatory properties of the murine S100 protein S100A8.

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.

Animals↗
PubMed1999

Overexpression, oxidative refolding, and zinc binding of recombinant forms of the murine S100 protein MRP14 (S100A9).

Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a cloned thrombin consensus sequence (. LVPRGS. ) located between glutathione S-transferase and mMRP14. Analysis of products of digestion by C4 reverse-phase HPLC and SDS-PAGE/Western blotting revealed two immunoreactive cleavage products with molecular weights around 13, 000. Masses of the two proteins determined by ESI/MS were 13,062 and 11,919 Da. The larger product corresponded to the expected mass of recombinant mMRP14 (13,061.9 Da). Analysis of the protein sequence of recombinant mMRP14 revealed a thrombin-like consensus sequence (. NNPRGH. ) located close to the C-terminus. The smaller protein corresponded to a truncated form of rec mMRP14 (rec MRP141-102) with a calculated mass of 11,918.6 Da. Optimization of the cleavage conditions resulted in >95% full-length rec mMRP14. Native mMRP14 contains one intramolecular disulfide bond between Cys79 and Cys90. The full-length recombinant protein was renatured and oxidized in ammonium acetate (pH approximately 7) for 96 h and formed >95% of the native intramolecular disulfide-bonded form. MRP141-102 bound substantially less 65Zn2+ compared to native mMRP14 or rec mMRP14 after transfer to polyvinylidene difluoride and incubation with 65ZnCl2, implicating the His residues located within the C-terminal domain in Zn2+ binding.

Amino Acid Sequence↗
PubMed1999

Increased plasma malondialdehyde levels in glomerular disease as determined by a fully validated HPLC method.

BACKGROUND: Reactive oxygen species and particularly free radical induced lipid peroxidative tissue damage have been implicated in the pathogenesis of various renal diseases. Lipid peroxidation is assessed indirectly by the measurement of secondary products, such as malondialdehyde (MDA), using the widely employed thiobarbituric acid reactive substances (TBARS) method. However, this method lacks sensitivity and specificity. We have therefore developed and validated an HPLC (high-performance liquid chromatography) method for measurement of MDA and applied this to a variety of plasma samples in renal patients. METHODS: The optimized method involves antioxidant treatment of the plasma sample, followed by a protein precipitation step using trichloroacetic acid, acid hydrolysis and formation of an MDA thiobarbituric acid complex. The MDA-(TBA)2 adduct is separated from other interfering compounds by C18 reverse-phase HPLC techniques, with visible detection at 532 nm. RESULTS: The assay was linear over the ranges 0.25-1.0 microM MDA and the detection limit was 0.06 microM MDA. Within-run precision was <4.5% and between-run precision was <10.0%. MDA plasma concentrations (mean+/-SD) were higher in ESRF diabetic patients (0.32 +/- 0.14 microM, n=20), non-diabetic ESRF patients (0.32 +/- 0.09 microM, n=20), and CRF patients (0.14 +/- 0.06 microM, n=40) compared to healthy controls (0.11 +/- 0.03 microM, n=40), (P < 0.001, P < 0.001 and P = 0.008). Levels were similar in healthy controls with normal renal function and transplanted patients (0.12 +/- 0.03 microM MDA, n=40), (P=NS). No correlation was observed between MDA and creatinine levels (r2 = 0.05, n=80), which suggests that MDA does not correlate with the degree of renal impairment. We matched CRF patients with glomerular and non-glomerular causes of renal failure for creatinine levels and found that MDA levels were higher in patients with glomerulonephritis (0.16 +/- 0.06 microM) than in those with CRF from non-glomerular causes (0.12 +/- 0.04 microM, P = 0.002). CONCLUSIONS: We have introduced a reliable and sensitive HPLC technique to enhance the specificity of MDA-(TBA)2 measurement, with a significant improvement in HPLC column life. Using this method, picomole quantities of MDA can be detected in plasma. We have shown that MDA levels are significantly raised in patients with CRF due to glomerulonephritis, regardless of serum creatinine, which suggests that there is oxidative injury independent of any possible MDA retention due to renal impairment.

Chromatography, High Pressure Liquid↗
PubMed1998

Identification of posttranslational modifications and cDNA sequencing errors in the rat S100 proteins MRP8 and 14 using electrospray ionization mass spectrometry.

MRP8 and 14 are S100 proteins expressed by myeloid cells and are predicted to have important functions in inflammation. The proteins were isolated from spleens from three rat strains. Electrospray ionization mass spectrometry indicated masses of 10,149 +/- 2 Da for MRP8 and 13,069 +/- 2 Da for MRP14 compared to masses calculated from proteins derived from their cDNA sequences of 10,211 and 13,214 Da, respectively, indicating posttranslational modifications and/or errors in the derived protein sequences. Several endoprotease digest peptides did not correspond to any theoretical digest products after comparison of ESI masses with those derived from the theoretical digest. Both proteins were N-terminally acetylated after deletion of the initiator Met, reducing the theoretical masses by 89 Da. A peptide with mass 28 Da greater than the theoretical was isolated from the Asp N digestion of MRP8. N-terminal sequencing indicated translated Val instead of the predicted Ala at position 72 of MRP8. A peptide 56 Da less than the theoretical was isolated from the chymotryptic digestion of MRP14, and the carboxyamidomethylated form was N-terminally sequenced and found to have translated Ser instead of the predicted Arg at position 105. In addition, His106 was methylated. The corrected theoretical masses, incorporating the posttranslational modifications and sequencing errors, are 10,149.4 and 13,069.9 Da for MRP8 and 14, respectively, in good agreement with the experimental masses.

Amino Acid Sequence↗
PubMed1998

Identification of noncovalent dimeric complexes of the recombinant murine S100 protein CP10 by electrospray ionization mass spectrometry and chemical cross-linking.

CP10 is a chemotactic S100 protein expressed by murine myeloid cells. A specific noncovalently linked dimeric complex of recombinant Ala43 CP10 was identified after electrospray ionization mass spectrometry using a nondenaturing solvent of 5-mM ammonium acetate (pH 6.5) and source temperature of 50 degrees C. With a low cone voltage (75 V), major ions were observed at approximately 2075, 2305, and 2613 Da, which were attributed to partially desolvated multiply charged noncovalently linked dimeric species (+10, +9, and +8 charge states, respectively). Deconvolution produced a broad peak centered around 20750 Da corresponding to the approximate mass of dimeric recombinant Ala43 CP10. Increasing the cone voltage, the collisionally activating the complex, gradually reduced the intensity of these dimeric ions, with a concomitant increase in higher and lower charge state monomeric ions. The intensities of these dimeric ions were greatly reduced in spectra recorded with a source temperature of 140 degrees C and cone voltage of 75 V, indicating a thermally unstable noncovalent complex. Similar spectra were obtained using recombinant CP10. Specific noncovalent S100 dimeric complexes were confirmed by chemically cross-linking recombinant Ala43 CP10 or CP10 with bis (sulfosuccinimidyl) suberate, followed by SDS/PAGE. The dominant silver-stained and CP10-immunoreactive component migrated at 20,000 suggested that the complex represents the major isoform in solution.

Animals↗
PubMed1997

Diagnostic contribution of renal allograft biopsies at various intervals after transplantation.

Renal allograft biopsy is the accepted gold standard for investigating episodes of graft dysfunction in the early posttransplant period. The situation is less clear in late transplant biopsies. Later renal biopsies performed for graft dysfunction or as part of a routine investigative protocol have not been subjected to detailed critical evaluation. Two hundred sixty-three consecutive renal allograft biopsies in a single center were evaluated. They were arbitrarily divided into three groups based on interval after transplantation: group 1, up to 3 months (n=117); group 2, 4-12 months (n=60); and group 3, greater than 12 months after transplantation (n=86). There were no significant differences in demographic factors among the groups. The mean interval after transplantation was 0.8+/-0.1 months in group 1, 6.1+/-0.3 months in group 2, and 40.1+/-3.4 months in group 3. There were six principal diagnostic categories: acute rejection (AR), chronic rejection (CR), cyclosporine (CsA) nephrotoxicity, acute tubular necrosis (ATN), normal, and others. A statistically significant decrease in the frequency of AR (P<0.001) was seen in group 3 (3%) compared with groups 1 (43%) and 2 (37%). In contrast, the frequency of CR was significantly higher (P<0.001) in group 3 (71%) compared with groups 1 (0) and 2 (10%). ATN was seen almost exclusively in group 1. All but one of the 37 patients with ATN were in this group. CsA nephrotoxicity remained an important cause of graft dysfunction in all three groups, with no significant difference in incidence among the three groups. The differences between groups with other histological types were not significant. Patient management was changed based on the biopsy report in 84 patients in group 1 (72%), 45 patients in group 2 (75%), and only 16 patients in group 3 (19%) (P<0.001). In only seven patients in group 3 did the change in management result in a significant change in serum creatinine. All of these seven patients had CsA nephrotoxicity on biopsy and also had a significantly higher level of CsA compared with those with AR or CR. Thus, the diagnosis might have been possible without the need for biopsy. We conclude that late renal allograft biopsies are only rarely helpful in patient management and as such should be an investigation of last resort.

Adolescent↗