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Biomedical subjects

C Stover

Publications and source records attributed to C Stover.

At least 19 recordsLinked to original sources
PubMed2005

Functional MASP2 single nucleotide polymorphism plays no role in psoriasis.

BACKGROUND: Psoriasis is a heritable disease and genome-wide scans have implicated several loci of susceptibility. The gene for MASP-2, a protease involved in complement activation, is located within one of these loci on chromosome 1p. OBJECTIVES: To assess whether partial or total MASP-2 deficiency is a risk factor for developing psoriasis. METHODS: We screened a cohort of patients affected by plaque psoriasis and their parents by restriction fragment length polymorphism analyses. RESULTS: We detected a single nucleotide polymorphism that leads to an amino acid exchange, which results in dissociation of MASP-2 from a carbohydrate recognition complex. CONCLUSIONS: We show that this mutant allele is not associated with psoriasis. There was no favoured transmission from parents to affected offspring. The calculated allele frequency in this psoriasis group (Scottish and English) was 0.0326, and in the unaffected group 0.0379.

Alleles↗
PubMed2001

The human gene for mannan-binding lectin-associated serine protease-2 (MASP-2), the effector component of the lectin route of complement activation, is part of a tightly linked gene cluster on chromosome 1p36.2-3.

The proteases of the lectin pathway of complement activation, MASP-1 and MASP-2, are encoded by two separate genes. The MASP1 gene is located on chromosome 3q27, the MASP2 gene on chromosome 1p36.23-31. The genes for the classical complement activation pathway proteases, C1r and C1s, are linked on chromosome 12p13. We have shown that the MASP2 gene encodes two gene products, the 76 kDa MASP-2 serine protease and a plasma protein of 19 kDa, termed MAp19 or sMAP. Both gene products are components of the lectin pathway activation complex. We present the complete primary structure of the human MASP2 gene and the tight cluster that this locus forms with non-complement genes. A comparison of the MASP2 gene with the previously characterised C1s gene revealed identical positions of introns separating orthologous coding sequences, underlining the hypothesis that the C1s and MASP2 genes arose by exon shuffling from one ancestral gene.

Base Sequence↗
PubMed2001

Role of the classical pathway of complement activation in experimentally induced polymicrobial peritonitis.

The complement system and the natural antibody repertoire provide a critical first-line defense against infection. The binding of natural antibodies to microbial surfaces opsonizes invading microorganisms and activates complement via the classical pathway. Both defense systems cooperate within the innate immune response. We studied the role of the complement system in the host defense against experimental polymicrobial peritonitis using mice lacking either C1q or factor B and C2. The C1q-deficient mice lacked the classical pathway of complement activation. The factor B- and C2-deficient mice were known to lack the classical and alternative pathways, and we demonstrate here that these mice also lacked the lectin pathway of complement activation. Using inoculum doses adjusted to cause 42% mortality in the wild-type strain, none of the mice deficient in the three activation routes of complement (factor B and C2 deficient) survived (mortality of 100%). Mortality in mice deficient only in the classical pathway of complement activation (C1q deficient) was 83%. Application of further dilutions of the polymicrobial inoculum showed a dose-dependent decrease of mortality in wild-type controls, whereas no changes in mortality were observed in the two gene-targeted strains. These results demonstrate that the classical activation pathway is required for an effective antimicrobial immune defense in polymicrobial peritonitis and that, in the infection model used, the remaining antibody-independent complement activation routes (alternative and lectin pathways) provide a supporting line of defense to gain residual protection in classical pathway deficiency.

Animals↗
PubMed2001

cDNA cloning, chromosome assignment, and genomic structure of a human gene encoding a novel member of the RBM family.

We have cloned and characterised a novel human gene mapping to chromosome 20q11.2. A partial transcript was initially isolated from a human cDNA library transcribed from RNA of the colon carcinoma cell line T-84. In order to determine the full coding sequence of this novel mRNA, we isolated seven cDNA clones from a human cDNA library transcribed from RNA of the acute monocytic leukemia cell line THP1 by colony hybridization. On Northern blot analysis of four human cell lines, the cDNAs isolated hybridize with an abundantly expressed mRNA species of 3.5 kb. A full-length cDNA transcript of this novel mRNA has an open reading frame of 2,796 bp encoding a protein with a calculated molecular weight of 97 kDa. Two repetitive structural consensus motifs are contained within the deduced protein sequence, namely five distinct RNA binding motifs and two proline rich regions. The derived protein sequence also contains putative transmembrane domains. These structural motifs identify this novel protein as a member of an expanding protein family containing RNA binding motifs (RBM). As seen from recently completed sequence of the genomic area encoding this novel mRNA by the Sanger Centre Human Genome Project, the coding region of this gene, RBM12, is intronless.

Amino Acid Motifs↗
PubMed2000

Crystallization and 1.1-A diffraction of chorismate lyase from Escherichia coli.

Chorismate pathway enzymes are important as producers of nonnucleotide aromatic compounds. The enzyme chorismate lyase from Escherichia coli has been crystallized in four distinct forms, three of which have been characterized by X-ray diffraction. Despite widespread screening, all four crystal forms grow from the same chemical conditions. The wild-type enzyme tends to aggregate, even in the presence of reducing agent, and yielded only one crystal form (monoclinic, form 1) that grew in intricate clusters. Chemical modification of the cysteines mitigated problems with aggregation and solubility but did not affect crystal growth behavior. Protein aggregation was largely eliminated by mutating the protein's two cysteines to serines. The double mutant retains full enzymatic activity and crystallizes in three new forms, one of which (triclinic) diffracts to 1.1-A resolution.

Bacterial Proteins↗
PubMed1997

Properdin, a positive regulator of complement activation, is released from secondary granules of stimulated peripheral blood neutrophils.

Properdin is an important regulatory constituent of the complement system. In contrast to most other components of complement, biosynthesis of properdin is restricted to a few cell types only, i.e., monocytes/macrophages and peripheral blood T cells. This report demonstrates the presence of properdin mRNA in peripheral blood granulocytes and shows that properdin is stored in the granules of human neutrophils and secreted upon stimulation with TNF-alpha, C5a, IL-8, or FMLP. Subcellular fractionation using Percoll density gradients and Western blot analyses revealed that the bulk of properdin is contained in the secondary granules. Moreover, flow cytometric analyses indicated that properdin is present on the surface of neutrophils. In contrast to alternative pathway components, components of the classical pathway of complement activation, such as C2 and C4, were not detected. Our findings suggest that neutrophils can actively stabilize and amplify the alternative activation pathway of complement by secretion of properdin as part of the innate defense against microorganisms.

Cell Compartmentation↗
PubMed1996

Golf for the senior player.

In no other sport does the senior player enjoy as much opportunity to participate and compete at various skill levels as in golf. No other sport encourages as much mixed competition. Because of their spending power, equipment and travel packages are often geared to the senior player. Advancing medical technology and specialized instruction effectively combat the physical effects of aging. These and other factors will ensure the continued popularity of the game of golf.

Exercise↗
PubMed1994

Cellular immunity and retrobulbar fibroblasts in Graves' ophthalmopathy.

In Graves' ophthalmopathy (GO), retrobulbar connective tissue is infiltrated by T cells whose role in the pathogenesis of the disease was investigated in the present work. The aims included firstly to characterize subsets of blood lymphocytes and of sessile lymphocytes cloned from a retroorbital tissue specimen. Second, in counterstimulation assays, the ability of patients' T cells to influence cultivated retrobulbar fibroblasts and in turn the enhancement of lymphocyte proliferation by retrobulbar fibroblasts was investigated. Blood lymphocytes of 16 GO patients and 12 controls isolated by density gradient centrifugation and retrobulbar fibroblasts obtained from orbital decompression were alternately exposed to irradiation of 4000 rad (to suppress proliferation of either cell type), then cocultivated for 48 h. Subsequently, the cells (250,000 lymphocytes, 5000 retrobulbar fibroblasts) were incubated with [3H]thymidine for 24 h. A stimulation index representing the degree of proliferation in comparison with a control was determined. Screening of 62 retrobulbar lymphocyte clones by cell-ELISA revealed a CD4/CD8 ratio of 8.2, contrasting with a normal ratio of 2.1 in peripheral lymphocytes (as obtained by FACS analysis). Incubation of patient's lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly elevated stimulation index (9.7) compared to incubation of lymphocytes with retrobulbar fibroblasts from controls (2.5). In another assay, the degree of stimulation amounted to 2.2 vs. -0.8. Retrobulbar fibroblasts were stimulated twice as much by lymphocytes of the same patient compared to retrobulbar fibroblasts from controls. The autologous reactions demonstrated suggest the presence of autoreactive T lymphocytes in GO patients directed against retrobulbar fibroblasts, which, in addition, are stimulated by these lymphocytes.

Adult↗
PubMed1993

Retrobulbar adipocytes and humoral immunity in Graves' ophthalmopathy.

As CT and MR-imaging revealed an enlargement of retrobulbar fat tissue in patients with Graves' ophthalmopathy, the role of the retrobulbar adipocytes in the pathogenesis of this disorder remains to be elucidated. To evaluate the in vitro influence of humoral immunity on retrobulbar adipose tissue, the effects of IgG and sera from 56 euthyroid ophthalmopathy patients and 53 controls on both porcine and human (patients' and controls') retrobulbar adipocytes were measured by means of several assays: An enzyme-linked immunosorbent assay was employed to reveal specific binding of antibodies to the adipocytes. Metabolic activity was determined by means of a colorimetric dimethyl thiazolium-diphenyl-tetrazolium bromide test which quantifies the activity of mitochondrial dehydrogenases; cell proliferation was measured by incorporation of [3H]-thymidine in 24 h, and activities of adipocyte specific enzymes, such as membrane-bound lipoprotein lipase and 1-glycerol-3-phosphate-dehydrogenase were determined. By means of these specific enzyme tests no distinctions could be made between patients and controls. Furthermore, a significant difference between patients' (untreated and treated) and controls' IgG to bind to, to activate or to stimulate the proliferation of porcine or human (patients' and controls') retrobulbar adipocytes could not be detected under the employed experimental conditions. The effects of patients' heat-inactivated and non-inactivated sera were indistinguishable from those of the controls. Incubation with autologous sera, however, led to an activation of the retrobulbar adipocytes which was higher than the median caused by the patients' group and that engendered by incubation with autologous IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocytes↗
PubMed1993

Glycosaminoglycan antibodies in endocrine ophthalmopathy.

An increased accumulation of glycosaminoglycans (GAG) in the orbita has been reported in endocrine ophthalmopathy (EO). In this study we investigated whether antibodies directed against GAG are present in the sera of 52 EO patients and 47 healthy controls. Three out of 52 patients exhibited low titers of antinuclear antibodies and all patients were negative for antibodies against extractable nuclear antigens. Isotype IgG antibodies were detected by means of an ELISA using hyaluronic acid and dermatan sulfate as antigens. Values were expressed as optical density at 405 nm. In comparison to the control group (0.445, 0.364, 0.588; median, 25th, 75th percentile) significantly (p < 0.001) higher hyaluronic acid antibody levels were found in EO patients (0.626, 0.473, 0.801). Untreated patients had significantly higher hyaluronic acid antibody values compared to controls (0.770 vs 0.437, median, p < 0.005). Thyroid status or antithyroid treatment did not influence binding of hyaluronic acid antibodies. When screening for dermatan sulfate antibodies no such difference could be observed between patients and controls. Thus, the detection of GAG antibodies in EO patients emphasizes the important role of glycosaminoglycans in the pathogenesis of thyroid eye disease.

Adolescent↗
PubMed1992

Relation of endocrine and cardiac findings in acromegalics.

Cardiac involvement in 32 acromegalics was related to endocrine parameters, clinical score and duration of the disease as well as compared to that of 50 controls free of cardiac disease. Stress ECG, 24h Holter monitoring and echocardiography revealed that supraventricular premature complexes did not occur more often in acromegalics than in controls, both prevalence and severity of ventricular arrhythmia, however, were higher in patients compared to controls: 15/32 (48%) acromegalics had complex ventricular arrhythmia as compared with 6/50 (12%) normal subjects (p less than 0.01). Repetitive ventricular arrhythmia was manifest in 10/32 (31%) patients but only in 4/50 (8%) controls (p less than 0.01). Furthermore, frequency of ventricular premature complexes increased with duration of acromegaly (p less than 0.01), the severity of the former being correlated with left ventricular mass and with clinical activity score (p less than 0.01). No correlation, however, was found between the degree of ventricular arrhythmia and hormone levels. Left ventricular muscle mass was increased (p less than 0.02) due to concentric hypertrophy. Thus, compared to controls, acromegalics show more frequent and complex ventricular arrhythmia and left ventricular hypertrophy. Duration of the disease rather than hormone levels seems to be relevant for these pathological changes.

Acromegaly↗
PubMed1992

Humoral immunity and retrobulbar fibroblasts in endocrine ophthalmopathy.

The exact role of retrobulbar fibroblasts in the immunopathogenesis of endocrine ophthalmopathy still remains to be elucidated. To evaluate the in vitro influence of humoral immunity on retrobulbar fibroblasts, the effects of immunoglobulin G as well as of the sera of 50 euthyroid patients with endocrine ophthalmopathy and 30 controls on both porcine and human (patients' and controls') retrobulbar fibroblasts were measured by means of several assays: a colorimetric test involving a heterocyclic chemical, a tetrazolium bromide, was applied to quantify the activity of mitochondrial dehydrogenases; the incorporation of 3H-thymidine was determined as a sensitive parameter for cell proliferation, and an enzyme-linked immunosorbent assay was to reveal specific binding of antibodies to the cells. There was consistently no significant difference between patients' (untreated or treated) and controls' IgG to bind to, to activate or to stimulate the proliferation of porcine and human (patients and controls) retrobulbar fibroblasts. The effects of patients' heat-inactivated and non-inactivated sera were indistinguishable from those of the controls. Incubation of autologous sera, however, led to an activation of retrobulbar fibroblasts which was both higher than the median caused by the patients' group and that engendered by incubation of autologous IgG. Yet, a significant role that humoral immunity might play directly on retrobulbar fibroblasts could not be detected in the experiments conducted in this study.

Adult↗
PubMed1992

In vitro synthesis of glycosaminoglycans in endocrine ophthalmopathy.

The effects of humoral and cell-mediated immunity on the glycosaminoglycan synthesis of retrobulbar fibroblasts was evaluated in patients with endocrine ophthalmopathy. After incubation with IgG and sera, secreted glycosaminoglycans, radiolabeled with D-6-3H-glucosamine and 35sulfate, were precipitated with cetylpyridinium chloride and ethanol. Hyaluronic acid synthesis of human retrobulbar fibroblasts after incubation with sera and IgG and after co-culture with lymphocytes was assessed by means of a radiometric test. Patients' IgG, compared to controls', accounted for a higher secretory stimulation of porcine retrobulbar fibroblasts (as measured by cetylpyridinium chloride precipitation) after 24 and 48 h. Contrasting with 24 h incubation time, glycosaminoglycan values after 48 h were increased two to threefold. Patients' and controls' sera caused earlier and stronger, yet indistinguishable glycosaminoglycan production. Non-sulfated hyaluronic acid was the preponderant glycosaminoglycan secreted into the media by retrobulbar fibroblasts. As assessed with the radiometric test, incubation with patients' and controls' sera and IgG did not reveal a significant difference in stimulating the hyaluronic synthesis of patients' and controls' retrobulbar fibroblasts. When measuring the hyaluronic acid synthesis of controls' and patients' retrobulbar fibroblasts after co-cultivation of lymphocytes, however, patients' lymphocytes had a marked ability to increase the hyaluronic acid concentration compared to controls' lymphocytes. The hyaluronic acid concentration after incubation of a patient's retrobulbar fibroblasts with autologous lymphocytes was markedly more elevated than the intrinsic hyaluronic acid production of retrobulbar fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Glycosaminoglycans in thyroid-associated ophthalmopathy.

Glycosaminoglycan (GAG) accumulation in the retrobulbar space of patients with thyroid-associated ophthalmopathy (TAO) has been documented in a number of immunohistochemical studies. In order to gain further insight into possible immunopathogenic mechanisms, the influence of humoral immunity on retrobulbar fibroblasts (RF) as GAG producing cells as well as on GAGs themselves was investigated. The effect of lymphocytes on hyaluronic acid (HA) synthesis of RF as well as in turn the influence of RF on lymphocytes were evaluated. In search of methods which would facilitate management of patients with TAO and allow assessment of disease activity, GAGs were determined in both urine and plasma. Immunoglobulin G (IgG) of patients with TAO were found to markedly stimulate the 3H-GAG secretion of RF. Patients with TAO exhibited significantly greater antibody values directed against HA than controls. Preliminary results concerning the influence of lymphocytes on RF indicate a tendency for patients' lymphocytes to increase the synthesis of HA. Furthermore, these lymphocytes in turn were stimulated more by irradiated autologous RF than by irradiated heterologous RF. Urine and plasma GAG determination proved to be suitable for the routine assessment of disease activity and outcome of therapy. In conclusion, GAGs seem to play an important role in the pathogenesis of the disease and their measurement may provide aid to the endocrinological evaluation of patients with TAO.

Antibody Formation↗