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Biomedical subjects

K Whaley

Publications and source records attributed to K Whaley.

At least 37 records · Page 2Linked to original sources
PubMed1993

Expression of the components and regulatory proteins of the alternative complement pathway and the membrane attack complex in normal and diseased synovium.

We have studied synthesis of the complement components and regulatory proteins of the alternative pathway and the membrane attack complex in synovial membrane. RNA was extracted from synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) as well as from normal synovial membrane. Dot blot analysis showed the presence of mRNAs for all the complement components and regulatory proteins (C3, factor B, factor D, C5, C6, C7, C9, factor H, factor I, S-protein, SP-40, 40, DAF, MCP, CR1, CD59), except for properdin, C8 alpha, C8 beta and C8 gamma in all three types of synovial membrane studied. In an attempt to determine which components were synthesised by each cell type, monocytes (mononuclear phagocytes), human umbilical vein endothelial cells (HUVEC), synovial membrane fibroblasts (from normal, OA and RA synovial membrane) and peripheral blood lymphocytes were cultured in vitro and secretion rates of individual components were measured and total cellular RNA analysed by northern blotting. Monocytes secreted properdin, C3, and factor H but not factor B, factor I, C5, C6, C7, C8 or C9. Fibroblasts and endothelial cells secreted factor B, factor H and factor I, but not properdin, C5, C6, C7, C8 or C9. Lymphocytes did not secrete any of these components. mRNAs encoding C3, factor B, factor H, S-protein, SP-40, 40, MCP and DAF were detected in all three other cell types (monocytes, fibroblasts and HU-VEC), but factor I and CD59 mRNAs were not detected in monocytes. C5, C6, C7, C8 alpha, C8 beta, CD8 gamma and C9 mRNAs were not detected in any of the cell types studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis, Rheumatoid↗
PubMed1993

Complement biosynthesis by mononuclear phagocytes.

Mononuclear phagocytes are an important in vivo source of a wide range of complement components. They are able to rapidly up-regulate or down-regulate complement synthesis in response to many different pharmacological and biological stimuli. This ability is likely to make a significant contribution to maintaining host defences particularly in peripheral tissues. The important role of molecular biology in the study of complement biosynthesis by mononuclear phagocytes will be emphasised.

Animals↗
PubMed1993

Human complement factor B: functional properties of a recombinant zymogen of the alternative activation pathway convertase.

The human complement factor B is a centrally important component of the alternative pathway activation of the complement system. Here we report the isolation, characterization and eukaryotic expression of the first full length cDNA transcript for human factor B. In a factor B dependent haemolysis assay, the recombinant human factor B generated by transient COS cell transfection was shown to reconstitute haemolytic activity of factor B depleted human serum. To study the biological activities assigned to factor B, the availability of recombinant polypeptides representing definite portions of the human factor B molecule is desirable.

Animals↗
PubMed1993

Carrier detection in families with properdin deficiency by microsatellite haplotyping.

Human properdin deficiency is an X-linked disorder strongly predisposing to meningococcal disease which has been recorded in over 50 cases of various ethnic origins. Immunochemically, total deficiency (type I), partial deficiency (type II), and deficiency due to a dysfunctional molecule (type III) can be differentiated. It is therefore most likely that the causative molecular defects will show considerable genetic heterogeneity. Analysis of the properdin locus at Xp11.3-Xp11.23 has led to the characterization of two polymorphic (dC-dA)n.(dG-dT)n repeats located approximately 15 kb downstream from the structural gene. Three families (two Scottish Caucasoid, one Tunisian Sephardic) with seven deficient individuals were investigated immunochemically and using a nonradioisotopic polymerase chain reaction-based method for microsatellite detection. Probable and definite carriers frequently showed properdin levels which were in the normal range. No recombinants between the microsatellite loci and properdin deficiency were detected, thus allowing identification of the defective allele through the generations in all three pedigrees. Haplotyping for these highly polymorphic microsatellites in close physical linkage to the properdin gene can provide rapid and nonradioactive detection of carrier status and prenatal diagnosis without extensive sequencing analysis.

Base Sequence↗
PubMed1993

Regulation of the synthesis of C1 subcomponents and C1-inhibitor.

We have investigated the synthesis of C1q, C1r, C1s and C1-inhibitor in HepG2 cells, human umbilical vein endothelial cells (HUVEC), fibroblasts (skin and synovial membrane), chondrocytes and monocytes. C1q was only synthesised by monocytes, although the mRNAs for the C1qA and C1qC chains were expressed in HUVEC. C1r, C1s and C1-inhibitor were synthesised by all cell types. The secretion rates of C1r and C1s were approximately equimolar in fibroblasts and chondrocytes whereas the secretion rate for C1s exceeded that for C1r in the other cell types. Molar ratios of C1s to C1r were approximately 2:1 for HepG2 cells, 5:1 for monocytes and 10:1 for HUVEC. Stimulation with interferon-gamma resulted in increased expression of all four proteins. The C1s:C1r ratio did not alter in chondrocytes or fibroblasts, but approached unity in HepG2, monocytes and HUVEC, due to relatively greater stimulation of C1r gene expression.

Carcinoma, Hepatocellular↗
PubMed1992

Effect of interferon-gamma on complement gene expression in different cell types.

We have studied the expression of the complement components C2, C3, factor B, C1 inhibitor (C1-inh), C4-binding protein (C4-bp) and factor H in human peripheral blood monocytes, skin fibroblasts, umbilical vein endothelial cells (HUVEC) and the human hepatoma cell line G2 (Hep G2) in the absence and the presence of interferon-gamma (IFN-gamma). E.l.i.s.a. performed on culture fluids, run-on transcription assays, Northern blot and double-dilution dot-blot techniques confirmed that monocytes expressed all six components, whereas fibroblasts, HUVEC and HepG2 each expressed five of the six components. Fibroblasts and HUVEC did not synthesize C4-bp, and Hep G2 did not produce factor H. In addition to these differences, the synthesis rates of C3, C1-inh and factor H were not the same in all cell types. However, the synthesis rates of C2 and factor B were similar in all four cell types. The half-lives of the mRNAs were shorter in monocytes than in other cell types. Monocyte factor H mRNA had a half-life of 12 min in monocytes, compared with over 3 h in fibroblasts and HUVEC. The instability of factor H mRNA in monocytes may contribute to their low factor H secretion rate. IFN-gamma produced dose-dependent stimulation of C2, factor B, C1-inh, C4-bp and factor H synthesis by all cell types expressing these proteins, but decreased C3 synthesis in all four cell types. Cell-specific differences in the response to IFN-gamma were observed. The increased rates of transcription of the C1-inh and factor H genes in HUVEC were greater than in other cell types, while the increased rate of transcription of the C2, factor B and C1-inh genes in Hep G2 cells was less than in other cell types. IFN-gamma did not affect the stability of C3, factor H or C4 bp mRNAs, but increased the stability of factor B and C1-inh mRNAs and decreased the stability of C2 mRNA. Although these changes occurred in all four cell types studied, the half-life of C1-inh mRNA in monocytes was increased almost 4-fold, whereas the increases in the other cell types were less than 30%. These data show that the constitutive synthesis rates of complement components may vary in the different cell types. They also show that the degree of change in synthesis rates in response to IFN-gamma in each of the cell types often varies due to differences in transcriptional response, sometimes in association with changes in mRNA stability.

Carrier Proteins↗
PubMed1992

Immunohistochemical and functional studies of glycoprotein 60 (gp60) in platelets.

We showed by immunofluorescence, immunoelectron microscopy and Western blot analysis that the plasma glycoprotein (gp60), an Fc gamma binding protein which inhibits complement-mediated prevention of immune precipitation, is present in platelets. The gp60 content of platelets in normal individuals and patients with rheumatoid arthritis was similar (mean 0.028 and 0.024 fg/platelet respectively). Immunoelectron microscopic studies showed that gp60 was present in the cytoplasm and the surface connecting structures but not in the alpha granules, dense granules or lysosomes. Using this technique gp60 was also found on platelet membranes, an observation which was confirmed by immunofluorescence. Activation of platelets with thrombin, calcium ionophore, and immune complexes (IC) resulted in the release of the contents of the alpha granules (beta-thromboglobulin), dense granules (5-hydroxytryptamine) and lysosomes (beta-glucuronidase) but did not induce gp60 secretion. The inability of Fab anti-gp60 to inhibit IC-mediated platelet aggregation and of F(ab')2 anti-gp60 to produce platelet aggregation suggested that IC-mediated platelet aggregation did not occur as a result of the interaction of IC with platelet gp60. However, as the preincubation of IC with purified gp60 produced dose-dependent inhibition of the ability of IC to aggregate platelets it is possible that fluid-phase plasma gp60 modulates the interaction of IC with platelets.

Blood Platelets↗
PubMed1992

The relative roles of C4A and C4B in prevention of immune precipitation, solubilisation and immune adherence.

C4A and C4B levels were measured in serum from 246 normal individuals. Complement-mediated solubilisation, assayed using alkaline phosphatase anti-alkaline phosphatase immune complexes (IC), correlated with both C4A and C4B levels. However, C4A and C4B levels showed no correlation with solubilisation of bovine serum albumin (BSA) ICs, or with the prevention of immune precipitation of BSA or alkaline phosphatase ICs, nor with immune adherence assayed using thyroglobulin and BSA ICs.

Antigen-Antibody Complex↗
PubMed1992

Dissociation of primary antigen-antibody bonds is essential for complement mediated solubilization of immune precipitates.

The role of dissociation of primary antigen-antibody bonds in the solubilization of immune complexes (IC) has been investigated using photo-affinity crosslinked IC comprising NAP15-BSA and murine monoclonal anti-DNP antibodies. Non-covalently linked IC were solubilized rapidly when incubated with normal human serum (NHS), whereas covalently-linked IC were solubilized poorly or not at all. The rate and extent of complement activation produced by incubating covalently-linked and non-covalently linked IC with NHS was similar as assessed by the production of the C1s:C1-inhibitor, C3:properdin and C5b-9 complexes and the anaphylatoxins C4a and C3a. Thus, the inability of serum to solubilize photo-affinity crosslinked IC must be due to failure of dissociation of primary antigen-antibody bonds.

Animals↗
PubMed1992

A single base deletion from the C1-inhibitor gene causes type I hereditary angio-oedema.

RFLP analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a C1-inhibitor gene mutation responsible for type I hereditary angio-oedema (HAE). A single base deletion (C-16698) from the eighth exon of the C1-inhibitor gene alters the reading frame of the exon and generates a premature translation termination codon. This represents the first report of this form of C1-inhibitor gene mutation in type I HAE.

Amino Acid Sequence↗
PubMed1992

Type II hereditary angio-oedema associated with two mutations in one allele of the C1-inhibitor gene around the reactive-site coding region.

The polymerase chain reaction and nucleotide sequence analysis have been used to characterise two point mutations in the eighth exon of one allele of the C1-inhibitor gene in a kindred with type II hereditary angio-oedema (HAE). The mutations comprise a G to A substitution at C1-inhibitor gene nucleotide 16789 and an upstream C to T substitution at nucleotide position 16765. This represents the first report of these two mutations in the same C1-inhibitor allele in type II HAE. The molecular genetic pathogenesis of HAE is discussed in the light of these findings.

Alleles↗
PubMed1991

Modulation of complement gene expression by glucocorticoids.

The addition of dexamethasone, prednisolone or cortisol (in order of efficacy) to human monocytes in culture produced dose-related increases in the synthesis rates of the complement components C1 inhibitor (C1-inh), factor B (B) and C2. In contrast, concentrations of C3 and lysozyme in the culture supernatants were decreased. Indomethacin stimulated synthesis of C1-inh, C2 and B, but had little effect on synthesis of C3 or lysozyme. The simultaneous addition of cycloheximide (2.5 micrograms/ml) abrogated the effects of dexamethasone on synthesis of C2, B and C1-inh, but the effect of indomethacin on the synthesis of these components was unchanged. These data suggest that protein synthesis is required for the effects of glucocorticoids on the synthesis of C2, B and C1-inh to occur. Dexamethasone and indomethacin increased the abundances of C1-inh mRNA, B mRNA and C2 mRNA in parallel with changes in the synthesis rates of these proteins. The changes in mRNA abundance were not transcriptional, but were shown to be due to increased mRNA stability. In contrast, dexamethasone decreased the expression of C3 and lysozyme by decreasing the rate of transcription of these genes. Indomethacin had no effect on transcription of the C3 and lysozyme genes. The half-lives of C3 mRNA, lysozyme mRNA and actin mRNA were not altered by dexamethasone or indomethacin. It is concluded that the effects of glucocorticoids on monocyte synthesis of C2, B and C1-inh are due to increased mRNA stability and may be related to inhibition of prostaglandin synthesis, as these effects are similar to those produced by indomethacin. The effects of dexamethasone on the synthesis of C3 and lysozyme differ from those on C2, B and C1-inh as they depend upon a decrease in gene transcription, which is not affected by indomethacin.

Cells, Cultured↗
PubMed1991

Human complement factor H. Tissue specificity in the expression of three different mRNA species.

Using cDNA clones H-19 and H-46, we have shown previously that three different mRNA species (4.3 kb, 1.8 kb and 1.4 kb) for complement factor H are expressed constitutively in human liver. Here we report data suggesting that the expression of these different factor-H mRNA species is regulated by tissue-specific control mechanisms. Total RNA and poly(A)-enriched RNA from various human tissues (heart, lung, temporal cortex, kidney, spleen, bone marrow and muscle) various cell lines (HepG2, HepG3, HepG4, Hep3B, H-4, Jurkat, Molt4, H-9, KHos24Os, A-431, U937, Mono Mac 6 and Raji) and from primary cultures of peripheral blood monocytes, fibroblasts and human umbilical vein endothelial cells (HUVEC) were investigated for the expression of factor-H mRNA. In RNA preparations from extrahepatic tissue, factor-H mRNA was only detected in biopsies from the lung. Using 20 micrograms total RNA isolated from all 13 cell lines it was not possible to detect any factor-H mRNA, while mRNA for factor H was expressed in monocytes, HUVEC and fibroblasts. When expressed in extrahepatic tissues, only the 4.3-kb and the 1.8-kb mRNA species were detected, while the 1.4-kb mRNA is expressed abundantly in liver. Interferon-gamma did not induce the expression of factor-H mRNA in any of the cell lines tested. On the other hand, tumour necrosis factor-alpha induced the expression of the 4.3-kb mRNA species in U937 cells. In HUVEC and fibroblasts the relative quantities of the 4.3-kb and the 1.8-kb mRNA species and the regulatory effects of interferon-gamma, interleukin-1, dexamethasone and retinoic acid on their expression showed significant tissue specificity.

Blotting, Northern↗
PubMed1991

An RNA splice site mutation in the C1-inhibitor gene causes type I hereditary angio-oedema.

Restriction fragment length polymorphism analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a single base substitution (G----T) at nucleotide 8863 in the C1-inhibitor gene. This destroys the 5' donor splice site recognition motif of the sixth intron. Family studies suggest that the mutation is responsible for type I hereditary angio-oedema in a studied kindred.

Angioedema↗
PubMed1991

Restriction fragment length polymorphism analysis of the C1-inhibitor gene in hereditary C1-inhibitor deficiency.

Four out of 12 kindreds with Type I hereditary angio-oedema (HAE) were shown to have unique disease-related restriction fragment length polymorphism (RFLPs) in one allele of the C1-inhibitor gene. These RFLPs were used to localise the gene mutations responsible for them in each family. The four mutations affected exon 4, exon 6, exon 7 and exon 8, respectively. Mutations in exon 6 and exon 8 have not been described previously in Type I HAE. The other two mutations which comprised an exon 4 deletion and an exon 7 deletion have already been documented by other investigators. In each family the mutation was seen to cosegregate with the disease. Detection of a disease-related RFLP in 30% of the Type I HAE kindred tested is higher than other published studies, and reflects the larger number of restriction enzymes employed. These results suggest that Type I HAE is likely to be associated with a multiplicity of gene mutations as is seen in other genetic diseases. A new C1-inhibitor gene-related RFLP in the normal population was also characterised. This may be useful as an indirect marker of the mutant C1-inhibitor allele in certain families with Type I HAE.

Angioedema↗
PubMed1991

Disseminated gonococcal infection associated with deficiency of the second component of complement.

A case of C2 deficiency presenting with disseminated gonococcal infection is described. The predisposition of C2-deficient individuals to infection in addition to the commoner problem of immune complex diseases is noted. Attention is drawn to the absence of documented cases of gonococcal infection associated with C2 deficiency. No other homozygous C2 deficient family members were identified. Lifelong penicillin prophylaxis was recommended for the patient.

Adult↗
PubMed1990

Interferon-mediated transcriptional and post-transcriptional modulation of complement gene expression in human monocytes.

The addition of lymphoblastoid interferon alpha, fibroblast interferon beta and recombinant interferon gamma to in vitro monocyte cultures produced dose-dependent increases in transcription rates of the genes encoding the second component of complement (C2), factor B (B) and C1 inhibitor, and the abundance of their respective mRNA. Interferon gamma was the most effective at stimulating transcription of the C1-inhibitor gene whereas interferons alpha and beta were more effective at increasing the transcription of the C2 and B genes. Transcription of the C3 gene was reduced by interferon gamma. None of these cytokines altered the level of transcription of the actin gene. Interferon-induced changes in the levels of transcription of the C2, B and C1-inhibitor genes occurred rapidly, with significant changes occurring within 30 min of exposure to these cytokines. Within 4 h of removal of the interferons from the culture fluid, the level of transcription of the C1-inhibitor, C2, B and C3 genes returned to control values, as did abundance of C2, B and C3 mRNA. However, the abundance of C1-inhibitor mRNA remained elevated in interferon-gamma-treated monocytes. Combinations of interferons produced less than additive effects on the stimulation of the transcription of C2, B and C1-inhibitor genes, whereas measurements of C1-inhibitor mRNA and B mRNA showed that interferon gamma acted synergistically with interferon gamma to increase the abundance of the mRNA. Their effects on C2 mRNA abundance were less than additive. The half-lives of C1-inhibitor, C2, B and C3 mRNA were not altered by interferon alpha, whereas interferon gamma shortened the half-life of C2 mRNA by approximately 50%, and prolonged the half-lives of B and C1-inhibitor mRNA approximately twofold and fivefold, respectively. The half-life of C3 mRNA was unaltered by either interferon. These results show that the large increase in C1-inhibitor synthesis which occurs in interferon-gamma-treated monocytes, is due to a combination of increased transcription and increased C1-inhibitor mRNA stability. They also suggest that the synergistic effects of interferon alpha together with interferon gamma on C1-inhibitor and factor B synthesis is also dependent upon increased transcription and increased mRNA stability.

Complement C1 Inactivator Proteins↗