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Biomedical subjects

M C Hillarby

Publications and source records attributed to M C Hillarby.

At least 19 recordsLinked to original sources
PubMed2003

Apoptosis of terminal hypertrophic chondrocytes in an in vitro model of endochondral ossification.

It is widely accepted that growth plate chondrocytes undergo apoptosis when they reach the terminal hypertrophic stage of their differentiation during the process of endochondral ossification in vivo. In this report, an established chondrocyte cell culture model of mammalian endochondral ossification was utilized to investigate the fate of chondrocytes after they had entered hypertrophy in vitro. Fetal bovine epiphyseal chondrocytes were treated with the demethylating agent, 5-azacytidine, for 48 h and then cultured under azacytidine-depleted conditions. There was evidence for apoptosis in azacytidine-treated cells, as demonstrated by nuclear condensation and fragmentation (days 27 and 35) using transmission electron microscopy, and the detection of exposed phosphatidylserine on the plasma membrane surface of apoptotic chondrocytes (day 27) using fluorescence-labelled annexin V. Treated cultures on days 10 and 20 and untreated cultures at all corresponding time-points showed no morphological characteristics of apoptosis. In situ hybridization studies of treated cultures revealed that expression of the apoptotic suppressor, bcl-2, remained consistently high throughout the culture period, whilst the apoptotic inducer, bax, was not expressed until day 23. Quantification of these data showed a gradual shift in the ratio of the expression level of bcl-2 and bax in favour of bax with time in culture, particularly from day 23 onwards. Taken together, the results indicate that azacytidine-treated epiphyseal chondrocytes entered terminal hypertrophy from day 23 onwards in culture and died by apoptosis. This study confirms this culture system as a successful recapitulation of the entire mammalian chondrocyte differentiation pathway, including apoptosis. The culture model will prove valuable for studies of the apoptotic fate of terminally differentiated chondrocytes in the growth plate with a view to providing a better understanding of the underlying mechanisms of skeletal malformations and other pathological disorders such as osteoarthritis.

Animals↗
PubMed2002

Increased gelatinase activity in systemic sclerosis dermal fibroblast cultures with unaltered gelatinase A mRNA expression.

Gelatinase A is one of the matrix metalloproteinases, the principle enzymes degrading extracellular matrix (ECM) and basement membrane components. The aim of this study was to study gelatinase expression in systemic sclerosis (SSc). Fibroblasts were grown from uninvolved and involved skin of SSc patients and from healthy controls. Gelatinase activity was assayed by degradation of tritium-labeled gelatin. Gelatinase A mRNA was quantitated by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). Gelatinase activity was significantly increased in both uninvolved and involved SSc cultures. However, gelatinase A mRNA was unaltered in both cases. Neither SSc nor control skin fibroblasts expressed gelatinase B, indicating that the increased gelatinase activity is not due to gelatinase B induction. Gelatinase A is a specific basement membrane degrading enzyme, so increased gelatinase activity may be related to the pathophysiology of SSc by initiating microvascular damage and leakage of substances capable of producing further endothelial cell damage or fibroblast activation. Increased gelatinase activity in SSc fibroblasts seems to be regulated at translational and/or post-translational level.

Adult↗
PubMed2001

Fas mRNA expression in blood is reduced during episodes of human corneal graft rejection.

BACKGROUND: Our purpose is to examine levels of Fas mRNA expression in blood during human corneal transplant rejection. METHODS: Fas mRNA expression was detected by reverse transcription-PCR in blood from normal controls, corneal recipients at the time of transplantation and during episodes of rejection. RESULTS: Samples taken at the time of a corneal rejection episode showed Fas mRNA levels were significantly lower in these patients than either normal controls (P = 0.017) or corneal transplant recipients not undergoing graft rejection (P = 0.00052). Serial samples from five patients who suffered an episode of rejection showed that the level of Fas mRNA is reduced during the rejection episode and subsequently recovers. CONCLUSIONS: These results indicate low levels of Fas mRNA in blood may have a role in corneal transplant rejection.

Adult↗
PubMed2001

A novel cell culture model of chondrocyte differentiation during mammalian endochondral ossification.

Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5-9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.

Animals↗
PubMed2000

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.

Amino Acid Sequence↗
PubMed1999

Corneal epithelial-specific cytokeratin 3 is an autoantigen in Wegener's granulomatosis-associated peripheral ulcerative keratitis.

PURPOSE: In a previous investigation it was demonstrated that circulating antibodies to a 66-kDa corneal epithelial antigen (BCEA-A) are associated with peripheral ulcerative keratitis (PUK) in patients with Wegener's granulomatosis (WG). The aim of this study was to identify BCEA-A. METHODS: The 66-kDa antigen was purified from a bovine corneal epithelial protein extract, using DE52 ion exchange chromatography. Purified protein was used to raise rabbit polyclonal antibodies. These antibodies were used to screen a bovine corneal epithelial cDNA expression library. Positive clones were purified and sequenced. Clones were identified by DNA sequence homology searches of the GenBank DNA database. RESULTS: A cDNA clone that demonstrated strong binding to both the rabbit polyclonal antibody and patient sera, showed 85% homology to rabbit cytokeratin 3 (K3). K3 is a basic cytokeratin specific to corneal epithelium. No bovine DNA sequence for K3 is available. However, bovine K3 is larger than rabbit K3, with a molecular weight of 66 kDa. Immunofluorescence using both patient sera and the rabbit antibody demonstrated a cytoplasmic binding pattern on human corneal epithelium. CONCLUSIONS: This evidence suggests that the 66-kDa autoantigen (BCEA-A) associated with PUK in WG is cytokeratin 3, and this may form the basis of a diagnostic/prognostic test.

Amino Acid Sequence↗
PubMed1998

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype.

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or 'dwarfed'. the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo.

Animals↗
PubMed1998

Serum levels of vascular endothelial growth factor (VEGF) are markedly elevated in patients with Wegener's granulomatosis.

OBJECTIVES: Necrotizing vasculitis and granuloma formation are the predominant features of Wegener's granulomatosis (WG). We have investigated the importance of vascular endothelial growth factor (VEGF) in monitoring disease activity in WG. METHODS: Serum VEGF levels were determined in 23 patients with active WG, 21 healthy controls and 25 patients with urinary infection, by ELISA using commercially available antibodies to VEGF. RESULTS: VEGF levels were enormously elevated in patients with WG compared to both controls and patients with urinary infection (P < 0.0001). Of the 23 patients, 21 (91.3%) had VEGF levels above the cut-off value (3.3 ng/ml, calculated as the mean of the controls + 2 S.D.). Further analysis of the data showed that VEGF levels did not correlate with age, sex, incidence of classic antineutrophil cytoplasmic antibodies (c-ANCA) or duration of the disease (P > 0.05), but there was correlation with disease activity (r = 0.51, P < 0.01). VEGF levels were higher in patients with major compared to those with minor disease activity (P < 0.01). However, there was no significant correlation between VEGF levels and the Birmingham scores for vascular activity and damage. CONCLUSION: VEGF levels are raised in WG patients compared to normal controls and may be a marker of disease activity. Further studies on serial blood samples from a large cohort of patients with WG and other systemic vasculitides are needed to evaluate the specificity and usefulness of VEGF levels in monitoring disease activity.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1998

Characterization of two corneal epithelium-derived antigens associated with vasculitis.

PURPOSE: In a previous investigation into corneal autoimmunity, it was demonstrated that a putative autoantigen, a protein of 66 kDa, present in bovine corneal epithelium, binds circulating autoantibodies in approximately 60% of patients with Wegener's granulomatosis (WG). The aim of the present study was to characterize and identify the 66-kDa protein. METHODS: A purification protocol was established for the 66-kDa protein using standard chromatography techniques. During the purification procedure it became clear that the 66-kDa protein detected in patients' sera was in fact two proteins, both running at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that eluted in different fractions on DE-52 chromatography columns. These two proteins have been labeled bovine corneal epithelial antigen-A and -B (BCEA-A and BCEA-B). Further investigations of antibody binding have demonstrated that patients' sera bind to either one or the other of these proteins with no cross-reactivity between them. Separated BCEA-A and BCEA-B protein extracts were immunoblotted with 27 WG patients' sera, 10 Churg-Strauss syndrome (CSS) patients' sera, 31 rheumatoid arthritis (RA) patients' sera, and 40 healthy control subjects' sera from the blood bank. RESULTS: Forty-six percent of WG patients' sera had antibodies to one of the 66-kDa antigens, whereas none of the healthy control subjects' sera had 66-kDa antibodies (P < 10(-5)). In the WG group, 31% were positive to BCEA-A (versus controls, P = 0.0023), and 15% were positive to BCEA-B. WG patients with peripheral ulcerative keratitis (PUK) had a significant association with anti-BCEA-A antibodies when compared with healthy control subjects (50%, P < 10(-6)). However, in the RA group with no eye disease there was an association with BCEA-A (25%, P = 0.011) but not in the RA group with PUK. The frequency of anti-BCEA-B antibodies was significantly increased in patients with CSS (60%, P < 10(-7)). CONCLUSIONS: In summary, it has been shown that vasculitis patients have antibodies to two 66-kDa corneal antigens and that autoantibodies to these antigens are mutually exclusive. It has also been shown that antibodies to BCEA-B are associated with CSS, whereas BCEA-A antibodies are associated with WG and RA.

Adult↗
PubMed1997

TNF microsatellite a2, b3 and d2 alleles are associated with systemic lupus erythematosus.

We have investigated TNF microsatellite polymorphisms in SLE and their association with both HLA class II alleles and disease expression. A total of 91 Caucasoid SLE and 109 matched Caucasoid controls were recruited for this study. TNF microsatellites a, b and d were typed using fluorescent based semi-automated gene scanning. TNF a2, b3 and d2 allele frequencies were significantly increased in the SLE group compared to controls. These alleles were found to be part of an extended HLA-DRB1*0301 haplotype and have previously been associated with high TNF-alpha production. When the SLE group was analyzed according to presentation of certain clinical features, photosensitivity and Raynaud's phenomenon, the frequency of these alleles (TNF a2, b3 and d2) were also significantly increased. No significant increase in the allele frequencies of TNF a2, b3 and d2 was demonstrated in the group of patients with renal involvement. These data suggest that TNF microsatellite alleles are not independent of HLA associations in SLE and may be important in the expression of certain clinical features in SLE.

Alleles↗
PubMed1997

A dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythematosus in a Spanish population.

OBJECTIVE: To determine dysfunctional mannose binding protein (MBP) status of Spanish patients with systemic lupus erythematosus (SLE) and to determine whether MBP and complement C4 null alleles contribute in an additive way to SLE susceptibility. METHODS: The frequencies of MBP alleles (characterized by polymorphisms at codon 54 and codon 57 of exon 1) were determined by the amplification refractory mutation system-polymerase chain reaction in 50 Spanish patients with SLE and 49 matched controls. Mutant genotypes for the codon 54 mutation were confirmed using a Ban I restriction enzyme digest method. Complement C4 allotyping was achieved by agarose gel electrophoresis of neuraminidase/carboxypeptidase B digested plasma samples followed by immunofixation and staining. RESULTS: At least one dysfunctional MBP allele, unable to activate complement, was present in 52% of patients with SLE and in 31% of controls (OR = 2.4, 95% CI 1.1-5.6). Complement C4 null alleles (either C4A or C4B) were present in 61% of patients and 43% of controls (OR = 2.1, 95% CI 0.9-4.9). A dysfunctional MBP allele and C4 null allele were present in 41% of patients and 16% of controls (OR = 3.2, 95% CI 1.2-8.1). CONCLUSION: The presence of a dysfunctional MBP allele is a risk factor for developing SLE in this Spanish population and may affect susceptibility in an additive way with C4 null alleles.

Acute-Phase Proteins↗
PubMed1995

Mannose-binding protein gene polymorphism in systemic lupus erythematosus.

OBJECTIVE: To determine whether an allelic form of mannose-binding protein (MBP) incapable of activating complement is associated with susceptibility to systemic lupus erythematosus (SLE). METHODS: MBP allele frequencies were determined by amplification refractory mutation system-polymerase chain reaction in 102 white SLE patients and 136 controls. RESULTS: The MBP allele that is unable to activate complement was present in 42 SLE patients (41%) and in 41 controls (30%) (P = 0.08, odds ratio [OR] = 1.6, 95% confidence interval [95% CI] 1.0-2.8). The gene frequency of this allele was 0.25 in SLE patients and 0.19 in controls (P = 0.08, OR = 1.5, 95% CI 1.0-2.3). CONCLUSION: Our results suggest that this allele of the MBP gene represents a minor risk factor for SLE.

Adolescent↗
PubMed1995

Relative contributions of HLA-DQA and complement C4A loci in determining susceptibility to systemic lupus erythematosus.

The objective of this study was to reassess the role of C4A null alleles in systemic lupus erythematosus (SLE) susceptibility after taking into account the association of DQA*0501 with this disease. The frequency of C4A null alleles in 82 SLE patients and 59 controls was determined using both immunofixation and a TaqI RFLP method. HLA-DQA and DQB alleles were identified by sequence-specific oligonucleotide typing. Empirical logistic analysis was used to assess the interactive effects of C4 and DQA alleles. It was found that the strongest association with SLE was for the combination of DQA*0501 and C4A*Q0 [odds ratio (OR) = 5.4, 95% confidence interval (CI) 2.5-11.7]. Both DQA*0501 (P = 0.02) and C4A*Q0 (P = 0.03) appeared to have significant individual effects on SLE susceptibility, with a significant statistical interaction between the two loci (P = 0.01). However, when anti-La antibody negative patients were examined only C4A*Q0 had a significant individual effect (P = 0.04). A significant statistical interaction between DQA*0501 and C4A*Q0 was again detected (P = 0.02). These results support the hypothesis that susceptibility to SLE is influenced by several genes with differing functions: HLA-DQA*0501 may predispose to autoantibody formation while C4A*Q0 impairs immune complex clearance.

Adolescent↗
PubMed1994

Tap gene associations in UK caucasoids.

The authors determined the allele frequencies of the TAP1 and TAP2 transporter genes in a healthy UK Caucasoid population by ARMS-PCR. TAP1A was the most frequent TAP1 allele by far, being present in 76% of subjects. TAP1 alleles could not be assigned in 24% of subjects, since the combinations TAP1A/1b and TAP1C/1D cannot be separated. TAP2A was the most frequent TAP2 alleles (75% of subjects) followed by TAP2B (43%), TAP2C (11%), TAP2D (8%) and TAP2E (6%). The authors also identified an individual with a previously undescribed TAP2 allele, TAP2H (isoleucine at amino acid [aa] 379, alanine at aa 565, alanine at aa 665). It was not possible to assign unequivocally TAP2 alleles in 15 individuals (9%) as TAP2A/D and TAP2C/E cannot be distinguished from each other. To address this problem a separate study of families of rheumatoid arthritis (RA) patients selected for this ambiguity were studied. In all five informative families, TAP2A/2D was confirmed as the combination present. In the population studied no evidence was found for linkage disequilibrium between TAP1 and TAP2 or between the TAP genes and HLA-DP. There was no evidence for extensive linkage disequilibrium between the TAP genes and HLA-DQR, although TAP2B was associated with DRI (delta = 0.056, corrected P < 0.01) and TAP2D with DR4 (delta = 0.018). In the RA families studied, TAP2D was found on DRB1*0401-bearing haplotypes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Polymorphisms of the TAP2 transporter gene in systemic lupus erythematosus.

OBJECTIVES: To determine whether the TAP2 transporter gene, which lies between HLA-DP and HLA-DQ, is involved in determining susceptibility to systemic lupus erythematosus (SLE). METHODS: TAP2 types were determined by ARMS-PCR in 89 white patients with SLE and 156 control subjects. RESULTS: No particular TAP2 dimorphism or allele was associated with SLE or with any clinical/immunological subgroup of SLE. Furthermore, there was no evidence for significant linkage disequilibrium between TAP2 and HLA-DQ/DR in SLE. CONCLUSIONS: These data suggest that TAP2 is not a disease susceptibility gene for SLE and that the disease-predisposing haplotypes do not extend as far as TAP2. This indicates that any HLA-DP association with SLE must be independent of other class II (DQ/DR) associations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗