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1.  Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain 
Neuroscience Letters  2012;520(1):104-109.
Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration.
PMCID: PMC3375179  PMID: 22634524
2.  Application of the Ibis-T5000 Pan-Orthopoxvirus Assay to Quantitatively Detect Monkeypox Viral Loads in Clinical Specimens from Macaques Experimentally Infected with Aerosolized Monkeypox Virus 
Monkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus, causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque (Macaca fascicularis) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units. Furthermore, the platform was able to distinguish MPXV and vaccinia viruses that were spiked into macaque blood samples at various concentrations. This platform provides a new tool for the diagnosis and monitoring of orthopoxviral loads during vaccine or antiviral studies, but also could provide rapid identification during natural outbreaks or bioterrorism attacks.
PMCID: PMC2813175  PMID: 20134011
3.  Heparanase Expression and Activity Influences Chondrogenic and Osteogenic Processes During Endochondral Bone Formation 
Bone  2008;43(4):689-699.
Endochondral bone formation is a highly orchestrated process involving coordination among cell-cell, cell-matrix and growth factor signaling that eventually results in the production of mineralized bone from a cartilage template. Chondrogenic and osteogenic differentiation occur in sequence during this process, and the temporospatial patterning clearly requires the activities of heparin binding growth factors and their receptors. Heparanase (HPSE) plays a role in osteogenesis, but the mechanism by which it does so is incompletely understood. We used a combination of ex vivo and in vitro approaches and a well described HPSE inhibitor, PI-88 to study HPSE in endochondral bone formation. In situ hybridization and immunolocalization with HPSE antibodies revealed that HPSE is expressed in the peri-chondrium, peri-osteum, and at the chondro-osseous junction, all sites of key signaling events and tissue morphogenesis. Transcripts encoding Hpse also were observed in the pre-hypertrophic zone. Addition of PI-88 to metatarsals in organ culture reduced growth and suggested that HPSE activity aids the transition from chondrogenic to osteogenic processes in growth of long bones. To study this, we used high density cultures of ATDC5 pre-chondrogenic cells grown under conditions favoring chondrogenesis or osteogenesis. Under chondrogenic conditions, HPSE/Hpse was expressed at high levels during the mid-culture period, at the onset of terminal chondrogenesis. PI-88 addition reduced chondrogenesis and accelerated osteogenesis, including a dramatic up-regulation of osteocalcin levels. In normal growth medium, addition of PI-88 reduced migration of ATDC-5 cells, suggesting that HPSE facilitates cartilage replacement by bone at the chondro-osseous junction by removing the HS component of proteoglycans, such as perlecan/HSPG2, that otherwise prevent osteogenic cells from remodeling hypertrophic cartilage.
PMCID: PMC2621444  PMID: 18589009
Heparanase; chondrogenesis; osteogenesis; bone; PI-88; migration
4.  Diabetes care provision and glycaemic control in Northern Ireland: a UK regional audit 
Archives of Disease in Childhood  2005;90(5):468-473.
Aims: To assess the care received, compared to national guidelines, and to investigate factors associated with glycaemic control in children and adolescents with type 1 diabetes attending clinics in Northern Ireland.
Methods: An audit of the care provided to all patients attending 11 paediatric diabetes clinics commenced in 2002. A research nurse interviewed 914 patients completing a questionnaire recording characteristics, social circumstances, and aspects of diabetes management, including the monitoring of complications and access to members of the diabetes team. Glycaemic control was measured by glycosylated haemoglobin (HbA1c), determined at a DCCT aligned central laboratory.
Results: The average HbA1c concentration was 8.8% (SD 1.5%), with 20% of patients achieving recommended HbA1c levels of less than 7.5%. In the year prior to the audit, 76% of patients were reviewed by a diabetes specialist nurse and 42% were tested for microalbuminuria. After adjustment for confounding factors, better glycaemic control was identified, particularly in patients who had attended exactly four diabetes clinics in the previous year, were members of the patient association Diabetes UK, and lived with both natural parents.
Conclusions: In Northern Ireland only a minority of patients achieved recommended HbA1c levels. Furthermore, children and adolescents with diabetes were reviewed by fewer specialists and were less intensively monitored for microvascular complications than recommended. There was evidence of better control in children who were members of Diabetes UK, suggesting that parental attitude and involvement could lead to benefits.
PMCID: PMC1720387  PMID: 15851427
5.  Perlecan Domain I Promotes Fibroblast Growth Factor 2 Delivery in Collagen I Fibril Scaffolds 
Tissue engineering  2005;11(1-2):76-89.
Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17, 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.
PMCID: PMC1635240  PMID: 15738663
6.  Perlecan: an important component of the cartilage pericellular matrix 
Perlecan (Pln) is a large proteoglycan that can bear HS (heparan sulfate) and chondroitin sulfate glycosaminoglycans. Previous studies have demonstrated that Pln can interact with growth factors and cell surfaces either via its constituent glycosaminoglycan chains or core protein. Herein, we summarize studies demonstrating spatially and temporally regulated expression of Pln mRNA and protein in developing and mature cartilage. Mutations either in the Pln gene or in genes involved in glycosaminoglycan assembly result in severe cartilage phenotypes seen in both human syndromes and mouse model systems. In vitro studies demonstrate that Pln can trigger chondrogenic differentiation of multipotential mouse CH310T1/2 stem cells as well as maintain the phenotype of adult human chondrocytes. Structural mapping indicates that these activities lie entirely within domain I, a region unique to Pln, and that they require glycosaminoglycans. We also discuss data indicating that Pln cooperates with the key chondrogenic growth factor, BMP-2, to promote expression of hypertrophic chondrocyte markers. Collectively, these studies indicate that Pln is an important component of human cartilage and may have useful applications in tissue engineering and cartilage-directed therapeutics.
PMCID: PMC1387052  PMID: 15758379
Perlecan; Heparan Sulfate Proteoglycan; Extracellular Matrix; Cartilage; Mouse
7.  Monitoring growth in asthmatic children treated with high dose inhaled glucocorticoids does not predict adrenal suppression 
Archives of Disease in Childhood  2004;89(8):713-716.
Aims: To determine whether routine outpatient monitoring of growth predicts adrenal suppression in prepubertal children treated with high dose inhaled glucocorticoid.
Methods: Observational study of 35 prepubertal children (aged 4–10 years) treated with at least 1000 µg/day of inhaled budesonide or equivalent potency glucocorticoid for at least six months. Main outcome measures were: changes in HtSDS over 6 and 12 month periods preceding adrenal function testing, and increment and peak cortisol after stimulation by low dose tetracosactrin test. Adrenal suppression was defined as a peak cortisol ⩽500 nmol/l.
Results: The areas under the receiver operator characteristic curves for a decrease in HtSDS as a predictor of adrenal insufficiency 6 and 12 months prior to adrenal testing were 0.50 (SE 0.10) and 0.59 (SE 0.10). Prediction values of an HtSDS change of –0.5 for adrenal insufficiency at 12 months prior to testing were: sensitivity 13%, specificity 95%, and positive likelihood ratio of 2.4. Peak cortisol reached correlated poorly with change in HtSDS (ρ = 0.23, p = 0.19 at 6 months; ρ = 0.33, p = 0.06 at 12 months).
Conclusions: Monitoring growth does not enable prediction of which children treated with high dose inhaled glucocorticoids are at risk of potentially serious adrenal suppression. Both growth and adrenal function should be monitored in patients on high dose inhaled glucocorticoids. Further research is required to determine the optimal frequency of monitoring adrenal function.
PMCID: PMC1720050  PMID: 15269067
8.  An assessment of pancreatic endocrine function and insulin sensitivity in patients with transient neonatal diabetes in remission 
Aims: To examine derived indices of ß cell function, peripheral insulin sensitivity, and the pancreatic response to intravenous glucose loading in children with a previous history of transient neonatal diabetes currently in remission, repeated after a period of two or more years.
Methods: The standard intravenous glucose tolerance test (IVGTT) was used to measure the first phase insulin response (FPIR) cumulatively at one and three minutes. In addition, fasting insulin and glucose values were used to estimate insulinogenic indices (ß cell function) and QUICKI (insulin sensitivity).
Patients: Six patients with known previous transient neonatal diabetes currently in remission with no exogenous insulin requirement were tested. Control data from 15 children of a similar age were available for derived fasting indices of ß cell functional capacity and insulin sensitivity.
Results: One child had a subnormal insulin secretory response to intravenous glucose that remained abnormal two and four years later. The other children had relatively normal or entirely normal responses over two years. Measures of ß cell function and insulin sensitivity in the fasting state showed comparable results to those obtained from normal controls.
Conclusions: Most children with transient neonatal diabetes in remission have no evidence of ß cell dysfunction or insulin resistance in the fasting state, although they might have been expected to show subtle defects given the tendency to relapse in adolescence. Measures of insulin response to intravenous glucose loading are often normal but suggest future recurrence if profoundly abnormal.
PMCID: PMC1721722  PMID: 15210671
9.  Hypopituitarism following cerebral oedema with diabetic ketoacidosis 
Archives of Disease in Childhood  2002;87(4):337-338.
PMCID: PMC1763040  PMID: 12244015
10.  Effect of genotype on changes in intelligence quotient after dietary relaxation in phenylketonuria and hyperphenylalaninaemia 
Archives of Disease in Childhood  2000;82(3):216-221.
BACKGROUND—Associations between genotype and intellectual outcome in patients with phenylketonuria are complicated because intelligence is influenced by many variables, including environmental factors and other genetic determinants. Intellectual changes with age, both on and after relaxation of diet, vary within the patient population. This study aims to determine whether a significant association exists between genotype and change in intelligence after relaxation of diet.
METHODS—125 patients with hyperphenylalaninaemia and phenylketonuria whose diet was relaxed after 8 years of age. Verbal, performance, and full scale intelligence quotients at 8, 14, and 18 years were expressed as standard deviation scores (IQ-SDS), and genotype as predicted residual enzyme activity (PRA) of phenylalanine hydroxylase.
RESULTS—IQ-SDS at 8, 14, and 18 years were significantly below normal; no association was found between PRA and IQ-SDS. Significant reductions in verbal and full scale IQ-SDS occurred between 8and 14 years and 8 and 18 years. There was a significant association between PRA and the reduction in verbal, performance, and full scale IQ between these years. Multiple regression analysis of 18 year results, using 8 year results as covariates, supported the association between PRA and IQ-SDS; after adjustment for phenylalanine control, both up to and after the age of 8 years, the full scale IQ-SDS at 14 and 18 years was 0.15 higher for each 10% increase in PRA.
CONCLUSIONS—Genotype might be useful in predicting the likelihood of intellectual change in patients with hyperphenylalaninaemia and phenylketonuria whose diet is relaxed after the age of 8years.

PMCID: PMC1718236  PMID: 10685924
11.  Toward Testing the Hypothesis that Group B Coxsackieviruses (CVB) Trigger Insulin-Dependent Diabetes: Inoculating Nonobese Diabetic Mice with CVB Markedly Lowers Diabetes Incidence 
Journal of Virology  2002;76(23):12097-12111.
Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.
PMCID: PMC136885  PMID: 12414951
12.  Confirming congenital hypothyroidism identified from neonatal screening. 
The Ulster Medical Journal  2002;71(1):38-41.
All patients identified in the neonatal screening programme for congenital hypothyroidism in Northern Ireland between 1983 and 1993 were reviewed. 131 infants were recalled because of TSH elevation of whom 85 proved to have true permanent congenital hypothyroidism, while 44 had transient TSH elevation and 2 cases died before the diagnosis could be confirmed. TSH elevation at presentation was milder in the transient group and these infants were more likely to be unwell and/or suffering from congenital malformation.
PMCID: PMC2475352  PMID: 12137163
14.  Combined Cisplatinum and Laser Thermal Therapy for Palliation of Recurrent Head and Neck Tumors 
In recent years endoscopically controlled laser-induced thermal therapy (LITT) has been increasingly accepted as a minimally invasive method for palliation of advanced or recurrent head and neck or gastrointestinal cancer. Previous studies have shown that adjuvant chemotherapy can potentiate endoscopic laser thermal ablation of obstructing tumors leading to improved palliation in advanced cancer patients. Eight patients with recurrent head and neck tumors volunteered to enroll as part of an ongoing phase II LITT clinical trial, and also elected to be treated with systemic chemotherapy (cisplatin, 80 mg/m2) followed 24 h later by palliative laser thermal ablation. Laser treatments were repeated in patients with residual disease or recurrence for a total of 27 LITT sessions. Four of the 8 patients treated with laser thermal chemotherapy remained alive after a median follow-up of 12 months. Of the 12 tumor sites treated, complete responses were located in the oral cavity (3), oropharynx (1), hypopharynx (1), maxillary sinus (1), and median survival for these patients was 9.5 months. This initial experience with cisplatinum-based laser chemotherapy indicates both safety and therapeutic potential for palliation of advanced head and neck cancer but this must be confirmed by longer follow-up in a larger cohort of patients.
PMCID: PMC2362744  PMID: 18493516
15.  Expression of the Heparan Sulfate Proteoglycan, Perlecan, during Mouse Embryogenesis and Perlecan Chondrogenic Activity In Vitro  
The Journal of Cell Biology  1999;145(5):1103-1115.
Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.
PMCID: PMC2133131  PMID: 10352025
heparan sulfate proteoglycan; perlecan; chondrogenesis
16.  Necropsy findings in lysinuric protein intolerance. 
Journal of Clinical Pathology  1996;49(4):345-347.
Lysinuric protein intolerance (LPI) is a rare autosomal recessive inborn error of metabolism, characterised by defective transport of the cationic amino acids lysine, arginine and ornithine. To date there are few reported necropsy cases. This report describes the necropsy findings in a 21 year old female patient originally diagnosed as having LPI in 1973. Liver function tests deteriorated and immediately before death jaundice, hyperammonaemia, coma, metabolic acidosis, and a severe bleeding diathesis developed. At necropsy, there was micronodular cirrhosis of the liver with extensive fatty change in hepatocytes. The lungs showed pulmonary alveolar proteinosis. Immunofluorescence and electron microscopy revealed the presence of a glomerulonephritis with predominant IgA deposition. These necropsy findings reflect the spectrum of lesions reported in LPI, providing further evidence of an association between this condition and pulmonary alveolar proteinosis, cirrhosis and glomerulonephritis.
PMCID: PMC500465  PMID: 8655715
18.  Genetic bias in immune responses to a cassette shared by different microorganisms in patients with rheumatoid arthritis. 
Journal of Clinical Investigation  1997;100(3):658-663.
Rheumatoid arthritis (RA) is an autoimmune disease associated with HLA-DRbeta1 alleles which contain the QKRAA amino acid sequence in their third hypervariable region(s). The QKRAA sequence is also expressed by several human pathogens. We have shown previously that an Escherichia coli peptide encompassing QKRAA is a target of immune responses in RA patients. Here we address two questions: first, whether QKRAA may function as an "immunological cassette" with similar, RA-associated, immunogenic properties when expressed by other common human pathogens; and second, what is the influence of genetic background in the generation of these responses. We find that early RA patients have enhanced humoral and cellular immune responses to Epstein-Barr virus and Brucella ovis and Lactobacillus lactis antigens which contain the QKRAA sequence. These results suggest that the QKRAA sequence is an antigenic epitope on several different microbial proteins, and that RA patients recognize the immunological cassette on different backgrounds. ANOVA of immune responses to "shared epitope" antigens in monozygotic twin couples shows that, despite significantly elevated responses in affected individuals, a similarity between pairs is retained, thus suggesting a role played either by hereditary or shared environmental factors in the genesis or maintenance of these responses.
PMCID: PMC508234  PMID: 9239413
20.  The human immunoglobulin V(H) gene repertoire is genetically controlled and unaltered by chronic autoimmune stimulation. 
Journal of Clinical Investigation  1996;98(12):2794-2800.
The factors controlling immunoglobulin (Ig) gene repertoire formation are poorly understood. Studies on monozygotic twins have helped discern the contributions of genetic versus environmental factors on expressed traits. In the present experiments, we applied a novel anchored PCR-ELISA system to compare the heavy chain V gene (V(H)) subgroup repertoires of mu and gamma expressing B lymphocytes from ten pairs of adult monozygotic twins, including eight pairs who are concordant or discordant for rheumatoid arthritis. The results disclosed that the relative expression of each Ig V(H) gene subgroup is not precisely proportional to its relative genomic size. The monozygotic twins had more similar IgM V(H) gene repertoires than did unrelated subjects. Moreover, monozygotic twins who are discordant for RA also use highly similar IgM V(H) gene-subgroup repertoires. Finally, the V(H) gene repertoire remained stable over time. Collectively, these data reveal that genetic factors predominantly control V(H) gene repertoire formation.
PMCID: PMC507745  PMID: 8981926
21.  Genetic control of T cell receptor BJ gene expression in peripheral lymphocytes of normal and rheumatoid arthritis monozygotic twins. 
Journal of Clinical Investigation  1996;98(7):1594-1601.
The amino acids encoded at the junctions of T cell receptor (TCR) V and J genes directly interact with MHC bound peptides. However, the regulation of the human TCRBJ gene repertoire has been difficult to analyze, because of the potentially complex number of BJ gene rearrangements. To overcome this problem, we developed a PCR-ELISA method to study BJ gene expression, and compared peripheral T lymphocytes from 12 pairs of monozygotic twins, including 6 rheumatoid arthritis (RA) discordant pairs, and 5 normals. Analyses of the TCRBV5, 13 and 17 gene families, which have been reported to be increased in RA patients, showed: (a) the three TCRBV transcripts have common features of BJ gene usage; (b) TCR transcripts from each TCRBV family display a distinctive BJ gene profile, which is displayed better by CD4+ than CD8+ lymphocytes; (c) the BJ gene repertoires of monozygotic twins are more similar than those of unrelated individuals; and (d) the inflammation of RA does not induce specific changes in the genetically determined pattern of BJ expression. These results indicate that the frequency of expression particular TCRBV-TCRBJ recombinants in human lymphocytes is controlled genetically, and is maintained despite the presence of a chronic inflammatory disease.
PMCID: PMC507592  PMID: 8833908
22.  Population and family studies of three disease-related polymorphic genes in systemic lupus erythematosus. 
Journal of Clinical Investigation  1995;95(4):1766-1772.
The contribution to systemic lupus erythematosus (SLE) of three lupus-associated polymorphisms (involving the C4A2 complement component, Humhv3005 and the T cell antigen receptor alpha chain gene) are investigated in 81 individuals from 14 multiplex SLE families, 41 unrelated lupus patients, and 88 unrelated healthy controls. The results show a strong association between C4A deletion and SLE in these families. While the current study confirms the previously reported association between hv3005 deletion and sporadic SLE, the study fails to support this association in familial SLE patients. Moreover, no correlation is detected between the occurrence of hv3005 deletion and C4A null alleles in lupus patients, suggesting that the effects of these genetic polymorphisms on predisposition to lupus are independent. The previously reported lupus-associated T cell receptor (TCR) alpha chain polymorphism is not detected in any of the individuals studied here. The combined data suggest that C4A null alleles predispose strongly to development of lupus, whereas the influence of hv3005 deletion is relatively weak. The results also suggest that contributions of weak susceptibility genes such as hv3005 to disease predisposition may be obscured by the effects of stronger genetic factors and thus need to be examined in patients lacking these factors.
PMCID: PMC295700  PMID: 7706484
24.  Defining the genetic origins of three rheumatoid synovium-derived IgG rheumatoid factors. 
Journal of Clinical Investigation  1994;93(6):2545-2553.
A major diagnostic marker in most rheumatoid arthritis (RA) patients is the rheumatoid factor (RF), an autoantibody that binds to the Fc region of IgG. To delineate the Ig genes and the underlying mechanism for RF production in RA patients, we applied a systematic approach to define the genetic origins of three IgG RFs derived from the synovial fluid of two RA patients. The results show that two of three IgG RF have substantial numbers of somatic mutations in their variable (V) regions, ranging from 13 to 23 mutations over a stretch of 291-313 nucleotides, resulting in a frequency of 4.4-7.8%. However, one IgG RF has only one mutation in each V region. This result indicates that an IgG RF may arise from a germline gene by very few mutations. The mutations occur mainly in the complementarity-determining regions (CDRs), and the mutations in the CDRs often lead to amino acid substitutions. Five of the six corresponding germline V genes have been found to encode either natural autoantibodies or autoantibodies in other autoimmune disorders; and three of the six V genes have been found in fetal liver. Taken together with other results, the data show that (a) several potentially pathogenic RFs in RA patients arise from natural autoantibodies, and (b) only a few mutations are required to convert the natural autoantibodies to IgG RFs.
PMCID: PMC294479  PMID: 8200991
25.  Regulation of the mature human T cell receptor gamma repertoire by biased V-J gene rearrangement. 
Journal of Clinical Investigation  1993;91(1):171-178.
To delineate how gene rearrangement influences the expressed human gamma delta T cell repertoire, we generated T cell receptor gamma (TCR gamma) V domain-specific cDNA libraries from the peripheral lymphocytes of eight donors and sequenced a total of 232 TCR gamma gene transcripts. The libraries consisted of both in-frame and out-of-frame rearranged TCR gamma genes. The in-frame TCR gamma gene transcripts were used to determine the diversity of functional T cells, whereas the out-of-frame transcripts, primarily derived from alpha beta T cells, were used to assess the frequencies of TCR V gamma-J gamma rearrangements in progenitor T lymphocytes. The results showed that both sets of transcripts exhibited strikingly restricted V gamma-J gamma combinations. Only 11 of 40 potential V gamma-J gamma rearrangements were common ( > or = 3% of total). The pattern of gene usage in the functional and nonfunctional transcripts was similar and did not differ markedly among donors. The only exception was the predominance of V gamma 9-JP in potentially functional transcripts from seven of eight individuals. These results show that V gamma-J gamma rearrangement is nonrandom and suggest that the diversity of TCR gamma genes in the functional gamma delta T cell repertoire partly depends upon preferentially rearranged V gamma-J gamma gene combinations. However, the expansion of V gamma 9/V gamma 2 T cells in adult peripheral blood can only be explained by antigenic selection of relatively rare V gamma 9-JP recombinants.
PMCID: PMC330011  PMID: 7678601

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