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P Donaldson

Publications and source records attributed to P Donaldson.

At least 19 recordsLinked to original sources
PubMed1999

Connexins in the lens: are they to blame in diabetic cataractogenesis?

The pathohistology of the diabetic lens is an enigma. Under normal conditions the lens behaves as a functional syncitium, whereas the diabetic lens exhibits a localized zone of fibre cell swelling and rupture that is confined to the lens outer cortex. Because the lens fibre cells are extensively coupled by gap junction channels, it is believed that the abnormal closure of these channels is responsible for this phenomenon. New evidence concerning regional differences in gap junction gating supports this contention, and it is used to propose a new hypothesis that may explain the cellular changes observed in the diabetic lens.

Animals↗
PubMed1999

Human leukocyte antigen-DQB1* genotypes encoding aspartate at position 57 are associated with 3beta-hydroxysteroid dehydrogenase autoimmunity in premature ovarian failure.

Premature ovarian failure (POF) has an autoimmune pathogenesis in a significant proportion of cases. Autoantibodies to the steroid cell enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD) are present in one fifth of patients and may identify an autoimmune subgroup. As autoimmune diseases are associated with alleles of the human leukocyte antigen (HLA) genes, we examined the distribution of HLA-DRB1 and -DQB1 genotypes in 118 women with POF, of whom 21% had 3betaHSD autoantibodies, and 134 racially matched control subjects. Two HLA-DQB1 alleles, 0301 and 0603, were associated with 3betaHSD autoantibody positivity (P = 0.04 and P = 0.006, respectively). As the DQB1*0301 and -0603 genes share an identical codon at position 57 (aspartate, Asp), we analyzed the frequency of DQbeta-Asp57 encoding DQB1 genes in our series. Eighteen of 21 POF patients with 3betaHSD autoantibodies had DQbeta-Asp57-encoding genotypes (haplotype frequency, 27 of 42; 64%) compared with 92 of 134 control subjects (haplotype frequency, 109 of 268; 41%; P = 0.004), and 9 of 21 (43%) cases were homozygous for codon 57 genotypes compared with 17 of 134 (13%) control subjects (P = 0.0006). These probability values were not significant after correction for multiple testing, and these trends will therefore require confirmation in larger cohorts. HLA class II molecules present antigenic peptides to CD4+ T lymphocytes. DQbeta57 occupies a key site at the boundary of the peptide binding groove, with a major impact on peptide binding. Our preliminary demonstration of an association between POF, 3betaHSD autoimmunity, and a distinctive HLA-DQ molecule supports the hypothesis that autoantibodies to this steroid cell enzyme may be markers of autoimmune ovarian failure and suggests that presentation of autoantigenic or external peptides to T lymphocytes by HLA-DQ molecules with Asp57-beta-chains is important in the pathogenesis of this disease.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Role of HLA compatibility in pediatric liver transplantation.

BACKGROUND: The influence of HLA mismatching in liver transplantation remains controversial. To date, few studies have focused solely on the pediatric population, and none have investigated DR and DQ mismatches using molecular genotyping. We sought to investigate HLA-A, -B, -DR, and -DQ mismatches in a large series of primary pediatric liver transplant recipients. Living-related liver transplants were excluded. METHODS: A total of 138 consecutive first liver transplants performed between January 1991 and July 1996 were studied. Minimum follow-up was 1 year, and both patient and graft survival rates were assessed. The incidence of the most common complications was analyzed. HLA-A and -B phenotyping was performed by complement-dependent microcytotoxicity or polymerase chain reaction (PCR)-sequence-specific primer protocols in 133 of 138 patients. HLA-DR and -DQ genotyping was performed by standard PCR-sequence-specific oligonucleotide and/or PCR-sequence-specific primer protocols in 135 patients. RESULTS: Overall, there was no influence of HLA mismatching on either graft or patient survival rates. However, patients with two mismatches at the A locus showed a significantly lower incidence of acute rejection than those with one A mismatch (52% vs. 72%; P < 0.03) and patients with two B locus mismatches had a better graft survival rate at 5 years than those with one mismatch (76% vs. 62%), although this was of only borderline significance (P < 0.09). No differences were found in the severity of the episodes of rejection, incidence of chronic rejection, cytomegalovirus hepatitis, and other causes of graft loss. CONCLUSION: This study indicates that HLA-A, -B, -DR, and -DQ mismatches are not detrimental in primary pediatric liver transplantation.

Acute Disease↗
PubMed1998

Defining the outcome of immunosuppression withdrawal after liver transplantation.

Successful immunosuppression withdrawal should benefit the natural history of organ transplantation patients. To identify the clinical hazards of removing drug treatment and possible characteristics that predict a favorable outcome in long-term liver recipients, immunosuppression was withdrawn completely and the clinicopathological outcome documented in 18 liver recipients. Indication for transplantation, HLA matching, early rejection history, and presence of microchimerism were examined as predictors of outcome. Chimerism was determined by polymerase chain reaction-based examination for donor-specific HLA-DRB1 alleles and Y-gene-specific nucleotide sequences. At 3 years, 5 patients (28%) remained completely off immunosuppression; 12 patients (67%) experienced histological graft changes: acute rejection in 4, portal tract inflammation/hepatitis in 7, and necrosis in 1. Hepatitis B or C viral infections did not account for the nonrejection patterns. Unmasking of systemic disorders occurred. Chimerism, demonstrated in 7 patients (39%), with skin the optimal tissue, was not associated with tolerance. Parameters associated with successful drug withdrawal were transplantation for non-immune-mediated liver disorders, fewer donor-recipient HLA A, B, and DR mismatches, and a low incidence of early rejection. Immunosuppression withdrawal is a feasible option in a proportion of selected liver recipients, but identification of tolerant patients remains imprecise.

Adult↗
PubMed1998

Tumor necrosis factor genomic polymorphism and outcome of acetaminophen (paracetamol)-induced acute liver failure.

BACKGROUND/AIMS: High levels of tumor necrosis factor-alpha are associated with an increased risk of severe encephalopathy in acute liver failure, and experimental studies suggest that tumor necrosis factor-alpha plays a role in the development of acetaminophen (paracetamol)-induced liver injury and associated multiple organ failure. Inter-individual variations in the production of tumor necrosis factor-alpha have been linked to genomic polymorphisms within the tumor necrosis factor-alpha locus. This study examined whether specific tumor necrosis factor polymorphisms are associated with variations in the severity of clinical features in acetaminophen-induced acute liver failure. METHODS: Genotypes at the -308 tumor necrosis factor A and tumor necrosis factor B Nco1 polymorphic sites were determined in 97 patients with severe acetaminophen-induced hepatotoxicity and 109 controls, using polymerase chain reaction and restriction fragment length polymorphism. The relationship between liver injury, multiple organ failure and encephalopathy, determined retrospectively from clinical notes and genotype, was examined. RESULTS: No significant association was found between either tumor necrosis factor A or B genotype and parameters for multiple organ failure or liver injury. The tumor necrosis factor B1B1 genotype was significantly under-represented in those patients developing severe encephalopathy (p=0.03) and a multivariate logistic regression analysis confirmed the influence of tumor necrosis factor B genotype (p<0.01). The association was independent of the HLA class II allele DRB1*03, which is closely linked to the TNFB locus. CONCLUSIONS: The development of acute liver failure is unlikely to be primarily sepsis driven. However, the apparent protective effect of the tumor necrosis factor B1B1 genotype on the development of severe encephalopathy may be related to the effects of this genotype on tumor necrosis factor-alpha production in sepsis.

APACHE↗
PubMed1998

Spatial differences in gap junction gating in the lens are a consequence of connexin cleavage.

Gap junctions in the vertebrate lens exhibit spatial differences in pH gating: those in the cortical fibre cells close upon tissue acidification while those in the core region do not. It has been speculated that this difference in channel gating is a consequence of the cleavage of the connexins (Cx) that form the gap junction channels. We report the construction of a truncation mutant of ovine Cx50 which mimicks the cleavage in the intact lens. The construct when expressed in Xenopus oocytes results in the formation of functional channels. Comparison with full-length Cx50 revealed a significant reduction in the pH-sensitivity of the truncated form. This is the first evidence linking the non-uniform gating of gap junction channels in the lens with connexin cleavage. It also reveals how fibre cells in the core region remain connected despite the acidic environment caused by elevated lactate levels.

Animals↗
PubMed1998

A distinct membrane current in rat lens fiber cells isolated under calcium-free conditions.

PURPOSE: To investigate whether lens fiber cells isolated and maintained under calcium-free conditions exhibit distinct membrane currents. METHODS: Fiber cells were isolated from the cortical portion of neonate rat lenses using a trypsin digestion protocol and were maintained in EDTA-buffered Ringer's solutions. Membrane currents were recorded from fiber bundles using the whole-cell patch-clamp technique. RESULTS: Cortical fiber cells of up to 600-microm length were viable and amenable to whole-cell patch-clamp recording. The major current recorded under these conditions was a slowly activating, voltage-dependent current that was markedly increased on membrane depolarization. This current appeared to be fiber cell specific and had similar properties to currents elicited by gap junction hemichannels previously recorded by others in Xenopus oocytes. CONCLUSIONS: This is the first report of whole-cell patch-clamp recordings from intact elongated fiber cells. Fiber cells kept in calcium-free bath medium appear to be electrically "leaky" and exhibit a distinct membrane current that has not been described previously for lens cells. This current is unlikely to be active in the normal lens but may play a role in the depolarized cataractogenic lens.

Animals↗
PubMed1998

Expression profiles of P2-receptor isoforms P2Y1 and P2Y2 in the rat lens.

PURPOSE: To verify at the molecular level that P2 receptors are expressed in the lens and to determine their expression profiles. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) was used to screen rat lens epithelial and fiber cells for the expression of the ionotropic P2X2 receptor and the G-protein-linked receptor isoforms P2Y1 and P2Y2. Northern blot analysis was used to confirm the level of expression of P2Y1 and P2Y2. The profile of P2-receptor isoform expression in the lens was identified using an indirect RT-PCR in situ hybridization procedure on paraffin sections of whole rat eyes. RESULTS: P2X2-receptor transcripts could not be detected in lens epithelial or fiber cells. P2Y1- and P2Y2-receptor transcripts were detected in lens fiber cells but not in the epithelial cells. Their expression profiles were maximal in the lens cortex and mostly overlapped each other, except that transcripts for the P2Y2-receptor isoform appeared earlier in the not yet fully elongated fiber cells in the lens bow region. CONCLUSIONS: The molecular data support physiological evidence previously reported by others that P2Y receptors are expressed in the lens and identifies cortical fiber cells as the principal site of expression.

Animals↗
PubMed1997

Gap junction channels: new roles in disease.

The importance of intercellular communication to complex cellular processes such as development, differentiation, growth, propagation of electrical impulses and diffusional feeding has long been appreciated. The realization that intercellular communication is mediated by gap junction channels, which are in turn comprised of a diverse family of proteins called the connexins, has provided new tools and avenues for studying the role of intercellular communication in these important cellular processes. The identification of different connexin isoforms has not only enabled the development of specific reagents to study connexin expression patterns, but has also allowed the functional properties of the different connexin isoforms and how they interact with each other, to be explored. Increasingly, the knowledge gained from studying connexin diversity is being used to investigate the role played by gap junction channels in a number of diseases. In this article we highlight selected cases where gap junction channels have been shown or are believed to be directly involved in the disease process.

Animals↗
PubMed1997

Processing of the gap junction protein connexin50 in the ocular lens is accomplished by calpain.

Gap junction channel forming connexins share a common membrane topology which has four transmembrane spanning segments with the amino- and carboxy termini both located on the cytoplasmic side. Both, mutation and truncation of the carboxyl tail of some connexins have been shown previously to have profound effects on channel function. Truncation of the carboxyl tail of connexin50 (Cx50) and connexin46 (Cx46) occurs naturally during the maturation of fiber cells in the mammalian lens. This system therefore offers the unique opportunity to study not only the cleavage process but also the functional role played by the cleaved domain, in a physiologically relevant context. As a first step, we now report on the cleavage of the 70 kDa ovine isoform of Cx50. The calcium-activated neutral protease calpain (EC 3.4.22.17) was identified as the enzyme which removed a 32 kDa carboxyl portion from the Cx50 molecule in mature lens fiber cells. The amino-terminal 38 kDa portion remained embedded in the plasma membrane and was isolated and visualized as channel structures. The amino-terminal sequence of the cleaved 32 kDa portion matched an interior portion of the published amino acid sequence of the ovine Cx50 isoform. Thus, two closely spaced calpain cleavage sites were identified in the Cx50 molecule which were located carboxy-terminal from the predicted exit of the fourth transmembrane spanning segment by 62 or 72 amino acid residues, respectively. These data provide the basic information required for the future construction of Cx50 mutants to explore the functional consequences of this cleavage.

Amino Acid Sequence↗
PubMed1996

Repeat HLA-B and -DR loci mismatching at second liver transplantation improves patient survival.

The role of HLA matching in liver transplantation remains uncertain, and the effect of HLA mismatches on the outcome of second transplants has not been studied. In renal transplantation, if HLA mismatches are repeated in the second graft there is a greatly increased risk of immunological graft failure. In the present study, 78 patients who had received second liver transplants were studied. HLA typing was performed using standard complement-dependent microcytotoxicity for class I (A and B) antigens, and a combination of restriction fragment length polymorphism and serology for HLA-DR. Patient survival at follow-up for those with a repeat B locus mismatch was improved (79% compared with 43%, P < 0.02), and a similar effect was noted for repeat DR mismatches (67% vs. 47%, P = 0.06). In the subgroup of patients who received a second transplant for graft rejection, 90% of patients with a repeat B mismatch were alive at follow-up compared with 46% without B mismatches (P = 0.02). This improvement in patient survival was evident during the first 2 months after the second transplant. In this study, repeat HLA-B and -DR mismatching improves survival after second transplantation.

Adolescent↗
PubMed1996

Evidence that the HLA DQA1*03 allele confers protection from chronic HCV-infection in Northern European Caucasoids.

In the search for factors which may influence susceptibility to and outcome from chronic hepatitis C virus (HCV) infection, few studies have considered the influence of host genes. In the present investigation we have performed HLA DRB1, DQA1, DQB1, and DPB1 genotyping on 104 northern European patients with chronic HCV infection and 177 racially and geographically matched controls. Three HLA class II alleles, DRB1*0403, DQA1*03, and DQB1*0302 were present at a significantly lower frequency in patients compared with controls (4.9% vs. 13%, 20.7% vs. 41.2%, and 11.4% vs. 30.5%, respectively) though only two DQB1*0302 and DQA1*03 were significant after correction for multiple testing (pc = 0.038, and pc = 0.046, respectively). No further HLA associations with chronic HCV infection were observed and there was no correlation between stage of disease and HLA genotype. These data provide the first suggestion that susceptibility to chronic HCV infection may be influenced by the hosts' HLA DQ alleles.

Alleles↗
PubMed1996

Liquefaction of cortical tissue in diabetic and galactosemic rat lenses defined by confocal laser scanning microscopy.

PURPOSE: To investigate whether a histologic link exists between osmotic fiber cell swelling and cortical tissue liquefaction in experimentally induced diabetic and galactosemic cataractogenesis of the rat lens. METHODS: Confocal laser scanning microscopy, in conjunction with specific membrane labels and correlative transmission electron microscopy, was used to image large cortical areas with precise definition of the individual cells. RESULTS: In both cataract models, tissue liquefaction--defined as the disintegration of tissue and the appearance of large fluid-filled spaces--typically was limited to a discrete zone in the lens cortex. The borders of the liquefaction zone were characterized by transitions between normal-appearing cells and swollen cells, which gained in size as plasma membranes ruptured and cytoplasmic contents fused and ultimately burst, thereby contributing to the formation of large fluid-filled spaces. During cataractogenesis, before tissue liquefaction became evident, selected fiber cells appeared swollen and accumulated specifically in the zone destined for tissue liquefaction. With increasing duration of diabetes or galactosemia, swollen fiber cells in this zone became more frequent and enlarged, resulting first in tissue disorder and then in tissue disintegration and the formation of large fluid-filled spaces. CONCLUSIONS: New imaging protocols strongly support a direct involvement of lens fiber cell swelling in the liquefaction of cortical tissue. The appearance of swollen fiber cells in the lens cortex, therefore, can be used as an early indicator of the histopathology of sugar cataractogenesis.

Animals↗
PubMed1995

Ocular lens gap junctions: protein expression, assembly, and structure-function analysis.

Recent advances in understanding lens fiber gap junction formation are reviewed. These include studies of junctional protein expression in the embryonic lens, and of age related changes affecting gap junction structure and composition in the adult lens. An in vitro assembly system based on detergent solubilized pore complexes and endogenous lipids has been developed to provide information on the molecular interactions involved in gap junction formation and to provide material for structure analysis. Important information on the electrical properties of lens gap junction channels is obtained using electrophysiological techniques including planar lipid bilayer analysis and patch clamping.

Animals↗
PubMed1994

The molecular genetics of autoimmune liver disease.

The dual observations that human leukocyte antigens have an antigen-binding groove and that the polymorphism we study as human leukocyte antigen types is largely related to amino acid substitutions in and around that groove have provided a new focus for immunogenetic studies. In autoimmune liver disease, recent studies have described specific amino acid substitutions in the antigen-binding groove of human leukocyte antigen DR molecules that may determine both disease susceptibility, through their direct influence on antigen binding, and the severity of the disease. In autoimmune hepatitis, lysine residues at DR beta position 71 in European subjects and arginine or histidine residues at DR beta position 13 in Japanese subjects may be responsible for much human leukocyte antigen-encoded disease susceptibility. Similar claims have been made for leucine residues at DR beta 38 in primary sclerosing cholangitis and for leucine residues at DP beta 35 in Japanese patients with primary biliary cirrhosis. To date, our knowledge of genetic susceptibility to autoimmune liver disease is incomplete. Other genes may contribute to susceptibility to autoimmune liver disease--for example the contribution of TAP genes, upstream promoter sequences and class III genes on chromosome 6 and the T-cell receptor genes and complement genes elsewhere in the human genome is currently unclear. Additional information concerning the immunogenetic contribution to disease severity is needed to complete the picture.

Autoimmune Diseases↗
PubMed1994

Reconstitution of native-type noncrystalline lens fiber gap junctions from isolated hemichannels.

Gap junctions contain numerous channels that are clustered in apposed membrane patches of adjacent cells. These cell-to-cell channels are formed by pairing of two hemichannels or connexons, and are also referred to as connexon pairs. We have investigated various detergents for their ability to separately solubilize hemichannels or connexon pairs from isolated ovine lens fiber membranes. The solubilized preparations were reconstituted with lipids with the aim to reassemble native-type gap junctions and to provide a model system for the characterization of the molecular interactions involved in this process. While small gap junction structures were obtained under a variety of conditions, large native-type gap junctions were assembled using a novel two-step procedure: in the first step, hemichannels that had been solubilized with octylpolyoxyethylene formed connexon pairs by dialysis against n-decyl-beta-D-maltopyranoside. In the second step, connexon pairs were reconstituted with phosphatidylcholines by dialysis against buffer containing Mg2+. This way, double-layered gap junctions with diameter < or = 300 nm were obtained. Up to several hundred channels were packed in a noncrystalline arrangement, giving these reconstituted gap junctions an appearance that was indistinguishable from that of the gap junctions in the lens fiber membranes.

Animals↗