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Biomedical subjects

M D Gorrell

Publications and source records attributed to M D Gorrell.

At least 19 recordsLinked to original sources
PubMed2002

Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection.

BACKGROUND: Activated hepatic stellate cells (HSCs), recognised by their alpha smooth muscle actin immunoreactivity, are primarily responsible for liver fibrosis. However, the presence of alpha smooth muscle actin positive HSCs is not always associated with the development of liver fibrosis. Recently, other markers of human HSCs including the gelatinase fibroblast activation protein (FAP) and glial fibrillary acidic protein have been identified. AIMS: We examined the relationship between the expression of these HSC markers and the severity of liver injury in patients with chronic hepatitis C virus infection. METHODS: Liver tissue from 27 patients was examined using immunohistochemistry. Linear correlation analysis was used to compare staining scores with the stage and grade of liver injury. RESULTS-CONCLUSIONS: FAP expression, seen at the tissue-remodelling interface, was strongly and significantly correlated with the severity of liver fibrosis. A weaker correlation was seen between glial fibrillary acidic protein expression and fibrosis stage. This contrasted with the absence of a relationship between alpha smooth muscle actin and the fibrotic score. A correlation was also observed between FAP expression and necroinflammatory score. In summary, FAP expression identifies a HSC subpopulation at the tissue-remodelling interface that is related to the severity of liver fibrosis.

Actins↗
PubMed2001

Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac.

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats.

Animals↗
PubMed2001

CD26: a multifunctional integral membrane and secreted protein of activated lymphocytes.

CD26 has proved interesting in the fields of immunology, endocrinology, cancer biology and nutrition owing to its ubiquitous and unusual enzyme activity. This dipeptidyl aminopeptidase (DPP IV) activity generally inactivates but sometimes alters or enhances the biological activities of its peptide substrates, which include several chemokines. CD26 costimulates both the CD3 and the CD2 dependent T-cell activation and tyrosine phosphorylation of TCR/CD3 signal transduction pathway proteins. CD26 in vivo has integral membrane protein and soluble forms. Soluble CD26 is at significant levels in serum, these levels alter in many diseases and soluble CD26 can modulate in vitro T-cell proliferation. CD26, being an adenosine deaminase binding protein (ADAbp), functions as a receptor for ADA on lymphocytes. The focus of this review is the structure and function of CD26 and the influence of its ligand binding activity on T-cell proliferation and the T cell costimulatory activity of CD26.

Adenosine Deaminase↗
PubMed2001

Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.

BACKGROUND: Primary biliary cirrhosis (PBC) is an autoimmune disease in which the pathogenesis of progressive liver injury is poorly understood. AIM: To provide novel insights into the pathogenesis of PBC related liver injury using cDNA array analysis, which simultaneously examines expression of many genes. METHODS: Utilising cDNA arrays of 874 genes, PBC was compared with primary sclerosing cholangitis (PSC) associated cirrhosis and non-diseased liver. Differential expression of 10 genes was confirmed by real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signalling, apoptosis, and oxidative stress. PBC was associated with increased expression of both Th1 and Th2 type molecules of the immune response. Fibrosis related gene expression featured upregulation of connective tissue growth factor and transforming growth factor beta3. Many more apoptosis associated molecules exhibited increased expression, consistent with apoptosis being a more active and regulated process, in PSC associated cirrhosis than in PBC. Increased expression of many genes of the Wnt and notch pathways implicated these highly conserved and linked pathways in PBC pathogenesis. The observed increases in expression of c-jun, c-myc, and c-fos related antigen 1 are consistent with increased Wnt pathway activity in PBC. Differential expression of four components of the Wnt pathway, Wnt-5a, Wnt-13, FRITZ, and beta-catenin, was confirmed by quantitative RT-PCR. CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis, and regeneration were upregulated in PBC cirrhosis. In particular, increased expression of a number of Drosophila homologues was seen in PBC.

Apoptosis↗
PubMed2000

Post-proline-cleaving peptidases having DP IV like enzyme activity. Post-proline peptidases.

DP IV has been studied extensively in disease and in the immune system by the use of enzyme assays which detect hydrolysis of Gly-Pro or Ala-Pro substrate. In addition many studies have used inhibitors of DP IV enzyme activity. The characterisation of a novel DP IV like protein, DPP4R, and of other proteases which have a substrate specificity similar to DP IV or that bind DP IV inhibitors suggests that these studies require further evaluation.

Animals↗
PubMed2000

Relating structure to function in the beta-propeller domain of dipeptidyl peptidase IV. Point mutations that influence adenosine deaminase binding, antibody binding and enzyme activity.

Point mutations in human CD26/DP IV were analysed for adenosine deaminase (ADA) binding, monoclonal antibody (mAb) binding and DP IV enzyme activity. Point mutations at either Leu294 or Val341 ablated ADA binding. Binding by mAbs that inhibit ADA binding was found to involve both Leu340 to Arg343 and Thr440/Lys441. Glu205 and Glu206 were found to be essential for enzyme activity. All residues of interest were mapped onto a model of the beta-propeller domain of DP IV. These data led us to suggest that in DP IV and related peptidases ligand and antibody binding sites are non-linear and that enzyme activity depends on charged sidechains that surround the entrance to the central tunnel of the beta-propeller.

Amino Acid Substitution↗
PubMed2000

Molecular pathogenesis of liver disease: an approach to hepatic inflammation, cirrhosis and liver transplant tolerance.

The hallmarks of chronic liver diseases are chronic inflammation, cellular damage, regeneration and fibrosis. An appreciation of intrahepatic molecular expression patterns in normal and diseased liver provides clues for understanding pathogenic pathways whilst studies of the structure and function of molecules implicated in liver disease provide insights into their potential as therapeutic targets. We have examined the expression, function, molecular structure and structure-function relationships of type IV dipeptidyl aminopeptidases. In particular, the roles of CD26/DPPIV in T-cell proliferation and chemotaxis and of fibroblast activation protein in human cirrhosis are discussed. We have investigated the pathogenesis of liver disease by characterising patterns of cytokine and growth factor expression in experimental and human cirrhosis. We have quite recently expanded this approach to use differential gene expression analyses to elucidate overall pathways of gene activation and suppression in human cirrhosis. In addition, our detailed molecular and cellular studies of the mechanisms of spontaneous liver transplant tolerance have generated novel insights into this process. This review touches on these diverse aspects of liver function and disease.

Adenosine Deaminase↗
PubMed2000

Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8.

Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.

Amino Acid Sequence↗
PubMed1999

Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity.

Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N-terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp-Trp-(Val/Ile/Leu)-Tyr-Glu-Glu-Glu (DW(V/I/L)YEEE) in the predicted beta propeller domain of the DPP IV-like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser-Asp-His catalytic triad.

Amino Acid Sequence↗
PubMed1999

Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis.

Fibroblast activation protein (FAP) is a cell surface-bound protease of the prolyl oligopeptidase gene family expressed at sites of tissue remodelling. This study aimed to delineate the expression of FAP in cirrhotic human liver and examine its biochemical activities. Seventeen cirrhotic and 8 normal liver samples were examined by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Hepatic stellate cells (HSC) were isolated and immunostained. Recombinant FAP and immunopurified, natural FAP were analyzed for protease activities and similarities to dipeptidyl peptidase IV (DPPIV), a structurally related enzyme. FAP-specific messenger RNA and immunoreactivity were detected in cirrhotic, but not normal, livers. FAP immunoreactivity was most intense on perisinusoidal cells of the periseptal regions within regenerative nodules (15 of 15 cases); this pattern coincides with the tissue remodelling interface. In addition, human FAP was expressed by cells within the fibrous septa (10 of 15 cases). Cell morphology, location, and colocalization with glial fibrillary acidic protein (GFAP) indicated that FAP is present on HSC in vivo. Similarly, isolated HSC expressed FAP in vitro. Both natural FAP from cirrhotic liver and recombinant FAP were shown to have gelatinase and dipeptidyl peptidase activities. FAP is a cell-bound, dual-specificity dipeptidyl peptidase and gelatinase expressed by activated HSC at the tissue remodelling interface in human cirrhosis. FAP may contribute to the HSC-induced extracellular matrix (ECM) changes of cirrhosis.

Actins↗
PubMed1999

Binding to human dipeptidyl peptidase IV by adenosine deaminase and antibodies that inhibit ligand binding involves overlapping, discontinuous sites on a predicted beta propeller domain.

Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.

Adenosine Deaminase↗
PubMed1997

Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)-mediated cleavage.

CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV activity but whose natural substrates and immunological functions have not been clearly defined. Several chemo-kines, including RANTES (regulated on activation, normal T cell expressed and secreted), have now been shown to be substrates for recombinant soluble human CD26. The truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3-68) showed a reduced activity, relative to that of RANTES(1-68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. Our results indicate that CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

Calcium↗
PubMed1997

Langerhans cells in the development of skin cancer: a qualitative and quantitative comparison of cell markers in normal, acanthotic and neoplastic ovine skin.

The distribution of Langerhans cells in normal, acanthotic and neoplastic ovine epithelium was examined using the enzyme marker Acetylcholinesterase (AchE) and monoclonal antibodies (MoAb) to CD1 (20.27) and MHC Class II (49.1 and 28.1) molecules. In normal skin, where Langerhans cells were regularly spaced within the basal layer, qualitative observations and direct pairwise testing showed that AChE was superior to the MoAb in detecting these cells. Significantly more (P < 0.01) dendritic cells were also detected with MoAb 49.1 than MoAb 20.27 or 28.1, suggesting differential expression of MHC Class II subsets and the presence of CD1- MHC Class II+ granule- dendritic cells in sheep analogous to indeterminate cells of man. In acanthotic skin, compared to normal skin, Langerhans cells were less numerous, irregular and more suprabasal in distribution and their morphology was occasionally swollen and indistinct. No difference was seen in the ability of AChE and MoAb in detecting Langerhans cells, however pairwise testing of markers did demonstrate that significantly more (P < 0.05) cells without dendritic processes were stained with MoAb 49.1 than with 20.27 or 28.1. In all squamous cell carcinomas examined dendritic cells that stained for AChE, CD1 or MHC Class II antigens were concentrated at the peripheral areas of neoplastic epithelium. Many dendritic cells were detected with MoAb to MHC Class II antigens, whereas CD1 and AChE positive dendritic cells were rare in tumor bearing tissue. The quantitative differences in the immunohistochemical staining of Langerhans cells between normal, acanthotic and neoplastic epithelium were consistent with ultrastructural studies. When compared with those of a newborn lamb, which had had very little exposure to antigens or ultraviolet radiation (UVR), the Langerhans cells of the aged sheep were deformed and contained far fewer Birbeck granules. The abnormalities were progressively more severe in acanthotic and neoplastic skin. These observed changes may have resulted from UVR induced damage and may be indicative of impaired function involved in the development of skin cancer.

Acantholysis↗
PubMed1997

Immunization with nonstructural proteins promotes functional recovery of alphavirus-infected neurons.

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.

Animals↗
PubMed1995

The distribution of lymphocyte subpopulations in normal and acanthotic ovine skin.

The prevalence and distribution of lymphocyte subpopulations in normal and acanthotic ovine skin were investigated using monoclonal antibody immunocytochemistry. CD8+ cells were predominant in the epidermis of both normal and acanthotic skin, but were CD8+ cells, CD4+ cells and T19+ cells infrequent in normal epidermis. Within the dermis of normal skin, there were significantly greater numbers of CD4+ and T19+ cells situated around the superficial dermal vessels than in any other region examined. The majority of the CD8+ cells adjoined vessels, but the proportion that did not was greater for CD8+ than for CD4+ or T19+ cells. The CD4+ and CD8+ subsets were represented equally in adnexa. T cells were of memory phenotype. B cells and naive T cells, both of which express the CD45RA antigen, were rarely seen and tended to be associated with vessels in both normal and acanthotic skin. None of the T19+ cells (which are gamma delta+) resembled the dendritic gamma delta cells seen in murine epidermis. Acanthotic skin was strikingly different to normal skin. There was a greater abundance of T cells, particularly CD4+ cells, in acanthotic epidermis and the numbers of CD8+ and T19+ cells, and to a greater extent CD4+ cells, were greater at the dermal-epidermal junction. There were more CD4+ and CD8+ cells in the superficial dermal stroma of acanthotic skin. Within the dermis of acanthotic skin, T cells were concentrated near vessels but the apportioning of T cells between stromal/adnexal and vessel-associated sites differed from normal. Such observations suggest that migration away from perivascular sites and into the stroma may be controlled separately for subregions of skin and for each T cell subset. The role of this altered nonrandom migration of T cells in skin chronically exposed to ultra violet radiation is uncertain.

Acantholysis↗
PubMed1995

Major histocompatibility complex antigens in normal, acanthotic and neoplastic ovine skin: an association been tumor invasiveness and low level MHC class I expression.

The distribution and density of ovine MHC class I and class II antigens in normal, acanthotic and malignantly transformed ovine skin was investigated using monoclonal antibodies and an immunoperoxidase technique. The subjects were sheep that had been exposed to high levels of sunlight for more than 6 years. The expression of MHC class II antigens in the plasma membrane of cells within the normal epidermis was restricted to basally located dendritic and mononuclear cells. Normal keratinocytes did not express MHC class II antigens. However, we observed low levels of intracellular MHC class II expression in both acanthotic and neoplastic keratinocytes. Expression of MHC class I antigens was variable in normal and acanthotic epithelium; it was usually present, but of low intensity in very early ovine squamous cell carcinoma and was increased in small, but morphologically typical, tumors. Tumors originating on the nose, which are more invasive than those on the ear, were found to express significantly less MHC class I (P < 0.05). Thus, an association between tumor invasiveness and low level expression of MHC class I was apparent. This may have diagnostic value and highlights a mechanism by which neoplastic cells may evade immune surveillance by T cells.

Acanthosis Nigricans↗