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D L Simmons

Publications and source records attributed to D L Simmons.

At least 55 records · Page 3Linked to original sources
PubMed1997

A distinct profile of six soluble adhesion molecules (ICAM-1, ICAM-3, VCAM-1, E-selectin, L-selectin and P-selectin) in rheumatoid arthritis.

Soluble forms of ICAM-1, VCAM-1, E-selectin, L-selectin, P-selectin and, more recently, ICAM-3 are known to exist in human serum and have elevated levels in numerous diseases. Previous studies have demonstrated that in rheumatoid arthritis (RA) the levels of circulating sICAM-1 and sE-selectin are elevated relative to healthy controls. We have compared the serum profiles of these six soluble adhesion molecules in patients with RA (n = 22) to those seen in healthy controls (n = 10) using sandwich ELISA. In the patients, there were significant elevations of serum sICAM-1 (P < 0.0001), sICAM-3 (P = 0.0327), sVCAM-1 (P = 0.0025), sL-selectin (P = 0.0194) and sP-selectin (P = 0.0025), but not E-selectin (P = 0.0672). However, only sP-selectin was found to correlate with disease activity in the patients (r = 0.461, P < 0.05). Thus, there is a distinct profile of soluble adhesion molecules in RA of which only sP-selectin correlates with disease activity.

Adult↗
PubMed1997

Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin alpha(v)beta3 in vitro.

The integrin alpha(v)beta3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified alpha(v)beta3 in the absence of heparan sulfate and other cell surface components. In this assay, FMDV (O1BFS) successfully replicated authentic ligand binding by cellular alpha(v)beta3 in terms of its high affinity, dependence on divalent cations, and activation by manganese ions. Virus binding to this preparation of alpha(v)beta3 was exquisitely sensitive to competition by short RGD-containing peptides (50% inhibition at < 10(-8) M peptide), and this inhibition was highly sequence specific, with the equivalent RGE peptide being at least 10(4) fold less effective as a competitor. Representative viruses of the other six serotypes of FMDV bound to alpha(v)beta3 in a similar RGD-specific manner, although significant differences in sensitivity to RGD peptides suggest that the affinity of the different FMDV serotypes for alpha(v)beta3 is influenced, in part, by the variable amino acid residues in the VP1 G-H loop on either side of the RGD.

Aphthovirus↗
PubMed1996

Phorbol ester augments butyrate-induced apoptosis of colon cancer cells.

Butyrate is a potentially selective therapeutic agent for many adenocarcinomas. Butyrate causes reversible growth arrest as well as some death of VACO 5 colon cancer cells. Combined treatment with butyrate and the phorbol ester TPA leads instead only to cell death, while TPA causes little death on its own. Cells dying during treatment with TPA and butyrate, as well as those dying in the presence of butyrate alone, exhibit features typical of apoptosis, including detachment, shrinkage and internucleosomal DNA cleavage. Pre-treating VACO 5 cell cultures with TPA for as little as 6 hr prior to butyrate addition led to a markedly diminished enhancement of butyrate-induced apoptosis. Treatment with a distinct PKC activator, bryostatin 1, was ineffective in enhancing butyrate-induced death and, furthermore, counteracted the death-enhancing actions of TPA. Such antagonism was apparent when bryostatin was added after 12 hr of TPA/butyrate treatment but was much less effective thereafter. The duration of TPA/butyrate treatment required for depressing cell survival by >95% was thereby estimated to be 24 hr. Other colon cancer cell lines were examined for the extent of cell death following treatment with TPA/butyrate. In each of these lines, butyrate inhibited cell replication in a reversible manner, similar to that seen in VACO 5. However, the combination of butyrate and TPA led to high levels of cell death in only a subset of these lines. TPA/butyrate-treated cultures of COLO 201 exhibited extensive apoptosis, similar in timing and magnitude to the response by VACO 5, whereas HCT 116 was reversibly growth-arrested. Our findings indicate that the PKC system plays a critical role in maintaining cell survival during butyrate-induced growth arrest.

Adenocarcinoma↗
PubMed1996

The deleted in colon cancer (DCC) gene is consistently expressed in colorectal cancers and metastases.

The DCC (deleted in colorectal cancer) gene was originally identified as a candidate tumour suppressor gene in colon carcinogenesis on the basis of allelic losses in chromosome 18q.21 in 70% of colon cancers. Reverse transcriptase polymerase chain reaction (RT-PCR) of DCC mRNA suggests that DCC expression may also be reduced in colon cancers. We have used monoclonal antibodies generated against the DCC immunoglobulin-like domain to investigate DCC isoforms and DCC protein expression during colon cancer progression. Normal mucosa and colonic tumour specimens representative of the range of colonic tumour progression from benign adenomatous polyps to metastases were compared by Western blot analyses. We show that while M(r) 194 000 DCC is present in normal colonic mucosa and adenomatous polyps, it is also similarly expressed in colorectal carcinomas and colonic metastases in the liver. The presence of DCC protein is consistent with the presence of DCC mRNA transcripts in the same tissue specimens. Notably DCC was not completely lost in any colonic tumour specimens examined, even those that had progressed to metastatic cancers. Quantitation of DCC protein expression in tissue specimens by densitometry demonstrated that both normal and malignant specimens exhibit a wide range of DCC protein levels and there was no significant correlation between diminished DCC protein expression and colon cancer progression. These results demonstrate the pattern of expression of the DCC gene product in colonic tumour progression and show that absence of DCC expression is not associated with colonic tumour progression.

Base Sequence↗
PubMed1996

Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.

Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.

Animals↗
PubMed1996

In vivo and in vitro expression of a non-mammalian cyclooxygenase-1.

Unlike cyclooxygenase 2 (COX-2), COX-1 has never been identified, purified or cloned in a non-mammalian species. Here we report the RT-PCR cloning of a chicken cDNA that encodes the amphipathic membrane binding region and parts of the dimerization and catalytic domains of COX-1-like enzyme. Sequence comparison showed this putative COX-1 to be evolutionarily less conserved than COX-2. Furthermore, whereas COX-1 in mammals is broadly expressed in tissues as a constitutive enzyme, the mRNA detected by our clone in chicken was almost absent in tissues and embryo fibroblasts (CEF). Highest expression was in brain and seminal vesicle. This transcript was not detectable during chick embryogenesis and, as is the case for mammalian COX-1, was not induced above background by mitogen stimulation. The identification of an avian COX-1 shows that COX-1 and COX-2 existed as separate catalysts for prostaglandin synthesis before the divergence of birds and mammals.

Amino Acid Sequence↗
PubMed1996

Alternatively spliced variants of the cell adhesion molecule CD44 and tumour progression in colorectal cancer.

Increased expression of alternatively spliced variants of the CD44 family of cell adhesion molecules has been associated with tumour metastasis. In the present study, expression of alternatively spliced variants of CD44 and their cellular distribution have been investigated in human colonic tumours and in the corresponding normal mucosa, in addition to benign adenomatous polyps. The expression of CD44 alternatively spliced variants has been correlated with tumour progression according to Dukes' histological stage. CD44 variant expression was determined by immunohistochemisty using monoclonal antibodies directed against specific CD44 variant domains together with RT-PCR analysis of CD44 variant mRNA expression in the same tissue specimens. We demonstrate that as well as being expressed in colonic tumour cells, the full range of CD44 variants, CD44v2-v10, are widely expressed in normal colonic crypt epithelium, predominantly in the crypt base. CD44v6, the epitope which is most commonly associated with tumour progression and metastasis, was not only expressed by many benign colonic tumours, but was expressed as frequently in normal basal crypt epithelium as in malignant colonic tumour cells, and surprisingly, was even absent from some metastatic colorectal tumours. Expression of none of the CD44 variant epitopes was found to be positively correlated with tumour progression or with colorectal tumour metastasis to the liver, results which are inconsistent with a role for CD44 variants as indicators of colonic cancer progression.

Alternative Splicing↗
PubMed1996

Regulation of hen granulosa cell prostaglandin production by transforming growth factors during follicular development: involvement of cyclooxygenase II.

PGs are important physiological regulators of granulosa cell proliferation during ovarian follicular development. The rate-limiting step in the synthesis of PGs from arachidonic acid is catalyzed by cyclooxygenase (COX). Although two isoforms of COX (COX I and COX II) have been identified in the ovary, the nature and physiological significance of their regulation by transforming growth factor (TGF) alpha and TGF beta are unclear. The object of the present studies was to investigate the regulation of hen granulosa cell PG production by TGF alpha and TGF beta and the role of COX II in this process. Granulosa cells from the first (F1) and fifth and sixth (F5-F6) largest hen preovulatory follicles were cultured for up to 21 h in the presence of TGF alpha (0-10 ng/ml) and/or TGF beta (0-20 ng/ml). COX protein and messenger RNA (mRNA) levels were determined by Western blot and Northern analysis, respectively. Granulosa cell PGF and PGE secretion was increased by TGF alpha but suppressed by TGF beta in vitro. The increase in PG secretion was accompanied by an elevation of COX II content, which was dose and time dependent, with maximum response observed within 6-12 h. Maximal increase in COX II mRNA abundance was evident at 4 and 8 h in cells from F1 and F5-F6, respectively. Although TGF alpha-stimulated PG secretion was higher in cells from mature follicle (F1), the magnitude of change in COX II mRNA abundance and protein content was, however, not dependent on follicular maturation. TGF beta significantly suppressed basal and TGF alpha-induced COX II transcript levels. COX I transcript, however, was undetectable irrespective of the presence of TGF alpha, duration of culture or the stage of follicular development. In conclusion, the mitogenic response of granulosa cells to TGF alpha is mediated by changes in PG secretion, which are regulated at the level of COX II. Unlike the control of PG secretion, the regulation of COX II by TGF alpha is independent of follicular maturation. Whereas COX II may be important in the signaling cascade for TGF alpha in the regulation of granulosa cell proliferation, the mechanism(s) of regulation of follicular stage-dependent PG secretion by the growth factor is complex and requires further investigation.

Animals↗
PubMed1996

Identification of alpha v beta 3 as a heterotypic ligand for CD31/PECAM-1.

CD31 (PECAM-1) is a member of the immunoglobulin gene superfamily (IgSF) and has an important role in a number of endothelial cell functions including angiogenesis, inflammation, integrin activation and cell-cell adhesion. CD31 has both homotypic and heterotypic adhesive properties and in common with other IgSF members contains multiple functional domains. Using chimaeric fusion proteins of CD31 and a panel of haematopoietic cell lines we show that CD31 can bind cells in a predominantly homotypic or heterotypic manner depending on the cell line used. Heterotypic binding was found to be cation and temperature dependent and enhanced by Mn2+: all features of integrin mediated binding. Using a panel of anti-CD31 and anti-integrin antibodies we show that alpha v beta 3 is a ligand for CD31 on the monocytic cell line U937. The specificity of the interaction between alpha v beta 3 and CD31 was further confirmed by solid phase binding assays and the use of alpha v beta 3 transfected cells which bound CD31 specifically. Furthermore, we have mapped the binding site for alpha v beta 3 to domains 1 and 2 of CD31. The interaction of CD31 with alpha v beta 3 may be important in many aspects of endothelial function including leukocyte-endothelial transmigration and angiogenesis.

Amino Acid Sequence↗
PubMed1995

Nonsteroidal antiinflammatory drugs cause apoptosis and induce cyclooxygenases in chicken embryo fibroblasts.

Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.

Animals↗
PubMed1995

Increased expression of CD44v6 and CD44v3 in ulcerative colitis but not colonic Crohn's disease.

Immune mechanisms, possibly involving cell-surface molecules such as CD44, have been invoked to explain the pathogenesis of inflammatory bowel disease. We used monoclonal antibodies against epitopes encoded within the variable region of CD44 to investigate CD44 isoform expression in colon, small intestine, and liver in patients with various intestinal disorders and in controls. Biopsy samples from patients with ulcerative colitis showed significantly increased epithelial expression of CD44 isoforms containing the v6 and v3 epitopes, detected with antibodies 2F10 and 3G5, respectively. CD44v6 was detected on colonic crypt epithelial cells in 23 of 25 ulcerative colitis samples compared with 3 of 18 colonic Crohn's disease samples (p = 3.0 x 10(-6); odds ratio 57.5 [95% CI 6.83-702]) and 3 of 52 controls (22 normal colon, 10 infective colitis, 2 radiation colitis, and 18 colonic Crohn's disease; p < 1 x 10(-8); odds ratio 199 [25.5-2294]). No significant expression of CD44v6, CD44v3, or CD44v8/9 was found in samples of normal proximal colon from 4 patients with distal ulcerative colitis, whereas samples from the affected area showed staining for CD44v6 and CD44v3. No expression of CD44 variants was found in 15 samples of normal small intestine, 11 small-bowel pouchitis, 8 coeliac disease, 3 small-bowel Crohn's disease, 6 normal liver, 6 primary biliary cirrhosis, or 9 primary sclerosing cholangitis. The high intensity of CD44v6 and v3 epitope expression on crypt epithelial cells in ulcerative colitis suggests that CD44 isoforms may have an important role in ulcerative colitis. Their detection could have diagnostic potential in differentiating ulcerative colitis from other forms of colonic inflammation including Crohn's disease.

Antibodies, Monoclonal↗
PubMed1995

Analysis of the binding site on intercellular adhesion molecule 3 for the leukocyte integrin lymphocyte function-associated antigen 1.

Intercellular adhesion molecule 3 (ICAM-3, CD50) is a member of the immunoglobulin superfamily and is a constitutively expressed ligand for the leukocyte integrin LFA-1 (CD11a/CD18). ICAM-3 is expressed at high levels by all resting leukocyte populations and antigen presenting cells and is a major ligand for LFA-1 in the resting immune system. ICAM-3 is a signal transducer and may play a key role in initiating immune responses. Mutant ICAM-3 Fc-chimeric proteins were quantitatively analyzed for their ability to bind COS cells expressing human LFA-1. The LFA-1-binding site on ICAM-3 is located in the N-terminal 2 Ig domains. Domains 3-5 do not significantly contribute to adhesion. The binding site has been further resolved by rational targeting of 14 point mutations throughout domains 1 and 2, coupled with modeling studies. Within domain 1 a cluster of residues (Glu37, Leu66, Ser68, and Gln75), that are predicted to lie on the CC'FG face of the Ig fold, play a dominant role in LFA-1 binding.

Amino Acid Sequence↗
PubMed1995

Epitope mapping and functional properties of anti-intercellular adhesion molecule-3 (CD50) monoclonal antibodies.

Intercellular adhesion molecule-3 (ICAM-3, CD50), a member of the immunoglobulin gene superfamily, is a major ligand for the lymphocyte function-associated antigen 1 (LFA-1, CD18/CD11a) in the resting immune system and plays a role as a signaling and costimulatory molecule on T lymphocytes. In this study we have generated a large panel of anti-ICAM-3 monoclonal antibodies (mAb) and show that the biological effects of these antibodies are critically dependent on the epitope recognized. By using an adhesion assay employing COS cells expressing LFA-1 binding to recombinant chimeric ICAM-3-Fc proteins (which overcomes the confounding effects of interleukocyte LFA-1/ICAM binding events), we have been able to examine the effects of these antibodies in blocking LFA-1/ICAM-3 adhesion. Our data suggests that only a small minority of ICAM-3 mAb, recognizing a distinct epitope, are able to mimic the effects of LFA-1 binding to ICAM-3. Moreover these antibodies are functionally distinct as defined by their costimulatory activity and ability to elicit interleukin-2 production and cell proliferation in T lymphocytes.

Animals↗
PubMed1995

Intercellular adhesion molecule-3 is the predominant co-stimulatory ligand for leukocyte function antigen-1 on human blood dendritic cells.

Dendritic cells (DC) are potent stimulators of primary T lymphocyte responses to foreign antigen. The initial DC-T lymphocyte interaction involves the binding of the adhesion molecule leukocyte function antigen-1 (LFA-1; CD11a/CD18) on the T lymphocyte to an intercellular adhesion molecule (ICAM) on the DC. Although blood and tonsil DC express ICAM-1 (CD54) and ICAM-2 (CD102) on their surface, anti-ICAM-1 and anti-ICAM-2 monoclonal antibodies (mAb) have little inhibitory activity on the DC-stimulated mixed leukocyte reaction (MLR). We therefore examined the expression of the more recently identified LFA-1 ligand, ICAM-3 (CD50), in comparison to ICAM-1 and ICAM-2 on blood DC and sought a functional role for ICAM-3 in DC-mediated T lymphocyte responses. Resting blood DC expressed significantly more ICAM-3 than ICAM-1 or ICAM-2 as assessed by flow cytometry. Treatment of resting DC with interferon-gamma led to increased expression of ICAM-1; however, ICAM-2 and ICAM-3 levels remained relatively constant. Solid-phase recombinant chimeric molecules ICAM-1-, ICAM-2- and ICAM-3-Fc were able to co-stimulate CD4+ T lymphocyte proliferation in conjunction with suboptimal solid-phase CD3 mAb 64.1. However, the anti-ICAM-3 mAb CAL 3.10 inhibited a DC-stimulated MLR to a greater extent than anti-ICAM-1 or anti-ICAM-2 reagents and appeared to act by blocking the DC ICAM-3- T lymphocyte LFA-1 interaction. As ICAM-3 is the predominant LFA-1 ligand on resting blood DC, we postulate that DC may utilize ICAM-3 for initial DC- T lymphocyte interactions, and that ICAM-1, which is up-regulated upon DC activation, and/or ICAM-2, may contribute to DC migration or later phases of the T lymphocyte activation process.

Antigens, CD↗
PubMed1995

Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts.

CD31 is a member of the immunoglobulin superfamily consisting of six Ig-related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti-CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM-1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31-CD31 interactions are critically important only in the initial phases of intercellular adhesion.

Antigens, Differentiation, Myelomonocytic↗
PubMed1995

Bacterial lipopolysaccharide-mediated fetal death. Production of a newly recognized form of inducible cyclooxygenase (COX-2) in murine decidua in response to lipopolysaccharide.

Maternal infection is a cause of spontaneous abortion and preterm labor in humans, but the pathophysiology is unclear. We hypothesized that eicosanoids play an important role in infection-driven pregnancy loss. To investigate this hypothesis, we administered lipopolysaccharide (LPS) to pregnant C3H/HeN mice and found that LPS administration caused fetal death in a dose-dependent fashion. Pretreatment with indomethacin significantly decreased the proportion of fetal death from 83% to < 25% in mice injected with 10 micrograms of LPS. Also, decidual explants from LPS-treated mice produced significantly more inflammatory eicosanoids, including prostaglandins E2 and F2 alpha and thromboxane B2, than controls. We investigated the regulatory mechanisms responsible for increased decidual prostanoid production in response to LPS. Western and Northern blots demonstrated that decidual protein and mRNA levels of a recently recognized highly inducible form of cyclooxygenase, COX-2, were substantially increased in mice treated with LPS. Induction of COX-2 was rapid: mRNA was detected 30 min after LPS injection. In contrast, another form of cyclooxygenase, COX-1, was only minimally induced in response to LPS. Our data indicate that LPS induces decidual prostanoid production via increased COX-2 expression. Since LPS-mediated fetal death is markedly diminished by pretreatment with indomethacin, COX-2-mediated eicosanoid production is likely a key pathophysiologic event in LPS-mediated fetal death.

Abortion, Spontaneous↗