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Biomedical subjects

C H Evans

Publications and source records attributed to C H Evans.

At least 19 recordsLinked to original sources
PubMed1996

Leukoregulin induction of prostaglandin-endoperoxide H synthase-2 in human orbital fibroblasts. An in vitro model for connective tissue inflammation.

Several proinflammatory cytokines can increase prostaglandin E2 (PGE2) synthesis in a variety of cell types, constituting an important component of the inflammatory response. We demonstrate here that leukoregulin, a 50-kDa product of activated T lymphocytes, dramatically increases PGE2 synthesis in cultured human orbital fibroblasts. This up-regulation is mediated through an induction of prostaglandin-endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Steady-state levels of PGHS-2 mRNA are increased within 1.5 h of leukoregulin addition and are near maximal by 6 h, when they are 50-fold or higher above basal levels. The increase in PGHS-2 mRNA levels is partially blocked by cycloheximide, suggesting de novo synthesis of an intermediate protein may be required for a maximal leukoregulin response. Nuclear run-on studies indicate PGHS-2 gene transcription is up-regulated by leukoregulin 2-fold after 2 and 6 h. PGHS-2 protein, as assessed by Western blotting and two-dimensional protein gel analysis, is increased dramatically in orbital fibroblasts. This lymphokine-dependent expression of PGHS-2 is blocked by dexamethasone, and the increase in PGE2 and cAMP levels following leukoregulin treatment is also blocked by indomethacin and by SC 58125, a newly developed PGHS-2-selective cyclooxygenase inhibitor. The dramatic increase in cAMP levels causes marked alteration in orbital fibroblast morphology. PGHS-2 expression in dermal fibroblasts is also increased by leukoregulin; however, the response is considerably less robust, and these cells do not undergo a change in morphology. Both orbital and dermal fibroblasts express high levels of PGHS-1 mRNA and protein, the other abundant form of cyclooxygenase. In contrast to its effects on PGHS-2 expression, leukoregulin fails to alter PGHS-1 levels in either orbital or dermal fibroblasts, suggesting that PGHS-1 is not involved in cytokine-dependent prostanoid production in human fibroblasts. The increased PGHS-2 expression elicited by leukoregulin in orbital fibroblasts may be a consequence of both transcriptional and post-transcriptional effects. These observations help clarify the pathogenic mechanism relevant to the intense inflammation associated with Graves' ophthalmopathy. Lymphocytes trafficked to orbital tissues have a putative role, through the cytokines they release, in the activation of fibroblasts in this autoimmune disease.

Blotting, Northern↗
PubMed1996

Suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy.

Gene therapy offers a novel approach to treating human joint diseases such as rheumatoid arthritis. In the present study, we have used the retrovirus, MFG-IRAP, to transfer the human IL-1 receptor antagonist protein (IRAP) gene to rabbits' knees and have assessed its impact on inflammatory and chondrodestructive aspects of the acute phase of antigen-induced arthritis in these joints. Surprisingly, intra-articular expression of IRAP was three- to fivefold higher in arthritic knees than in nonarthritic knees, accumulating to levels of over 20 ng/knee in the highest expressing joints. This level of expression produced a marked chondroprotective effect but a milder anti-inflammatory one. Both the increased cartilage matrix catabolism and the inhibition of matrix synthesis that occur in antigen-induced arthritis were abrogated in the presence of the IRAP gene; the latter effect was particularly strong. Of the indices of inflammation that were examined, only leukocyte influx into the joint space was inhibited, and this effect declined with time. Concentrations of rabbit IL-1 were reduced by the IRAP gene, suggesting inhibition of an autocrine induction loop. These data demonstrate that the course of arthritic disease in the rabbit knee can be altered by genetic manipulation, thus encouraging the further development of gene treatments for human joint diseases.

Animals↗
PubMed1996

Neurophysiologic changes in lumbar nerve root inflammation in the rat after treatment with cytokine inhibitors. Evidence for a role of interleukin-1.

STUDY DESIGN: The present study was designed to evaluate the effect of cytokine inhibitors in experimental allergic radiculitis. OBJECTIVE: Evaluation of the effect of cytokine inhibitors in experimental allergic radiculitis. SUMMARY OF BACKGROUND DATA: A number of cytokines are known to be involved in hyperalgesia and may play a role in radiculitis. Corticosteroids and other cytokine inhibitors antagonize their effects. METHODS: Experimental allergic radiculitis was induced in rats by injection of bovine myelin from the peripheral nervous system. The sham group subsequently received saline injections; the treatment groups received either prednisolone or interleukin-1 receptor antagonist. Treatment effect was assessed on the basis of motor performance and neurophysiologic parameters. RESULTS: Treatment ameliorated the symptoms of experimental allergic radiculitis. Prednisolone appeared to be somewhat more effective than interleukin-1 receptor antagonist. CONCLUSIONS: Because interleukin-1 receptor antagonist specifically blocks the effects of interleukin-1 at its receptors, the present results imply that interleukin-1 is a causal factor in the model of experimental radiculitis used. Its specificity and apparent lack of side effects make interleukin-1 receptor antagonist an attractive candidate treatment for the human disease.

Animals↗
PubMed1996

Effects of immortalization upon the induction of matrix metalloproteinases in rabbit synovial fibroblasts.

Induction of stromelysin and collagenase mRNAs in response to phorbol myristate acetate (PMA) and autocrine factors (CAF) was compared in primary cultures of lapine synovial fibroblasts and an immortalized line of these cells known as HIG-82. In both cell types, message induction was quicker for CAF than for PMA. Appearance of both stromelysin and collagenase mRNAs occurred earlier in HIG-82 cells and, unlike primary cells, HIG-82 cells partially resisted inhibition by cycloheximide. To determine whether differences in AP-1 activity could account for these observations, the induction of c-fos and c-jun mRNAs was studied in conjunction with gel shift assays for AP-1 binding. Both inducers increased the abundance of c-fos mRNA, although the response was weaker in HIG-82 cells. However, the increase in c-jun mRNA was more marked in HIG-82 cells; furthermore, this increase was sustained for over 6 h. Gel shift assays confirmed that in both types of cells PMA and CAF increased AP-1 binding activity. In primary cells, this activity was sensitive to cycloheximide, but in HIG-82 cells, there was only partial sensitivity to cycloheximide. The gel shift analyses and data from experiments using an AP-1-CAT reporter construct revealed, in many cultures, constitutive AP-1 activity in the absence of stromelysin and collagenase expression, suggesting that AP-1 alone is insufficient for matrix metalloproteinase induction. Antisense oligonucleotides to c-fos and c-jun strongly inhibited the induction of stromelysin mRNA in primary cells treated with PMA, but was only weakly active against message induction in HIG-82 cells. In neither primary cells nor HIG-82 cells did antisense oligonucleotides strongly inhibit stromelysin induction in response to CAF. These data suggest there may exist an AP-1-independent route to message induction or that factors other than c-FOS and c-JUN may be used in certain circumstances. Western blot analyses detected no marked difference between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in the kinetics and cycloheximide sensitivity of MMP induction may reside in their abilities to modify posttranslationally the relevant transcription factors.

Animals↗
PubMed1996

Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2.

STUDY DESIGN: Herniated lumbar disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced. OBJECTIVES: Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in intervertebral disc degeneration and in the pathophysiology of radiculopathy. SUMMARY OF BACKGROUND DATA: Low back pain with or without radiculopathy is a significant clinical problem, but the etiology of low back pain and the exact pathophysiology of radiculopathy remain elusive. The biochemical events that occur with intervertebral disc degeneration and, in particular, the role of biochemical mediators of inflammation and tissue degradation have received sparse attention in the literature. There is some preliminary evidence that inflammatory mediators may have an important role in the pathophysiology of radiculopathy. METHODS: Eighteen herniated lumbar discs were obtained from 15 patients undergoing disc surgery. The specimens were cultured and incubated for 72 hours, and the media were collected subsequently for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, eight lumbar disc specimens were obtained from four patients undergoing anterior surgery for scoliosis and traumatic burst fractures, and similar biochemical analyses were performed. RESULTS: The culture media from the herniated lumbar discs showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated discs compared with the control discs. Interleukin 1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of either the herniated or control discs. CONCLUSIONS: Herniated lumbar discs were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These products may be involved intimately in the biochemistry of disc degeneration and the pathophysiology of radiculopathy. Their exact roles certainly need further investigation, but their mere presence implicates biochemical processes in intervertebral disc degeneration.

Adult↗
PubMed1996

The role of nitric oxide in proteoglycan turnover by bovine articular cartilage organ cultures.

Monolayer cultures of articular chondrocytes synthesize large amounts of nitric oxide (NO) following exposure to IL-1. The latter has antianabolic and procatabolic activities on these cells, but little is known about the role, if any, of NO in the integrated metabolic pathways of the chondrocyte. In the present study, the role of endogenously produced NO in both the synthesis and degradation of proteoglycans was investigated for the first time. Bovine articular cartilage slices exposed to 20 U/ml human rIL-1 beta (hrIL-1 beta) synthesized large amounts of NO for 1 to 2 days, after which production fell to a steady state level approximately 20% of the peak value for the remainder of the 14-day incubation. The NO synthase inhibitor, N-monomethyl L-arginine (L-NMA, 1 mM), blocked NO production and enhanced the acute catabolic effects of hrIL-1 beta in cartilage derived from both cartilage derived from both calves and adult animals. However, in late cultures, release of proteoglycans was reduced in the presence of L-NMA. The proteolytic activity in conditioned medium of these cultures (measured as caseinolytic activity) was enhanced by L-NMA; however, this inhibitor did not affect the rates of synthesis of proteoglycans. Although NO is widely assumed to be a mediator of cartilage catabolism, our data suggest that it may instead have an acute protective effect. Whether this effect is maintained chronically is less clear.

Animals↗
PubMed1996

Interleukin-1 receptor antagonist suppresses neurotrophin response in injured rat brain.

Traumatic brain injury (TBI) induces astrocytic and microglial activation and proliferation and augmented production of the cytokine interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF). The increase in NGF temporally follows the increase in IL-1 beta, suggesting that the IL-1 beta up-regulation after trauma directly induces the increase in NGF. We examined the effect of IL-1 receptor antagonist protein (IL-1ra) on microglial proliferation and NGF production in rat cortex, following two different models of TBI. Rabbit fibroblasts infected with a retroviral vector containing the human IL-1ra gene were implanted into the wound cavity immediately following a cortical stab wound or 6 hours after a weight drop-induced trauma. Both microglial proliferation and NGF up-regulation were decreased significantly in animals receiving IL-1ra-expressing cells compared with animals receiving naive (untransfected) fibroblasts. These data demonstrate that the increase in NGF after central nervous system trauma is directly mediated through IL-1 beta and that blocking IL-1 beta following brain injury leads to suppression of an NGF-mediated reparative response. Such blockade of inflammation, however, may prove to be of significant therapeutic benefit in human brain injury and other inflammatory states.

Animals↗
PubMed1996

Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo.

OBJECTIVE: To assess the abilities of various vectors to transfer genes to the synovial lining of joints. METHODS: Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). RESULTS: Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. CONCLUSION: None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.

Adenoviridae↗
PubMed1996

Pathways to gene therapy in rheumatoid arthritis.

Gene therapy offers novel possibilities for the treatment of rheumatoid arthritis. Present research is directed toward harnessing gene transfer technology to deliver genes whose products possess antiarthritic properties; the current emphasis is on transferring genes encoding secreted proteins. Genes may be delivered locally to individual diseased joints or systemically to extra-articular sites where the secreted gene products may enter the circulation. Local delivery is more laborious and unlikely to address systemic components of rheumatoid arthritis but should avoid side effects. Either ex vivo or in vivo strategies may be used to deliver the genes to the target tissues. Ex vivo techniques are more cumbersome but safer, because all genetic manipulations occur outside the body and cells may be extensively screened prior to implantation. A variety of vectors, including retrovirus, adenovirus, herpes simplex virus, and liposomes, as well as naked DNA, have been tested for their ability to deliver genes to joints. At the present stage of vector development, adenovirus seems best suited for in vivo gene delivery to synovium, but several authors have noted an inflammatory response, resulting in loss of gene expression. Ex vivo gene transfer using a retrovirus encoding human interleukin-1 receptor antagonist has succeeded in obtaining high intra-articular transgene expression with promising antiarthritic effects in animal models. Based on these data, the first human gene therapy trial for arthritis has been approved by the US Food and Drug Administration and will begin shortly.

Arthritis, Rheumatoid↗
PubMed1995

Herniated cervical intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2.

STUDY DESIGN: Herniated cervical disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced. OBJECTIVES: Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in cervical intervertebral disc degeneration and in the pathophysiology of cervical radiculopathy. SUMMARY OF BACKGROUND DATA: Neck pain with or without radiculopathy is a common clinical problem, but the etiology of neck pain and the exact pathophysiology of radiculopathy remain uncertain. We have previously reported the production of various biochemical agents by herniated lumbar disc specimens in vitro. Because of a lack of such studies in the literature with respect to the cervical spine, the purpose of this study was to determine whether similar biochemical agents of inflammation and tissue degradation were being produced by herniated cervical disc specimens. METHODS: Eighteen herniated cervical discs were obtained from 15 patients undergoing anterior disc surgery. The specimens were cultured and incubated for 72 hours, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, six cervical discs specimens were obtained from three patients undergoing anterior surgery for traumatic burst fractures, and similar biochemical analyses were performed. RESULTS: The culture media from the herniated cervical disc specimens showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated disc specimens compared with the control discs. Interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of the herniated or control discs. CONCLUSIONS: Herniated cervical disc specimens were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These results were similar to those obtained in herniated lumbar disc specimens that we have previously reported. These products may be intimately involved in the biochemistry of disc degeneration and the pathophysiology of radiculopathy.

Adult↗
PubMed1995

Effect of growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments.

Growth factors have been shown to stimulate fibroblast division and thus may influence ligament healing. We analyzed the effects of individual growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments of the rabbit in vitro in order to identify growth factors that might enhance proliferation of fibroblasts and to compare the responses of the fibroblasts from the two ligaments to these growth factors. Through measurement of the uptake of [3H]-thymidine into DNA, fibroblasts from these ligaments that had been treated with epidermal growth factor and basic fibroblast growth factor were found to proliferate nearly eight times more than control fibroblasts. Additionally, the fibroblasts of both ligaments proliferated at similar rates when exposed to platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, insulin-like growth factor-1, and interleukin-1-alpha. However, epidermal growth factor and transforming growth factor-beta caused the fibroblasts from the medial collateral ligament to proliferate at a rate 1.3-1.4 times greater than that of fibroblasts from the anterior cruciate ligament. The reverse was true with acidic fibroblast growth factor, which stimulated the fibroblasts from the anterior cruciate ligament to proliferate at a rate 1.3-1.6 times greater than that of fibroblasts from the medial collateral ligament. This study demonstrated that growth factors can stimulate cell division in ligaments and may be effective in enhancing ligament healing but that these differences were not great enough to explain fully the clinical differences observed between healing of the medial collateral and anterior cruciate ligaments.

Animals↗
PubMed1995

Nitric oxide: what role does it play in inflammation and tissue destruction?

Large amount of nitric oxide (NO) are produced at sites of inflammation through the action of inducible nitric oxide synthase (iNOS) present in both infiltrating leucocytes and activated, resident tissue cells. However, the role of NO in inflammation remains unclear. NO is a vasodilator, which inhibits the adhesion of neutrophils to the vascular endothelium; it reduces the production of IL-6 by Kupffer cells and chondrocytes, and the production of gamma-IFN and TNF-alpha by splenocytes. The literature provides contradictory information on the effect of NO on vascular leakiness, chemotaxis, prostaglandin production and tissue damage. Increasingly, data suggest that NO is immunosuppressive. Inhibitors of NOS have potent prophylactic activity in several but not all, animal models of inflammatory disease. However, in rat adjuvant arthritis, therapeutic activity is weak. Whether inhibitors of iNOS will be therapeutically useful in human inflammatory diseases cannot be predicted on the basis of present information.

Amino Acid Oxidoreductases↗
PubMed1995

Faculty development in a changing academic environment.

Faculty development--thought of not merely as improving teaching skills, but more broadly as nurturing the growth of trainees into accomplished faculty--is critical to the existence of academic medical centers. Profound changes in academic medicine and health care delivery are making the traditional model of faculty development obsolete. That model is now inadequate and untenable in the face of shrinking government support, changes in the sophistication, costs, and funding of biomedical research, changes in the reimbursement systems for hospitals and physicians, a general loss of respect for the medical and scientific professions, and radical changes in the structure of the health care delivery system. The changes have implications for the type of faculty development needed for this new environment, and a new model is outlined.

Career Mobility↗
PubMed1995

Leukoregulin is a potent inducer of hyaluronan synthesis in cultured human orbital fibroblasts.

Leukoregulin, a 50-kDa glycoprotein lymphokine, can regulate the extracellular matrix in dermal fibroblasts. Here we investigate the effects of leukoregulin on the synthesis of glycosaminoglycans in human orbital fibroblasts. We demonstrate that leukoregulin enhances the incorporation of [3H]glucosamine into glycosaminoglycans. The effect is dose dependent in the concentration range tested (0.1-2 U/ml), is maximal at 1 U/ml, and is time dependent. [3H]glycosaminoglycan accumulation is enhanced 7.67 +/- 1.23-fold (SE, n = 7) in orbital fibroblast strains. Pulse-chase studies indicate that this enhanced accumulation is not a result of a decreased rate of macromolecular degradation. Radiolabeled material induced by leukoregulin is sensitive to Streptomyces hyaluronidase digestion. Dexamethasone (10(-8) M) and cycloheximide (10 micrograms/ml) can block the cytokine's stimulation of hyaluronan synthesis. [35S]sulfate incorporation into glycosaminoglycan is unaffected by leukoregulin. In dermal fibroblasts, leukoregulin increased hyaluronan synthesis 3.66 +/- 0.37-fold (n = 5 strains, P < 0.02 compared with orbit). The increase in hyaluronan synthesis in orbital fibroblasts is substantially greater than that observed previously with other cytokines, making leukoregulin a candidate molecular trigger in Graves' ophthalmopathy.

Antineoplastic Agents↗