Biomedical subjects

J Steven Alexander

Publications and source records attributed to J Steven Alexander.

34 records · Page 2Linked to original sources

PubMed2004

Pathogenesis of brain and spinal cord atrophy in multiple sclerosis.

For more than a century, multiple sclerosis was viewed as a disease process characterized by oligodendrocyte and myelin loss, and research into the pathogenesis of multiple sclerosis was mainly focused on the mechanisms of inflammation. However, with development of more sophisticated neuroimaging and molecular biology techniques, attention has shifted to new aspects of pathogenesis of multiple sclerosis: axonal loss and neurodegeneration. Evidence is increasing that tissue destruction, primarily axonal loss and neurodegeneration, is a key element in the pathogenesis of multiple sclerosis. In addition, it is now known that brain and spinal cord atrophy begins early in the disease process of multiple sclerosis and advances relentlessly throughout the course of the disease. Cumulative data suggest that axonal loss is the major determinant of progressive neurologic disability in patients with multiple sclerosis. Magnetic resonance imaging and magnetic resonance spectroscopy in patients with multiple sclerosis for < 5 years indicate brain atrophy and loss of axonal integrity. Neurodegeneration and axonal loss in patients with multiple sclerosis are initially accompanied by a local response from oligodendrocyte progenitor cells and some remyelination. However, these repair mechanisms eventually fail, and patients typically develop generalized brain atrophy, cognitive decline, and permanent disability. Although the exact mechanisms underlying central nervous system atrophy in patients with multiple sclerosis are largely unknown, evidence exists that atrophy may represent an epiphenomenon related to the effects of dynamic inflammation within the central nervous system, including demyelination, axonal injury, neuronal loss, Wallerian degeneration, and possibly iron deposition. This article summarizes the potential mechanisms involved in central nervous system atrophy in patients with multiple sclerosis.

Apoptosis↗

PubMed2004

Cerebral cortical aquaporin-4 expression in brain edema following cardiac arrest in rats.

OBJECTIVES: Brain edema occurs following clinical as well as experimental cardiac arrest (CA) and predicts a poor neurologic outcome. The objective of this study was to determine the expression of cerebral cortex aquaporin (AQP)-4, a member of a family of membrane water-channel proteins, in brain edema formation following normothermic or hypothermic CA. METHODS: Twenty-four rats were subjected to time-matched normothermic (N-Sham, 37.5 degrees C +/- 0.5 degrees C, n = 6) or hypothermic (H-Sham, 34 degrees C +/- 0.5 degrees C, n = 6) sham experiments and normothermic (N-CA, n = 6) or hypothermic (H-CA, n = 6) CA induced by asphyxiation for 8 minutes. Hypothermia was induced before CA. The animals were resuscitated with cardiopulmonary resuscitation, ventilation, and epinephrine administration. Brain edema was determined by brain wet-to-dry weight ratio at one hour of resuscitation. AQP4 immunoactivity in the cerebral cortex was determined using immunohistochemical staining and was semiquantified as an intensity of staining with an automated cell imaging system. RESULTS: Mild hypothermia in the sham experiments did not alter cerebral cortex AQP4 immunoactivity (mean +/- SD) (55.0 +/- 3.7 in H-Sham vs. 53.3 +/- 1.7 in N-Sham, p > 0.05). N-CA resulted in a significant increase in AQP4 immunoactivity (61.8 +/- 4.5) compared with N-Sham (p = 0.01) and H-Sham (p = 0.03). H-CA attenuated AQP4 compared with N-CA (53.4 +/- 1.3, p = 0.01). Brain wet-to-dry weight ratios were 4.41 +/- 0.07 in N-Sham, 4.40 +/- 0.08 in H-Sham (p > 0.05 vs. N-Sham), 4.55 +/- 0.04 in N-CA (p = 0.004 vs. N-Sham; p = 0.005 vs. H-Sham), and 4.43 +/- 0.09 in H-CA (p = 0.02 vs. N-CA; p > 0.05 vs. N-Sham and H-Sham). CONCLUSIONS: Cerebral cortical AQP4 expression is up-regulated after normothermic CA, which is attenuated by hypothermia induced before CA.

Animals↗

PubMed2003

Increased disease activity in eNOS-deficient mice in experimental colitis.

Oral dextran sodium sulfate (DSS, 3%) produces experimental colitis with many features of human inflammatory bowel disease (IBD), (leukocyte extravasation, cachexia, and histopathology). Previous studies suggest that the inducible nitric oxide synthase (iNOS) in blood cells or in the endothelium contribute to this injury. However, until now no study has been performed to directly evaluate the role of endothelial nitric oxide synthase (eNOS) in IBD. We compared disease activity in wild-type (eNOS+/+) and eNOS-deficient (eNOS-/-) mice in the DSS model of colitis. Administration of DSS induced weight loss, stool blood, and overt histopathology in both mouse strains. Disease activity was dramatically increased in eNOS-/- mice compared to wild types. Histologically, eNOS-deficient mice had greater leukocyte infiltration, gut injury, and expressed higher levels of the mucosal addressin, MAdCAM-1. These results demonstrate that eNOS plays an important role in limiting injury to the intestine during experimental colitis and altered eNOS content and/or activity may contribute to human IBD.

Animals↗

PubMed2003

Tight junction peptide antagonists enhance neutrophil trans-endothelial chemotaxis.

Occludin is an integral membrane protein within tight junctions. Previous studies suggest it functions as a sealing element, which promotes barrier in endothelial and epithelial cell layers. Here, we examine the role of occludin in neutrophil chemotaxis, using cyclic occludin peptide antagonists that incorporate a conserved occludin cell adhesion recognition (CAR) sequence. Human umbilical vein endothelial cells were pre-treated with occludin specific cyclic peptide antagonists to examine effects on neutrophil migration towards a chemotactic gradient of 10(-7) M fMLP. The spatial organization of occludin and VE-cadherin were also assessed in control and occludin peptide-treated monolayers by immunofluorescent staining. The cyclic peptide, peptide B, which contains the CAR sequence of occludin, increased neutrophil chemotaxis in a time and dose dependent manner. Scrambled sequence peptide controls and linear peptides did not. The cyclic occludin antagonist, peptide B, disorganized junctional occludin, but apparently not VE-cadherin as assessed by immunofluorescence. The correlation between diminished occludin organization and increased neutrophil trans-endothelial chemotaxis provides additional support for occludin in the maintenance of the tight junctional barrier.

Adult↗

PubMed2003

Transfection of IL-10 expression vectors into endothelial cultures attenuates alpha4beta7-dependent lymphocyte adhesion mediated by MAdCAM-1.

BACKGROUND: Enhanced expression of MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is associated with the onset and progression of inflammatory bowel disease. The clinical significance of elevated MAdCAM-1 expression is supported by studies showing that immunoneutralization of MAdCAM-1, or its ligands reduce inflammation and mucosal damage in models of colitis. Interleukin-10 (IL-10) is an endogenous anti-inflammatory and immunomodulatory cytokine that has been shown to prevent inflammation and injury in several animal studies, however clinical IL-10 treatment remains insufficient because of difficulties in the route of IL-10 administration and its biological half-life. Here, we examined the ability of introducing an IL-10 expression vector into endothelial cultures to reduce responses to a proinflammatory cytokine, TNF-alpha METHODS: A human IL-10 expression vector was transfected into high endothelial venular ('HEV') cells (SVEC4-10); we then examined TNF-alpha induced lymphocyte adhesion to lymphatic endothelial cells and TNF-alpha induced expression of MAdCAM-1 and compared these responses to control monolayers. RESULTS: Transfection of the IL-10 vector into endothelial cultures significantly reduced TNF-alpha induced, MAdCAM-1 dependent lymphocyte adhesion (compared to non-transfected cells). IL-10 transfected endothelial cells expressed less than half (46 +/- 6.6%) of the MAdCAM-1 induced by TNF-alpha (set as 100%) in non-transfected (control) cells. CONCLUSION: Our results suggest that gene therapy of the gut microvasculature with IL-10 vectors may be useful in the clinical treatment of IBD.

Adhesiveness↗

PubMed2003

Randomized, placebo-controlled, double blind, crossover design trial of the efficacy and safety of Zestra for Women in women with and without female sexual arousal disorder.

Zestra for Women is a botanical feminine massage oil formulated to enhance female sexual pleasure and arousal when applied to the vulva. We conducted this randomized, double-blinded, crossover study to evaluate the efficacy and safety of Zestra for Women compared to placebo oil in 10 women with and 10 women without female sexual arousal disorder (FSAD) in conditions of home use in conjunction with sexual activities. Subjects were screened by physical examination, sex therapist interviews, and questionnaires. We randomized qualified subjects to treatment paths and gave them 5 doses of test article and diaries to use at home. At Visit 2, we assessed them by questionnaires and gave them 5 doses of crossover test article and diaries to use at home. At the final visit, we assessed them with questionnaires. We assessed safety by adverse event reports and primary efficacy by responses to a diary question regarding satisfaction with arousal. Secondary efficacy instruments included remaining diary questions, recall-based questionnaires, global assessment questions, and a consumer-testing questionnaire. All 20 subjects completed the study. Three subjects reported single incidences of mild genital burning sensations lasting 5-30 min after use of Zestra for Women. Both normal and FSAD women showed statistically significant improvements, relative to placebo, in level of arousal, level of desire, satisfaction with arousal, genital sensation, ability to have orgasms, and sexual pleasure. Although FSAD women showed greater magnitude of response, the presence of FSAD had no effect on response rates. Zestra for Women was just as effective in women using selective serotonin reuptake inhibitor antidepressants as in women not using antidepressants. Zestra for Women improved sexual function in normal and FSAD women under conditions of home use.

Administration, Topical↗

PubMed2003

The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin reduces disease activity and inflammation in dextran-sulfate induced colitis.

The dextran sulfate (DSS) model of colitis causes intestinal injury sharing many characteristics with inflammatory bowel disease, e.g., leukocyte infiltration, loss of gut epithelial barrier, and cachexia. These symptoms are partly mediated by entrapped leukocytes binding to multiple endothelial adhesion molecules (MAdCAM-1, VCAM-1, ICAM-1, and E-selectin). Pravastatin, an 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, has anti-inflammatory potency in certain inflammation models; therefore, in this study, we measured the effects of pravastatin in DSS-induced colitis. The administration of pravastatin (1 mg/kg) relieved DSS-induced cachexia, hematochezia, and intestinal epithelial permeability, with no effect on serum cholesterol. Histopathologically, pravastatin prevented leukocyte infiltration and gut injury. Pravastatin also blocked the mucosal expression of MAdCAM-1. DSS treatment promoted mucosal endothelial nitric-oxide synthase (eNOS) mRNA degradation, an effect that was blocked by pravastatin. Importantly, the protective effects of pravastatin in DSS-induced colitis were not found in eNOS-deficient mice. Our results demonstrate that HMG-CoA reductase inhibitors preserve intestinal integrity in colitis, most likely via increased eNOS expression and activity, independent of cholesterol metabolism.

Animals↗

PubMed2003

Serum from patients with multiple sclerosis downregulates occludin and VE-cadherin expression in cultured endothelial cells.

Disruption of the blood brain barrier (BBB) and transendothelial migration of inflammatory cells are crucial steps in the development of demyelinating lesions in multiple sclerosis (MS). Occludin and vascular endothelial-cadherin (VE-cadherin) are two major components of the tight junctions (TJs) in the brain microvasculature that help to create the BBB. In the present study, we investigated the effect of serum from MS patients on the expression of these two junctional markers and on the endothelial integrity. Serum from six MS patients in exacerbation, six in remission, and six normal controls (10% by volume) was incubated with cultured endothelial cells, and the expression of occludin and VE-cadherin was measured by immunoblotting. Serum from MS patients in exacerbation significantly reduced the expression of occludin and VE-cadherin compared with patients in remission and normal controls. This disintegrating effect was more pronounced for occludin than for VE-cadherin. We assume that the elevation in cytokines or other serum-soluble factors in MS patients in exacerbation likely provokes downregulation of occludin and VE-cadherin. This downregulation of TJs proteins may, therefore, contribute to the disruption of the BBB in this condition.

Adult↗

PubMed2003

Blood-brain barrier disruption in multiple sclerosis.

The blood-brain barrier (BBB) is a complex organization of cerebral endothelial cells (CEC), pericytes and their basal lamina, which are surrounded and supported by astrocytes and perivascular macrophages. Collectively these cells separate and form the compartments of the cerebral vascular space and the cerebral interstitium under normal conditions. Without the BBB, the 'interior milieu' of the central nervous system (CNS) would be flooded by humoral neurotransmitters and formed blood elements that upset normal CNS functions and lead to vascular/neural injury. Dysregulation of the BBB and transendothelial migration of activated leukocytes are among the earliest cerebrovascular abnormalities seen in multiple sclerosis (MS) brains and parallel the release of inflammatory cytokines/chemokines. Mechanisms for breakdown of the BBB in MS are incompletely understood, but appear to involve direct effects of these cytokines/ chemokines on endothelial regulation of BBB components, as well as indirect cytokine/chemokine-dependent leukocyte mediated injury. Unique endothelial structural features of the BBB include highly organized endothelial tight junctions, the absence of class II major histocompatibility complex, abundant mitochondria and a highly developed transport system in CEC. Exposure of endothelium to proinflammatory cytokines (IFN-gamma, TNF-alpha and IL-1beta) interrupts the BBB by disorganizing cell-cell junctions, decreases the brain solute barrier, enhances leukocyte endothelial adhesion and migration as well as increases expression of class II MHC and promotes shedding of endothelial 'microparticles' (EMP). In this review we examine interactions between cytokines/chemokines, activated leukocytes, adhesion molecules and activated CEC in the pathogenesis of BBB failure in MS.

Blood-Brain Barrier↗

PubMed2003

Gamma knife irradiation increases cerebral endothelial expression of intercellular adhesion molecule 1 and E-selectin.

OBJECTIVE: Alterations in multiple functions of the microvasculature occur in response to gamma irradiation and are thought to contribute to radiation-induced end organ damage by inducing inflammatory responses, particularly leukocyte infiltration into the affected area. Endothelial cell adhesion molecules (ECAMs) mediate leukocyte adhesion and migration. Here, we validate a method to study the effect of Leksell gamma knife stereotactic radiosurgery on the expression of ECAMs on human cerebral endothelium at 0, 24, 48, and 72 hours after irradiation. METHODS: A human brain endothelial cell line (IHEC) was cultured on 12-mm coverslips and subjected to 50 Gy of collimated gamma irradiation with the Leksell gamma knife (Elekta Instruments, Inc., Atlanta, GA). Lactate dehydrogenase release was measured at 24, 48, and 72 hours after irradiation and caspase-3 at 24, 48, 72, 96, and 120 hours. ECAM expression was measured at postirradiation intervals of 0, 24, 48, and 72 hours by cell enzyme-linked immunoabsorbent assay. We used a cell irradiator composed of two chambers. The upper chamber holds the coverslips firmly in place while they are immersed in media. The lower chamber is connected to a peristaltic pump, which pumps water into the chamber and maintains the media in the upper chamber at 37 degrees C through convection. RESULTS: None of the ECAMs tested was significantly elevated compared with the control basally. Twenty-four hours after irradiation, intercellular adhesion molecule 1 was significantly elevated on brain endothelial cells but there was no significant elevation of E-selectin. Vascular cell adhesion molecule 1 was increased slightly but not significantly and decreased at 48 hours. At 72 hours, E-selectin expression was significantly increased; intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were not altered relative to sham controls. CONCLUSION: Increased ECAM expression and lactate dehydrogenase release support the idea that the cerebral microvasculature undergoes an inflammatory response after Leksell gamma knife stereotactic radiosurgery.

Apoptosis↗

PubMed2002

Devic disease: clinical course, pathophysiology, and management.

Devic disease is characterized by an attack or attacks of optic neuritis and necrotizing myelitis without clinical or magnetic resonance imaging (MRI) evidence of brain involvement or presence of oligoclonal bands in the cerebrospinal fluid (CSF). More than a century after its first systematic study by the French physician, Eugene Devic, its cause as well as its pathogenesis, remains unknown. Devic disease is both a diagnostic and therapeutic challenge to clinicians. The authors present the latest review on the nature, course, possible mechanisms, and therapeutic options of Devic disease.

Journal Article↗

PubMed2002

Identification of an occludin cell adhesion recognition sequence.

The molecular mechanisms by which the tight junction integral membrane protein, occludin promotes cell adhesion and establishes an endothelial monolayer permeability barrier have not been elucidated. In particular, the amino acid sequences of the occludin cell adhesion recognition (CAR) sites have not been determined. Here we demonstrate that a cyclic peptide containing the sequence LYHY, which is found in the second extracellular domain of occludins in all mammalian species, inhibits the establishment of endothelial cell barriers in vitro and in vivo. This cyclic peptide also prevents the aggregation of fibroblasts stably transfected with cDNA encoding occludin. The data suggest that the LYHY motif is an occludin CAR sequence.

Amino Acid Sequence↗

PubMed2002

Extravascular transport of fluorescently labeled albumins in the rat mesentery.

OBJECTIVE: Fluorescently labeled albumin is used frequently as a tracer when monitoring microvascular permeability. Several fluorescent dyes are available for labeling protein, including fluorescein isothiocyanate (FITC) and Texas Red (TR). Because differences in leakage of dye-labeled proteins have been reported, the objective of the present study was to compare the accumulation of these two tracers in interstitium and lymph after the inflammatory event of ischemia-reperfusion. METHODS: Anesthetized rats were injected intravenously with FITC-labeled albumin (FITC-alb) and TR-labeled albumin (TR-alb) before 30 minutes of mesenteric ischemia. Because the tracers leaked out of the microcirculation after reperfusion, accumulation in the surrounding buffer-superfused tissue, and in separate experiments, accumulation in lymph vessels, was defined as the ratio of tissue-to-plasma and lymph-to-plasma fluorescence. RESULTS: Reperfusion induced a significant increase in tissue-to-plasma fluorescence of FITC-alb; however, no increase was observed for TR-alb. In contrast, lymph-to-plasma fluorescence of TR-alb tended to be greater than FITC-alb. Reperfusion-induced increases in tissue-to-plasma fluorescence of TR-alb occurred only when the superfusate was replaced with mineral oil, in which case tissue-to-plasma TR-alb fluorescence tended to be greater than FITC-alb. CONCLUSIONS: Measurement of fluorescently labeled albumin leakage from mesenteric venules depends on the dye used to label the albumin and requires an assessment of losses from the extravascular measuring region.

Albumins↗

PubMed2002

Endothelial junctional protein redistribution and increased monolayer permeability in human umbilical vein endothelial cells isolated during preeclampsia.

OBJECTIVE: To examine monolayer permeability and junctional protein distribution and expression in endothelial cells isolated from women with preeclampsia and from women with normal pregnancies. We hypothesized that increased endothelial monolayer permeability in preeclampsia reflects altered monolayer barrier properties produced by disorganization of endothelial cell junction proteins. STUDY DESIGN: Endothelial cells were isolated from umbilical veins from women with normal pregnancies (n = 9) and from women with preeclampsia (n = 9) immediately after delivery. In the first passage of endothelial cells, the permeability was determined by measurement of horseradish peroxidase passage through confluent cell monolayers grown on transwell filters, and the distribution and protein expression of vascular endothelial cadherin and occludin were evaluated by use of immunofluorescent staining of the proteins and Western blot analysis. The distribution of vascular endothelial cadherin was also evaluated in the second and third passage endothelial cells. Messenger ribonucleic acid expression of vascular endothelial cadherin and occludin were examined by reverse transcriptase polymerase chain reaction. Data were expressed as mean values (+/- SE) and were analyzed by use of an unpaired t test. RESULTS: The relative monolayer permeability was significantly increased in the endothelial cells isolated from women with preeclampsia compared with those isolated from women with normal pregnancies (0.271 +/- 0.049 versus 0.086 +/- 0.031 for DeltaOD(470); P <.05). Vascular endothelial cadherin expression for normal endothelial cells showed a continuous staining of the junctional protein that surrounded cell borders. In comparison, vascular endothelial cadherin in endothelial cells from preeclamptic pregnancies exhibited disorganized staining and vascular endothelial cadherin fibrils were retracted, with gaps present at the cell borders. The expression of occludin showed a pattern similar to that of vascular endothelial cadherin in both normal and preeclamptic conditions. Western blot results for expression of vascular endothelial cadherin and occludin also showed decreased expression of junctional proteins. The altered endothelial cell junctional protein distribution and expression of vascular endothelial cadherin and occludin observed in the first passage of endothelial cells from preeclamptic pregnancies was restored to normal by the time cells reached the third passage in vitro. There was no statistical difference in mRNA expression for the vascular endothelial cadherin and occludin between normal endothelial cells and those from preeclamptic pregnancies. CONCLUSION: In preeclampsia, increased endothelial cell monolayer permeability appears to reflect disorganized and diminished expression of endothelial cell junctional proteins. The latter response is mediated at the posttranscriptional level. These findings provide new insights about the cellular and molecular basis for altered endothelial cell integrity and barrier dysfunction that are associated with preeclampsia.

Antigens, CD↗

PubMed2002

Is the gastric epithelial barrier regulated by cadherin recruitment?

While still highly controversial, regulation of junctional structure remains an important feature in many physiological/pathological conditions. Mizuno et al. here report a mechanism in stomach epithelia where additional copies of cadherins appear to be recruited to the cell surface, apparently to maintain the solute barrier. This phenomenon may represent an important novel mechanism which epithelia may use to enhance barrier.

Cadherins↗

PubMed2002

Mild hypothermia induced before cardiac arrest reduces brain edema formation in rats.

OBJECTIVES: The mechanisms by which hypothermia improves cardiac arrest (CA)-induced brain damage are unclear. The authors hypothesized that mild hypothermia induced before CA attenuates brain edema formation by preventing neutrophil-mediated dysfunction of the endothelial cell junction proteins. METHODS: Eighteen rats were randomized to normal control surgery (group 1, n = 6), normothermic (37.5 degrees C) CA (group 2, n = 6), or hypothermic (34 degrees C) CA (group 3, n = 6). Hypothermia was induced with external cooling before CA in group 3. Cardiac arrest was induced by 8 minutes of asphyxiation. Brain edema was determined by wet-to-dry weight ratio and cerebral spinal fluid pressure (CSFP). Brain neutrophil content was determined by myeloperoxidase (MPO) activity, and occludin degradation was assessed by western blotting. RESULTS: Normothermic CA significantly increased brain wet-to-dry weight ratio from 4.52 +/- 0.04 in group 1 to 4.80 +/- 0.04 in group 2 (p = 0.0003) and CSFP from 3.6 +/- 0.9 in group 1 to 8.9 +/- 0.9 mm Hg in group 2 (p = 0.004). Mild hypothermia before CA in group 3 significantly reduced brain wet-to-dry weight ratio (4.68 +/- 0.03, p = 0.008 vs. group 2) and CSFP (3.8 +/- 0.5 mm Hg, p = 0.004 vs. group 2). Cardiac arrest increased brain MPO from 0.07 +/- 0.025 in group 1 to 0.16 +/- 0.02 units/gram brain weight in group 2 (p = 0.006) that was not decreased by hypothermia before CA (0.12 +/- 0.02 in group 3 (p = 0.07 vs. group 2). There was no occludin proteolysis in any group. CONCLUSIONS: Mild hypothermia before CA decreases CA-induced brain edema. The hypothermia-elicited reduction in brain edema does not appear to be neutrophil-dependent and the early brain edema formation may not involve the proteolysis of occludin.

Analysis of Variance↗