Robert E. Wolf

Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide.

Leukocyte-endothelial cell adhesion and an altered metabolism of endothelial cell-derived nitric oxide (NO) have been implicated in the microvascular dysfunction associated with ischemia/reperfusion (I/R). The objective of this study was to determine whether NO donors can attenuate the reperfusion-induced increase in venular albumin leakage via an effect on leukocyte-endothelial cell adhesion. Leukocyte adherence and emigration as well as albumin extravasation were monitored in single postcapillary venules in rat mesentery subjected to 20 minutes of ischemia followed by 30 minutes of reperfusion. This I/R protocol elicits significant leukocyte adherence and emigration as well as a profound albumin leakage response. Superfusion of the mesenteric microcirculation with the NO donors sodium nitroprusside, spermine-NO, and SIN1 significantly reduced the I/R-induced leukocyte adherence/emigration and albumin leakage in postcapillary venules, whereas neither spermine nor the NO synthase inhibitor NG-nitro-L-arginine methyl ester affected the I/R-induced responses. Platelet-leukocyte aggregation and mast cell degranulation were also observed in the postischemic mesentery, and the responses were also attenuated by the NO donors. Plasma nitrate/nitrite levels in the superior mesenteric vein were significantly reduced by I/R. The results of this study indicate that I/R-induced microvascular dysfunction (albumin leakage) is attenuated by NO and that the protective effect of NO donors may be related to their ability to reduce leukocyte-endothelial cell and leukocyte-platelet interactions and/or mast cell degranulation.

Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease

The inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.The inflammatory bowel diseases (IBD; Crohns disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.

Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 μM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 μM of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (ΔΨm) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of ΔΨm. This drop in ΔΨm was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation → excess ROS production → GSH depletion → oxidative stress → disruption of ΔΨm → release of cytochrome C and other apoptosis related proteins to cytosol → apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.

T-Lymphocytes Modulate the Microvascular and Inflammatory Responses to Intestinal Ischemia-Reperfusion

Objective: The overall objective of this study was to define the contribution of T‐lymphocytes to the microvascular and inflammatory responses of the intestine to ischemia/reperfusion (I/R).

Design and implementation of a system-based course in musculoskeletal medicine for medical students.

BACKGROUNDnThe amount of time devoted to musculoskeletal medicine in the typical undergraduate curriculum is disproportionately low compared with the frequency of musculoskeletal complaints that occur in a general practice. Consequently, whether because of the quantity or quality of the education, the competence level of graduating physicians regarding musculoskeletal problems is inadequate. Our purposes were to design a self-contained, system-based course in musculoskeletal medicine for medical students in the preclinical years and to measure the level of competence achieved by a class of first-year medical students who took the course.nnnMETHODSnThe course was formulated by faculty from the departments of orthopaedic surgery, anatomy, and rheumatology and included elements of both objectives-based and problem-centered curricular models. The clinical lectures were preceded by pertinent anatomy lectures and dissections to provide a context for the clinical information. The lectures on basic science were designed to rationalize and explicate clinical practices. Small-group activities were incorporated to permit engagement of the students in critical thinking and problem-solving. A general musculoskeletal physical examination was taught in two two-hour-long small-group sessions with the orthopaedic residents serving as instructors. Cognitive competency was evaluated with use of comprehensive anatomy laboratory and written examinations, the latter of which included a validated basic competency examination in musculoskeletal medicine. Process-based skills were evaluated in the small-group meetings and in a timed, mock patient encounter in which each students ability to perform the general musculoskeletal physical examination was assessed.nnnRESULTSnThe course lasted six weeks and consisted of forty-four lecture hours, seventeen hours of small-group meetings, and twenty-eight hours of anatomy laboratory. The average student score on the basic competency examination was 77.8%, compared with 59.6% for a historical comparison group (p < 0.05). Each student demonstrated the ability to adequately perform a general musculoskeletal physical examination in twenty minutes. The survey of student opinion after the course indicated a high level of student satisfaction.nnnCONCLUSIONSnThe main features of the course were: (1) an emphasis on both cognitive and process-based knowledge; (2) more contact hours and broader content than in previously described courses in musculoskeletal medicine; (3) the use of small groups to focus on problem-solving and physical examination competencies; (4) basic-science content directly related to clinical goals. These features might be used at other institutions that employ a system-based curriculum for the preclinical years to help improve competence in musculoskeletal medicine.

Prevalence of Active Hepatitis C Virus Infection in Patients with Systemic Lupus Erythematosus

Objective:Hepatitis C virus (HCV) infection is associated with various autoimmune disorders and can mimic systemic lupus erythematosus (SLE) clinically and serologically. There are few reports of prevalence of HCV infection in patients with SLE. The aim of this study was to determine the prevalence of HCV viremia by polymerase chain reaction (PCR) in patients with SLE. Methods:We tested sera from 40 consecutive patients with SLE collected from1993 to 2000. All of the patients had HCV viral load measured by PCR. The results were compared with the prevalence of HCV viremia in a control group of blood donors in our geographic area as well as in United States general population. Results:HCV was detected in 4 of 40 patients (10%). The prevalence of HCV in our area blood donors is 130 cases per 100,000 persons (0.13%; P < 0.0001). The prevalence of HCV infection in the United States general population, screened by PCR, is 1330 cases per 100,000 people (1.33%; P = 0.002). The prevalence of HCV infection was significantly higher in our SLE patients than in our area blood donors. The frequency of HCV infection was also higher than that of the United States general population. Conclusion:Our observations support those of other investigators who have reported an increased prevalence of HCV infection in SLE patients. Further detailed investigation of this association may help in understanding the pathogenesis of SLE. HCV infection should be tested when the diagnosis of SLE is considered.

NFκB Signaling in Posthypoxic Endothelial Cells: Relevance to E-Selectin Expression and Neutrophil Adhesion

Our previous studies have implicated the nuclear transcription factor ĸB (NFĸB) in the regulation of adhesion molecule expression in endothelial cells exposed to anoxia-reoxygenation (A/R) or a redox imbalance. The objectives of this study were (1) to define the kinetics of NFĸB activation by examining IĸBα degradation and the nuclear translocation of p65 in response to A/R or redox imbalance (induced by treatment of cells with diamide and buthionine sulfoximine) and (2) to determine whether the signal for IĸBα degradation, nuclear translocation of p65, and E-selectin-mediated neutrophil adhesion is related to the activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTPase) and/or protein kinase C (PKC). The results demonstrate that both A/R and redox imbalance led to IĸBα degradation within 30 min and the concomitant appearance of p65 in the nucleus, consistent with rapid cytosolic activation of NFĸB and subsequent nuclear translocation of the activated p65 subunit. Inhibition of PKC blocked IĸBα degradation and p65 translocation in A/R-challenged, but not redox-altered, endothelial cells. However, both A/R- and redox-induced NFĸB activation was blocked by inhibition of PTK. Similarly, A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion were blocked by inhibition of PKC or PTK, while only PTK inhibited the redox-induced adhesion response. Pretreatment of cells with N-acetyl cysteine effectively blocked A/R- or redox-induced IĸB degradation and significantly attenuated the respective neutrophil adhesion responses. Collectively, these findings indicate that A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion are mediated by both PKC and PTK, which signal rapid activation of NFĸB. This A/R-induced NFĸB signaling response appears to be mediated, at least in part, by intracellular redox imbalance.

Postanoxic T Lymphocyte–Endothelial Cell Interactions Induce Tumor Necrosis Factor-α Production and Neutrophil Adhesion Role of Very Late Antigen-4/Vascular Cell Adhesion Molecule-1

The objective of this study was to define the influence of postanoxic T-lymphocyte-endothelial cell interactions on anoxia-reoxygenation (A/R)-induced neutrophil-endothelial cell adhesion and cell adhesion molecule (CAM) expression on human umbilical vein endothelial cells (HUVECs). HUVEC monolayers were exposed to 60 minutes of anoxia, followed by 24 hours of reoxygenation, wherein freshly isolated human T lymphocytes were added at 6 hours during reoxygenation. After an additional 18 hours of incubation (ie, total of 24 hours of reoxygenation), the T-cell/endothelial cell (TC/EC) coculture media were collected and added to naive HUVEC monolayers incubated with neutrophils. Although the A/R-conditioned media per se had no effect on neutrophil adhesion, the media from TC/EC cocultures significantly increased the adhesion response. This enhanced adhesive interaction was associated with significant increases in tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) levels in the TC/EC coculture media and was accompanied by a pronounced increase in endothelial E-selectin expression. Treatment of the TC/EC coculture media with anti-TNF-alpha or anti-IL-8 antibodies reduced the media-induced neutrophil adhesion response. The enhanced neutrophil adhesion and the elevated medium levels of TNF-alpha, but not IL-8, were markedly reduced by inserts that prevented direct TC/EC contact and by monoclonal antibodies directed against vascular cell adhesion molecule-1 (VCAM-1) or very late antigen-4 (VLA-4). Collectively, these findings show that VLA-4-/VCAM-1-mediated interactions between T lymphocytes and postanoxic endothelial cells stimulates TNF-alpha production, which in turn elicits endothelial cell adhesion molecule expression and a corresponding increase in neutrophil adhesion.

Gap junctions in human synovial cells and tissue

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated‐patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 ± 150 MΩ. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2‐nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap‐junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function. J. Cell. Physiol. 184:110–117, 2000.

ICAM-3 (CD50) cross-linking augments signaling in CD3-activated peripheral human T lymphocytes.

ICAM‐3 is a pan‐hematopoietic, constitutive adhesion molecule. ICAM‐3 binds to LFA‐1 on antigen‐presenting cells (APC) stabilizing the T cell‐APC interaction, facilitating signaling through the CD3/TCR complex. However, recent evidence using cultured and transformed T cells suggests ICAM‐3 may also function in signaling. Because ICAM‐3 is constitutively expressed on resting T cells, we postulated that signaling through ICAM‐3 in resting T cells represents an important costimulatory mechanism in these cells. In purified resting human T cells, cross‐linking both ICAM‐3 and CD3 with plate‐bound antibodies resulted in a marked increase in cell size (consistent with blastogenesis), synergistically increased surface expression of CD25 and CD69, and increased T cell metabolism. Similarly, concomitant ICAM‐3 and CD3 stimulation significantly (P < 0.001) increased resting human T cell phosphatidylinositol hydrolysis and phospholipase C‐γ1 phosphorylation. These results indicate that ICAM‐3 augments signaling through CD3, functioning as a costimulatory molecule for resting T cells in the initial activation step. J. Leukoc. Biol. 65: 867–874; 1999.