Ocular neuroprotection by siRNA targeting caspase-2.

Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss.

The roles of hyperglycaemia and oxidative stress in the rise and collapse of the natural protective mechanism against vascular endothelial cell dysfunction in diabetes.

Vascular endothelial cell (VEC) dysfunction in diabetes has been associated with hyperglycaemia-induced intra- and extracellular glycation of proteins and to overproduction of glucose-derived free radicals. VEC protect their intracellular environment against an increased influx of glucose in face of hyperglycaemia by reducing the expression and plasma membrane abundance of their glucose transporter-1 (GLUT-1). We investigated the hypothesis that glucose-derived free radicals induce this down-regulatory mechanism in VEC, but proved the contrary. In fact, pro-oxidants significantly increased the expression and plasma membrane abundance of GLUT-1 and the rate of glucose transport in VEC while abolishing high-glucose-induced down-regulation of the hexose transport system. The resulting uncontrolled influx of glucose followed by overproduction of glucose-derived ROS further up-regulates the rate of glucose transport, and vice versa. This perpetuating glycoxidative stress finally leads to the collapse of the auto-regulatory protective mechanism and accelerates the development of dysfunctional endothelium in blood vessels.

Cyclooxygenase-2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in an insulin- and AMPKalpha-independent manner.

AIMS/HYPOTHESIS: Some cyclooxygenase-2 (COX2, also known as prostaglandin-endoperoxide synthase 2 [PTGS2]) inhibitors have been shown to increase insulin sensitivity in man or induce hypoglycaemic episodes when overconsumed or taken in combination with oral hypoglycaemic drugs. These side-effects and their impact on patients are not always recognised in routine clinical practice. We investigated whether these side-effects of COX2 (PTGS2) inhibitors result from stimulation of the glucose transport system in skeletal muscle cells. MATERIALS AND METHODS: L6 myotube cultures were used to study effects of COX2 (PTGS2) inhibitors on the glucose transport system and their relationship to PTGS2 expression, insulin action and AMP-activated protein kinase alpha (AMPKalpha) activity. RESULTS: The inhibitors niflumic acid, nimesulide and rofecoxib increased the rate of hexose uptake in L6 myotubes in the absence of insulin and in a dose- and time-dependent manner. They did this by increasing the total cell content of member 4 of the solute carrier family 2 (SCLC2A4, previously known as glucose transporter 4 [GLUT4]) (but not SCLC2A1 [previously known as GLUT1]) mRNA and protein and the amount of it in the plasma membrane. AMPKalpha was not involved in the latter effect since the inhibitors did not activate it. In addition, none of the inhibitors modulated the rate of hexose transport in vascular endothelial and smooth muscle cells expressing PTGS2 and SCLC2A1. Prostaglandin-endoperoxide synthase 1 (also known as cyclooxygenase 1) inhibitors (acetylsalicylic acid and indomethacin) did not alter the rate of hexose uptake and SCLC2A4 subcellular distribution in L6 myotubes. CONCLUSIONS/INTERPRETATION: This study suggests that certain COX2 (PTGS2) inhibitors can alter glucose homeostasis in vivo by stimulating glucose uptake in skeletal muscles that express PTGS2.

Delayed autoregulation of glucose transport in vascular endothelial cells.

AIMS/HYPOTHESIS: We aimed to characterise the development of autoregulation of glucose transport in vascular endothelial cells and its relationship to 12-lipoxygenase (12-LO) expression. METHODS: Bovine aortic endothelial cells were exposed to 5.5 and 23.0 mmol/l glucose for up to 48 h. The rates of glucose transport, GLUT-1 and 12-LO expression and of 12-hydroxyeicosatetraenoic acid (12-HETE) production were determined. RESULTS: We showed high glucose-dependent downregulation of glucose transport and transporter in vascular endothelial cells within 36-48 h. A similar time-dependent increase in the expression of 12-LO and the generation of its product 12-HETE was also observed. This downregulatory process was prevented when lipoxygenase activity was inhibited. CONCLUSIONS/INTERPRETATION: Vascular endothelial cells, which were previously thought to be "glucose-blind", do in fact downregulate GLUT-1 expression and the rate of glucose transport in response to extended exposure to high glucose concentrations. This slow development of glucose-induced downregulation in vascular endothelial cells is related to the slower basal rate of glucose transport in these cells and the slow induction of 12-LO. These data are interesting in view of current hypotheses that attribute vascular endothelial cell dysfunction in diabetes to the lack of a glucose-induced autoregulatory response.

Leukoreduction and acute rejection in liver transplantation: an interim analysis.

BACKGROUND: Little is known about the effect of blood transfusions and leukoreduction on acute rejection in liver transplantation. The purpose of this study was to assess the impact of leukoreduction on the occurrence of early rejection episodes in liver transplantation. METHODS: In 1999, mandatory leukoreduction was implemented in our program. Data from 339 consecutive liver transplant recipients were analyzed with attention to the time period as a proxy for leukoreduction, the number of transfusions, the wait list status, the hepatitis B or C status, the recipient age, and the type of immunosuppression. RESULTS: Using an early (6-month) rejection-free graft survival model, we observed that introduction of leukoreduction was independently associated with fewer rejection episodes (P =.001). Despite the lower rejection rate, due to a regimen of tacrolimus and antithymocyte globulin, the effect of implementation of leukoreduction remained significant (P =.021). CONCLUSION: The use of leukoreduction is associated with fewer early rejections, irrespective of the type of immunosuppression. These data support an exploration of the immunomodulatory effect of leukoreduction.

Do biliary endoprostheses decrease biliary complications after liver transplantation?

AIM: Most technical complications after orthotopic liver transplantation (OLT) are related to the biliary tree. This report reviews the role of routine intraoperative placement of stents to reduce biliary complications. METHODS: We retrospectively analyzed 396 consecutive OLTs. We reviewed rates of biliary complications after hepaticojejunostomy (HJA) as well as following choledochocholedochostomy (CCA) groups: "experimental" group (routine intraoperative biliary stenting, last 10 months), "recent" control group (nonstented, previous 10 months), "historical" control group (prior to that period of time). RESULTS: All groups were matched for donor/recipient characteristics and for graft cold/warm ischemia time. The overall prevalence of biliary complications was 30.7% after CCA versus 35% after HJA. In the experimental group 21 patients had a 4.8% biliary complication rate compared to the recent control and historical groups, where biliary complication rates were 30% and 32.6%, respectively (P <.05). CONCLUSIONS: The intraoperative use of biliary stents is feasible and appears to decrease the rate of biliary complications. These results support the need for a prospective randomized trial.

Efficacy of mycophenolate mofetil combined with very low-dose cyclosporine microemulsion in long-term liver-transplant patients with renal dysfunction.

BACKGROUND: Cyclosporine (CsA)-induced renal dysfunction is common after liver transplantation. We evaluated the efficacy of tapering CsA to a very low dose and introducing mycophenolate mofetil (MMF) in long-term liver-transplant recipients with renal dysfunction. In addition, we assessed the impact of this strategy on calcineurin inhibition and on transforming growth factor (TGF)-beta levels. METHODS: We prospectively enrolled 19 adult, long-term (>1 year) liver-transplant recipients with a decreased creatinine clearance greater than 25% compared with the first month posttransplant. MMF was introduced, and CsA was tapered to 25 mg twice daily. Calcineurin inhibition and TGF-beta were measured at baseline and 3 months thereafter. RESULTS: The CsA dose was tapered over 13+/-3 weeks. At 1-year follow-up, serum creatinine decreased from 141+/-24 to 105+/-22 micromol/L (P=0.002), creatinine clearance increased from 53+/-9 to 71+/-19 ml/min (P=0.02), and glomerular filtration rate increased from 40+/-13 to 64+/-18 mL/min (P=0.002). The incidence of acute rejection was 29%. Antihypertensive medications were discontinued in 71% of the patients. Although CsA levels decreased significantly, serum TGF-beta did not differ from normal controls, and calcineurin inhibition remained stable. The incidence of gastrointestinal side-effects and leukopenia was 18% and 24%, respectively. CONCLUSION: In long-term liver-transplant recipients with renal dysfunction, the introduction of MMF followed by tapering of CsA to a very low dose resulted in a significant improvement in renal function. However, this strategy maybe associated with a risk of acute rejection. The clinical pertinence of measuring serum TGF-beta levels and calcineurin inhibition remains to be determined.

Elevation of CA-125 level is due to abdominal distension in liver transplantation candidates.

BACKGROUND: CA (cancer antigen) 125 is a serologic marker used in the monitoring of ovarian cancer. Elevated levels are also reported in cirrhosis. We evaluated the range of serum CA 125 levels seen before and after liver transplantation, and examined possible factors associated with CA 125 elevation. METHODS: We examined prospectively 57 consecutive patients with cirrhosis who underwent liver transplantation. CA 125 levels were also measured in two patients with polycystic liver disease. RESULTS: The mean serum CA 125 level before transplantation was 352+/-549 u/ml, compared with 46+/-49 u/ml after transplantation (P<0.001). Multivariate analysis identified the degree of ascites as the only significant predictive variable of preoperative CA 125 level. In five patients who underwent abdominal paracentesis, the mean ascites CA 125 level (951+/-322 u/ml) was higher than that of the serum (619+/-290 u/ml) (P<0.003). In 16 hepatectomy specimens, the grade of staining for CA 125 was 0.8+/-1.4 for the mesothelium of patients with a normal serum CA 125 level, compared with 1.5+/-1.1 in patients with elevated serum levels (P=0.37). Two patients with severe abdominal distension due to polycystic liver disease but without ascites had elevated serum CA 125 levels. DISCUSSION: CA 125 concentration is elevated in the majority of patients with cirrhosis and normalizes after liver transplantation. It is a reflection of the abdominal distention seen in these patients. Therefore, an elevation in CA 125 should not be considered a contraindication to liver transplantation in the absence of evidence of malignancy.

Use of older donor livers is associated with more extensive ischemic damage on intraoperative biopsies during liver transplantation.

Initial poor graft function is associated with increased morbidity and graft loss after liver transplantation. Donor age is a risk factor for the development of initial poor function. The severity of ischemic damage on intraoperative postreperfusion (0Post) allograft biopsy specimens is predictive of subsequent initial poor function. This study was performed to assess whether donor age is a risk factor for the development of ischemic damage on 0Post biopsy specimens. The records of 94 liver transplantations were reviewed. 0Post biopsy specimens were obtained after complete allograft revascularization. The severity of ischemic damage was graded as follows: 0, none; 1, minimal; 2, mild; 3, moderate; and 4, severe. Grafts were defined as older when donor age was 50 years or older. Other independent variables examined included donor cause of death, length of hospital stay, acidosis, serum alanine aminotransferase level, graft cold ischemia time, and degree of steatosis. Older grafts were associated with higher grades of ischemic damage than younger grafts (2.3 +/- 1.0 v 1.3 +/- 1.1; P =.003). Univariate and multivariate analysis identified donor age of 50 years or older as the only significant predictive variable of the severity of ischemic damage. In 16 transplantations involving older grafts, there was no statistically significant association between the severity of ischemic damage and incidence of initial poor function and graft loss. The use of older liver grafts is associated with more extensive ischemic damage immediately after graft reperfusion. Whether this early lesion identifies among older graft recipients those at risk for a worst outcome remains to be determined.

CD44 involvement in experimental collagen-induced arthritis (CIA).

CD44 is a pro-inflammatory cell surface molecule that supports cell migration and cell lodgment in target organs. Therefore, CD44 targeting with specific monoclonal antibodies (mAbs) should be useful for the inhibition of collagen-induced arthritis (CIA) as well as other autoimmune diseases that are dependent on inflammatory cells. In the present paper, we confirm and expand previous reports showing the anti-arthritogenic effect of anti-CD44 mAbs directed against constant epitopes of the CD44 receptor. We demonstrate that such anti-CD44 mAbs can induce resistance to CIA after disease onset. Even accelerated disease developed after two injections of type II collagen was markedly inhibited by IM7.8.1 anti-CD44 mAb. Although KM81 anti-CD44 mAb is a less efficient anti-arthritogenic reagent than IM7.8.1, its Fab' fragments partially inhibit CIA. This finding implies that the antibody blocks CD44 function rather than modulating CD44 cell surface expression or mediating Fc-dependent activities. Histopathological analysis revealed that the anti-CD44 mAb markedly reduces the synovial inflammatory cellular response and the consequent damage to the joint. As CD44 is an alternatively spliced multistructural molecule, similar anti-arthritogenic effects may be achieved by mAbs directed against CD44 isoforms expressed on the pathological cells in question, but not on normal cells, thus leaving the physiological functions intact.

Family violence curricula in U.S. medical schools.

INTRODUCTION: Family violence (adult domestic violence, child abuse/neglect, and elder abuse) is endemic. Victims of family violence are seen in every venue of health care, yet physicians do not routinely inquire about abuse, even when patients present with obvious clinical characteristics. Although a comprehensive health care response is key to a coordinated community-wide approach to family violence, most practicing physicians have never received education in any aspect of family violence, including child abuse. This paper reports the results of a survey of family violence instruction in medical schools. METHODS: A written survey of medical school deans and student representatives of all 126 U.S. medical schools was conducted to (1) determine curriculum content in family violence, (2) assess differences between deans' and students' perceptions of curricular offerings, and (3) compare the results of the current survey with those of an earlier curriculum survey conducted in 1987. RESULTS: The majority of deans reported existing curriculum in all three topic areas of family violence. Compared to the 1987 survey, more deans reported existing curriculum in family violence. However, neither total instructional time nor curriculum during clinical training increased. Moreover, student and dean responses were discrepant regarding awareness of curriculum in domestic violence and elder abuse. CONCLUSION: Despite an increase in the number of schools reporting curriculum in family violence, there does not appear to be increased attention to this problem, at least as measured by time devoted to teaching. Insights from this descriptive survey can promote ongoing efforts toward comprehensive curriculum development in family violence.

First-year medical students' knowledge of, attitudes toward, and personal histories of family violence.

PURPOSE: To assess first-year students' knowledge of, attitudes toward, and personal histories of family violence. METHOD: An anonymous, self-administered, 70-item questionnaire was developed and distributed in the first six months of medical school to 390 first-year students at three New England medical schools in 1991-92. The students were tested on knowledge and asked questions about their personal histories. Attitude questions were scored on a five-point Likert scale. Attitude scales were developed by dividing questions into three content groups (education about family violence, the physician as advocate, acceptability of violent behaviors) and removing items for which inter-item coefficients with all other items were less than .20. Two-tailed t-tests were performed on continuous variables and chi-square tests on categorical variables. Ninety-five percent Cls were calculated for point estimates. RESULTS: In all, 370 students (95%) responded. Of these, 139 (38%) reported personal histories of abuse. The mean knowledge score was 11.3 (of 16 questions). One-third of the students answered more than 75% of these questions correctly; 12% answered less than half of the questions correctly. The women felt more strongly than the men about the need for violence education (p < .001). The students who reported histories of abuse more strongly favored education (p < .05) and advocacy roles (p < .001) for physicians. In addition, the students who had histories of family violence were more likely to report histories of suicidal thoughts (p < .0001). CONCLUSION: The students lacked knowledge but felt a need to learn more about family violence. Family violence curricula should be better integrated into medical education. These curricula should be sensitive to students' attitudes, given the reported high prevalence of personal histories of family violence among students.

Family violence: an overview.

This article reviews the definitions and epidemiology of the several forms of interpersonal violence in family and intimate relationships. Interpersonal violence includes both fatal and nonfatal violence where physical force, or other means, is used by one person with the intent of causing harm, injury, or death: family violence includes child maltreatment, adult intimate-partner violence, and elder mistreatment; abuse refers to a pattern of behaviors organized around the international use of power by one person to control another; and child maltreatment involves the abrogation of adult responsibilities for the care and protection of children, and includes child abuse, child sexual abuse, and child neglect. Violence is a major public health problem in the United States. Half of assault and homicide victims are related to or acquainted with their assailants, as are two-thirds of rape victims. Children and adolescents are at particular risk of violence. The study of interpersonal violence is a complex and evolving held, and is increasingly a part of training and medical practice in academic settings.