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To improve therapeutic responses in patients with glioma, new combination therapies that exploit a mechanistic understanding of the inevitable emergence of drug resistance are needed. Intratumoral heterogeneity enables a low barrier to resistance in individual patients with glioma. We reasoned that targeting two or more fundamental processes that gliomas are particularly dependent upon could result in pleiotropic effects that would reduce the diversity of resistant subpopulations allowing convergence to a more robust therapeutic strategy. In contrast to the cytostatic responses observed with each drug alone, the combination of the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induced apoptosis of adult and pediatric glioma cell lines at clinically achievable doses. Resistance that developed was examined using RNA-sequencing and pharmacologic screening of resistant versus drug-naïve cells. Quinolinic acid phosphoribosyltransferase (QPRT), the rate-determining enzyme for de novo synthesis of NAD+ from tryptophan, exhibited particularly high differential gene expression in resistant U87 cells and protein expression in all resistant lines tested. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective and targetable dependency for the panobinostat–bortezomib resistance phenotype. Pharmacologic inhibition of either NAD+ biosynthesis or processes such as DNA repair that consume NAD+ or their simultaneous inhibition with drug combinations, specifically enhanced apoptosis in treatment-resistant cells. Concomitantly, de novo vulnerabilities to known drugs were observed. Implications: These data provide new insights into mechanisms of treatment resistance in gliomas, hold promise for targeting recurrent disease, and provide a potential strategy for further exploration of next-generation inhibitors.

MALT1 is the effector protein of the CARMA/Bcl10/MALT1 (CBM) signalosome, a multiprotein complex that drives pro-inflammatory signaling pathways downstream of a diverse set of receptors. Although CBM activity is best known for its role in immune cells, emerging evidence suggests that it plays a key role in the pathogenesis of solid tumors, where it can be activated by selected G protein–coupled receptors (GPCR). Here, we demonstrated that overexpression of GPCRs implicated in breast cancer pathogenesis, specifically the receptors for Angiotensin II and thrombin (AT1R and PAR1), drove a strong epithelial-to-mesenchymal transition (EMT) program in breast cancer cells that is characteristic of claudin-low, triple-negative breast cancer (TNBC). In concert, MALT1 was activated in these cells and contributed to the dramatic EMT phenotypic changes through regulation of master EMT transcription factors including Snail and ZEB1. Importantly, blocking MALT1 signaling, through either siRNA-mediated depletion of MALT1 protein or pharmacologic inhibition of its activity, was effective at partially reversing the molecular and phenotypic indicators of EMT. Treatment of mice with mepazine, a pharmacologic MALT1 inhibitor, reduced growth of PAR1+, MDA-MB-231 xenografts and had an even more dramatic effect in reducing the burden of metastatic disease. These findings highlight MALT1 as an attractive therapeutic target for claudin-low TNBCs harboring overexpression of one or more selected GPCRs. Implications: This study nominates a GPCR/MALT1 signaling axis as a pathway that can be pharmaceutically targeted to abrogate EMT and metastatic progression in TNBC, an aggressive form of breast cancer that currently lacks targeted therapies.

Agonist CD40 antibodies are under clinical development in combination with chemotherapy as an approach to prime for anti-tumor T cell immunity. However, treatment with anti-CD40 is commonly accompanied by both systemic cytokine release and liver transaminase elevations which together account for the most common dose-limiting toxicities. Moreover, anti-CD40 treatment increases the potential for chemotherapy-induced hepatotoxicity. Here, we report a mechanistic link between cytokine release and hepatotoxicity induced by anti-CD40 when combined with chemotherapy and show that toxicity can be suppressed without impairing therapeutic efficacy. We demonstrate in mice and humans that anti-CD40 triggers transient hepatotoxicity marked by increased serum transaminase levels. In doing so, anti-CD40 sensitizes the liver to drug-induced toxicity. Unexpectedly, this biology is not blocked by depletion of multiple myeloid cell subsets, including macrophages, inflammatory monocytes, and granulocytes. Transcriptional profiling of the liver after anti-CD40 revealed activation of multiple cytokine pathways including TNF and interleukin (IL)-6. Neutralization of TNF, but not IL-6, prevented sensitization of the liver to hepatotoxicity induced with anti-CD40 in combination with chemotherapy without impacting anti-tumor efficacy. Our findings reveal a clinically feasible approach to mitigate toxicity without impairing efficacy in the use of agonist CD40 antibodies for cancer immunotherapy.

Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle formation confirmed structural integrity. Group 2 HA stem antigen designs were identified that, when displayed on ferritin nanoparticles, activated B cells expressing inferred unmutated common ancestor (UCA) versions of human antibody lineages associated with cross-group-reactive, broadly neutralizing antibodies (bNAbs). Immunization of mice led to protection against a lethal homosubtypic influenza virus challenge. These candidate vaccines are now being manufactured for clinical evaluation., Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans.

Diffuse intrinsic pontine gliomas (DIPG) are incurable brain tumors with an aggressive onset. Apart from irradiation, there are currently no effective therapies available for patients with DIPG, who have a median survival time of less than one year. Most DIPG cells harbor mutations in genes encoding histone H3 (H3K27M) proteins, resulting in a global reduction of H3K27 trimethylation and activation of oncogenic signaling pathways. Here we show that the H3K27M mutations contribute to RAS pathway signaling, which is augmented by additional RAS activators including PDGFRA. H3K27M mutation led to increased expression of receptor tyrosine kinases (RTK). A RAS pathway functional screen identified ERK5, but not ERK1/2, as a RAS pathway effector important for DIPG growth. Suppression of ERK5 decreased DIPG cell proliferation and induced apoptosis in vitro and in vivo. In addition, depletion or inhibition of ERK5 significantly increased survival of mice intracranially engrafted with DIPG cells. Mechanistically, ERK5 directly stabilized the proto-oncogene MYC at the protein level. Collectively, our data demonstrate an underappreciated role of H3K27M in RAS activation and reveal novel therapeutic targets for treating DIPG tumors. Significance: These findings identify the H3K27M mutation as an enhancer of RAS activation in DIPG and ERK5 as a novel, immediately actionable molecular target.

Low-grade gliomas (LGGs) are the most common brain tumor affecting children. We recently reported an early phase clinical trial of a peptide-based vaccine, which elicited consistent antigen-specific T cell responses in pediatric LGG patients. Additionally, we observed radiologic responses of stable disease (SD), partial response (PR), and near-complete/complete response (CR) following therapy. To identify biomarkers of clinical response in peripheral blood, we performed RNA sequencing on PBMC samples collected at multiple time points. Patients who showed CR demonstrated elevated levels of T cell activation markers, accompanied by a cytotoxic T cell response shortly after treatment initiation. At week 34, patients with CR demonstrated both IFN signaling and Poly-IC:LC adjuvant response patterns. Patients with PR demonstrated a unique, late monocyte response signature. Interestingly, HLA-V expression, before or during therapy, and an early monocytic hematopoietic response were strongly associated with SD. Low IDO1 and PD-L1 expression before treatment and early elevated levels of T cell activation markers were associated with prolonged progression-free survival. Furthermore, we identified that genes associated with dendritic cell activation of T-cells correlated with IFNγ ELISPOT counts. Currently we are validating the observed response patterns and biomarkers in high-grade glioma patients treated with peptide vaccine immunotherapy. Overall, our data support the presence of unique peripheral immune patterns in LGG patients associated with different radiographic responses to our peptide vaccine immunotherapy. Future clinical trials, including our ongoing phase II LGG vaccine immunotherapy, should monitor these response patterns.

Low-grade gliomas (LGGs) are the most common brain tumor affecting children. We recently reported an early phase clinical trial of a peptide-based vaccine which elicited consistent antigen-specific T-cell responses in pediatric LGG patients. Additionally, we observed radiologic responses of stable disease (SD;n=7), partial response (PR;n=3), and near-complete/complete response (CR;n=2) following therapy. To identify biomarkers of clinical response in peripheral blood, we performed RNA-sequencing on PBMC samples (n=54) collected at multiple time points. Patients who showed CR demonstrated elevated levels of T-cell activation markers accompanied by a cytotoxic T-cell response shortly after treatment initiation. At week 34, CR patients demonstrated both interferon signaling and poly-IC:LC adjuvant response patterns. Patients with PR demonstrated a unique, late monocyte response signature. Interestingly, HLA-V expression, before or during therapy, and an early monocytic hematopoietic response were strongly associated with SD. Finally, low IDO1 and PD-L1 expression before treatment and early elevated levels of T-cell activation markers were associated with prolonged PFS. Overall, our data support the presence of unique peripheral immune patterns in LGG patients associated with different radiographic responses to our peptide vaccine immunotherapy. Future clinical trials, including our ongoing Phase II LGG vaccine immunotherapy, should monitor these response patterns.

Low-grade gliomas (LGGs) are the most common brain tumor affecting children. We recently reported an early phase clinical trial of a peptide-based vaccine, which elicited consistent antigen-specific T cell responses in pediatric LGG patients. Additionally, we observed radiologic responses of stable disease (SD), partial response (PR), and near-complete/complete response (CR) following therapy. To identify biomarkers of clinical response in peripheral blood, we performed RNA sequencing on PBMC samples collected at multiple time points. Patients who showed CR demonstrated elevated levels of T cell activation markers, accompanied by a cytotoxic T cell response shortly after treatment initiation. At week 34, patients with CR demonstrated both IFN signaling and Poly-IC:LC adjuvant response patterns. Patients with PR demonstrated a unique, late monocyte response signature. Interestingly, HLA-V expression, before or during therapy, and an early monocytic hematopoietic response were strongly associated with SD. Finally, low IDO1 and PD-L1 expression before treatment and early elevated levels of T cell activation markers were associated with prolonged progression-free survival. Overall, our data support the presence of unique peripheral immune patterns in LGG patients associated with different radiographic responses to our peptide vaccine immunotherapy. Future clinical trials, including our ongoing phase II LGG vaccine immunotherapy, should monitor these response patterns.

Pediatric High-Grade Gliomas (PHGG), which include Diffuse Midline Gliomas (DMG), are a leading cause of brain tumor death in children. Our recent work has identified extracellular signal-regulated kinase 5 (ERK5) as a critical mediator of cell survival in PHGG. Suppression of ERK5 genetically or pharmacologically leads to decreased cell proliferation and increased apoptosis both in vitro and in vivo in multiple PHGG and H3K27M mutant DMG cell lines. Mechanistically, we show that ERK5 directly stabilizes the proto-oncogene MYC at the protein level, providing rationale to clinically target ERK5. ERK5 contains both a kinase domain (KD) and a transactivation domain (TAD), unlike all other ERKs. Unexpectedly, we found that our ERK5 depleted cells could be partially rescued by an ERK5 kinase domain dead (ERK5-KDD) but TAD intact construct. Additionally, persistent ERK5 depletion does not result in complete growth inhibition and therefore we set out to determine potential adaptation or resistance mechanisms in response to ERK5 loss. To address this, we performed RNA sequencing of DMG cells, comparing control cells to ERK5 knockdown cells, and performed gene-ontology (GO) pathway analysis to identify transcriptional changes that occur in response to ERK5 depletion. We identified 105 differentially expressed genes, and GO analysis identified alternative receptor tyrosine kinase (RTK) gene-expression as one of the top biological processes upregulated in response to ERK5 loss. We validated our top targets at the RNA and the protein level. Our top targets were Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4) and Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), both clinically actionable targets. Our future work will focus on functional validation of these RTKs as potential resistance mechanisms to ERK5 loss. Identification of resistance mechanisms to ERK5 loss will have both biological and translational relevance and may lead to effective therapeutic combinations.

Infants are at high risk of developing chronic, life-threatening disease as a result of hepatitis B virus infection. Universal vaccination of infants against hepatitis B virus, before discharge from the hospital after delivery is recommended as a measure to eradicate infection and associated mortality and morbidity. The purpose of this study was to determine rates of perinatal hepatitis B vaccine (Hep B) administration at a tertiary care center in Iowa and to assess the impact of maternal factors on Hep B uptake.Data concerning mother-infant pairs that received care at one institution from 1/2009 to 4/1/2013 were extracted from the system's electronic medical record. Characteristics of study participants were compared using chi-square tests. Multivariate logistic regression was used to assess the association between each factor and vaccination status, controlling for other characteristics.Of 5663 mother-infant pairs, 5175 (91.4%) infants received Hep B within 7 days after delivery. The majority of those not vaccinated had a medical indication to delay vaccination. Single women were significantly more likely to have an infant not vaccinated, after adjustment for all other factors. Women of minority groups were significantly less likely to have an infant who lacked Hep B at hospital discharge than Caucasian women.Significant improvements have occurred in Hep B rates in the state and region. Infants of single mothers may be at the greatest risk for lacking vaccination at hospital discharge.

Heavy chain diseases are rare B‐cell disorders that are characterized by an overproduction of abnormal and structurally incomplete monoclonal immunoglobulin (Ig) heavy chains and are devoid of light chains. We describe a case of a 62 year‐old African‐American woman with a long history of poorly controlled type 2 diabetes and subsequent probable diabetic nephropathy, hypertension, and recent onset of peripheral neuropathy involving all extremities. Routine laboratory testing revealed a distinct beta spike by urine protein electrophoresis (UPEP). No serum abnormality was noted on serum protein electrophoresis (SPEP). Serum and urine immunofixation demonstrated an IgG heavy chain protein devoid of any corresponding light chains. IgG subclasses identified IgG1 as the predominant IgG component but when we added all the subclasses, the sum, 683.4 mg/dL, failed to come close to our total IgG of 1,770 mg/dL. Therefore, a urine IgG subclass determination was performed in‐house and we identified a subclass 3 gamma chain. In conclusion, we portray a patient with an underlying monoclonal gamma heavy chain disease (HCD) who presented with a complex medical history. The evaluation of IgG subclasses in the context of a HCD may be limited by the capability of the test to recognize the particular IgG fragment. J. Clin. Lab. Anal. 22:146–150, 2008. © 2008 Wiley‐Liss, Inc.

ObjectiveRenal insufficiency continues to be complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. One proposed mechanism of renal injury after suprarenal aortic clamping (above the superior mesenteric artery) and reperfusion (SMA-SRACR) is the loss of microvascular renal blood flow with subsequent loss of renal function. This study examines the hypothesis that the loss of medullary and cortical microvascular blood flow following SMA-SRACR is due to oxygen-derived free radical down-regulation of endogenous medullary and cortical nitric oxide synthesis.MethodsAnesthetized male Sprague-Dawley rats (about 350 g) either had microdialysis probes or laser Doppler fibers inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored. The microdialysis probes were connected to a syringe pump and perfused in vivo at 3 μL/min with lactated Ringer’s solution. The animals were subjected to SMA-SRACR (or sham) for 30 minutes, followed by 60 minutes of reperfusion. Laser Doppler blood flow after the 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion was compared with the time zero (basal) and with the corresponding sham group and reported as percent change compared with the time zero baseline. The microdialysis fluid was collected at time zero (basal) and compared with the dialysis fluid collected after 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion as well as the corresponding sham group. The microdialysis dialysate was analyzed for total nitric oxide (μM) and prostaglandin E2 (PGE2), 6-keto-PGF1α (PGI2 metabolite), and thromboxane B2 synthesis. The data are reported as percent change compared with the baseline time zero. The laser Doppler blood flow and microdialysis groups were treated with either saline carrier, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (30 mg/kg, nitric oxide synthesis inhibitor), L-arginine (400 mg/kg, nitric oxide precursor), superoxide dismutase (SOD, 10,000 U/kg, oxygen-derived free radical scavenger), L-NAME + SOD, or L-arginine + SOD. SOD was given 30 minutes before the reperfusion, and the other drugs were given 15 minutes before reperfusion. The renal cortex and medulla were separated and analyzed for inducible nitric oxide synthase (iNOS), cyclooxygenase-2, prostacyclin synthase, and PGE2 synthase content by Western blot.ResultsSuperior mesenteric artery-SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical nitric oxide synthesis. These changes were further accentuated by L-NAME treatment but restored toward sham levels by L-arginine treatment after SMA-SRACR. The kidney appeared to compensate for these changes by increasing cortical and medullary PGE2 synthesis and release. SOD treatment restored renal cortical and medullary nitric oxide synthesis and blood flow in the ischemia-reperfusion group and in the ischemia-reperfusion group treated with L-NAME.ConclusionsThese data show that nitric oxide is important in maintaining renal cortical and medullary blood flow and nitric oxide synthesis. These data also support the hypothesis that the loss of medullary and cortical microvascular blood flow following SRACR is due in part to oxygen-derived free radical downregulation of endogenous medullary and cortical nitric oxide synthesis.Clinical RelevanceThis study suggests that clinically relevant cortical and medullary vasodilators (nitric oxide and vasodilator prostanoids) are required to maintain microvascular renal cortical and medullary blood flow. This study combines in vivo techniques of microdialysis and laser Doppler flow probes to show that oxygen-derived free radicals are one of the mediators that downregulate endogenous cortical and medullary nitric oxide synthesis, contributing to decreased cortical and medullary blood flow that occurs in the SMA-SRACR model. Prevention or inhibition of oxygen-derived free radical production during SMA-SRACR should be one of the treatment strategies that could help maintain renal microvascular blood flow during the treatment of complex aortic pathology that requires SMA-SRACR.

In a significant number of patients, platelets do not respond to aspirin and/or clopidogrel. Furthermore, this lack of response has recently been shown to affect cardiovascular outcome. At the time of the CAPRIE, CURE, CREDO, and MATCH studies, no in vitro assessment was made to determine platelet response. In vitro platelet response should be considered as an important correctable risk factor for atherosclerotic events.

Many air pollutants which are considered important in ambient (outdoor) air are also found, sometimes at higher levels, in indoor air. With demanding standards having been set for many of these pollutants, both in the workplace and ambient air, consideration of the problems posed by indoor pollution is gaining pace. Studies on exposure to pollutants found in the indoor domestic environment are increasing and are contributing to an already significant compilation of datasets. Improvement in monitoring techniques has helped this process. Documented reports of fatalities from carbon monoxide poisonings are still worrying. However, studies on health effects of non-fatal, long term, low dose, indoor exposure to carbon monoxide and other pollutants, are still inconclusive and too infrequently documented. Of particular concern are the levels of air pollutants found in the domestic indoor environment in developing countries, despite simple interventions such as vented stoves having shown their value. Exposure to biomass smoke is still a level that would be considered unacceptable on health grounds in developed countries. As in the occupational environment, steps need to be taken to control the risks from exposure to the harmful constituents of indoor air in the home. However, the difficulty regarding regulation of the domestic indoor environment is its inherent privacy. Monitoring levels of pollutants in the home and ensuring regulations are adhered to, would likely prove difficult, especially when individual behaviour patterns and activities have the greatest influence on pollutant levels in indoor air. To this end, the Department of Health is developing guidance on indoor air pollution to encourage the reduction of pollutant levels in indoor domestic air. The importance of the effects of domestic indoor air on health and its contribution to the health of the worker are increasingly appreciated. Occupational physicians, by training and interest, are well placed to extend their interests into the environmental field and to focus on this important area.

Objective This study examined the hypothesis that clamping the aorta above the superior mesenteric artery (SMA) followed by suprarenal aortic clamping and reperfusion (SRACR) decreases microvascular blood flow by loss of endogenous medullary and cortical nitric oxide (NO) and prostaglandin (PG) E 2 synthesis. Study Design Anesthetized male Sprague-Dawley rats (350 g) had either microdialysis probes or laser Doppler fibers inserted into the renal cortex to a depth of 2 mm and into the renal medulla at 4 mm. Laser Doppler blood flow was continuously monitored (data reported as percentage of change compared to basal), and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 μL/min with lactated Ringer solution. Dialysate fluid was collected at basal time zero, following 30 minutes of suprarenal aortic clamping (ischemia) followed by 60 minutes of reperfusion and compared to a sham operation. Both groups were treated with saline carrier, indomethacin (INDO) (10 mg/kg, a cyclooxygenase [COX] inhibitor), N G -nitro-l-arginine methyl ester (l-NAME) (20 mg/kg, a NO synthase [NOS] inhibitor), or l-arginine (200 mg/kg, an NO precursor). Dialysate was analyzed for total NO (μM) and PGE 2 (pg/mL) synthesis. The renal cortex and medulla were analyzed for inducible NOS (iNOS) and COX-2 content by Western blot. All data are reported as mean ± SEM, N > 5 and analyzed by analysis of variance. Results SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical NO synthesis. Treatment with l-NAME further decreased blood flow and NO synthesis in the medulla and cortex. l-Arginine restored medullary and cortical NO synthesis and blood flow in the cortex but not the medulla. SRACR did not alter renal medullary or cortical PGE 2 ; however, addition of INDO, COX inhibitor, caused a concomitant decrease in medullary and cortical PGE 2 synthesis and blood flow. Conclusions NO is an important endogenous renal vasodilator that, when maintained can help preserve cortical blood flow following SRACR. These data also suggest that avoidance of COX-2 inhibitors can help maintain endogenous renal cortical and medullary PGE 2 synthesis and thus contribute to maintaining normal blood flow. Clinical Relevance This study is the first to combine in vivo physiologic assays to simultaneously identify clinically relevant intrarenal vasodilators (cortical and medullary) that are required to maintain microvascular blood flow. Identification of endogenous renal cortical and medullary vasodilators responsible for maintaining renal microvascular blood flow will allow development of treatment strategies to preserve these vasodilators following SRACR. Successful preservation of endogenous intrarenal vasodilators will help maintain renal microvascular blood flow and renal function in the treatment of complex aortic pathology that requires SRACR.

Introduction : This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE 2 release by up-regulation of COX-2. Methods : BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto- PGF 1 α , PGE 2 and TxB 2 by EIA. Results : BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE 2 and 6-keto- PGF 1 α by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. Conclusions : BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.

Background: The variable course of renal disease in type 2 diabetes mellitus in part may reflect associated atherosclerotic nephropathy. Methods: To determine the influence of subcritical (

Objective: To test the hypothesis that the physicochemical properties of perfluorochemical liquid used in partial liquid ventilation can influence ventilatory requirements, pulmonary mechanics, microvascular permeability, and vasoactive mediator release in the abnormal lung. Design: Prospective, controlled animal study. Setting: Research laboratory in a university setting. Subjects: Male Sprague-Dawley rats: sham and intestinal ischemia/reperfusion injury. Interventions: Treatment with perfluorochemical partial liquid ventilation (PLV: PP-5 or H-130) or conventional mechanical ventilation (CMV) over 60 mins of superior mesenteric artery occlusion and 60 mins of reperfusion. Measurements and Main Results: Gas exchange, ventilatory requirements, and pulmonary mechanics were measured in vivo. Subsequently, pulmonary vascular resistance, microvascular permeability, and thromboxane were measured by using the isolated perfused lung preparation. PLV with PP-5 required significantly (p

Objective. The aim of this study was to determine whether the prostaglandin synthase inhibitor indomethacin reverses the inflammation and abnormal gallbladder contractility that occur after common bile duct ligation (CBDL), a model of acute cholecystitis. Methods. Gallbladder muscle contractility was studied in vitro in normal, CBDL, and sham-operated guinea pigs. Animals were treated with saline or indomethacin in vivo. Acetylcholine (ACh) was used to directly contract the muscle and electric field stimulation (EFS) to activate intrinsic nerves. Hematoxylin and eosin-stained slides of muscle strips were scored for inflammation. Results. CBDL in saline-treated animals increased the inflammation score and decreased gallbladder muscle contractility to ACh and EFS. Indomethacin decreased the inflammation score and partly reversed the smooth muscle contractile response to ACh 6 and 24 h after CBDL, but not at 48 h. Indomethacin did not reverse the CBDL-induced decrease in nerve-evoked contractions. Conclusion. Gallbladder inflammation and contractile dysfunction after CBDL are partly reversed with indomethacin at 6 and 24 h, but not at 48 h. This suggests that, early in the course of CBDL, the inflammation and contractile dysfunction are, in part, prostaglandin-mediated.

Background. Impaired smooth muscle contractility is important in the pathophysiology of acalculous cholecystitis. Common bile duct ligation (CBDL) is a model of acalculous cholecystitis, producing acute inflammatory changes and decrease in gallbladder smooth muscle contractility. The aim of this study was to determine whether there is coexistent dysfunction of neural efferent motor pathways of the gallbladder after CBDL. Materials and methods. Gallbladder muscle contractility was studied in vitro in normal, CBDL, and sham-operated guinea pigs. Electric field stimulation (EFS; 2–16 Hz) was used to activate intrinsic nerves and exogenous acetylcholine (ACh) was used to directly stimulate the muscle. H&E-stained slides of muscle strips were scored for inflammatory changes. Results. After CBDL, there was a progressive increase in the inflammation score and decrease in gallbladder muscle contractility to ACh. There was also a progressive decline in EFS-induced contractility when expressed as absolute force or normalized to the maximal muscle contractile response to ACh. The nitric oxide synthase inhibitor l -NNA (10 μM) increased EFS-induced contractions by 50 ± 25% (P = 0.05) in CBDL animals but had no effect in sham surgical controls. Conclusions. CBDL with its acute gallbladder inflammation affects gallbladder contractility by two mechanisms: (1) decreased smooth muscle contractility, and (2) decreased neurally mediated contractions. The neurally mediated alterations result from dysfunction of cholinergic excitatory nerves and upregulation of nitric-oxide-mediated inhibition of smooth muscle contractility.

Background. Our previous studies have shown that acute gallbladder (GB) inflammation increases endogenous bradykinin (BK)-stimulated prostaglandin (PG) release and inhibits guinea pig (GP) GB contractility. This study examines the hypothesis that exaggerated PG release following BK stimulation in the inflamed guinea pig GB is due to new protein synthesis of cyclooxygenase 1 (COX-1) and prostacyclin synthase (PS). Materials and methods. Male Hartley GPs (450–550 g) were anesthetized and underwent common bile duct ligation (BDL, a model of acute inflammation). GBs were harvested after 3 days from BDL and control groups. Tissue slices were prepared and placed in oxygenated tissue culture medium at 37°C for 1 h (basal) and for a second hour in medium alone (carrier, Car), medium plus 10 −6 M BK, or medium plus 10 −6 M BK plus cycloheximide 100 μg/ml (BK + CX). The medium was assayed for net release of 6-keto-PGF 1α (PGI 2 metabolite), thromboxane B 2 (TxB 2 ), PGE 2 , leukotriene B 4 (LTB 4 ), and C 4 (LTC 4 ) by enzyme immunoassay and data are reported as nanograms per milligram of protein. GB tissue from control and BDL groups was examined for COX-1, COX-2, PS, and inducible nitric oxide synthase (iNOS) content by Western blot analysis, analyzed by densitometry, and reported as densitometry units. Results. All data were analyzed by ANOVA and t test and reported as means ± SEM, N ≥ 5. BK increased the release of PGI 2 and PGE 2 from the control group and markedly exaggerated release of PGI 2 and PGE 2 from the BDL GP gallbladder. This exaggerated PGI 2 and PGE 2 release was greatly diminished by inhibition of new protein synthesis with cycloheximide. TxB 2 , LTB 4 , and LTC 4 showed no significant differences between any groups. COX-1 and PS contents were significantly elevated in the BDL group compared with control. COX-2 and iNOS were not present in control or BDL GBs. Conclusions. These data suggest that the enhanced BK-stimulated PG release seen in the acutely inflamed GP gallbladder is due to the synthesis of new COX-1 and PS enzymes.

Gallbladder motility is impaired in chroniccholelithiasis but has not been studied in acuteacalculous cholecystitis. The aim of this study was todetermine the effects of acute acalculous inflammation on gallbladder contractility using the commonbile duct ligation (CBDL) model in guinea pigs. Threegroups of guinea pigs were studied: CBDL, normal, andsham surgical controls. Gallbladder dimensions were measured, and muscle strips were used forhistology and in vitro contractility studies. CBDLresulted in progressive gallbladder distension,increased serum bilirubin, and gallbladder inflammation.There was a progressive decline in musclecontractility in the CBDL group as evidenced by adecrease in the contractile response to potassium andbethanechol with the duration of CBDL. In conclusion,CBDL in the guinea pig produces acute gallbladderinflammation and decreased gallbladder musclecontractility. Direct inhibition of muscle function isindicated by impaired contractile responses to potassiumdepolarization and bethanechol stimulation. Although themechanism of the decrease in contractility with CBDL isunknown, we speculate that impaired muscle contractilityis secondary to inflammation and may play a role in the clinicopathology of acute acalculouscholecystitis.

A 65-year-old female presented with upper gastrointestinal hemorrhage thirty years following an aorta-to-right renal artery bypass constructed with saphenous vein. Upper endoscopy demonstrated a duodenal ulcer, and a CAT scan demonstrated aneurysmal degeneration of her renal artery bypass with duodenal impingement. Laparotomy demonstrated erosion of the aneurysm through the posterior wall of the duodenum; extra-anatomic renovascular reconstruction and primary duodenal repair was performed. Although aneurysmal degeneration of intraabdominal saphenous vein grafts is well described and rupture likewise reported, this report represents the first description of an intraabdominal autogenous vein graft aneurysm presenting with gastrointestinal erosion and fistula.

Background: This study examines the hypothesis that specific inhibition of the inducible isoform of nitric oxide synthase (iNOS) will attenuate intestinal reperfusion-induced pulmonary microvascular dysfunction. Methods: Sprague-Dawley rats underwent intestinal ischemia-reperfusion (IR) or sham operation (SHAM). Before injury, the animals received a selective inhibitor of iNOS (S-methylisothiourea sulfate, SMT; L-N 6 -[1-iminoethyl] lysine, L- NIL), a nonselective inhibitor of NOS (N G -nitro-L -arginine methylester, L -NAME) or vehicle (0.9% saline). IR-induced changes in pulmonary microvascular permeability were assessed by quantitating the extravasation of Evans blue dye (EBD)-bound protein into the lung. Pulmonary iNOS activity and content were assessed by radiochemical analysis and Western blot, respectively. Results: There was 60% more EBD within the lungs of animals sustaining IR when compared with controls ( P P > .05). There was significantly greater iNOS activity and enzyme content within the lungs of animals sustaining IR compared with controls. Conclusions: These data are consistent with the hypothesis that the release of nanomolar quantities of nitric oxide generated by iNOS contributes to IR-induced pulmonary microvascular dysfunction. (Surgery 1998;124:457-63.)

Intestinal reperfusion (IR)-induced pulmonary edema has been related to endogenous pulmonary thromboxane A 2 CTxA 2 ) release. This study examines the hypothesis that alveolar macrophages (aMOs) activated during IR are an important cellular source of TxA 2 in this model. Anesthetized Sprague Dawley rats underwent 120 min of intestinal ischemia and 60 min of reperfusion (IR) or sham operation (Sham). aMOs were isolated by bronchoalveolar lavage and incubated in Krebs buffer for 30 min, after which the supernatant was analyzed for TxB 2 (metabolite of TxA 2 ) and prostaglandin E 2 . Other parameters of aMO activation measured included lysosomal enzyme release (β-glucuronidase), superoxide (O 2 - ) release, and procoagulant activity. aMOs from animals sustaining IR generated more than twice as much TxA 2 and prostaglandin E 2 as did those isolated from controls (p

Estrogen has been proposed as a negative risk factor for development of peripheral vascular disease yet mechanisms of this protection are not known. This study examines the hypothesis that estrogen stimulates rat aortic endothelial cell (RAEC) release of PGI2. Male Sprague-Dawley rat abdominal aortic 1-mm rings were placed on 35 mm matrigel plates, and incubated for 1 week. The cells were transferred to a Primaria 60-mm dish and maintained from passage 3 in RAEC complete media and experiments performed between passages 4-10. Cells were incubated with Krebs-Henseleit buffer (pH 7.4) containing carrier or increasing concentrations of beta-estradiol or testosterone for 60 min. The effluent was analyzed for eicosanoid release of 6-keto-PGF1 alpha (6-keto, PGI2 metabolite), PGE2 and thromboxane B2 (TXB2) by EIA (hormone stimulated-basal). Cells were analyzed for total protein by the Bradford method and for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PS) content by Western blot analysis and densitometry. Testosterone did not alter RAEC 6-keto-PGF1 alpha release, whereas estrogen increased RAEC 6-keto-PGF1 alpha release in a dose-related manner. Estrogen preincubation (10 ng/ml) decreased COX-1 and PS content by 40% suggesting that the estrogen-induced increase in male RAEC PGI2 release was not due to increased synthesis of COX-1 or PS. These data support the hypothesis that estrogen stimulation can increase endogenous male RAEC release of PGI2.

Chronic mesenteric ischemia caused by thrombosis of large visceral arteries due to cocaine abuse are reported in two young women. Both cases were managed successfully with visceral revascularization.

Purpose: Elevated lipoprotein (a) (Lp[a]) lipoprotein, total homocysteine, and hypercoagulable states (HCS) have all been implicated as risk factors for premature-onset atherosclerosis. This study was performed to determine the prevalence of these abnormalities in young men with chronic lower extremity ischemia (peripheral vascular disease [PVD]) and to determine their relative strengths as risk factors for premature peripheral atherosclerosis. Methods: We analyzed 50 young white men (age 45 years or younger at onset of symptoms) and compared them with 45 age-matched white male control subjects. Results: Atherosclerotic risk factors were similar in both groups. The mean (±SEM) Lp(a) lipoprotein level was 36±6 mg/dl among the study patients, compared with 14±2 mg/dl among control subjects ( p =0.02, Mann-Whitney). Twenty (40%) study patients and seven (16%) control subjects had Lp(a) lipoprotein levels of 30 mg/dl or greater (atherosclerotic risk threshold) ( p =0.01, odds ratio=3.62, confidence interval (CI) 1.4 to 9.5). Positive HCS panels (antiphospholipid antibodies or deficiencies in antithrombin III, protein C, or protein S) were nearly twice as prevalent in study patients (n=15,30%) as in controls (n=8,18%), but this difference did not achieve statistical significance. The mean total plasma homocysteine level among the study patients was 15.9±0.9 μmol/L, which was not significantly different from the mean control value of 14.7±0.7 μmol/L. Lp(a) lipoprotein was related to risk of premature PVD through a linear logistic relationship ( p =0.003, odds ratio per each 1 mg/dl Lp(a) change was 1.03, CI 1.0 to 1.1). Multivariate analysis with stepwise logistic regression selected two variables: Lp(a) lipoprotein≥30 mg/dl ( p =0.01, odds ratio=3.6, CI 1.3 to 9.9) and family history ( p =0.07, odds ratio=2,2, CI 0.9 to 5.3). Tests of interaction demonstrated no effect between Lp(a) lipoprotein, HCS, and homocysteine. Conclusions: Lp(a) lipoprotein of 30 mg/dl or greater is an independent risk factor for premature peripheral atherosclerosis in men. None of the other examined variables exhibited a significant association with premature PVD.

Background. The natural history of peripheral atherosclerosis in young adults appears to be unfavorable compared with that in older patients. No universally accepted definition of “premature” atherosclerosis exists, however, making comparison of clinical studies difficult. This study examined age-related differences in distribution of atherosclerotic lesions and determined an age threshold at which such differences became apparent. Such a threshold may provide a definition of premature atherosclerosis. Methods. Arteriograms of all patients 49 years of age and younger undergoing evaluation of lower extremity ischemia during the past 5 years were reviewed and the findings were tabulated. Medical records were reviewed to obtain demographic data, assess risk factors, and confirm disease etiology. Exclusion criteria included normal arteriograms (three patients), history of acute or remote trauma (six patients), unclear cause of ischemic symptoms (three patients), arteritis (four patients), aneurysmal disease (one patient), and acute ischemia without prior chronic symptoms (12 patients). For comparison we also reviewed arteriograms performed during the same period in 140 patients older than 50 years of age who had chronic lower extremity ischemia caused by atherosclerosis. Results. The mean age of the 59 study patients was 43.4±5.8 years (median age, 46 years; range, 25 to 49 years). Arteriograms were available in all cases; medical records were available in 54 (92%). Atherosclerosis involved only the aortoiliac segment in 25 patients (42%), the femoropopliteal-tibial arteries alone in 21 (36%), and both levels in 13 (22%). Patients with distal atherosclerosis had a higher prevalence of diabetes than those with proximal atherosclerosis (p=0.004). Ninety-two (66%) of the 140 patients older than 50 years of age had atherosclerosis confined to a single arterial segment, which was not significantly different from the prevalence of single-level disease in the study group. However, 25 (54%) of the 46 study patients with single-level atherosclerosis had aortoiliac disease compared with only 15 (16%) of 92 patients older than 50 years of age with single-level disease (p Conclusions. In contrast to the pattern of disease in older adults, atherosclerosis in young, nondiabetic patients most commonly involves the aortoiliac segment. Differences in lesion distribution become increasingly apparent with age but are most striking between those 49 years of age and younger and those 50 years of age and older. Accordingly, we propose that premature peripheral atherosclerosis be defined as beginning at or before the age of 49 years.

This study examines the hypothesis that neutrophils isolated from animals sustaining intestinal reperfusion (IIR) induce pulmonary microvascular dysfunction. Lungs were isolated from normal Sprague-Dawley rats and perfused with a physiologic buffer in vitro. Neutrophils (2 x 10(6)) isolated from animals sustaining IIR (n = 5) or sham operation (SHAM; n = 6) were infused into the isolated lung model. A third group of lungs underwent in vitro perfusion without exposure to neutrophils (n = 5). Lung injury was assessed by measuring wet to dry weight ratios and pulmonary artery pressure (PAP). Pulmonary ultrastructure was assessed by electron microscopy. The wet:dry ratio of lungs from animals sustaining IIR was greater than that of lungs exposed to SHAM neutrophils (p = .03) or perfusate alone (p = .02). The PAP of lungs exposed to IIR neutrophils was nearly 10 times greater than that of lungs exposed to SHAM neutrophils (p = .003) or buffer alone (p = .006). Ultrastructural examination of lungs exposed to IIR neutrophils demonstrated interstitial edema with occasional focal disruptions in the alveolar capillary endothelial cell membrane whereas lungs exposed to SHAM neutrophils were normal. These experiments provide important in vitro correlation of prior in vivo studies suggesting that neutrophils are important pathogenic mediators of IIR-induced lung injury.

This study examines the hypothesis that pulmonary inducible nitric oxide synthase (iNOS) activity is up-regulated during intestinal reperfusion and that inhibition of NO generation exacerbates pulmonary microvascular dysfunction.Sprague-Dawley rats underwent intestinal ischemia and reperfusion (IIR) or sham operation (SHAM). Pulmonary iNOS activity was measured by quantitating the conversion of L-arginine (L-Arg) to L-citrulline. Another set of animals undergoing IIR or SHAM received an inhibitor of NOS (NG-nitro-L-arginine methylester; L-NAME; 20 mg/kg intravenously), substrate for NO generation (L-Arg; 300 mg/kg intravenously), or vehicle (normal saline solution; 3 ml). Pulmonary microvascular dysfunction was then quantitated by measuring the extravasation of Evans blue dye (EBD) into the lung.Inducible NOS activity was six times greater in the lungs of animals sustaining IIR when compared with SHAM (p = 0.0005). The concentration of EBD within the lungs of animals sustaining IIR was 30% greater than SHAM (p0.05). Inhibiting NOS with L-NAME significantly increased pulmonary EBD concentration of both IIR and SHAM groups when compared with normal saline solution-treated animals (p0.0001). Treatment with L-Arg prevented this IIR-induced increase in pulmonary dye extravasation.These data suggest that pulmonary iNOS activity is up-regulated in animals sustaining IIR and that this may serve as a compensatory protective response to remote organ injury.

Background This study examines the hypothesis that oxygen radicals down-regulate renal nitric oxide synthesis and contribute to renal vasoconstriction after hemorrhage/reperfusion injury. Methods Arterial pressure and renal artery blood flow were measured in anesthetized rats subjected to sham or hemorrhage (30 nun Hg for 30 minutes) followed by blood reperfusion without or with superoxide dismutase, an oxygen radical scavenger. Animals were sequentially injected with 10 mg/kg NG-nitro- l -arginine methyl ester ( l -NAME), a nitric oxide synthase inhibitor, and 200 mg/kg and 400 mg/kg l -arginine, a nitric oxide precursor. Results The l -NAME treatment increased arterial pressure and decreased renal artery blood flow whereas l -arginine decreased arterial pressure and decreased renal blood flow in the sham animals. Hemorrhage/reperfusion injury attenuated the pressure and renal blood flow changes following l -NAME and l -argininene treatment, which was reversed by superoxide dismutase. Conclusions These data suggest that oxygen radicals contribute to the regulation of renal nitric oxide synthesis, contributing to renal artery vasoconstriction following hemorrhage/reperfusion injury.

This study examines the hypothesis that reduced splanchnic blood flow during intestinal reperfusion (IR) is associated with impaired release of the vasodilatory prostanoid PGI 2 . Sprague-Dawley rats underwent occlusion of the superior mesenteric artery (SMA) for 120 min and reperfusion for up to 60 min. SMA blood flow was measured by transonic flow probe and radiolabeled microspheres ( 141 Ce and 103 Ru). Sham-operated animals served as controls (SHAM). Splanchnic eicosanoid release was quantitated by measuring thromboxane B 2 (TxB 2 , stable metabolite of TxA 2 ), 6-keto-PGF 1a (6-keto, stable metabolite of PGI 2 ), and PGE 2 within the portal vein (PV) and inferior vena cava (IVC) of animals sustaining IR and SHAM. SMA flow in IR animals was

Microvascular dysfunction is a prominent feature of the lung injury associated with intestinal reperfusion (IR). This study examines the hypothesis that IR induces pulmonary thromboxane A 2 (TxA 2 ) release, which contributes to pulmonary microvascular dysfunction. Sprague-Dawley rats underwent 120 min of intestinal ischemia and 60 min of reperfusion (IR). Sham-operated animals served as controls (SHAM). Following IR or SHAM, the lungs were perfused in vitro with a modified Krebs buffer and ventilated with room air. Eicosanoid levels within the pulmonary venous effluent and bronchoalveolar lavage (BAL) fluid were determined (TxB 2 , 6-keto-PGF 1a , and PGE 2 ). Pulmonary artery pressure (PAP) was measured continuously and expressed as change from baseline in mm Hg. The dominant eicosanoid generated by the lungs in response to IR was TxB 2 . TxB 2 levels in the pulmonary venous effluent of IR lungs were 75% greater than controls (P = 0.005). Similarly, TxB 2 levels in the BAL were more than 2.5 times controls (P = 0.001). The change in PAP of lungs from IR animals was significantly greater than that of controls (4.1 ± 1.5 vs 0.3 ± 0.54 mm Hg, IR vs SHAM, P = 0.01). The increased PAP associated with IR lungs was prevented by cyclooxygenase inhibition with indomethacin (-1.28 ± 0.29 mm Hg, P < 0.05) and thromboxane synthetase inhibition with imidazole (-1.75 ± 0.95 mm Hg, P < 0.05). These experiments support the hypothesis that IR up-regulates endogenous pulmonary TxA 2 release. Furthermore, the local release of TxA 2 by the lung may contribute to the microvascular dysfunction characteristic of IR-induced lung injury. © 1995 Academic Press, Inc.

Purpose: The purpose of this study was to determine whether aortic size influences late patency of aortofemoral reconstructions in men and women with premature atherosclerosis.Methods: We studied 37 consecutive young women (mean age ± SEM, 44 ± .7 years) and 36 young men (mean age 44 ± .8 years) who underwent elective operations for aortoiliac occlusive disease during the past 15 years. Clinical data from patients with occluded versus patent grafts were studied, and angiographic findings in patients with occluded versus patent grafts and in young adult patients in a control group (n = 50) who had nonatherosclerotic conditions were compared.Results: Twenty (54%) women and 17 (47%) men had limb occlusions within a mean of 31 ± 6 months. These occlusions resulted in major amputations in 17 (23%) patients. When patients with occluded versus patent grafts were compared no differences were found in age, sex, symptoms, type or number of atherosclerotic risk factors, or operative details. As a whole, patients in the study group had smaller infrarenal aortas than did patients in the control group (p = 0.009). Women with limb occlusions had smaller infrarenal aortas than did women with patent grafts (p = 0.03) or healthy female patients in the control group (p = 0.002). Men with limb occlusions had smaller infrarenal aortas than did men with patent grafts (p = 0.017) or male patients in the control group (p < 0.001). Angiographic outflow scores were not different in men or women with occluded versus patent grafts. Among all variables studied proportional hazards regression analysis indicated that only mean infrarenal aortic diameter was predictive of graft patency.Conclusions: These data suggest that late graft failure after aortofemoral reconstruction is common in young adults. Patients with premature atherosclerosis have smaller infrarenal aortas compared with young adults in a control group, making them more vulnerable to symptoms from atherosclerotic lesions. Size of the infrarenal aortic segment is a critical determinant of late graft patency regardless of sex. (J VASC SURG 1995;21:296-306.)

Rabbit common bile duct ligation has been shown to concomitantly increase levels of gallbladder taurodeoxycholic acid and gallbladder eicosanoid release. This study examines the hypothesis that taurodeoxycholic acid, a known chemical mediator of gallbladder inflammation, stimulates endogenous gallbladder eicosanoid release. Male New Zealand white rabbits were anesthetized, gallbladders removed and perfused in vitro with Krebs-Henseleit buffer (pH 7.4, 37 degrees C) at 1 ml/min with increasing doses of taurodeoxycholic acid (0, 10, 30 and 100 mM) added to the perfusate. The effluent was collected at 15, 30, 60 and 120 min of perfusion and assayed for 6-keto-PGF1 alpha (PGI2 metabolite), PGE2, and thromboxane B2 (TXB2) by enzyme immunoassay. Taurodeoxycholic acid increased gallbladder eicosanoid release in a dose-related manner with 6-keto-PGF1 alpha and PGE2 release 10-fold higher than TXB2. Indomethacin (1.5 mM) decreased gallbladder eicosanoid release by 50% in the gallbladders perfused with 30 mM taurodeoxycholic acid, demonstrating that the increased gallbladder eicosanoid release was due to de novo synthesis. These findings suggest that the increased release of gallbladder PGI2 and PGE2 described in animal models of cholecystitis may, in part, be related to increased gallbladder bile levels of taurodeoxycholic acid.

This study examines the hypothesis that hydroxyl radical (OH.) generation during intestinal reperfusion activates the complement system forming the potent chemotaxin C5a. Anesthetized Sprague-Dawley rats underwent 120 min of intestinal ischemia and 60 min of reperfusion (IIR). Complement (C) activation was assessed by measuring total plasma C activity and C5a-related chemotaxis and leukoaggregation. Dimethylthiourea and the iron chelator deferoxamine were utilized to assess the role of the OH. in the activation of C in this model. Sham-operated animals served as controls. Total plasma C activity of animals sustaining IIR was 64% of controls (p < .05). Plasma of animals sustaining IIR induced greater chemotaxis and leukoaggregation than plasma from sham-operated groups (p < .05). Treatment of IIR plasma with anti-C5a antibody ameliorated the enhanced leukoaggregation characteristic of IIR plasma. Pretreatment with dimethylthiorea and deferoxamine prevented reperfusion-induced activation of complement and inhibited the chemotactic activity of plasma from IIR animals. These data are consistent with the hypothesis that IIR activates complement and that the OH. generated during reperfusion may be one mechanism by which C is activated in this injury model.

The stimulus for increased gallbladder eicosanoid synthesis during cholecystitis is unknown. This study examines the hypothesis that increased intragallbladder pressure stimulates endogenous gallbladder eicosanoid release. Rabbit gallbladders were perfused in vitro at 1 ml/minute with oxygenated Krebs-Henseleit buffer and subjected to 0, 12 or 24 mm Hg of intraluminal gallbladder pressure. Release of 6-keto-PGF1 alpha infinity PGE2 and thromboxane B2 were measured in all groups after 15 and 30 and 60 minutes of perfusion by enzyme immunoassay and gallbladders were examined histologically. Increasing intraluminal gallbladder pressure concomitantly increased gallbladder 6-keto-PGF1 alpha release 2 fold or more at all time of perfusion and altered gallbladder mucosal architecture by increasing basolateral edema in the submucosal space. Infusion of indomethacin (10 micrograms/ml in the perfusion media) decreased 6-keto-PGF1 alpha release from the in vitro perfused gallbladders subjected to 24 mm Hg by 70%. Increased gallbladder eicosanoid release during early cholecystitis may in part be related to the physical force of increased gallbladder intraluminal pressure on the gallbladder mucosa.

The role of endogenous splanchnic eicosanoids in mediating splanchnic vasoconstriction induced by the leukotriene C4 (LTC4) was examined during mild hemorrhage/reperfusion injury. Male Sprague-Dawley rats were anesthetized and subjected to sham or acute hemorrhage for 30 minutes, to 30 mm Hg, followed by blood reperfusion (SK+R). The superior mesenteric artery was cannulated and removed with its end-organ intestine (SV+SI preparation) and perfused in vitro with oxygenated Krebs-Henseleit buffer. Perfusion pressure was constantly recorded. Net SV+SI release of 6-keto-PGF1 alpha, PGE2 and thromboxane B2 were analyzed by enzyme immunoassay after LTC4 stimulation. Leukotriene C4 increased perfusion pressure and decreased the ratio of 6-keto-PGF1 alpha to thromboxane release (but not PGE2 to thromboxane B2) in the sham group. Hemorrhage/reperfusion increased perfusion pressure and decreased the ratio of 6-keto-PGF1 alpha to thromboxane B2. Mild hemorrhage/reperfusion increased LTC4-induced splanchnic vasoconstriction in part by decreasing the release ratio of endogenous splanchnic PGI2 to thromboxane B2.

Context Ethnic/racial minority and nonminority families' perceived barriers to discussing preemptive living related kidney transplantation (LRKT) and their views on the potential value of health care professionals trained to address barriers are unknown. Objective, Setting, and Participants To collect pilot data for evaluating perceived barriers to preemptive LRKT and to inform the development of a culturally sensitive intervention to improve families' consideration of LRKT. In 4 structured group interviews of African American and non—African American patients (2 groups) with progressing chronic kidney disease and their family members (2 groups), participants' perceived barriers to initiating LRKT discussions and their views regarding the value of social workers to support discussions were explored. Results Patients' barriers included concerns about their (1) ability to initiate discussions, (2) discussions being misinterpreted as donation requests, (3) potential burdening of family members, (4) uncertainty about when to initiate discussions, and (5) inducing guilt or coercing family members. Family members' barriers included (1) feeling overwhelmed by patients' illness, (2) patients' denial about their illness, (3) caregiver stress, and (4) uncertainty about their own health or the health of other family members who might donate or need a kidney in the future. Participants reported that social workers could facilitate difficult or awkward discussions and help families understand the LRKT process, address financial concerns, and cope emotionally. Themes were similar between African Americans and non—African Americans. Conclusions Families identified several barriers to discussing preemptive LRKT that could be addressed by social workers. Further research must be done to determine whether social workers need to tailor interventions to address families' cultural differences.

Purpose: This study was designed to determine the prevalence of unsuspected renal artery stenoses (RAS) in patients undergoing arteriography for evaluation of aneurysmal or occlusive vascular disease and whether symptomatic coronary artery disease (CAD) is more prevalent among patients with unsuspected RAS. Methods: We reviewed the arteriograms and medical records of 346 consecutive patients with aortic aneurysms or occlusive disease in whom RAS was unsuspected on clinical grounds. Results: Aortography revealed unsuspected RAS (50% or greater diameter loss) in 98 patients (28%). Patients with RAS had a higher prevalence of mild, controlled hypertension ( p p p = 0.002). The correlation between the prevalence of CAD and RAS severity was highly significant ( p p = 0.001), aortic aneurysm disease ( p = 0.01), and hypertension ( p = 0.001). RAS measuring 75% or greater diameter loss was associated with the highest estimated odds ratio: patients with this degree of RAS had a fourfold increase in the prevalence of clinically overt CAD. We also evaluated the relationship between RAS, mesenteric artery stenosis, and CAD; although RAS was more frequent among patients with mesenteric artery stenoses, mesenteric artery stenoses were not associated with CAD. Conclusions: Unsuspected RAS is common among patients with peripheral vascular disease and should be considered an independent marker for CAD. (J VASC SURG 1993;18:433-40.)

Purpose: This study was performed to determine the natural history of patients with symptoms of claudication and systolic toe pressures (TP) of 40 mm Hg or less. Methods: We followed the clinical course of 56 men with stable claudication and TP of 40 mm Hg or less. All TP measurements were performed on at least two occasions 6 months apart. Primary end points included development of rest pain, tissue loss, or gangrene. The clinical course of 56 case controls with TP greater than 40 mm Hg matched for age, sex, and race was used for comparison. Results: During a mean (±SD) follow-up time of 31 ± 4 months, 37 (66%) patients with TP of 40 mm Hg or less remained stable, and 19 (34%) had ulceration ( n = 10), rest pain ( n = 6), or gangrene ( n = 3). Nine (24%) of the 37 stable patients had gradual improvement of TP values greater than 40 mm Hg. Among the 19 patients whose conditions deteriorated, eight (42%) patients underwent successful bypasses, and five (26%) patients required amputations. Two patients who had rest pain had spontaneous resolution, and three patients who had ulcerations healed without intervention. In contrast, five (9%) of the case controls with TP greater than 40 mm Hg had rest pain ( n = 2) or gangrene ( n = 3) ( p = 0.003). Among patients with TP of 40 mm Hg or less, there were no statistically significant differences between the stable patients and patients with deteriorating conditions in age, ankle-brachial indexes, or risk factors (including diabetes mellitus). However, diabetes conferred a higher probability of clinical deterioration ( p = 0.005, Kaplan-Meier). Conclusions: In patients with symptoms of intermittent claudication, TP of 40 mm Hg or less portends clinical deterioration. Patients with diabetes in this group have a significantly higher risk of development of critical ischemia. Close scrutiny is warranted. (J VASC SURG 1993;18:506-11.)

Gallbladder explants from control rabbits and rabbits subjected to bile duct ligation (BDL) for 24 and 72 h (cholecystitis model) were placed in cell culture to determine the source for increased gallbladder prostanoid synthesis during cholecystitis. Cultures from control and 24 h BDL gallbladders grew spindle shaped fibroblasts which did not exhibit increased prostanoid synthesis. 72 h BDL gallbladder cell cultures grew large polygonal shaped cells which appeared to be ‘stimulated fibroblasts’ by light and electron microscopy and were associated with increased basal and bradykinin stimulated 6-keto-PGF1α release and increased content of prostacyclin synthase when measured by enzyme immunoassay and protein immunoblot analysis respectively. Use of bradykinin antagonists showed that the bradykinin BK2 subtype receptor was the most prominent in the 72 h BDL cell cultures. The ‘stimulated fibroblasts’ were the source of bradykinin stimulated gallbladder 6-keto-PGF1α synthesis in the inflamed rabbit gallbladder which was mediated by the bradykinin B2 subtype receptor.

OBJECTIVE: This study evaluated the morbidity, mortality, and intermediate term follow-up of patients undergoing replacement of their aortoiliac-femoral systems with lower extremity deep and superficial veins. SUMMARY BACKGROUND DATA: The most commonly used treatment for aortic prosthetic infection is ectopic bypass and removal of the prosthesis. The overall mortality rate with this approach is approximately 20%, with an amputation rate of 10% to 14%. Other limitations include thrombosis of the ectopic bypass leading to limb loss, reinfection of the ectopic bypass, and aortic stump blowout. Dissatisfaction with this approach has led the authors to develop the following. METHODS: A neo-aortoiliac system (NAIS) was fashioned from lower extremity deep veins (DV), greater saphenous veins (GSV), or both in patients with infected aortobifemoral prosthesis (n = 17) and other complex aortic problems (n = 3). Removal of infected prosthetic material, harvest of vein, and creation of NAIS was performed as a single-staged procedure. RESULTS: The in-hospital mortality and amputation rates were 10% each. The mean (+/- standard deviation [SD]) operative time was 6.5 +/- 1.8 hours and the blood transfusion requirement was 4 +/- 3 units. Four patients experienced postoperative gastrointestinal complications with peritonitis and sepsis; NAIS vein graft resisted infection and remained intact. The mean follow-up time was 22.5 +/- 16 months. NAISs constructed from GSVs were prone to the development of focal stenoses requiring intervention or diffuse neointimal hyperplasia leading to occlusion. In contrast, all NAISs from larger caliber DVs have remained widely patent. The failure rate of GSV NAISs was 64%, compared to 0% for DV NAISs (p = 0.006). Despite the high failure rate in patients with GSV NAISs, none has required amputation. In patients who had DVs harvested for NAIS reconstruction, limb edema and other signs of venous hypertension have been minimal. CONCLUSION: NAIS reconstruction from lower extremity veins is a successful option in patients with extensive aortic prosthetic infection and other complex aortic problems.

This study examined the hypothesis that burn injury inhibits the release of splanchnic PGI2, a potent endogenous vasodilator of the splanchnic vascular bed. Male Hartley Guinea Pigs (350 grams) were anesthetized, shaved and subjected to a full thickness scald burn comprising 45% of the total body surface area (or sham burn). The animals were treated with intravenous or topical lazaroid, a 21-aminosteroid U75412E. After recovery for 24 hours, the animals were anesthetized, and the superior mesenteric artery was cannulated and removed with its intact intestine (SV + SI). The SV + SI was perfused in vitro with oxygenated Krebs buffer. The venous effluent was assayed for 6-keto-PGF1 alpha, PGE2 and thromboxane B2 by enzyme immunoassay. Acute burn injury decreased SV + SI release of 6-keto-PGF1 alpha by 57% but did not alter release of PGE2 or thromboxane B2. Treatment of the animals with topical lazaroid and not intravenous lazaroid restored SV+SI 6-keto-PGF1 alpha release to control levels. These data showed that topical lazaroid therapy maintained endogenous splanchnic PGI2 release following acute burn injury. Maintaining endogenous splanchnic PGI2 release by topical lazaroid treatment may be one strategy to avoid splanchnic ischemia following acute thermal injury.

Renal artery stenoses (RASs) that are unsuspected on clinical grounds are common in patients with peripheral vascular disease. These lesions may be missed in patients with abdominal aortic aneurysms (AAAs) who undergo arteriography based on selective clinical indications alone. We reviewed 98 consecutive patients with AAAs to determine how often selective arteriography would fail to diagnose unsuspected RAS. The location and degree of RASs were noted on preoperative arteriograms, which were routinely obtained in all patients considered for AAA repair during the study period. Medical records were studied to determine the presence of selective clinical indications for preoperative arteriography (moderate to severe hypertension or renal insufficiency). Twenty-four patients had a significant (or = 50% diameter loss) RAS, and 10 patients had a severe (or = 75% diameter loss) RAS or renal artery occlusion. Patients with significant RAS had a higher incidence of hypertension (p = 0.035) and renal insufficiency (p = 0.018). All 10 patients with severe RASs required at least two antihypertensive medications to control their hypertension compared with 22 of 88 patients who did not have a severe RAS (p0.001). Forty-three patients had an indication for arteriography according to selection criteria (renal insufficiency, moderate or severe hypertension, or both). No severe RASs were found in patients who did not meet the selection criteria for arteriography. Using arteriography based on the presence of hypertension requiring two or more medications for control will detect the vast majority of severe, unsuspected RASs in AAA patients.