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R D Hurst

Publications and source records attributed to R D Hurst.

At least 19 recordsLinked to original sources
PubMed2005

In vitro models for the blood-brain barrier.

The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

Animals↗
PubMed2001

A comparison of the induction of immortalized endothelial cell impermeability by astrocytes.

The suitability of various commercially available endothelial cell lines in studies of astrocytic/endothelial cell interactions was assessed. The endothelial-like cell line ECV304 was compared with T24/83, Eahy929, and b.End5 and rat cerebral endothelial cells in their ability, when co-cultured with rat (C6) glioma cells, to form a transendothelial electrical resistance (TEER), an indicator of tight junction formation which is an important property of the blood-brain barrier. As reported previously, the basal TEER of ECV304 cell monolayers was significantly enhanced upon co-culture, an effect reproduced by human 1321N1 astrocytes and primary rat astrocytes. T24/83 cells formed a patchy, gapped monolayer, which produced a poor basal TEER with little in the way of an increase upon co-culture. Similarly, all the other cell monolayers analysed demonstrated poor TEERs that were only moderately increased upon co-culture. These data confirm that while no endothelial cell line with ideal features is available, ECV304 cells remain an appropriate choice especially for studies of astrocyte/endothelial cell interactions.

Animals↗
PubMed2001

Nitric-oxide-induced inhibition of glyceraldehyde-3-phosphate dehydrogenase may mediate reduced endothelial cell monolayer integrity in an in vitro model blood-brain barrier.

The process of nitric-oxide (NO)-induced cellular toxicity may involve energy deprivation since the radical is reported to prevent both mitochondrial oxidative phosphorylation and glycolysis. In order to determine whether these processes are important in NO-induced blood-brain barrier (BBB) dysfunction, we used a cell culture model of the BBB and compared the effects of gaseous NO, potassium cyanide (KCN, a mitochondrial respiratory chain inhibitor) and iodoacetate [IA, an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] on endothelial cell ATP content, GAPDH activity and barrier integrity. NO lead to a rapid breakdown in model barrier integrity and resulted in a reduction in endothelial cell ATP content and GAPDH activity. KCN had no effect on endothelial cell ATP content or barrier integrity, while IA, at a concentration that completely blocked endothelial cell GAPDH activity, resulted in a rapid decline in ATP content but did not lead to a decline in barrier integrity until at least 2 h of exposure. These results indicate that inhibition of endothelial cell GAPDH activity rather than mitochondrial respiration causes an energy deficiency and delayed barrier dysfunction. However, the rapid detrimental effects of gaseous NO on barrier integrity cannot be fully explained by endothelial cell energy depletion and may be related to the actions of the free radical and its products on cellular lipids.

Adenosine Triphosphate↗
PubMed2001

Primary closure of complicated perineal wounds with myocutaneous and fasciocutaneous flaps after proctectomy for Crohn's disease.

BACKGROUND: The purpose of this study is to detail the use of advanced tissue transfer techniques to achieve primary closure of the perineal wound after proctectomy for Crohn's disease. METHODS: Between October 1984 and March 2000, we performed proctectomy with permanent intestinal stoma in 97 patients with Crohn's disease. Twelve of these patients (12.4%) required at least 1 myocutaneous flap to achieve primary closure of the perineal wound. Details of each patient's perioperative course were recorded prospectively. RESULTS: All 12 patients had fistulizing perineal Crohn's disease combined with Crohn's proctitis. Two patients had a simultaneous anal adenocarcinoma. Indications for flap closure included management of large perineal skin defects (n = 11), reconstruction of the posterior vaginal wall (n = 2), and the need to fill a large pelvic dead space (n = 3). (Three patients had a combination of the previous indications). In total, 6 rectus abdominis, 5 gluteus maximus, 1 posterior thigh, 3 chimeric posterior thigh, and 1 latissimus dorsi flaps were performed. Six patients required more than 1 flap. Three patients had complications develop related to the flaps (2 wound hematomas and 1 seroma). Complete perineal healing was achieved in all patients. CONCLUSIONS: Complex tissue flap closure of the perineal wound after proctectomy for perineal complications of Crohn's disease should be considered when simple closure is not possible or when reconstruction of the posterior wall of the vagina is necessary.

Adenocarcinoma↗
PubMed2000

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures.

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Surgical treatment of anorectal complications in Crohn's disease.

BACKGROUND: The purpose of our study was to elucidate features, surgical procedures, and long-term results in patients with anorectal complications of Crohn's disease. METHODS: Physical findings, surgical treatment, and long-term outcome were recorded prospectively for 224 patients who had anorectal complications of Crohn's disease between October 1984 and May 1999. RESULTS: Presenting complications included abscess (n = 36), fistula-in-ano (n = 51), rectovaginal fistula (n = 20), anal stenosis (n = 40), anal incontinence (n = 11), or a combination of features (n = 66). Twenty-four patients did not undergo surgical treatment; the remaining 200 patients underwent 284 procedures. Ultimately, 139 patients (62%) retained anorectal function; reasons for proctectomy in the remaining 85 patients included disease (n = 66), extensive fistular disease (n = 15), fecal incontinence (n = 2), and tight anal stenosis (n = 1). Patients with rectal disease had a significantly higher rate of proctectomy than patients with rectal sparing (77.6% vs. 13.6%, respectively, P<.0001). In the absence of rectal involvement, patients with multiple complications had a significantly higher rate of proctectomy than patients with single complications (23% vs. 10%, P<.05). CONCLUSIONS: A wide spectrum of surgical techniques is required for the management of the diverse anorectal complications of Crohn's disease. Complete healing and control of sepsis can be achieved in the majority of patients. Active rectal disease and multiple complications significantly increase the need for proctectomy.

Abscess↗
PubMed2000

Side-to-side isoperistaltic strictureplasty in extensive Crohn's disease: a prospective longitudinal study.

OBJECTIVE: To report on the results of a prospective longitudinal study of a new bowel-sparing procedure (side-to-side isoperistaltic strictureplasty [SSIS]) in patients with extensive Crohn's disease. METHODS: Between January 1992 and April 1999, the authors operated on 469 consecutive patients for Crohn's disease of the small bowel. Seventy-one patients (15.1%) underwent at least one strictureplasty; of these, 21 (4.5%; 12 men, 9 women; mean age 39) underwent an SSIS. The long-term changes occurring in the SSIS were studied radiographically, endoscopically, and histopathologically. RESULTS: The indication for surgical intervention was symptomatic partial intestinal obstruction in each of the 21 patients. Fourteen SSISs were constructed in the jejunum, four in the ileum, and three with ileum overlapping colon. The average length of the SSIS was 24 cm. Performance of an SSIS instead of a resection resulted in preservation of an average of 17% of small bowel length. One patient suffered a postoperative gastrointestinal hemorrhage. All patients were discharged on oral feedings after a mean of 8 days. In all cases, SSIS resulted in resolution of the preoperative symptoms. With follow-up extending to 7.5 years in 20 patients (one patient died of unrelated causes), radiographic, endoscopic, and histopathologic examination of the SSIS suggests regression of previously active Crohn's disease. CONCLUSIONS: SSIS is a safe and effective procedure in patients with extensive Crohn's disease. The authors' results provide radiographic, endoscopic, and histopathologic evidence that active Crohn's disease regresses at the site of the SSIS.

Adult↗
PubMed2000

The role of laparoscopy and strictureplasty in the management of inflammatory bowel disease.

The surgical management of Crohn's disease has always been a challenging issue for physicians because of concerns of the historical need for repeated surgeries over time, the physiological limitations of a shortened small bowel, and the transmural, fistulizing, and/or skip-lesion nature of the disease. Patients are fearful of the potentially disfiguring results, especially the need for a permanent ostomy. The challenge has been to develop surgical approaches that are bowel sparing and/or minimally invasive. Stricutureplasty has been used with relatively good results as a bowel-sparing procedure for patients with small-bowel Crohn's disease, potentially sparing patients of a short-bowel syndrome. Laparoscopic approaches to Crohn's disease have thus far been mostly limited to ileocecal resections in selected patients, but as more expertise is developed, will hopefully be extended to other surgical procedures in patients with Crohn's disease in the future. Patient criteria, success rates, complications, and economic implications are discussed for each procedure.

Constriction, Pathologic↗
PubMed1999

Upregulation of intercellular adhesion molecule-1 expression on human endothelial cells by tumour necrosis factor-alpha in an in vitro model of the blood-brain barrier.

Adhesion molecules on the endothelial surface of the blood-brain barrier (BBB) play an important role in the pathogenesis of many encephalopathies, including multiple sclerosis (MS) and cerebral malaria (CM). The expression of four surface molecules of relevance to MS and CM on the immortalized human umbilical vein endothelial cell line, ECV304, was investigated using immunofluorescence flow cytometry. We found that ECV304 cells express intercellular adhesion molecule-1 (ICAM-1) and low levels of CD36, but not vascular cell adhesion molecule-1 (VCAM-1) or E-selectin. This expression pattern was unaltered on ECV304 cells which were co-cultured with C6 glioma cells; conditions under which the endothelial cells display enhanced barrier formation. Tumour necrosis factor-alpha (TNF-alpha), which is elevated in MS and CM, decreased the integrity of the barrier in co-cultured endothelial cells and upregulated the expression of ICAM-1 nine-fold. The significance of elevated ICAM-1 expression in relation to the binding of parasitised erythrocytes at the BBB in CM is discussed.

Animals↗
PubMed1999

Butyric acid mediated induction of enhanced transendothelial resistance in an in vitro model blood-brain barrier system.

Previously we reported that the co-culture of non-brain vascular endothelial cells with glioma cells leads to the induction of a more differentiated endothelial cell phenotype which exhibits important properties of the blood-brain barrier (BBB). Recognising the potential for improving the model barrier system with agents known to modify the growth and differentiation of cells in culture we examined the effects of four differentiating agents (butyric acid, dexamethasone, retinoic acid, and dimethyl sulfoxide) on barrier function. Of these agents only butyric acid and dexamethasone resulted in an enhancement (depending on the dose used) of transendothelial electrical resistance (barrier function). The greatest effect was observed with butyric acid in a dose-dependent manner and was slow in onset and only occurred in the endothelial/glial cell co-cultures. These data indicate that butyric acid may be a beneficial agent in optimising conditions necessary for induction of BBB properties in in vitro barrier systems.

Blood-Brain Barrier↗
PubMed1998

Increased inducible nitric oxide synthase protein but limited nitric oxide formation occurs in astrocytes of the hph-1 (tetrahydrobiopterin deficient) mouse.

It has been suggested that decreased tetrahydrobiopterin (BH4) availability may be a useful tool for limiting excessive nitric oxide (NO) formation. In order to test this hypothesis we utilised cultured astrocytes derived from the brain of the hph-1 (BH4 deficient) mouse. In response to treatment with lipopolysaccharide and interferon-gamma (LPS/gammaIFN) levels of BH4 doubled in both wild type and hph-1 astrocytes. However, levels of BH4 in hph-1 astrocytes remained only 25% of the wild type astrocytes. Nitric oxide formation, measured with an NO-electrode, was 45% less from LPS/gammaIFN stimulated hph-1 astrocytes compared with wild type stimulated astrocytes. In contrast, iNOS specific activity and iNOS protein were enhanced in hph-1 stimulated astrocytes by 40 and 60%, respectively when compared with wild type. In conclusion it appears that whilst a decrease in BH4 may limit NO release per se, the possibility and consequences of long term 'over' induction of iNOS protein requires further consideration.

Animals↗
PubMed1998

Decreased endothelial cell glutathione and increased sensitivity to oxidative stress in an in vitro blood-brain barrier model system.

Using a cell culture model of the blood-brain barrier (BBB) we have evaluated the role of endothelial cell glutathione in protecting barrier integrity against nitric oxide (NO)-induced oxidative stress. The co-culture of human umbilical vein endothelial cells (ECV304) with rat (C6) glioma cells, or incubation with glioma cell or primary astrocytic conditioned medium, resulted in a decline in endothelial cell glutathione. Exposure to a single addition of NO gas induced a rapid breakdown in model barrier integrity in endothelial/glioma co-cultures. Addition of NO gas or tumour necrosis factor-alpha (TNF-alpha) also resulted in a loss of membrane integrity, as measured by an enhanced release of lactate dehydrogenase, only from endothelial cells treated with glioma conditioned medium. Furthermore, assessment of viability in endothelial cells grown alone or treated with glioma conditioned medium, by propidium iodide labelled flow cytometry. demonstrated no difference in the number of positively stained cells after NO exposure. These results indicate that when enhanced endothelial monolayer barrier formation occurs via astrocytic-endothelial interactions, cellular glutathione levels are decreased. This renders the barrier cells, under these conditions, more susceptible to oxidative stress but does no necessarily lead to greater cell death.

Animals↗
PubMed1998

The implications of acute pouchitis on the long-term functional results after restorative proctocolectomy.

A prospective study was conducted to determine the implications of acute pouchitis on the long-term functional results of restorative proctocolectomy with J-pouch ileoanal anastomosis (IPAA). Between July 1988 and June 1996, 137 consecutive patients underwent IPAA for treatment of ulcerative colitis. 127 patients (93%) have been available for follow-up. All patients completed diaries detailing bowel habits over a 7-day period at 3, 6, 9, 12, 18, 24 months, and yearly after reestablishment of intestinal continuity. Diaries were completed only during time periods in which patients were not suffering from acute symptomatic pouchitis. Patients with chronic pouchitis (n = 7) were excluded from this study leaving 120 patients for analysis. Fifty patients suffered at least one episode of pouchitis (Pouchitis Group). Seventy patients never had pouchitis (No Pouchitis Group). Patients with a history of pouchitis having significantly more bowel movements per day were more likely to ever have minor incontinence (75% vs. 45%, p < 0.005) or major incontinence (37% vs. 17%, p < 0.02). The stools of Pouchitis Group were less likely to be formed (24% vs. 31%, p < 0.001). Pouchitis Group patients also were more likely to wear a protective pad during the day (21% vs. 7% p < 0.04) or during the night (40% vs. 13%, p < 0.001). Even in the absence of clinically active pouchitis, patients who have suffered at least one episode of pouchitis have a poorer long-term functional result after IPAA. The results of this study suggest that ileal pouchitis may represent a chronic condition that displays episodic symptomatic exacerbations.

Acute Disease↗
PubMed1998

Strictureplasty for Crohn's disease: techniques and long-term results.

Strictureplasty for treatment of symptomatic intestinal strictures secondary to Crohn's disease is being performed with increasing frequency. To determine the overall clinical results after strictureplasty for Crohn's disease, all patients undergoing this procedure were prospectively studied. Between 6/1/89 and 2/1/97, 57 Crohn's disease patients underwent 60 operations utilizing strictureplasties. A total of 109 strictureplasties were performed (90 Heineke-Mikulicz, 6 Finney, and 13 side-to-side isoperistaltic). The 30-day perioperative morbidity was 12%, with complications being less common for patients undergoing elective versus unscheduled operations (p < 0.002). Recurrence of Crohn's disease requiring operation was seen in seven patients after a mean follow-up of 38 months. The estimated cumulative recurrence rate after 2 years was 15 +/- 6% (+/- standard error) and 22 +/- 10% at 5 years. A recurrence developed at the site of the previous strictureplasty in only five cases. Strictureplasty is a safe, effective means of providing long-term surgical palliation to selected patients with Crohn's disease. Perioperative complication rates are comparable to those seen with standard surgical treatment, and recurrences are not excessive.

Adolescent↗
PubMed1998

Alterations in transendothelial electrical resistance by vasoactive agonists and cyclic AMP in a blood-brain barrier model system.

We have previously reported that the co-culture of endothelial and glioma cell lines provides an in vitro model for investigating properties of the blood-brain barrier (BBB). To characterise the model system further we have investigated the effects of vasoactive substances implicated in increases in BBB permeability. Additionally, we have also examined whether activation of cyclic AMP signalling pathways, which elevate cerebral endothelial cell barrier function, similarly modulate our model system. ATP, histamine, bradykinin, and serotonin significantly decreased model BBB transendothelial electrical resistance and manipulations which elevate cyclic AMP enhanced culture resistance. These data indicate that our model BBB system responds in a manner characteristic of cerebral microvascular endothelial cells and the BBB in vivo. These data further emphasize the usefulness of our model system.

Blood-Brain Barrier↗