The L-arginine/nitric oxide pathway--biological properties and therapeutic applications.
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Biomedical subjects
Publications and source records attributed to B J Rowlands.
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Systemic endotoxemia consistently occurs in jaundiced patients undergoing surgery. Kupffer cell dysfunction is implicated in the development of endotoxemia and its postoperative complications. A novel in situ single-pass hepatic perfusion technique using a fluorescein isothiocyanate-labeled latex probe was developed for measuring Kupffer cell clearance capacity and was applied in an animal model of biliary obstruction. Control rats and rats jaundiced for 1, 2, 3, and 4 weeks' duration were studied. Kupffer cell clearance capacity, plasma bilirubin, endotoxin, and anticore glycolipid concentrations were measured. Maximal hyperbilirubinemia preceded reduced Kupffer cell clearance capacity. Rats jaundiced for greater than 2 weeks had a significantly decreased Kupffer cell clearance capacity but significantly higher endotoxin and anticore glycolipid concentrations. Anticore glycolipid concentrations correlated strongly with systemic endotoxemia and both were inversely correlated with duration of jaundice. Impairment of Kupffer cell clearance capacity may contribute to endotoxemia associated with cholestasis.
The indigenous intestinal flora and an intact mucosa are vital components of body defences against luminal pathogenic bacteria. Disruption of these defences in inflammatory bowel disease may permit bacterial translocation and contribute to disease severity. Support for this hypothesis comes from this study of a hapten-induced rat model of colitis. Induction of colitis was associated with a significantly increased colonic Gram-negative aerobic bacilli count. The results, expressed as log10 [colony-forming units per gram tissue] were: colitic 6.97-8.86 versus control 4.90-6.69 (P < 0.05). Colitis was also associated with a decreased Gram-positive cocci count at 4.00-8.04 versus control 6.45-8.30 (P < 0.05). Bacteria translocated to the mesenteric lymph nodes in five of eight colitic rats (P = 0.01), to the spleen in four (P = 0.04) and to the liver in five (P = 0.01) but to these organs in none of the eight control animals. There was a positive correlation between the severity of colonic inflammation and extent of bacterial translocation in colitic animals (rs = 0.86, P = 0.007).
Jaundiced patients undergoing invasive diagnostic and therapeutic procedures are at increased risk of complications and death. Despite the large number of clinical and experimental investigations carried out to identify relevant risk factors, no single parameter has been found to be consistently useful in predicting morbidity or mortality. Biliary decompression was initially employed by surgeons and subsequently by interventional radiologists. More recently, endoscopic retrograde cholangiopancreatography has provided an alternative route for decompression of the biliary tree and preliminary data using this method are encouraging. Although there are enthusiastic proponents of various therapeutic techniques, controlled trials have not been convincing in highlighting the benefits of biliary drainage or in determining the best approach. This article reviews the literature pertaining to this complex surgical problem; an attempt has been made to balance the advantages and disadvantages of biliary decompression as palliation and/or preliminary treatment for extrahepatic biliary obstruction.
Thirteen nuclear and cellular morphometric variables were measured in 312 cases of colorectal adenocarcinoma. All variables, except nuclear shape factors, differed significantly (P < 0.001) between normal colorectal and tumor tissue. In adenocarcinomas, epithelial nuclei in well-differentiated mucosa tended to be elliptic, while those in poorly differentiated mucosa were more spheric. Increasing values of maximum nuclear and elliptic diameter were associated with progression from none to simple tubule configuration (P < 0.001), none to easily discerned nuclear polarity (P < 0.001), and expanding growth pattern (P < 0.001). Univariate survival analysis revealed that none of the morphometric variables was significantly related to patient survival. Multivariate regression analysis showed that no morphometric variable could add significantly to a model containing the variables of patient age, Dukes stage, and tumor differentiation. Morphometry may be useful in distinguishing malignant from normal tissue and degrees of differentiation, but it is of little prognostic value in colorectal adenocarcinoma.
The resting membrane potential (RMP) of skeletal muscle cells was investigated in a hyperdynamic animal model of sepsis. Reduced membrane potential is known to parallel the clinical course of sepsis and hemorrhagic shock in animal models. Pentoxifylline (PTF), a dimethyl xanthine derivative of the theophylline family, may improve the metabolic effects of sepsis. A cecal ligation and puncture (CL&P) sepsis model in Wistar rats was used in which RMP was measured with differing administration regimes of PTF. The RMP decreased less rapidly in our study when PTF was administered after CL&P and this effect was most noticeable after 18 hours. Administration of PTF before CL&P had little effect on membrane potential fall-off. These results suggest a potential role for PTF in the management of sepsis.
Ischaemia of the large bowel occasionally occurs following abdominal aortic aneurysm repair and may lead to multiple system organ failure (MSOF). Intramucosal acidosis of the sigmoid colon is a good indicator of sigmoid colonic ischaemia. Intramucosal pH of the sigmoid colon was measured using the silicone tonometer in 21 patients undergoing abdominal aortic aneurysmectomy. Samples were taken for plasma endotoxin, tumour necrosis factor (TNF) and interleukin-6 (IL-6) measurements preoperatively, half-hourly during the operation, 2-hourly for the next 12 h, 4-hourly for a further 48 h and 8-hourly thereafter until the fifth day. The intramucosal pH of the sigmoid colon fell to less than 7.00 peri-operatively in 10 patients, four of whom developed diarrhoea; in comparison, this did not occur in any of the 11 whose pH remained greater than 7.00 (p = 0.036). Higher peak concentrations of endotoxin, TNF and IL-6 were found in those patients whose intramucosal pH fell to less than 7.00 compared to those whose pH remained greater than 7.00 (mean +/- S.E.M. pg/ml, endotoxin = 112 +/- 24 vs. 58 +/- 6, p < 0.05; TNF = 26 +/- 8 vs. 7 +/- 2, p < 0.05; IL-6 = 213 +/- 59 vs. 87 +/- 12, p = 0.09). In the two patients who died, both from the group with pH level less than 7.00, concentrations of IL-6 were considerably higher than that in most of the other patients.(ABSTRACT TRUNCATED AT 250 WORDS)
We examined the role of free radical induced lipid peroxidation in lower limb swelling in patients following femoro-popliteal bypass grafting. In 20 patients undergoing this operation blood samples were taken from the femoral vein via a cannula before the femoral artery clamp was applied, just prior to and immediately after clamp release and at 10 min intervals thereafter for 1 h for measurements of malondialdehyde (MDA) and vitamin E. The concentration of MDA was significantly elevated at 40 min after reperfusion (mean +/- S.E.M., 573 +/- 83 pmol/ml) compared to just before clamp release (359 +/- 41 pmol/ml; p < 0.01). This was associated with a corresponding fall in the concentration of vitamin E at the time of peak MDA rise (5.68 +/- 0.28 to 5.29 +/- 0.28 mumol/mM cholesterol, p < 0.05) suggesting its utilisation as an antioxidant. The degree of oedema was related to the changes in MDA and vitamin E. Thus, in the 15 patients with greater than 10% increase in lower limb volume the rise in the concentration of MDA was 364 +/- 44 to 693 +/- 76 pmol/ml (p = 0.0001) while that in the five, whose swelling was less than 10%, was 344 +/- 40 to 559 +/- 243 pmol/ml (p = 0.25). A significant fall in vitamin E was found only in the group with greater than 10% lower limb oedema (5.90 +/- 0.33 to 5.40 +/- 0.34 mumol/mM cholesterol, p < 0.01), in comparison to those with less than 10% swelling (5.01 +/- 0.35 to 5.04 +/- 0.50 mumol/mM cholesterol).(ABSTRACT TRUNCATED AT 250 WORDS)
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The intestinal mucosa protects the body from a large reservoir of intraluminal pathogenic bacteria and endotoxins. This mucosal barrier is disrupted by the inflammation and ulceration of inflammatory bowel disease and may permit the absorption of toxic bacterial products. Systemic endotoxaemia has been demonstrated in ulcerative colitis and Crohn's disease and correlates with the extent and activity of disease. In this study the efficacy of absorbents as antiendotoxin agents in a hapten induced rat model of colitis is investigated. Induction of colitis was associated with systemic endotoxaemia. Enteral administration of terra fullonica and kaolin, but not of charcoal, significantly reduced systemic endotoxaemia (terra fullonica 4.2 (1.40) pg/ml; kaolin 5.29 (1.86) pg/ml; charcoal 32.7 (16.6) pg/ml; water 39.8 (12.6) pg/ml). Data expressed as mean (SE). With increasing severity of colitis, there was a decreasing ability of adsorbent therapy (terra fullonica) to control systemic endotoxaemia. Enteral administration of adsorbents controls gut derived systemic endotoxaemia in experimental colitis in animals and may be a useful antiendotoxin treatment in patients with inflammatory bowel disease.
DNA analysis was assessed by densitometry for 281 cases of colorectal adenocarcinoma. Detection of aneuploidy in a single case rose from 65% if one, to 92.5% when three or more sections, were analysed. Although aneuploid tumours had significantly larger nuclear areas than near diploid tumours (p = 0.009), densitometric measurements showed no association with clinicopathological variables. DNA content determined by densitometry was compared with that from flow cytometry on 465 tissue sections from 241 cases. Aneuploidy assessed by flow cytometry was significantly associated with that determined by densitometry (p < 0.01 for all comparisons), ploidy state being similar in 381 sections (82%, kappa = 0.63, p < 0.001), and 187 cases (77.6%, kappa = 0.57, p < 0.001). Univariate survival analysis showed that DNA densitometric variables had no significant association with survival in (a) all cases, (b) cases without lymph node metastases, or (c) cases without distant metastases. Multivariate regression analysis of densitometric and clinicopathological variables identified Dukes's stage, patient age, and tumour differentiation as the combination of variables most closely related to survival. Densitometric measurement of DNA content could not significantly improve on the prognostic model containing these three variables. It is concluded that, although the assessment of DNA content by densitometry is comparable with that of flow cytometry, conventional histological variables remain the best predictors of prognosis in colorectal cancer.
Flow cytometry was performed upon 312 patients with adenocarcinoma of the colon and rectum, satisfactory results being obtained with 275 (108 diploid, 130 aneuploid and 37 tetraploid). The proportion of nondiploid instances increased from 28 percent if one, to 80 percent when six specimens were assessed per patient. Reproducibility of the technique showed substantial agreement in the assessment of deoxyribonucleic acid ploidy (Kappa value equals 0.74). Increasing values of cells in the diving (G2/M) phase of the cell cycle were associated with little lymphocytic tumor infiltration (p = 0.0002) and extensive tumor fibrosis (p = 0.003). Univariate survival analysis revealed that, although diploid tumors tended to have a better prognosis than nondiploid tumors (p = 0.06), no flow cytometric variable was significantly related to survival. Flow cytometry similarly was not of prognostic value in instances without lymph node metastases or without distant metastases. Multivariate regression analysis of flow cytometric and clinicopathologic variables identified Dukes' stage, patient age and tumor differentiation as the combination of variables most closely related to survival. No flow cytometric variable could significantly improve on the prognostic model containing these three variables. It is concluded that conventional histologic variables remain the best predictors of prognosis in carcinoma of the colon and rectum.
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The prognostic power of the extent of tumour invasion is indisputable; Dukes' classification has repeatedly been proven to be strongly correlated with patient survival. Modifications have led only to confusion, resulting in caution being required in the classification of patients with Dukes' A tumours. In the UK, the American tumour node metastasis and Australian clinicopathological systems are frequently considered too complex for routine clinical use. Meanwhile, Jass's classification may be complicated by observer variation between pathologists, and recent evidence suggests that it offers no advantage over that of Dukes. All the conventional staging systems also fail to take the skill of the surgeon into account when determining outcome. Attempts at quantifying tumour structure have not heralded the expected major advance. For instance, the expense and uncertain prognostic value of tumour DNA content assessed by flow cytometry are likely to restrict widespread use of this technique. It may soon be possible, however, to provide optimum treatment for patients based on individual tumour doubling times. Classification using knowledge of how a small number of cells in the tumour have the ability to invade locally, enter blood vessels and metastasize would also provide important prognostic information on which treatment could be based. Until then, the ease of use and high prognostic power of Dukes' classification ensure that, after 60 years, it is still the 'gold standard' against which all other prognostic classifications in colorectal cancer should be assessed.
A protocol was developed for the analysis of volatile short chain fatty acids in microsamples of feces, short chain fatty acid (SCFA) extraction was from fecal samples using ethanol incorporating n-hexanoic acid as an internal standard. The SCFAs were converted to pentafluorobenzyl esters with alpha-2,3,4,5,6-pentafluorotoluene and analyzed on a gas-liquid chromatograph equipped with an electron capture detector. One hundred milligrams of sample was routinely used but analysis could be carried out on 20 mg of sample.