Biomedical subjects
G Carter
Publications and source records attributed to G Carter.
Roughening and ripple instabilities on ion-bombarded Si.
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Transgenic animals demonstrate a role for the IL-1 receptor in regulating IL-1beta gene expression at steady-state and during the systemic stress induced by acute pancreatitis.
Interleukin-1 (IL-1) gene expression is selectively induced in tissues involved in multisystem organ failure during acute pancreatitis, suggesting a role in the pathogenesis of distant organ dysfunction. This study was undertaken to investigate the mechanism of pancreatitis-induced end organ cytokine production and to better understand the processes by which IL-1 production is regulated. Seventy adult male transgenic mice in which the type 1 IL-1 receptor had been deleted by gene targeting in embryonic stem cells were utilized (homozygous -/- IL-1R knockout). Acute pancreatitis was induced by one of two methods: (A) IP injections of caerulein (50 microgram/kg/hr x 4) with animals sacrificed at 0, .5, 1, 2, 4, 6, and 8 hr; (B) 48-hr exposure to a choline deficient ethionine supplemented (CDE) diet with animals sacrificed at 0 and 72 hr. Knockout animals were compared to strain-specific control mice expressing the normal wild-type IL-1 receptor gene in which pancreatitis was similarly induced. The severity of pancreatitis was stratified by serum amylase, lipase, and blind histologic grading. IL-1 mRNA production was determined within the pancreas, lungs, liver, and spleen by quantitative differential RT-PCR. Deletion of the IL-1R1 attenuated the severity of pancreatitis, reaching statistical significance in the less severe edematous model. There was little or no constitutive expression of IL-1 mRNA within any of the tissues examined from wild-type animals; however, knockout animals showed elevated steady-state levels in each tissue. IL-1 mRNA became detectable in all tissues of wild-type animals shortly after either form of pancreatitis became apparent and increased significantly with worsening pancreatitis. Despite the attenuated pancreatitis, knockout animals produced significantly greater levels of IL-1 mRNA in each tissue, typically demonstrating a 30-50% increase over time matched IL-1 mRNA production in wild-type animals which was not pancreatitis model dependent. We conclude that genetic deletion of IL-1 receptors results in the overproduction of IL-1 mRNA in organs known to produce cytokines during pancreatitis even when the severity of pancreatitis is lessened. This suggests that a negative feedback loop exists between the IL-1 receptor and IL-1 gene expression.
Timing of tumor necrosis factor antagonism is critical in determining outcome in murine lethal acute pancreatitis.
BACKGROUND: Tumor necrosis factor (TNF) is produced in large amounts within the pancreas, lungs, and liver during severe acute pancreatitis and is believed to mediate many of the detrimental consequences typical of this disease. Investigations into the benefit of TNF antagonism have suggested that TNF may also mediate processes that are protective to the host. METHODS: With the hypothesis that timing plays a role in these dissenting views, TNF was antagonized either prophylactically or therapeutically with a recombinant form of the soluble type I TNF receptor (TNFbp) during a lethal model of necrotizing pancreatitis induced by feeding a choline-deficient diet. Mortality was determined for 10 days in 390 female mice divided into three groups: control, TNFbp early (time, 0 to 5 days), and TNFbp late (time, 1.5 to 5 days). Pancreatitis severity and cytokine production were assessed daily. RESULTS: Animals in the control group had a 75% mortality rate that was significantly decreased by prophylactic TNF blockade (64%, p < 0.05). Delaying TNF antagonism until serum cytokines were elevated and pancreatitis was manifest decreased mortality to 42% (p < 0.001 versus control, p < 0.01 versus early). Early and late TNF blockade decreased pancreatic edema and serum amylase, lipase, interleukin-1, and interleukin-6 (all p < 0.05) but not TNF. Late antagonism typically resulted in the greatest attenuation of all these parameters. CONCLUSIONS: Blockade of TNF by the administration of a soluble TNF receptor attenuates the severity of pancreatitis, decreases the production of associated inflammatory cytokines, and significantly improves survival. Delaying antagonism until pancreatitis is manifest and circulating cytokines are elevated but not yet maximal appears to be more protective than simple prophylactic TNF antagonism.
Tissue-specific regulation of glutamine synthetase gene expression in acute pancreatitis is confirmed by using interleukin-1 receptor knockout mice.
BACKGROUND: Acute pancreatitis causes a pronounced depletion of plasma and muscle glutamine pools. In several other catabolic disease states expression of the enzyme glutamine synthetase (GS) is induced in lung and muscle to support glutamine secretion by these organs. The hormonal mediators of GS induction have not been conclusively identified. We used mice deficient for the expression of the type 1 interleukin-1 receptor (IL-1R1 knockout mice) to investigate the expression of GS during acute edematous pancreatitis. METHODS: Acute edematous pancreatitis was induced in adult male wild-type and IL-1R1 knockout mice by means of the intraperitoneal administration of cerulein, and their conditions were monitored. Five organs, including lung, liver, gastrocnemius muscle, spleen, and pancreas, were assayed for relative GS messenger RNA (mRNA) content by Northern blotting. RESULTS: The ultimate severity of pancreatitis was reduced by IL-1R1 deficiency. GS mRNA levels increased during progression of pancreatitis in lung, spleen, and muscle tissue from each group. No consistent increase in GS mRNA level was observed in liver. IL-1R1 deficiency did not affect GS mRNA expression in lung tissue but consistently retarded GS induction in the spleens of knockout animals. IL-1R deficiency altered the kinetics of GS induction in muscle. CONCLUSIONS: Cerulein-induced experimental pancreatitis causes an induction in GS mRNA levels in a tissue-specific fashion. IL-1R1 deficiency reduced the ultimate severity of the condition and altered the induction of GS mRNA in the spleen and muscle.
Active interleukin-1 receptor required for maximal progression of acute pancreatitis.
OBJECTIVE: The authors' aim was to determine the requirement for an active interleukin (IL)-1 receptor during the development and progression of acute pancreatitis. SUMMARY OF BACKGROUND DATA: Interleukin-1 is a pro- inflammatory cytokine that has been shown to be produced during acute pancreatitis. Earlier animal studies of moderate and severe pancreatitis have shown that blockade of this powerful mediator is associated with attenuated pancreatic destruction and dramatic increases in survival. The exact role played by IL-1 and the requirement for activation of its receptor in the initiation and progression of pancreatitis is unknown. METHODS: Conventional and IL-1 receptor "knockout" animals were used in parallel experiments of acute pancreatitis induced by intraperitoneal injection of cerulean (50 microg/kg every 1 hour X 4). The conventional mouse strain had the IL-1 receptor blocked prophylactically by means of a recombinant IL-1 receptor antagonist (10 mg/kg injected intraperitoneally every 2 hours). The second mouse strain was genetically engineered by means of gene targeting in murine embryonic stem cells to be devoid of type 1 IL-1 receptor (IL-1 receptor knockout). Animals were killed at 0, 0.5, 1, 2, 4, and 8 hours, with the severity of pancreatitis determined by serum amylase, lipase, and IL-6 levels and blind histologic grading. Strain-specific controls were used for comparison. RESULTS: The genetic absence of the IL-1 receptor or its pharmacologic blockade resulted in significantly attenuated pancreatic vacuolization, edema, necrosis, inflammation, and enzyme release. Serum IL-6, a marker of inflammation severity, was dramatically decreased in both groups. CONCLUSIONS: Activation of the IL-1 receptor is not required for the development of pancreatitis but apparently is necessary for the maximal propagation of pancreatic injury and its associated inflammation.
Changes in bone density and biochemical markers of bone turnover in pregnancy-associated osteoporosis.
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A simple measure of insulin resistance.
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Antidepressants and suicide. Study analyses were flawed.
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A screen for RAS mutations in individuals at risk of secondary leukaemia due to occupational exposure to petrochemicals.
Occupational exposure to petrochemicals, in particular benzene, has been identified as a risk factor in the development of acute leukaemia. A cohort of exposed (n = 44) and non-exposed individuals (n = 19) from the same petrochemical installation were screened by polymerase chain reaction (PCR) followed by oligonucleotide hybridization (ONH) for the presence of mutations in the H, K, and NRAS cellular proto-oncogenes. A KRAS mutation was detected in one individual from the exposed group who was haematologically normal at the time of sampling. The presence of this mutation was confirmed by nude mouse tumorigenicity assay and positively identified as a K13 Gly-Asp substitution by cloning and sequencing.
Raised serum 11-deoxycortisol in men with persistent acne vulgaris.
OBJECTIVE: Acne vulgaris is androgen dependent but the hormonal mechanisms are unclear. Although there have been many studies of serum hormones in women with acne there are few studies in men and the results are conflicting. We have therefore carried out a further study in men. DESIGN AND PATIENTS: Fifty men with acne vulgaris were age-matched against 50 normal men. MEASUREMENTS: Serum levels of dehydroepiandrosterone sulphate (DHEAS), 17 alpha-hydroxyprogesterone, 11-deoxycortisol, androstenedione and testosterone were measured by radioimmunoassay, sex hormone binding globulin (SHBG) by immunoradiometric assay and LH, FSH and oestradiol by automated ELISA. RESULTS: The acne patients had higher levels of androstenedione, median 7.35 nmol/l, (interquartile range 2-7) vs 6.05 (2.3), P = 0.004; testosterone, 21.7 nmol/l (7.5) vs 17.55 (7.7), P = 0.04; and free androgen index (FAI) 78.26 (40) vs 65.06 (20), P = 0.007, but also had higher levels of 11-deoxycortisol, 13.65 nmol/l (4.3) vs 12.0 (4.3), P = 0.022. The LH, FSH, 17 alpha-hydroxyprogesterone, DHEAS, oestradiol and SHBG levels were not significantly different. Examination of the Spearman rank correlation coefficient matrices for the serum levels of 17 alpha-hydroxyprogesterone, androstenedione and 11-deoxycortisol showed that the strongest correlation was between androstenedione and 11-deoxycortisol. CONCLUSION: Although there was overlap between the results of the acne patients and controls the acne patients tended to have higher levels of androstenedione, testosterone, free androgen index and 11-deoxycortisol. The higher levels of 11-deoxycortisol are suggestive of 11 beta-hydroyxlase dysfunction which could be due to a primary adrenal defect or a consequence of raised androgens. Also, a pathway between androstenedione and 11-deoxycortisol has been described in sheep and, although unsubstantiated in man, requires consideration.
In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors.
Selective type II (inducible) nitric oxide synthase (NOS) inhibitors have several potential therapeutic applications, including treatment of sepsis, diabetes, and autoimmune diseases. The ability of two novel, selective inhibitors of type II NOS, S-ethylisothiourea (EIT) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), to inhibit type II NOS function in vivo was studied in lipopolysaccharide (LPS) treated rats. Type II NOS activity was assessed by measuring changes in plasma nitrite and nitrate concentrations ([NOx]). Both EIT and AMT elicited a dose-dependent and > 95% inhibition of the LPS-induced increase in plasma [NOx]. The ED50 values for EIT and AMT were 0.4 and 0.2 mg/kg, respectively. In addition, the administration of LPS and either NOS inhibitor resulted in a dose-dependent increase in animal mortality; neither compound was lethal when administered alone. Pretreatment with L-arginine (but not D-arginine) prevented the mortality, while not affecting the type II NOS-dependent NO production, suggesting the toxicity may be due to inhibition of one of the other NOS isoforms (endothelial or neuronal). Thus, although EIT and AMT are potent inhibitors of type II NOS function in vivo, type II NOS inhibitors of even greater selectivity may need to be developed for therapeutic applications.
Raised serum androgens and increased responsiveness to luteinizing hormone in men with acne vulgaris.
We have investigated the hormone profiles and the relationship between serum testosterone and luteinizing hormone in men with long-standing acne vulgaris. In the controls, age correlated with the free androgen index and with serum dehydroepiandrosterone-sulphate, so for the purposes of comparison 33 men with acne vulgaris were age-matched against 33 controls. Compared with controls, the acne patients had higher medians of serum androstenedione, testosterone and free androgen index. In both groups there was a significant correlation between testosterone and luteinizing hormone levels. For a given level of luteinizing hormone the patients with acne tended to have higher levels of serum testosterone than normal controls. These results indicate that in this group of men with long-standing acne there was either increased responsiveness of the target organ to luteinizing hormone or there was an increased amount of androgen-secreting tissue.
Antidepressants and side effects.
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Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: pharmacological evaluation of nitric oxide synthase inhibitors.
The benefits of nitric oxide synthase (NOS) inhibitors in the treatment of endotoxemia or sepsis presumably arise from inhibition of the type II (inducible) NOS. However, inasmuch as the effect of these inhibitors on NOS function in vivo is rarely assessed, NOS activity was evaluated in rats and mice by measuring changes in plasma nitrite and nitrate concentrations ([NOx]) after administration of lipopolysaccharide (LPS). In both species, [NOx] peaked at 20 hr, returning to base line by 48 to 72 hr. The ED50 values (dose that elicited a 50% inhibition of the LPS-dependent increase in [NOx] 6 hr after LPS administration) for L-NG-monomethylarginine acetate, L-NG-nitroarginine methyl ester and aminoguanidine (administered 3 hr after LPS) were 34, 21 and 19 mg/kg in the rat and 32, 5 and 4 mg/kg in the mouse. These compounds also decreased the survival of LPS-challenged animals, which in the case of L-NG-nitroarginine methyl ester was reversed by L-arginine. Dexamethasone (which prevents the induction of type II NOS) also inhibited the LPS-dependent increase in [NOx] with ED50 values of 0.05 mg/kg (rat) and 1 mg/kg (mouse), but did not lead to decreased survival. Thus, inhibition of the type I (neuronal) or type III (endothelial) NOS, rather than the type II isoform, may be a possible mechanism for the animal mortality. These models provide a simple and reproducible means for assessing the in vivo inhibition of type II NOS by various compounds.
The nitric oxide synthase inhibitor, L-NG-monomethylarginine, reduces carrageenan-induced pleurisy in the rat.
Nitric oxide (NO) is a biological mediator that, when produced by the type II (inducible) nitric oxide synthase (NOS), has been implicated in the pathophysiology of inflammatory diseases. To examine this putative role of NO, pleural inflammation was elicited in rats by the intrapleural injection of carrageenan (1 mg). A pleural exudate and cellular influx developed, which peaked at 24 h and generally resolved by 72 h. The cellular influx was primarily composed of polymorphonuclear cells during the first 24 h, followed by macrophages during the subsequent 24 h. Inflammatory cell-associated NOS activity and pleural exudate nitrite (NO2-) + nitrate (NO3-) (NOx) also increased, peaking at 6 h and 24 h, respectively. Cell-associated NOS activity was calcium-independent, indicating the presence of the type II NOS isoform; NOS activity in the pleural cavity and polymorphonuclear cells influx were temporally correlated. Administration of L-NG-monomethylarginine (L-NMA) (200 mg/kg/day) attenuated the pleural exudation, cellular influx, pleural exudate NOx, and cell-associated NOS activity. The relative composition of the pleural cavity cellular infiltrate was not changed by L-NMA, indicating the influx of individual cell types were affected equally. L-Arginine (500 mg/kg/day) completely prevented the effects of L-NMA on pleural exudation and cellular influx and partially prevented the inhibition of pleural exudate NOx accumulation by L-NMA. These data implicate NO as a modulator of the pleural inflammatory response and support a future clinical role for NOS inhibitors in the treatment of inflammatory disease.
RAS and FMS mutations following cytotoxic therapy for childhood acute lymphoblastic leukaemia.
Patients who have received cytotoxic therapy for primary neoplastic disease are at an increased risk of developing secondary (therapy-related) acute myeloid leukaemia (AML) or myelodysplasia (MDS). RAS and FMS mutations have been observed in patients with AML and MDS. It has been suggested that the mutational status within these genes may be predictive of early secondary leukaemic disease. In this study we have screened 50 haematologically normal patients in complete remission from childhood acute lymphoblastic leukaemia (ALL) for activating point mutations in the RAS and FMS proto-oncogenes. Such patients may be considered at risk of therapy-related disease. Codons 12, 13 and 61 were screened in RAS and codon 969 in FMS using the polymerase chain reaction (PCR) followed by oligonucleotide hybridization (ONH). Three of the 50 patients (6%) were found to harbour N12 RAS mutations. One of these three patients (2%) had both a N12 RAS and FMS 969 mutation. Upon sequencing the RAS mutations, substitutions of serine, cysteine and aspartic acid for glycine were identified. The FMS 969 mutation was also confirmed, by sequencing, as a histidine substitution. RAS mutations were not detected in presentation samples indicating that these lesions have been somatically acquired presumably subsequent to cytotoxic therapy for the primary disease. Continued follow-up of these patients may indicate a role for these mutations in the development of secondary malignancies.
Sensitive liquid chromatographic method for the determination of a specific M1 agonist, LY246708, an investigational agent with potential for the treatment of Alzheimer's disease, in human plasma.
A reversed-phase HPLC method is reported for the determination of a new M1 agonist, LY246708, in human plasma. The compound and an internal standard were extracted from plasma with hexane at basic pH. The organic extract was evaporated to dryness and the residue was reconstituted with mobile phase [0.5% diethylamine (pH 3, adjusted with phosphoric acid)-acetonitrile (70:30, v/v)]. The analytes were separated from endogenous substances on a Zorbax CN column; the effluent was monitored by measuring its absorbance at 296 nm. The limit of quantification was determined at 1.5 ng/ml and the response was linear from 1.5 to 20 ng/ml. Validation studies showed the method to be both repeatable and reproducible. Its robustness was demonstrated by transfer between analytical laboratories and continued use in support of pharmacokinetic studies and therapeutic monitoring of the compound.