Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor.

Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe-->Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.

Cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs (etodolac, meloxicam, celecoxib, rofecoxib, etoricoxib, valdecoxib and lumiracoxib) for osteoarthritis and rheumatoid arthritis: a systematic review and economic evaluation.

OBJECTIVES: To review the clinical effectiveness and cost-effectiveness of cyclooxygenase-2 (COX-2) selective non-steroidal anti-inflammatory drugs (NSAIDs) (etodolac, meloxicam, celecoxib, rofecoxib, etoricoxib, valdecoxib and lumiracoxib) for osteoarthritis (OA) and rheumatoid arthritis (RA). DATA SOURCES: Electronic databases were searched up to November 2003. Industry submissions to the National Institute for Health and Clinical Excellence (NICE) in 2003 were also reviewed. REVIEW METHODS: Systematic reviews of randomised controlled trials (RCTs) and a model-based economic evaluation were undertaken. Meta-analyses were undertaken for each COX-2 selective NSAID compared with placebo and non-selective NSAIDs. The model was designed to run in two forms: the 'full Assessment Group Model (AGM)', which includes an initial drug switching cycle, and the 'simpler AGM', where there is no initial cycle and no opportunity for the patient to switch NSAID. RESULTS: Compared with non-selective NSAIDs, the COX-2 selective NSAIDs were found to be equally as efficacious as the non-selective NSAIDs (although meloxicam was found to be of inferior or equivalent efficacy) and also to be associated with significantly fewer clinical upper gastrointestinal (UGI) events (although relatively small numbers of clinical gastrointestinal (GI) and myocardial infarction (MI) events were reported across trials). Subgroup analyses of clinical and complicated UGI events and MI events in relation to aspirin use, steroid use, prior GI history and Helicobacter pylori status were based on relatively small numbers and were inconclusive. In the RCTs that included direct COX-2 comparisons, the drugs were equally tolerated and of equal efficacy. Trials were of insufficient size and duration to allow comparison of risk of clinical UGI events, complicated UGI events and MIs. One RCT compared COX-2 (celecoxib) with a non-selective NSAID combined with a gastroprotective agent (diclofenac combined with omeprazole); this included arthritis patients who had recently suffered a GI haemorrhage. Although no significant difference in clinical GI events was reported, the number of events was small and more such studies, where patients genuinely need NSAIDs, are required to confirm these data. A second trial showed that rofecoxib was associated with fewer diarrhoea events than a combination of diclofenac and misoprostol (Arthrotec). Previously published cost-effectiveness analyses indicated a wide of range of possible incremental cost per quality-adjusted life-year (QALY) gained estimates. Using the simpler AGM, with ibuprofen or diclofenac alone as the comparator, all of the COX-2 products are associated with higher costs (i.e. positive incremental costs) and small increases in effectiveness (i.e. positive incremental effectiveness), measured in terms of QALYs. The magnitude of the incremental costs and the incremental effects, and therefore the incremental cost-effectiveness ratios, vary considerably across all COX-2 selective NSAIDs. The base-case incremental cost per QALY results for COX-2 selective NSAIDs compared with diclofenac for the simpler model are: celecoxib (low dose) 68,400 pounds; celecoxib (high dose) 151,000 pounds; etodolac (branded) 42,400 pounds; etodolac (generic) 17,700 pounds; etoricoxib 31,300 pounds; lumiracoxib 70,400 pounds; meloxicam (low dose) 10,300 pounds; meloxicam (high dose) 17,800 pounds; rofecoxib 97,400 pounds; and valdecoxib 35,500 pounds. When the simpler AGM was run using ibuprofen or diclofenac combined with proton pump inhibitor (PPI) as the comparator, the results change substantially, with the COX-2 selective NSAIDs looking generally unattractive from a cost-effectiveness point of view (COX-2 selective NSAIDs were dominated by ibuprofen or diclofenac combined with PPI in most cases). This applies both to 'standard' and 'high-risk' arthritis patients defined in terms of previous GI ulcers. The full AGM produced results broadly in line with the simpler model. CONCLUSIONS: The COX-2 selective NSAIDs examined were found to be similar to non-selective NSAIDs for the symptomatic relief of RA and OA and to provide superior GI tolerability (the majority of evidence is in patients with OA). Although COX-2 selective NSAIDs offer protection against serious GI events, the amount of evidence for this protective effect varied considerably across individual drugs. The volume of trial evidence with regard to cardiovascular safety also varied substantially between COX-2 selective NSAIDs. Increased risk of MI compared to non-selective NSAIDs was observed among those drugs with greater volume of evidence in terms of exposure in patient-years. Economic modelling shows a wide range of possible costs per QALY gained in patients with OA and RA. Costs per QALY also varied if individual drugs were used in 'standard' or 'high'-risk patients, the choice of non-selective NSAID comparator and whether that NSAID was combined with a PPI. With reduced costs of PPIs, future primary research needs to compare the effectiveness and cost-effectiveness of COX-2 selective NSAIDs relative to non-selective NSAIDs with a PPI. Direct comparisons of different COX-2 selective NSAIDs, using equivalent doses, that compare GI and MI risk are needed. Pragmatic studies that include a wider range of people, including the older age groups with a greater burden of arthritis, are also necessary to inform clinical practice.

Methods of prediction and prevention of pre-eclampsia: systematic reviews of accuracy and effectiveness literature with economic modelling.

OBJECTIVES: To investigate the accuracy of predictive tests for pre-eclampsia and the effectiveness of preventative interventions for pre-eclampsia. Also to assess the cost-effectiveness of strategies (test-intervention combinations) to predict and prevent pre-eclampsia. DATA SOURCES: Major electronic databases were searched to January 2005 at least. REVIEW METHODS: Systematic reviews were carried out for test accuracy and effectiveness. Quality assessment was carried out using standard tools. For test accuracy, meta-analyses used a bivariate approach. Effectiveness reviews were conducted under the auspices of the Cochrane Pregnancy and Childbirth Group and used standard Cochrane review methods. The economic evaluation was from an NHS perspective and used a decision tree model. RESULTS: For the 27 tests reviewed, the quality of included studies was generally poor. Some tests appeared to have high specificity, but at the expense of compromised sensitivity. Tests that reached specificities above 90% were body mass index greater than 34, alpha-foetoprotein and uterine artery Doppler (bilateral notching). The only Doppler test with a sensitivity of over 60% was resistance index and combinations of indices. A few tests not commonly found in routine practice, such as kallikreinuria and SDS-PAGE proteinuria, seemed to offer the promise of high sensitivity, without compromising specificity, but these would require further investigation. For the 16 effectiveness reviews, the quality of included studies was variable. The largest review was of antiplatelet agents, primarily low-dose aspirin, and included 51 trials (36,500 women). This was the only review where the intervention was shown to prevent both pre-eclampsia and its consequences for the baby. Calcium supplementation also reduced the risk of pre-eclampsia, but with some uncertainty about the impact on outcomes for the baby. The only other intervention associated with a reduction in RR of pre-eclampsia was rest at home, with or without a nutritional supplement, for women with normal blood pressure. However, this review included just two small trials and its results should be interpreted with caution. The cost of most of the tests was modest, ranging from 5 pounds for blood tests such as serum uric acid to approximately 20 pounds for Doppler tests. Similarly, the cost of most interventions was also modest. In contrast, the best estimate of additional average cost associated with an average case of pre-eclampsia was high at approximately 9000 pounds. The results of the modelling revealed that prior testing with the test accuracy sensitivities and specificities identified appeared to offer little as a way of improving cost-effectiveness. Based on the evidence reviewed, none of the tests appeared sufficiently accurate to be clinically useful and the results of the model favoured no-test/treat-all strategies. Rest at home without any initial testing appeared to be the most cost-effective 'test-treatment' combination. Calcium supplementation to all women, without any initial testing, appeared to be the second most cost-effective. The economic model provided little support that any form of Doppler test has sufficiently high sensitivity and specificity to be cost-effective for the early identification of pre-eclampsia. It also suggested that the pattern of cost-effectiveness was no different in high-risk mothers than the low-risk mothers considered in the base case. CONCLUSIONS: The tests evaluated are not sufficiently accurate, in our opinion, to suggest their routine use in clinical practice. Calcium and antiplatelet agents, primarily low-dose aspirin, were the interventions shown to prevent pre-eclampsia. The most cost-effective approach to reducing pre-eclampsia is likely to be the provision of an effective, affordable and safe intervention applied to all mothers without prior testing to assess levels of risk. It is probably premature to suggest the implementation of a treat-all intervention strategy at present, however the feasibility and acceptability of this to women could be explored. Rigorous evaluation is needed of tests with modest cost whose initial assessments suggest that they may have high levels of both sensitivity and specificity. Similarly, there is a need for high-quality, adequately powered randomised controlled trials to investigate whether interventions such as advice to rest are indeed effective in reducing pre-eclampsia. In future, an economic model should be developed that considers not just pre-eclampsia, but other related outcomes, particularly those relevant to the infant such as perinatal death, preterm birth and small for gestational age. Such a modelling project should make provision for primary data collection on the safety of interventions and their associated costs.

Systematic reviews of the clinical effectiveness and cost-effectiveness of proton pump inhibitors in acute upper gastrointestinal bleeding.

OBJECTIVES: To evaluate the clinical effectiveness and cost-effectiveness of proton pump inhibitors (PPIs) in the prevention and treatment of acute upper gastrointestinal (UGI) haemorrhage, as well as to compare this with H2-receptor antagonist (H2RA), Helicobacter pylori eradication (in infected patients) or no therapy, for the prevention of first and/or subsequent bleeds among patients who continue to use non-steroidal anti-inflammatory drugs (NSAIDs). Also to evaluate the clinical effectiveness of PPI therapy, compared with other treatments, for the prevention of subsequent bleeds in patients who had previously experienced peptic ulcer (PU) bleeding. DATA SOURCES: Electronic databases and major conference proceedings were searched up to February 2006. REVIEW METHODS: Data were collected from the systematic reviews addressing each research objective. These were then entered into an economic model to compare the costs and quality-adjusted life-days of alternative management strategies over a 28-day period for patients who have had UGI bleeding. A Markov model with a Monte Carlo simulation used data from the systematic reviews to identify the most cost-effective treatment strategy for the prevention of UGI bleeding (first and subsequent) among NSAID users using an outcome of costs per quality-adjusted life-years (QALYs) over a lifetime from age 50 years. RESULTS: PPI treatment initiated after endoscopic diagnosis of PU bleeding significantly reduced re-bleeding and surgery compared with placebo or H2RA. Although there was no evidence of an overall effect of PPI treatment on all-cause mortality, PPIs significantly reduced mortality in subgroups when studies conducted in Asia were examined in isolation or when the analysis was confined to patients with high-risk endoscopic findings. PPI treatment initiated prior to endoscopy in UGI bleeding significantly reduced the proportion of patients with stigmata of recent haemorrhage (SRH) at index endoscopy compared with placebo or H2RA, but there was no evidence that PPI treatment affected clinically important outcomes. Giving oral PPI both before and after endoscopy, with endoscopic haemostatic therapy (EHT) for those with major SRH, is preferred to all others on cost-effectiveness grounds at any threshold over 25,000 pounds per QALY, even if only short-term effects are taken into account, and at any threshold over 200 pounds per life-year gained if long-term effects are included. The risk of NSAID-induced endoscopic gastric and duodenal ulcers was reduced by standard doses of PPI and misoprostol, and double doses of H2RAs. Standard doses of H2RAs reduced the risk of endoscopic duodenal ulcers. PPIs reduced NSAID-induced dyspepsia. PPIs were superior to misoprostol in preventing recurrence of NSAID-induced endoscopic duodenal ulcers, but PPIs were comparable to misoprostol in preventing the recurrence of NSAID-induced endoscopic gastric ulcers. Full-dose misoprostol reduced bleeding, perforation or gastric outlet obstruction due to NSAID-induced ulcers, but misoprostol was poorly tolerated and associated with frequent adverse effects. H. pylori eradication treatment was equally effective with PPI treatment for the primary or secondary prevention of endoscopic ulcers in NSAID users. H. pylori eradication treatment was more effective than placebo for the primary prevention of endoscopic PU and for the prevention of re-bleeding from PU in NSAID users. With regard to primary and secondary prevention of bleeding PU in NSAID users, the two most cost-effective strategies are H. pylori eradication alone, and H. pylori eradication followed by misoprostol (substituted by a PPI, if misoprostol is not tolerated) at an additional 4810 pounds per QALY. In patients who had previously experienced a bleed from a PU, re-bleeding was less frequent after H. pylori eradication therapy than after non-eradication antisecretory therapy, whether or not the latter was combined with long-term maintenance antisecretory therapy. CONCLUSIONS: PPI treatment compared with placebo or H2RA reduces mortality following PU bleeding among patients with high-risk endoscopic findings, and reduces re-bleeding rates and surgical intervention. PPI treatment initiated prior to endoscopy in UGI bleeding significantly reduces the proportion of patients with SRH at index endoscopy but does not reduce mortality, re-bleeding or the need for surgery. The strategy of giving oral PPI before and after endoscopy, with EHT for those with major SRH, is likely to be the most cost-effective. Treatment of H. pylori infection was found to be more effective than antisecretory therapy in preventing recurrent bleeding from PU. H. pylori eradication alone or eradication followed by misoprostol (with switch to PPI, if misoprostol is not tolerated) are the two most cost-effective strategies for preventing bleeding ulcers among H. pylori-infected NSAID users, although the data cannot exclude PPIs also being cost-effective. Further large randomised controlled trials are needed to address areas such as PPI administration prior to endoscopic diagnosis, different doses and administration of PPIs, as well as the primary and secondary prevention of UGI bleeding.

Sinusitis in children: the importance of diagnosis and treatment.

The complications from untreated sinusitis in a 10-month-old male infant, though at the more severe end of the spectrum, brings to light the importance of diagnosis and treatment even in the very young patient. Acute sinusitis should be diagnosed using established guidelines. Appropriate pharmacologic and osteopathic manipulative treatment should be initiated on diagnosis. Initial antibiotic therapy is a 14-day course of amoxicillin. If the sinusitis fails to resolve, a trial of a second-line antibiotic should be considered. The use of adjunctive medications such as antihistamines, decongestants, and nasal steroids remains controversial. If the patient fails maximal medical therapy, a computed tomography scan and referral to an otolaryngologist for possible surgical intervention should be considered.

Secretion of biologically active human proapolipoprotein A-I in a baculovirus-insect cell system: protection from degradation by protease inhibitors.

Studies of the structure and function of apolipoprotein A-I (apoA-I) often require its purification by delipidation of high density lipoprotein isolated from large quantities of human plasma and separation of apoA-I from other plasma apolipoproteins. To reduce the need for extensive purification procedures, we have developed an insect cell/baculovirus expression system for the production and secretion of human proapoA-I. The recombinant baculovirus containing full-length human apoA-I cDNA, when introduced into Spodoptera frugiperda, directs the synthesis of preproapoA-I, which is subsequently secreted into the growth medium as proapoA-I, indicating correct processing of the signal peptide during secretion. To prevent the extensive degradation of secreted proapoA-I, leupeptin and pepstatin A were added to the serum free cell culture medium. The protein was simply purified by filtration of the medium, which contained up to 80 mg/l proapoA-I, followed by chromatography on phenyl-sepharose CL-4B. The resultant proapoA-I was found to bind lipid and to activate lecithin:cholesterol acyltransferase as effectively as apoA-I from human plasma. The advantage of this expression system is the ease of purification of intact, biologically active apoA-I in high yield.

Molecular cloning of human cardiac troponin T isoforms: expression in developing and failing heart.

Troponin T, which links the troponin complex to tropomyosin, is found as multiple isoforms in the hearts of many animal species. Changes in isoform composition have been correlated with variation in myofilament sensitivity to calcium. In order to determine the origin of diversity of the cardiac troponin T (cTnT) isoforms indicated by existing protein data, we have determined the sequences and patterns of expression of mRNAs encoding troponin T in fetal and adult heart and those present in adult heart in end-stage failure. Three main regions of alternative splicing within the cTnT coding region were identified using reverse-transcriptase polymerase chain reaction (RT-PCR). Alternatively spliced RNAs are developmentally regulated and some of the fetal forms are expressed in adult failing heart. The molecular structure of the spliced regions was determined from cloned cDNAs and RT-PCR products. In the 5' region of the mRNA, isoforms are generated by the inclusion or exclusion of 15-, 3- and 27-nucleotide (nt) sequences and by the inclusion or exclusion of a separate 3-nt sequence. In the 3' region of the mRNA, alternative splicing involves a 9-nt sequence which can be present in full, in part or not at all. A further splicing site was identified in the central region involving a 234-nt sequence and resulting in rare but detectable mRNAs. This work demonstrates the complexity of cTnT RNA composition in human heart and provides the information necessary to address the function of cTnT isoforms in contraction.

Biliary sludge after liver transplantation: 2. Treatment with interventional techniques versus surgery and/or oral chemolysis.

OBJECTIVE: Interventional treatment of biliary sludge in liver transplant recipients includes transhepatic biliary drainage and saline irrigation, catheter chemolysis and/or basket extraction, and endoscopic intervention. The purpose of this study was to compare these interventional procedures with oral chemolysis and with surgical treatment of biliary sludge in order to evaluate the effectiveness of interventional procedures as an alternative to surgery in the treatment of this complication. MATERIALS AND METHODS: We retrospectively evaluated the outcome of several forms of treatment for biliary sludge occurring after liver transplantation in 49 cases. Treatments included oral chemolysis with chenodeoxycholic acid (n = 35), percutaneous transhepatic biliary drainage (n = 13) followed by irrigation with heparinized saline solution (n = 4), intraluminal chemolysis with glycero-octanoate-carnosine and bile salts-EDTA (n = 3) and/or basket extraction (n = 5), and endoscopic intervention (n = 2) or surgery (n = 26). Oral chemolysis was attempted in all cases of biliary sludge if no other complications were present. If this conservative treatment failed and the sludge was limited to the main bile ducts, interventional procedures were attempted. Surgical removal of the sludge (n = 15) or retransplantation (n = 5) without any attempt at prior nonsurgical treatment was performed if concomitant complications were present (n = 14) or if the extent of the sludge was considered too time-consuming for an interventional attempt (n = 6). The six patients in whom nonsurgical treatment failed underwent surgery. Treatment was considered successful if cholangiograms obtained after therapy showed no more evidence of sludge. Treatment was considered a failure if biliary sludge was shown after therapy by means of cholangiography, surgery, or autopsy. RESULTS: Complete disappearance of biliary sludge as a result of oral chemolysis was achieved in 14 (40%) of 35 cases. Interventional procedures were performed in 15 of the patients in whom oral treatment failed. After percutaneous transhepatic biliary drainage, the sludge was successfully removed by chemolysis with glycero-octanoate-carnosine in three cases, by basket extraction in one case, and by a combination of chemolysis and basket extraction in three cases. In two other cases, underlying recurrent tumor was treated palliatively with percutaneous transhepatic biliary drainage or endoscopic stenting. Irrigation with heparinized saline solution failed in four cases, and percutaneous or endoscopic basket extraction failed in one case each. Surgical treatment was successful in 18 (86%) of 21 cases, and retransplantation was successfully done in five patients. In all, interventional techniques were used in 43% of the patients with biliary sludge who could not be treated successfully with oral chemolysis, and the overall success rate was 60%. CONCLUSION: Interventional techniques are effective therapeutic alternatives for treating biliary sludge occurring after liver transplantation and should be considered before surgical procedures. An indication for interventional procedures in biliary sludge is lack of success of oral chemolysis and an absence of other complications that require surgery or retransplantation.

[Correlation of the x-ray morphology with the histopathological staging and grading of colorectal cancer]. / Korrelation der Röntgenmorphologie mit dem histopathologischen Staging und Grading kolorektaler Karzinome.

In a retrospective study stage grouping as well as histopathological grading (G category) of colorectal carcinomas was compared to radiographic findings at double contrast barium enema. A good correlation of tumour stage according to Dukes and the pT category and histopathological grading could be established to radiographic morphology. Apple core lesions and eccentrically obstructing neoplasms as well as saddle-like tumours exhibited worse staging and grading categories in comparison to polypoid lesions; plaque-like tumours are probably in between. It can therefore be assumed that the radiographic appearance of colorectal carcinomas may be a valuable tool to indicate stage group and G-category and thus offer information about patient prognosis.

[X-ray diagnosis of renal osteopathy in dialysis patients with reference to clinical aspects]. / Röntgendiagnostik der renalen Osteopathie bei Dialysepatienten unter Berücksichtigung klinischer Aspekte.

Pathophysiological histological and radiological findings in renal osteodystrophy are described. Special emphasis is laid on secondary hyperparathyroidism. Preliminary results of the authors' investigations show a good correlation between radiological findings in the phalanges of the hand and the concentration of parathyroid hormone (PTH) in 14 patients. The concentration of the hormone in the blood was measured by a new "two-site" immunoradiometric assay, which is specific for the intact, biologically active hormone. Patients with high concentrations of PTH in the blood tended to have more severe radiological changes. In 4 patients for whom radiographs of the hands revealed no pathologic findings, normal PTH concentrations in the blood were measured by this method, whereas the conventional assay gave elevated hormone concentrations for the same patients. This is due to the lack of specificity of the conventional method for the intact, biologically active hormone. Nevertheless, further investigations are needed to confirm these findings.