Magnetic Resonance Imaging as a Valid Noninvasive Tool for the Assessment of Pancreatic Fibrosis.

OBJECTIVES: The aim of this study was to evaluate the utility of magnetic resonance imaging (MRI) for the noninvasive assessment of pancreatic fibrosis (PF). METHODS: Fifty-two patients who underwent surgical resection of the pancreas, histological examination of resection margins, preoperative abdominal MRI, and fecal elastase-1 test were enrolled in the study. Pancreatic tissue was identified on the MRI T1-, T2-, and diffusion-weighted imaging sequences. Apparent diffusion coefficient (ADC) was measured at the expected resection margin of the pancreas. RESULTS: There was a significant negative correlation between the ADC mean and histologically determined PF (r = -0.752, P = 0.001). For equal to or greater than 25% of PF, the ADC cutoff value was 1.331 or less, with a sensitivity of 77% and specificity of 88%. The unenhanced T1-weighted signal intensity ratio (T1SI) cutoff value was 172.1 or less. For equal to or greater than 50% of PF, the ADC cutoff value was 1.316 or less with a sensitivity of 85% and specificity of 88%. The highest sensitivity was obtained by combining ADC and T1SI values. CONCLUSIONS: Combining both the ADC and T1SI measurement allows the detection of early PF with good sensitivity and specificity. Magnetic resonance imaging has the advantage of being noninvasive and widely used in the clinical setting, thus making our results easily transferable to routine clinical practice.

Pleiotropy and the low cost of individual traits promote cooperation.

The evolution of cooperation is thought to be promoted by pleiotropy, whereby cooperative traits are coregulated with traits that are important for personal fitness. However, this hypothesis faces a key challenge: what happens if mutation targets a cooperative trait specifically rather than the pleiotropic regulator? Here, we explore this question with the bacterium Pseudomonas aeruginosa, which cooperatively digests complex proteins using elastase. We empirically measure and theoretically model the fate of two mutants--one missing the whole regulatory circuit behind elastase production and the other with only the elastase gene mutated--relative to the wild-type (WT). We first show that, when elastase is needed, neither of the mutants can grow if the WT is absent. And, consistent with previous findings, we show that regulatory gene mutants can grow faster than the WT when there are no pleiotropic costs. However, we find that mutants only lacking elastase production do not outcompete the WT, because the individual cooperative trait has a low cost. We argue that the intrinsic architecture of molecular networks makes pleiotropy an effective way to stabilize cooperative evolution. Although individual cooperative traits experience loss-of-function mutations, these mutations may result in weak benefits, and need not undermine the protection from pleiotropy.

Pancreatic function assessment.

Several non invasive tests are available to assess pancreatic function, but no one is routinely used in clinical practice to diagnose chronic pancreatitis, due to their poor sensitivity in diagnosing mild pancreatic insufficiency. (13)C breath tests share the same limits of the other non invasive functional tests, but the mixed triglyceride breath test seems to be useful in finding the correct dosage of enzyme substitutive therapy to prevent malnutrition in patients with known pancreatic insufficiency.

Mechanical assessment of elastin integrity in fibrillin-1-deficient carotid arteries: implications for Marfan syndrome.

AIMS: Elastin is the primary component of elastic fibres in arteries, which contribute significantly to the structural integrity of the wall. Fibrillin-1 is a microfibrillar glycoprotein that appears to stabilize elastic fibres mechanically and thereby to delay a fatigue-induced loss of function due to long-term repetitive loading. Whereas prior studies have addressed some aspects of ageing-related changes in the overall mechanical properties of arteries in mouse models of Marfan syndrome, we sought to assess for the first time the load-carrying capability of the elastic fibres early in maturity, prior to the development of ageing-related effects, dilatation, or dissection. METHODS AND RESULTS: We used elastase to degrade elastin in common carotid arteries excised, at 7-9 weeks of age, from a mouse model (mgR/mgR) of Marfan syndrome that expresses fibrillin-1 at 15-25% of normal levels. In vitro biaxial mechanical tests performed before and after exposure to elastase suggested that the elastic fibres exhibited a nearly normal load-bearing capability. Observations from nonlinear optical microscopy suggested further that competent elastic fibres not only contribute to load-bearing, they also increase the undulation of collagen fibres, which endows the normal arterial wall with a more compliant response to pressurization. CONCLUSION: These findings support the hypothesis that it is an accelerated fatigue-induced damage to or protease-related degradation of initially competent elastic fibres that render arteries in Marfan syndrome increasingly susceptible to dilatation, dissection, and rupture.

Evaluation of micro-CT for emphysema assessment in mice: comparison with non-radiological techniques.

OBJECTIVES: To define the potential, limitations and synergies of micro-CT and other non-radiological techniques for the quantification of emphysema and related processes in mice, by performing a complete characterization of the elastase-induced emphysema model. MATERIALS AND METHODS: Ninety A/J mice (45 treated and 45 controls) were studied at different time points using breath-hold gated micro-CT, functional test parameters, RT-PCR for RNA cytokine expression, Luminex technology for cytokine plasma concentration and histomorphometry. RESULTS: Both histomorphometry and micro-CT imaging reflect rapid initial emphysema progression followed by steady-state development at decreasing rates. Cytokine measurements reveal an acute inflammatory response within the first 24 h that disappears after the first week. Limited systemic effect was observed based on plasma cytokine concentration. Lung compliance decreases during the acute inflammation phase and increases afterwards. CONCLUSION: Histomorphometry is the most sensitive technique since it detects airspace enlargement before the other methods (1 h after treatment). Micro-CT correlates well with histology (r2 = 0.63) proving appropriate for longitudinal studies. Functional test parameters do not necessarily correlate with the extent of emphysema, as they can be influenced by acute inflammation. Finally, cytokine measurements correlate with the presence of inflammation in histology but not with emphysema.

Pancreatic function, quality of life and costs at long-term follow-up after acute pancreatitis.

AIM: To evaluate long-term endocrine and exocrine pancreatic function, quality of life and health care costs after mild acute pancreatitis and severe acute pancreatitis (SAP). METHODS: Patients prospectively included in 2001-2005 were followed-up after 42 (36-53) mo. Pancreatic function was evaluated with laboratory tests, the oral glucose tolerance test (OGTT), fecal elastase-1 and a questionnaire. Short Form (SF)-36, was completed. RESULTS: Fourteen patients with a history of SAP and 26 with mild acute pancreatitis were included. Plasma glucose after OGTT was higher after SAP (9.2 mmol/L vs 7.0 mmol/L, P = 0.044). Diabetes mellitus or impaired glucose tolerance in fasting plasma glucose and/or 120 min plasma glucose were more common in SAP patients (11/14 vs 11/25, P = 0.037). Sick leave, time until the patients could take up recreational activities and time until they had recovered were all longer after SAP (P < 0.001). No significant differences in SF-36 were seen between the groups, or when comparing with age and gender matched reference groups. Total hospital costs, including primary care, follow-up and treatment of complications, were higher after SAP (median €16 572 vs €5000, P < 0.001). CONCLUSION: Endocrine pancreatic function was affected, especially after severe disease. SAP requires greater resource use with long recovery, but most patients regained a good quality of life.

Prevalence of pancreatic insufficiency in inflammatory bowel diseases. Assessment by fecal elastase-1.

Pancreatic insufficiency (PI) may be an extraintestinal manifestation of inflammatory bowel diseases (IBD). We report the results of a cross-sectional study that was carried out to investigate both the prevalence of PI in IBD patients and its clinical course over a 6-month follow-up period. In total, 100 Crohn's disease (CD) patients, 100 ulcerative colitis (UC) patients, and 100 controls were screened for PI by the fecal elastase-1 (FE-1) test. The decision limits employed were: < or =200 microg/g stool for PI and < or =100 microg/g for severe PI. Patients with abnormal FE-1 values were re-tested after 6 months. Odds ratios (OR) for PI were estimated by unconditional logistic regression analysis. PI was found in 22 UC and 14 CD patients. The OR for the FE-1 test < or =200 microg/g was 10.5 [95% confidence interval (CI): 2.5-44.8] for IBD patients compared to the controls. The risk of PI was related to three or more bowel movements per day (OR = 25.0), the passage of loose stools (OR = 7.7), and previous surgery (OR = 3.7). At the 6-month follow-up, FE-1 values became normal in 24 patients and showed persistently low concentrations in 12. These patients had a larger number of bowel movements per day (OR = 5.4), previous surgery (OR = 5.7), and a longer duration of the disease (OR = 4.2). PI is frequently found in IBD patients, particularly in those with loose stools, a larger number of bowel movements/day and previous surgery. PI is reversible in most patients, and persistent PI is not associated with clinically active disease.

Faecal elastase-1 test is superior to faecal lipase test in the assessment of exocrine pancreatic function in cystic fibrosis.

BACKGROUND AND AIMS: Direct tests are characterized by the highest sensitivity and specificity. However, their practical use, especially in children, is limited. Among the indirect tests, the highest sensitivity and specificity was documented for faecal elastase-1 test, yet the value of faecal lipase test in cystic fibrosis (CF) has not been defined. Therefore, the aim of the present study was to compare the sensitivity and the specificity of the faecal lipase test to the faecal elastase-1 test in the assessment of exocrine pancreatic function in children with CF. METHODS: The study comprised 90 CF patients and 95 healthy subjects (HS). In all subjects, faecal elastase-1 concentrations (ELISA) and lipase activities (ELISA) were measured. The presence of pancreatic insufficiency was documented by the determination of faecal fat excretion in 78 pancreatic insufficient and by the secretin-cholecystokinin test in 12 CF patients without steatorrhoea. Sensitivity and specificity of the faecal elastase-1 test and faecal lipase test were analysed and, in 50 HS, sample-to-sample and day-to-day variations were determined. RESULTS: With cut-off levels providing the same specificity for both tests (95.8%), the sensitivity of the faecal elastase-1 test (91.1%) was significantly higher (p < 0.0036) than that of the faecal lipase test (76.7%). Sample-to-sample (mean +/- SEM: 13.2 +/- 1.2% vs 23.4 +/- 2.2%) and day-to-day variations (mean +/- SEM: 16.3 +/- 1.2% vs 32.5 +/- 2.6%) were significantly lower (p < 0.0001) for elastase-1 than for lipase measurements. CONCLUSION: Among indirect tests, faecal elastase-1 test is superior to faecal lipase test in the assessment of exocrine pancreatic function in cystic fibrosis.

A prospective, comparative study of the para-aminobenzoic acid test and faecal elastase 1 in the assessment of exocrine pancreatic function.

BACKGROUND: The assessment of exocrine pancreatic insufficiency is part of the routine work-up of patients with persistent diarrhoea or suspected steatorrhoea. Direct and indirect tests for the diagnosis of exocrine pancreatic insufficiency have their drawbacks. Measurement of faecal elastase 1 by enzyme-linked immunoabsorbent assay is a simple, non-invasive, robust test for exocrine pancreatic insufficiency. METHODS: We performed a prospective comparison of the para-aminobenzoic acid test and faecal elastase 1 test in 45 patients being investigated for diarrhoea or suspected steatorrhoea. Details of clinical suspicion, imaging and response to treatment were recorded. RESULTS: Exocrine pancreatic function was normal in 20 patients with normal para-aminobenzoic acid and faecal elastase 1 levels. Eight patients had exocrine pancreatic insufficiency with low para-aminobenzoic acid and faecal elastase 1 levels, which improved with enzyme supplementation. In 14 of the 15 patients with low or borderline low para-aminobenzoic acid and normal faecal elastase 1 levels, a non-pancreatic cause was found; one patient had a false positive para-aminobenzoic acid test. Two had normal para-aminobenzoic acid but low faecal elastase 1 levels. One improved with pancreatic supplementation, and imaging revealed chronic pancreatitis. The other had a false positive faecal elastase 1 test related to profuse diarrhoea. CONCLUSIONS: Faecal elastase 1 estimation is a simple, non-invasive, robust test of exocrine pancreatic insufficiency, performed on an out-patient stool sample. Its diagnostic performance is superior to that of the para-aminobenzoic acid test in investigating patients with diarrhoea or suspected steatorrhoea.

Assessment of the antielastase activities in bronchoalveolar lavage fluid: effect of assay buffer ionic strength.

OBJECTIVE: To evaluate the effect of assay buffer ionic strength on assessment of the antielastase activities in bronchoalveolar lavage fluid. METHOD: An improved assay protocol in which elastase (in Tris-HCI buffer) is added to increasing volumes of test samples (made up to equal volume with phosphate-buffered saline) was used. RESULTS: The percent NE activity inhibited by BALF decreased with increasing NaCl concentration of the buffer. Inhibition of pancreatic elastase (PE) was not affected. One hundred percent inhibition of NE by pure AAT and SLPI standards occurred at molar ratios of 0.91 +/- 0.03 for AAT-to-NE and 0.83 +/- 0.02 for SLPI-to-NE when assayed in buffer with < or = 0.15 mol NaCl/L, compared to ratios of 0.99 +/- 0.02 and 1.06 +/- 0.02, respectively, for assays in buffers with 0.50-1.00 mol NaCl/L (p < 0.05 for AAT-to-NE; p < 0.02 for SLPI-to-NE). The AAT-to-PE molar ratio at 100% inhibition of PE was not affected. Assays in buffer with < or = 0.15 mol NaCl/L indicated that 86.9 +/- 4.1% of AAT and 100.9 +/- 4.9% of SLPI in BALF were active against NE, while assays in buffer with 0.50-1.00 mol NaCl/L showed that 84.4 +/- 3.5% of AAT and 81.6 +/- 5.9% of SLPI present were active. AAT inhibited NE and PE equally only in buffer with 0.50-1.00 mol NaCl/L. CONCLUSIONS: The results of assays of BALF antielastase activities depend on the assay buffer NaCl concentration, which may account for the conflicting reports in the literature. The buffer with 0.50-1.00 mol NaCl/L appear to be optimal for valid quantitation of anti-NE activities in BALF.

Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design.

The results of a protein design project are used to compare different predictive strategies with respect to protein-protein interactions. We have been able to generate variants of human pancreatic secretory trypsin inhibitor (hPSTI) optimized with respect to the affinity and specificity for human leukocyte elastase relative to trypsin and chymotrypsin, and in particular chymotrypsin. The extremely strong and specific human leukocyte elastase inhibitors were thus developed in three rounds of mutagenesis and two rounds of 3-D modelling; only 24 variants in total were synthesized, although variations at seven different amino acid positions were involved (i.e. from 20(7) possible variants). An excellent elastase inhibitor could be designed with the minimum of two amino acid exchanges. The value of structural modelling and actual structure determination is discussed in the light of the experimental results of the designed protein variants and the results of tertiary structure determinations of the free variant and the inhibitor-protease complex. Particular reference is given to the strategy to be followed in protein design projects in general and to the development of protease inhibitors in particular.

Inflammatory mediators and cytokines--new aspects of the pathophysiology and assessment of severity of acute pancreatitis?

Most attacks of acute pancreatitis are mild and self-limiting. In 10-20% of the cases, however, severe disease with multiple systemic complications develops. During the last few years it has been recognized that activated leukocytes have an important role in the multisystem involvement during acute pancreatitis. Activated leukocytes are thus a pathogenetic factor in the severity of the disease. Activation of polymorphonuclear granulocytes (PMNs) and of monocytes/macrophages is an early event during severe acute pancreatitis. Factors released by activated leukocytes therefore reflect the severity of the disease. Three independent studies have shown that released PMN-elastase is a reliable early prognostic marker that permits correct classification of 80-95% of the patients within the first 24-48 hours. Interleukin-6 (IL-6), mainly secreted by activated monocytes/macrophages, is also an early prognostic parameter (shown in one study), but is not superior to PMN-elastase. Leukocyte activation markers are more reliable than multiple scoring systems in the assessment of the severity of acute pancreatitis. Compared with PMN-elastase or IL-6, increased plasma concentrations of such acute-phase proteins as alpha-1-antitrypsin or CRP, and consumption of the protease inhibitor alpha-2-macroglobulin, are later events that can be detected only 1 to 4 days later. Comparison of the various inflammatory parameters suggests that PMN-elastase is the best early and reliable prognostic marker in acute pancreatitis. The reviewed data underscore the role of activated leukocytes in the pathogenesis of complicated acute pancreatitis.

Tumour necrosis factor-alpha, resting energy expenditure and cachexia in cystic fibrosis.

1. We investigated the relationship between circulating tumour necrosis factor-alpha concentrations, resting energy expenditure, cachexia and altered intermediary metabolism in patients with cystic fibrosis and chronic pulmonary infection. 2. Twenty adult patients with cystic fibrosis and chronic bronchial sepsis covering a spectrum of severity of lung disease (forced expiratory volume in 1 s 30-100% of predicted) were compared with 10 age matched, healthy, non-cystic fibrosis subjects. 3. Circulating tumour necrosis factor-alpha, C-reactive protein and neutrophil elastase-alpha 1-antiproteinase complex concentrations were determined simultaneously with glycerol, non-esterified fatty acids, catecholamines, anthropometric indices and resting energy expenditure (ventilated hood method). 4. Weight, body mass index and arm muscle mass were reduced in patients with cystic fibrosis compared with healthy control subjects (P < 0.01), whereas mean resting energy expenditure was increased [121 versus 101% predicted, mean difference 19.2% (95% confidence interval 11.0-27.4%), P < 0.001]. Circulating concentrations of glycerol (P < 0.01), non-esterified fatty acids (P < 0.01), adrenaline (P < 0.05), tumour necrosis factor-alpha, C-reactive protein and neutrophil elastase-alpha 1-antiproteinase complex (P < 0.01) were increased in patients compared with control subjects [tumour necrosis factor-alpha 96.9 versus 24.7 pg/ml, mean difference 72.2 pg/ml [95% confidence interval 27.7-116.7 pg/ml), P < 0.001]. Resting energy expenditure was significantly related to tumour necrosis factor-alpha levels and forced expiratory volume in 1 s. 5. In patients with cystic fibrosis and chronic pulmonary sepsis changes in resting energy expenditure, body composition and intermediary metabolism are consistent with the systemic effects of the host inflammatory response, which may be responsible for cachexia in adult patients. In particular these changes are consistent with the action of tumour necrosis factor-alpha, which was detected in the circulation during a period of apparent clinical stability.

Cartilage degradation by polymorphonuclear leucocytes: in vitro assessment of the pathogenic mechanisms.

Polymorphonuclear leucocytes (PMNs), which predominate in inflammatory synovial fluid, can degrade cartilage. This was measured by a novel in vitro model; PMNs were incubated for up to one hour with 2 or 3 microns sections of cartilage and the glycosaminoglycan loss determined by microdensitometry after alcian blue staining. Glycosaminoglycan loss could be as a result of damage from reactive oxygen species, proteolytic enzymes, or a combination of the two. The relative contributions of these mechanisms were evaluated using selective inhibitors. The results show that activated PMNs will degrade cartilage and that this degradation is due to proteolytic enzymes and not reactive oxygen species. There is a specificity involving elastase but not other serine proteases. It is suggested that enzyme inhibition may play a part in reducing PMN mediated cartilage damage.

Scintigraphic assessment of leukocyte infiltration in acute pancreatitis using technetium-99m-hexamethyl propylene amine oxine as leukocyte label.

The infiltration of leukocytes has been linked to the pathophysiology of complicated or severe pancreatitis. We have tested the ability of leukocyte scintigraphy using technetium-99m-hexamethyl propylene amine oxine (HM-PAO) as label to demonstrate the localization of leukocytes in the pancreas during acute pancreatitis. Twenty-eight patients with acute pancreatitis (eight with biliary, 13 with alcoholic, and seven with unknown origin) were studied with leukocyte scintigraphy using planar imaging and single photon emission computed tomography (SPECT). Fourteen patients had a mild (group I), II a severe (group II), and three a lethal outcome (group III) of pancreatitis. All patients of group III, six of group II, and two of group I had a positive leukocyte scan. Thus, the sensitivity of leukocyte scintigraphy for the detection of a lethal course of acute pancreatitis was 100%, of a severe course 54%, and of a severe or lethal course 64%. The specificity of a negative scan for a mild pancreatitis was 86%. Comparison of the results of leukocyte scintigraphy with those of contrast enhanced CT showed that six of eight patients with pancreatic necrosis in CT had a positive leukocyte scan, but only five of 20 patients without detectable pancreatic necrosis in CT. In summary, leukocyte infiltration into the pancreas during pancreatitis can be demonstrated by noninvasive leukocyte scintigraphy using technetium-99m-HM-PAO as label. A correlation between the severity of the disease and leukocyte infiltration exists.

Pathogenesis of emphysema. Assessment of basic science concepts through clinical investigation.

It is clear that alpha 1AT deficiency leads to early onset pulmonary emphysema. With the lead provided by the deficiency state, studies aimed at the linkage between alpha 1AT and its target enzyme, neutrophil elastase, have provided useful information about the pathogenesis of emphysema due to cigarette smoking. alpha 1AT represents the predominant antielastase of the lower respiratory tract. This observation implicates neutrophil elastase as the enzyme responsible for lung destruction, since affinity studies demonstrate that alpha 1ATs physiologically relevant function is the inhibition of neutrophil elastase. However, because of the inexorably slow nature of the emphysema process, demonstration of the protease-antiprotease imbalance in the lungs of smokers has been difficult. Studies using sensitive assays for alpha 1AT function and for neutrophil elastase's presence have added new support for the protease-antiprotease theory, and evaluation of related disorders such as the adult respiratory distress syndrome and cystic fibrosis have provided corraborative evidence. Finally, studies that have indicated that the major site of the protease-antiprotease imbalance is the microenvironment of protease-producing cells offer a new direction for future research into the pathogenesis of emphysema.

Assessment of the role of pancreatic proteases in human abdominal aortic aneurysms and occlusive disease.

Previous studies on the pathogenesis of abdominal aortic aneurysms have shown both elastase-like activity in the aortic wall and a decreased elastin content. The present study, using specific radioimmunoassays for pancreatic elastase 2 (IRE2) and cationic trypsin(ogen) (IRCT), investigates the concentrations of these proteases which are known to circulate in blood, in abdominal aortic aneurysms. Aortic specimens were obtained from 32 patients with aneurysms and 21 patients with atherosclerotic occlusive disease. Aortic tissue, obtained at autopsy from young adults, served as controls. Elastase-like activity was 300% and 800% higher, respectively, in aortic homogenates from aneurysms in comparison to occlusive disease and control aortic tissue. This was associated with 1.4-fold higher level of IRE2 and 2.7-fold higher levels of IRCT as compared to occlusive disease. Although there was no significant difference in the aortic collagen concentration among all 3 groups, the elastin content of aneurysmal aorta was 85% and 74% lower, respectively, in comparison to control and occlusive aorta. The results of this investigation demonstrate the presence of pancreatic elastase 2 and cationic trypsin(ogen) in abdominal aortic aneurysmal tissue and suggest that circulating pancreatic proteases contribute to the pathophysiology of aneurysms of the infrarenal aorta.

[Recent results in emphysema research]. / Neue Ergebnisse der Emphysemforschung.

The incidence as well as the medical, social and economic importance of lung emphysema are evaluated. Results of experimental emphysema research, including the author's own investigations on rats are discussed. Inhalation of tobacco smoke is one of the most important etiologic factors. The pathogenetic role of endogenous enzymes, in particular protease-antiprotease imbalance, is elaborated. The survey is completed with the reference of prophylactic investigations and with an outlook on the main points of future research.