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Background: Alcohol-related pancreatitis is common and the gastrointestinal tract plays an important role in the regulation of pancreatic exocrine function. While the relationship between pancreatic proteolytic enzymes and insulin (as well as other pancreatic hormones) has been investigated in detail, little is known about the relationship between pancreatic proteolytic enzymes and gastrointestinal humoral factors. The aim of this study was to study the associations between trypsin, chymotrypsin, and a panel of gastrointestinal humoral factors in patients after an episode of alcohol-related versus non-alcohol-related pancreatitis., Methods: Fasting venous blood samples were analyzed for trypsin, chymotrypsin, cholecystokinin, gastrin, ghrelin, gastrin-related peptide, neuropeptide Y, peptide YY, secretin, and vasoactive intestinal peptide. Linear regression analysis was used in three statistical models, adjusting for covariates (age, sex, ethnicity, smoking, exercise, body mass index, dysglycemia, recurrence of pancreatitis, duration of pancreatitis, and severity of pancreatitis)., Results: The study included 21 patients with alcohol-related pancreatitis and 72 with non-alcohol-related pancreatitis. Gastrin, cholecystokinin, and vasoactive intestinal peptide were significantly associated with chymotrypsin in all three statistical models and resulted in a 1.06, 1.98, and 2.74 times higher chymotrypsin level in alcohol-related pancreatitis, respectively. Ghrelin was significantly associated with trypsin in all three statistical models and resulted in a 2.64 times higher trypsin level in alcohol-related pancreatitis. Other associations did not demonstrate a consistent significant pattern., Conclusion: In alcohol-related pancreatitis, several gut-related peptides are significantly associated with pancreatic exocrine function. Further studies to investigate the effect of alcohol on the interaction between cholecystokinin (as well as gastrin, ghrelin, and vasoactive intestinal peptide) and pancreatic exocrine function are warranted., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome., Methods: Intensive care patients with septic shock (n=29) or sepsis (n=6) and non-hospitalised subjects (n=9) were recruited as part of the prospective observational trial 'ShockOmics' (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects., Results: Evidence of systemic proteolysis was indicated by a larger number of circulating peptides in septic shock patients, compared with septic patients and non-hospitalised healthy subjects. The peptide count and abundance in the septic shock patients were greater in patients who died (n=6) than in survivors (n=23), suggesting an association between magnitude of proteolysis and outcome. In silico analysis of the peptide sequences and of the sites of cleavage on the proteins of origin indicated a predominant role for serine proteases, such as chymotrypsin, and matrix metalloproteases in causing the observed proteolytic degradation., Conclusions: Systemic proteolysis is a novel fundamental pathological mechanism in septic shock. Plasma peptidomics is proposed as a new tool to monitor clinical trajectory in septic shock patients., Clinical Trial Registration: NCT02141607., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The ubiquitin-proteasome system is relevant in the pathobiology of many haematological malignancies, including multiple myeloma. The assessment of proteasome concentration and chymotrypsin-like (ChT-L) activity might constitute a new approach to diagnosis, prognosis and monitoring of anticancer treatment of patients with haematological malignancies and other diseases. The aim of our study was to determine which material, plasma or serum, is better for measuring chymotrypsin-like (ChT-L) activity and proteasome concentration. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in 70 plasma and serum samples drawn from 28 patients at different treatment stages for multiple myeloma (MM) and 31 healthy volunteers. Proteasome ChT-L activity and concentration in multiple myeloma patients were significantly higher in plasma compared to serum. In this group we observed significant and positive correlations both between the plasma and serum proteasome ChT-L activity and plasma and serum proteasome concentration. The higher values of proteasome concentration and ChT-L activity in plasma than in serum and their better correlations with parameters of tumour load and prognosis suggest that plasma constitutes a better biological material for measuring ChT-L activity and proteasome concentration than serum in multiple myeloma patients.

Background: While the close morphological relationship between the exocrine and endocrine pancreas is well established, their functional interaction remains poorly understood. The aim of this study was to investigate the associations between circulating levels of pancreatic proteolytic enzymes and insulin, as well as other pancreatic hormones., Methods: Fasting venous blood samples were collected and analyzed for trypsin, chymotrypsin, insulin, glucagon, somatostatin, and pancreatic polypeptide. Linear regression analysis was used in unadjusted and two adjusted (accounting for prediabetes/diabetes, body mass index, smoking, and other covariates) statistical models., Results: A total of 93 individuals with a history of acute pancreatitis were included in this cross-sectional study. Chymotrypsin was significantly associated with insulin in the two adjusted models (p = 0.005; p = 0.003) and just missed statistical significance in the unadjusted model (p = 0.066). Chymotrypsin was significantly associated with glucagon in both unadjusted (p = 0.025) and adjusted models (p = 0.014; p = 0.015); as well as with somatostatin - in both unadjusted (p = 0.001) and adjusted models (p = 0.001; p = 0.002). Trypsin was not significantly associated with insulin in any of the models but was significantly associated with glucagon in both unadjusted (p < 0.001) and adjusted models (p < 0.001), and pancreatic polypeptide in both unadjusted (p < 0.001) and adjusted (p < 0.001) models., Conclusion: The state of hyperinsulinemia is characterized by a dysfunction of the exocrine pancreas. In particular, chymotrypsin is increased in the state of hyperinsulinemia and trypsin is significantly associated with glucagon and pancreatic polypeptide., (Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Pancreatic function tests are used to determine the presence of chronic pancreatitis, particularly in the early stage of the disease. Chymotrypsin is an indicator of pancreatic function and is thus related to pancreatic diseases. A new fluorescent biosensing method for assay of chymotrypsin activity was developed using DNA (dC 12 )-templated silver nanoclusters and graphene oxide (GO). A peptide probe was also designed using chymotrypsin-cleavable amino acid sequence and a cysteine terminus. The peptide probe formed Ag-S bond to dC 12 -AgNCs to enhance the fluorescence of dC 12 -AgNCs. After the addition of GO, the peptide was adsorbed to the negative GO surface and the fluorescence of dC 12 -AgNCs was quenched by FRET. The peptide was then degraded into amino acid fragments upon addition of chymotrypsin; these fragments were released from the GO surface, and the FRET was terminated. The developed label-free method features lower cost and higher sensitivity to chymotrypsin activity assay compared with conventional fluorescence analysis. The method can be used to analyze chymotrypsin (as low as 3ng/mL, signal/noise =3) across a dynamic range of 0.0-50.0ng/mL. The proposed biosensing strategy can also be extended to other proteases by using different peptide substrates., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many haematologic malignancies, including multiple myeloma. Under conditions of rapid cell turnover and growth rate, proteasomes are returned into circulation. The measurement of their levels or activity could offer a new approach to diagnosis, prognosis and monitoring of anticancer treatment in carcinoma patients. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in the plasma of 64 patients with a newly diagnosed multiple myeloma and 30 healthy volunteers. The values were found to be significantly higher in the studied patients and advanced disease stages compared to the control group, and decreased significant after chemotherapy. Both proteasome concentration and ChT-L activity correlated with adverse prognostic factors, such as lactate dehydrogenase and β2-macroglobulin. We also showed that proteasome concentration positively correlates with IL-6 level, as opposed to proteasome ChT-L activity. Of note, higher proteasome ChT-L activity, unlike the concentration, was proved to be an indicator of a shorter progression free survival, constituting thereby an important prognostic marker., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To determine the safety, target inhibition, and signals of clinical activity of tipifarnib in combination with bortezomib in patients with advanced acute leukemias., Experimental Design: In a "3 + 3" design, patients received escalating doses of tipifarnib (days 1-14) and bortezomib (days 1, 4, 8, 11) every 3 weeks until maximum tolerated dose was reached. Peripheral blood mononuclear cells (PBMC) were collected at days 1, 8, and 22 for measurement of chymotrypsin-like and farnesyltransferase activity. Purified bone marrow leukemic blasts were collected at baseline and at day 8 for measurement of NF-κB activity., Results: The combination was well-tolerated, and maximum tolerated dose was not reached. Dose-limiting toxicities included diarrhea, fatigue, and sensorimotor neuropathy. Chymotrypsin-like and farnesyltransferase activity within PBMCs were decreased in a majority of patients at day 8. NF-κB activity within leukemic blasts was decreased in a majority of patients at day 8. Complete response with incomplete count recovery was observed in 2 patients, and additional 5 patients had stable disease., Conclusions: Tipifarnib and bortezomib combination in patients with advanced leukemias was well-tolerated, demonstrated relevant target inhibition, and was associated with signals of clinical activity in patients with advanced and refractory acute leukemias. Future studies of this combination may be warranted in more selected groups of patients in whom these molecular targets are of particular importance., (©2011 AACR.)

The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 μL of 1× phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases.

In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin-specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.

Purpose: Cytogenetic abnormalities are currently the most important predictors of response and clinical outcome for patients with acute myeloid leukemia (AML) or advanced-stage myelodysplastic syndrome (MDS). Because clinical outcomes vary markedly within cytogenetic subgroups, additional biological markers are needed for risk stratification., Experimental Design: We assessed the utility of measuring pretreatment proteasome chymotrypsin-like, caspase-like, and trypsin-like activities in plasma to predict response and survival of patients with AML (n = 174) or advanced-stage MDS (n = 52)., Results: All three enzymatic activities were significantly (P < 0.001) increased in the plasma of patients with AML and MDS compared with normal controls. Both chymotrypsin-like and caspase-like activities, but not trypsin-like activity, correlated with outcome. Chymotrypsin-like and caspase-like activities, but not trypsin-like activity, predicted response in univariate analysis (P = 0.002). However, only chymotrypsin-like activity was independent predictor of response from age grouping (<70 versus > or =70 years), cytogenetics, and blood urea nitrogen in multivariate analysis. Similarly, both chymotrypsin-like and caspase-like activities, but not trypsin-like activity, were predictors of overall survival in univariate analysis (P < 0.0001), but only chymotrypsin-like activity was independent of cytogenetics, age, performance status, blood urea nitrogen, and beta(2)-microglobulin in multivariate Cox regression models. Chymotrypsin-like activity was also a strong independent predictor of survival in patients with intermediate karyotype (n = 124)., Conclusions: Measuring plasma chymotrypsin-like activity may provide a powerful biomarker for risk stratification in patients with AML and advanced-stage MDS, including those with normal karyotype.

Objective: We evaluated whether the proteasomal chymotrypsin-like (ChT-L) activity is increased in plasma of patients with acute lymphoblastic (ALL), acute myeloblastic (AML) and chronic lymphocytic (CLL) leukemias., Methods: The activity was assayed using the fluorogenic peptide substrate in the presence of an artificial activator sodium dodecyl sulfate (SDS) in the plasma of healthy donors (n=15) and ALL (n=15), AML (n=28) and CLL (n=22) patients., Results: The activity was significantly (P<0.001) higher in the plasma of ALL and AML patients at the diagnosis than in healthy subjects and decreased after therapy or remained unchanged or rose during relapse. By contrast, in CLL patients at the diagnosis, the activity did not differ significantly from the healthy controls. In each group, the activity positively correlated with the serum lactic dehydrogenase activity., Conclusions: Plasma proteasome ChT-L activity can be a useful bio-marker for patients with acute leukemia at the blast stage.

Background: The ubiquitin-proteasome pathway has been implicated in the pathogenesis of many hematologic malignancies., Methods: The authors measured proteasome peptidase activity levels in plasma samples from 225 patients with chronic lymphocytic leukemia (CLL) and correlated the results with clinical behavior. By using fluorogenic kinetic assays, the enzymatic activity levels of 3 proteasomes were measured: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L)., Results: All activity levels were significantly higher in patients who had CLL compared with the levels in a control group of healthy volunteers (P<.001). Rai stage was correlated with Ch-L activity (P<.001) but not with Cas-L or Tr-L activity. Levels of beta2 microglobulin (B2M) were correlated with Ch-L activity (correlation coefficient [R]=0.4; P<.001) and with Cas-L activity (R=0.25; P=.001) but not with Tr-L activity. Cas-L activity as a continuous variable was a strong predictor of survival. Ch-L and Cas-L activity levels as categorical variables both were strong predictors of survival; Cas-L activity was independent of B2M level but not of immunoglobulin variable heavy chain gene (IgVH) mutation status. However, the combination of elevated B2M levels (>3.2 mg/L) and Cas-L activity (>1.32 pmoL/second/mL plasma) was associated with significantly shorter survival independent of IgVH mutation status., Conclusions: The current results indicated that measuring plasma proteasome activity has prognostic value in CLL that, when combined with B2M, can be independent of IgVH mutation status., (Copyright (c) 2008 American Cancer Society.)

Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The Aalpha and Bbeta chains of fibrinogen, but not the gamma chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, approximately 100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100 degrees C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP(-) mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP(-)) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

Eight Angus steers (290 +/- 8 kg), surgically prepared with pancreatic pouch-duodenal reentrant cannulas and abomasal infusion catheters were used in a replicated 4 x 4 Latin square experiment to investigate the effects of abomasal infusion of starch hydrolyzate (SH) and/or casein on pancreatic exocrine secretion and plasma concentration of hormones. Steers were fed a basal diet of alfalfa (1.2 x NEm) in 12 equal portions daily. Abomasal infusion treatments (6-L total volume infused per day) were water (control), SH [2.7 g/(kg BW x d)], casein [0.6 g/(kg BW x d)], and SH + casein. Periods were 3 d for adaptation and 8 d of full infusion. Pancreatic juice and jugular blood samples were collected over 30-min intervals for 6 h on d 11. Weight and pH of pancreatic samples were measured, and a 10% subsample was composited and frozen until analysis of total protein and pancreatic enzyme activities. The remaining sample was returned to the duodenum. Plasma was harvested and frozen until analyzed. Pancreatic juice (67 mL/h) and protein (1.8 g/h) secretion rates were not affected by nutrient infusion. There were SH x casein interactions for all pancreatic enzyme secretions (U/h; alpha-amylase, P < 0.03; trypsin, P < 0.08; and chymotrypsin, P < 0.03) and plasma insulin concentration (P < 0.10). Secretion of pancreatic enzymes was increased by SH (trypsin) and casein (alpha-amylase, trypsin, and chymotrypsin) but not when SH + casein were infused together. Glucose (P < 0.10) and cholecystokinin octapeptide concentrations (CCK-8; P < 0.05) were increased by SH, but glucagon was decreased (P < 0.10). Casein decreased (P < 0.10) plasma CCK-8 concentrations. These data indicate that positive effects of postruminal casein on enzyme secretion were inhibited by SH, emphasizing the complexity of the regulatory mechanisms involved in dietary adaptation of pancreatic exocrine secretion. Changes in hormone concentration may not relate directly to changes in enzyme secretion.

Following the Cambridge and Marseilles Symposia, functional recovery of the pancreas occurs if the primary cause and complications of the disease have been eliminated. However, recent research showed contradictory results, owing to the difference in diagnostic methods and the proportion of patients studied in relation to the etiologic factor and severity of the disease, as well as the differences in the tests utilized. Sixty-three consecutive patients with acute biliary pancreatitis were prospectively studied. Seventeen were men (27%) and 46 were women (73%), with an average age of 62.3 years, 45 were mild cases and 18 were severe. All patients underwent a cholecystectomy. No patient in this series underwent necrosectomy. During the acute phase, severity was evaluated following the Atlanta criteria as well as the existence of necrosis and its percentage by means of dynamic computed tomography (CT). During the follow-up, different tests were used to assess the pancreatic exocrine function, 1 month, 6 months and 1 year after the acute pancreatitis (AP) episode. The possible existence of pancreatic exocrine insufficiency following biliary origin AP as well as whether this possible deficit was related to the severity of the episode was investigated. We found no such insufficiency 1 year after the episode, and no link with the severity of the episode., (Copyright 2002 S. Karger AG, Basel and IAP)

Trypsin and chymotrypsin readily bind to human erythrocyte ghosts and to resealed right-side-out ghosts, but not to intact erythrocytes, as followed with [3H]trypsin and [3H]chymotrypsin and with cold proteases in a caseinolytic assay. The proteases freely reacted with casein in the presence of intact cells. Trypsin activated trypsinogen over an 8-hr time course at a faster rate in the presence of erythrocytes than in the absence thereof, after a slight initial delay. Trypsinogen did not bind to intact erythrocytes, thereby behaving comparably to trypsin. These results suggest that different microenvironments exist about the erythrocyte ghosts and the intact erythrocytes, thereby permitting the proteases to bind to the former but not to the latter. Hence, in the absence of considerable ghosts in circulating blood, which may mask the binding site of the proteases, the proteases may be more readily accessible for interaction with circulating serpins, leading to inactivation of the proteases and protection from their degradative potential. The presence of the serpins in circulating blood may assist in the control of the degradative power of the pancreatic proteases in pancreatitis and may negatively modulate such processes as thrombosis, activation of the complement system, and vascular remodeling.

The aim of this study was to determine trypsin-lake proteinase activity, chymotrypsin-like proteinase activity, trypsin, alpha 1-antitrypsin and alpha 2-macroglobulin levels in blood serum at the children with gastroenterological pathology. These parameters did not chang at the children with functional disorder of stomach and duodenum. The stable balance between proteinases and inhibitors was determined only at the duration of the disease not more 5 years. The absence of normal levels these enzymes after traditional treatment was explain the necessity to continue the therapy at home with control of enzymes' levels.

Protease activities in serum and urine of 116 children with glomerulonephritis and 16 healthy children were tested using chromogenic peptide substrates Z-Dala&#95;Leu-Arg-pNA and Glp-Ala-ALa-Leu-pNa. We found the dependence between activity of serine proteinases and clinical, morphological forms of primary glomerulonephritis and hypertension.

The immunological system of patients with anorexia nervosa seems to react in a non typical and, up to now, obscure way. We do not observe greater ability to catch infectious diseases while damage due to other reasons leads as a rule to breakdown of resistance mechanisms thus giving increased receptivity to infections. The works concerning the immunological functions in anorexia nervosa are not heterogeneous and concern small groups of patients. The research was made upon 29 female patients aged 12 to 20 with the diagnosis of anorexia nervosa due to DSM-IV criteria. The research was made before starting the treatment and 6 weeks after the withdrawal of all anorexia nervosa symptoms. The research results showed that in the female patients there were no essential disturbances in the scope of electrophoresis of serum protein. Testing the protein of acute phase CRP as well as the estimation of their main microheterogeneity did not show significant deviation from the norm, which shows any features of early and later activation of the immunological system. However, a slight decrease of CD3 and low coefficient CD 4/8 during the disease were observed. After the symptoms retreated the coefficient became normal. Summing up we can state that the immunological system in patients with anorexia nervosa, in spite of great cachexia, did not show significant changes. The stated specific dysfunctions may be connected with a subtype of disease and other neurotransmitter mechanisms, which still requires to be confirmed in further research.

Candida albicans yeast cells bind soluble human plasma fibronectin (Fn) through a glycoprotein receptor (adhesin) located on the cell surface. This work demonstrates that a 120 kDa proteolytic fragment of Fn encompassing the cell binding domain binds more avidly to the yeast cell adhesin than does the parent Fn molecule. The presence of binding of Fn fragments containing heparin- and gelatin-binding domains of Fn could not be detected. The binding of the 120 kDa fragment is inhibited by a monoclonal antibody to the cell binding domain containing the amino acid sequence, Arginine-Glycine-Aspartic acid (RGD) as well as by an RGD-containing approximately 23-mer Fn peptide, but not with heparin or GRGDSPL. The fact that the cell binding domain of soluble Fn binds more avidly than does the parent molecule may explain the difference in the interaction of soluble Fn and immobilized Fn with Candida. It is possible that, upon immobilization, Fn may expose domains of the molecule previously unexposed when the molecule is in the soluble state.

Polymeric doxorubicin prodrugs were prepared linking monomethoxy poly(ethylene glycol), 5000 D molecular weight, to the doxorubicin amino group, using an amino acid or a peptide as a spacer arm. As spacers glycine, L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine were used. The conjugates showed enhanced stability to alkaline degradation compared to the free doxorubicin. Towards Ehrlich solid tumor in mice the glycin spaced derivative was devoid of activity, whereas the phenylalanine and tryptophan derivatives were 20% and 16% active and the tripeptide one 50% active with respect to free doxorubicin. On the other hand the derivatization was accompanied by a great decrease of toxicity in mice with respect to the free drug. Doxorubicin was not released from conjugates by chymotrypsin incubation or in plasma.

The analytical, within-subject and between-subject components of variation were estimated for serum albumin, transthyretin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, haptoglobin, beta 2-microglobulin and C-reactive protein in a cohort of 19 apparently healthy subjects over 20 weeks. Desirable analytical goals based on biological variation should be able to be met except for serum albumin and beta 2-microglobulin for which methodological improvement is warranted. All proteins showed marked individuality which casts doubt on the utility of conventional population-based reference values as interpretative criteria. The critical differences required for significance of changes in serial results differ markedly from protein to protein and the data presented allow generation of objective criteria for monitoring individuals.

The authors treated destructive forms of acute pancreatitis for the first time by means of specific plasma sorption of proteinases with the use of a proteinase plasma sorbent which is an acid-stable proteinase inhibitor immobilized on sepharose (ASI-Sepharose). Specific plasma sorption of proteinases on ASI-Sepharose was applied to the treatment of 29 patients with various forms of acute destructive pancreatitis. At the end of specific plasma sorption procedure the activity of blood plasma proteolytic enzymes in the patients reduced by 60-75%, which was attended by marked improvement of the condition in most cases; aggravation of the patients' condition was not encountered. The mortality in this group was 20.7% and was due to complications occurring during the development of the main disease. The mortality rate among patients treated by specific proteinase plasma sorption is much lower than that recorded in the USSR for destructive forms of acute pancreatitis which ranges from 30 to 75%.

Heparin cofactor II is a proteinase inhibitor which inhibits both chymotrypsin and thrombin, and displays great similarities with antithrombin III, the main inhibitor of thrombin in human plasma. Since acute pancreatitis is known to be associated with modification of the proteinase-antiproteinase equilibrium, we studied heparin cofactor II and antithrombin III as well as other biochemical and haematological parameters in 10 patients experiencing attacks of acute pancreatitis. Heparin cofactor II activity decreased during the first week of illness, while its antigen concentration remained subnormal. This discrepancy between antigen concentration and activity which persisted during the first week of illness was due both to complex formation of heparin cofactor II with its target proteinases and to partial proteolysis of the inhibitor. Heparin cofactor II was shown to form a complex with chymotrypsin in the plasma of such patients. Antithrombin III levels remained unchanged throughout the study, with no discrepancy between its activity and antigen concentration. No modification of haemostasis was shown either, except for a rise in the fibrinogen level during the first days of illness. It is concluded that, unlike antithrombin III, heparin cofactor II is involved in the proteinase-inhibitor equilibrium in patients with acute pancreatitis, and that heparin cofactor II might react as an inhibitor of pancreatic proteinases rather than an inhibitor of thrombin.

A new synthetic analogue of cholecystokinin, Thr28Nle31CCK25-33(CCK9) is compared with caerulein with regards to plasma bioactivity, degradation rate, side effects, and stimulation of pancreatic secretion. 24 healthy male volunteers were intubated with a double lumen Lagerlöf-tube. 30 min after correct positioning of the tube subjects received a continuous intravenous infusion of synthetic secretin (1 U/kg) together with either ceruletide (61.5 pM/kg) or CCK9 (30 pM/kg) both for 45 min. 30 pM/kg of CCK9 have been shown by others to cause maximal enzyme secretion (1). 61.5 pM/kg (= 100 ng) of caerulein are probably supramaximal but used by many centers for direct pancreatic function tests. Plasma CCK was measured by bioassay which compares amylase release of isolated rat pancreatic acini stimulated by plasma extracts with known standards of CCK8. Lipase, amylase, trypsin, chymotrypsin, and bicarbonate were measured in 15 min fractions after the onset of CCK infusion. Both drugs caused a similar stimulation of pancreatic enzyme secretion during infusion of the respective analogue which declined after termination. After the onset of CCK9 infusion plasma bioactivity reached a plateau around 20 pM at 15 min. Values declined after 30 min. After termination of infusion bioactivity rapidly declined within 3 min but still remained slightly elevated after further 15 min as compared to basal values (3.9 vs 0.6 pM). Plasma kinetics of caerulein were quite similar. However, despite a dose which was only twice as high as compared to CCK9, plasma bioactivity was six times higher with plateau values of about 120 pM.(ABSTRACT TRUNCATED AT 250 WORDS)

Various peptide fragments related to eglin c, which consists of 70 amino acid residues, were synthesized by a conventional solution method and their inhibitory effects on leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Among them, H-Arg-Glu-Tyr-Phe-OMe (eglin c 22-25) and H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe (eglin c 41-49) inhibited cathepsin G and alpha-chymotrypsin but not leukocyte elastase, while H-Thr-Asn-Val-Val-OMe (eglin c 60-63) inhibited leukocyte elastase but not cathepsin G or alpha-chymotrypsin, although eglin c potently inhibited leukocyte elastase, cathepsin G and alpha-chymotrypsin. These results indicated that the interaction sites of eglin c with leukocyte elastase, cathepsin G and alpha-chymotrypsin might be different.

Proteinase-inhibitor balance in human plasma before and after artificial blood circulation was examined. It was shown that trypsinlike and chemotrypsinlike proteinases activities after this procedure were 1.6 and 2.8 times higher and antitryptic activity 1.2 times lower than in the same patients before the procedure. The 40-50% decrease of proteinase activity in human plasma after artificial blood circulation was observed as a result of in vitro plasmasorption on immobilized human urinary trypsin inhibitor.

Reaction mixtures of increasing amounts of the pancreatic homologous proteases, anodal and cathodal chymotrypsin and trypsin, respectively, and normal rat serum were analyzed by immunoelectrophoretic methods in order to determine their distribution on serum protease inhibitors. This paper concerns three proteins occurring in normal serum and capable of binding protease viz. alpha1-macroglobulin, alpha1-antitrypsin and alpha1-inhibitor 3. The distribution of the enzymes among these protease inhibitors differed significantly from one protease to another. The distribution of the proteases among the serum protease inhibitors following intravenous injection of 125I-labelled proteases corresponded to that in vitro. Complexes formed with alpha1-macroglobulin and alpha1-inhibitor 3 were quickly eliminated irrespective of the enzyme species used, whereas those formed with alpha1-antitrypsin persisted much longer in the circulation.

The specificities of human neutrophile elastase and chymotrypsin-like protease towards oxidized insulin B chain were studied. The neutrophile elastase was found to differ from porcine pancreatic elastase in its specificity towards insulin B chain. The neutrophile elastase preferred mostly valine near the cleaved bond in contrast to pancreatic elastase which preferred alanine as well as valine near the cleaved bond. Human neutrophile chymotrypsin-like protease was found to cleave mostly bonds involving leucine and phenylalanine.

Serum lactoferrin concentrations were elevated in almost all children with meningococcal septicemia, in whom the disease had been clinically apparent for less than 18 hours, while the concentrations were normal or only moderately elevated in patients who had had the disease longer before being admitted. Concentrations of C-reactive protein (CRP) were markedly elevated, even with a time lapse of less than six hours, making this the most suitable parameter for the early diagnosis of severe meningococcal infection. Following an operative injury on children the lactoferrin concentrations changed very little. More than six hours after an operation, however, a marked increase in CRP-values was observed, possibly indicating differentiation of this response from that of bacterial infection. The concomitant study of serum alpha 1-antitrypsin, alpha 1-antichymotrypsin, orosomucoid and haptoglobin did not uncover results of great significance with regard to early changes.

Inactive renin (prorenin) can be activated by certain proteases in human blood, of which a possible source in vivo is polymorphonuclear leukocytes (PMNs). We extracted enzyme from PMNs using methods established for the recovery of neutral and acid protease fractions, and tested their effectiveness on plasma prorenin in vitro. Neutral protease preparations, possessing mainly chymotrypsin and elastase activity, produce no activation of prorenin. Exogenous pancreatic alpha-chymotrypsin does activate plasma prorenin, but less effectively than trypsin. From the quantity of PMNs extracted for neutral protease, and its failure to activate protenin, we deduce that this enzyme preparation, like exogenous chymotrypsin, is qualitatively unimportant. In contrast, the extracted PMN acid protease fraction, believed to be rich in cathepsin D, exhibited high prorenin activating ability, suggesting both quantitative and qualitative importance. The low pH requirement of this acid protease (near pH 4.0), together with its inactivity at neutral pH, argues against an important systemic role in the conversion of prorenin. However, it may contribute to systemic activation in partnership with other enzymes, or else play a specialized local role in situations where PMN concentration and activity increase, and the pH is on the acid side.

The serum proteins supposed to be indicative of the nutritional status, albumin, prealbumin and transferrin, as well as the serum proteinase inhibitors alpha 1-protease inhibitor (alpha 1-antichymotrypsin (Ach) and alpha 2-macroglobulin (alpha 2M) were measured in 14 Thai males suffering from uncomplicated falciparum malaria on the day of admission and after treatment with mefloquin on the 2nd, 28th and 63rd day. The same serum proteins had been determined from 31 healthy Thai males. Upon admission albumin and prealbumin concentrations had been lower and Ach higher in malaria patients compared with healthy Thai males. A significantly higher alpha 1 PI value was observed on the day of admission compared with the 28th day of the malaria patients. Only on the day of admission and only for the patients was a statistically significant negative linear regression found for albumin and prealbumin with Ach and a positive correlation for prealbumin with alpha 2 M as well as for albumin and transferrin correlated with alpha 1 PI. In well-nourished malaria patients the synthesis of the "acute phase reactants", alpha 1 PI and Ach, might be enhanced and in a reverse relationship the synthesis of albumin, pre-albumin and transferrin depressed.

Concentrations of 19 different proteins were measured after hospital admission, before hospital discharge and 3 months thereafter in 40 patients suffering from an acute episode of Crohn's disease. Serum levels of acute phase proteins (alpha 1-glycoprotein, alpha 1-antichymotrypsin, alpha 1-antitrypsin, CRP, haptoglobin) and immunoglobulin M corresponded to the severity of inflammatory symptoms and correlated significantly with CDAI (Crohn's disease activity index). Albumin and transferrin were characteristic for nutritional status of the patient under basal conditions and during nutritional therapy. Prealbumin and retinol-binding protein behaved similarly, but results were not significant. The measurement of the proteins mentioned give valuable clues in regard to the course of the disease and therapeutic success in Crohn's disease.

Data on biochemistry, physiological significance and clinical investigations of two main inhibitors of proteinases in human blood serum (alpha 1-antitrypsin and alpha 2-macroglobulin) are reviewed. Physico-chemical properties of the inhibitors, mechanisms of their interaction with proteinases and methods for determination are discussed. Modification of the specificity of some proteinases by alpha 2-macroglobulin, which probably plays a significant role in the regulation of enzymatic systems of proteolysis in blood, is considered. The use of the inhibitors of proteolysis for diagnostics of pathological processes is discussed. Data on the alpha 1-antitrypsin deficiency and its relation to the development of degenerative diseases in lungs are reviewed.

Corticosteroids caused a reduction in the ratio of sol-phase sputum concentration to serum concentration of albumin in 12 patients with chronic obstructive bronchitis, suggesting a reduction in protein transudation. Alpha-1-antitrypsin values followed the same pattern as those of albumin in both the control and treatment periods, confirming the similar behaviour of the two proteins. The alpha 1-antichymotrypsin ratios were on average three times higher than those of albumin in the control period, confirming the presence of local mechanisms in the lung for preferentially concentrating this protein. The sputum-to-serum ratio of alpha 1-antichymotrypsin, however, rose during steroid treatment with the result that there was a selective increase in this protease inhibitor, which may be of potential benefit to such patients.

The pancreas can be studied for obstructive disease by measuring serum lipase levels in the two stage provocative test. The test is nonspecific but noninvasive and applicable to all stages of pancreatic diseases. In this test, the pancreas is stimulated twice in two hour intervals before measuring the serum enzyme levels: first, with pancreozyin and secretin--the stage 1 test and, second, with pancreozymin, secretin, betazole hydrochloride and morphine sulfate--the stage 2 test. Among the pancreatic enzymes measured, lipase was most reliable. Serum lipase level elevation in the stage 1 test indicates a pancreatic abnormality and it completes the test. Patients who fail to respond to the stage 1 test have either a normal pancreas or pancreatic insufficiency and need the stage 2 test for differential diagnosis. In the stage 2 test, the serum lipase level is elevated in patients with a normal pancreas but not in those with pancreatic insufficiency. As a preliminary study, ten patients with carcinoma of the pancreas, two with pancreatitis and ten in the control group were studied. All patients with a known pancreatic disease demonstrated an abnormality in the test. Two of ten in the control group also had abnormal results. The two stage provocative test may be used prior to undertaking more invasive examinations, such as an arteriogram, in patients who are suspected of having pancreatic disease, yet other tests have failed to indicate it.

20 patients with chronic staphylococcal osteomyelitis were tested for 6 acute phase reactants: alpha 1-antitrypsin, orosomucoid, haptoglobin, ceruloplasmin, C-reactive protein (CRP) and antichymotrypsin during different phases of the disease. CRP was best correlated to clinical activity and in 3 cases CRP and ceruloplasmin increased a few weeks before a clinically apparent exacerbation of the osteomyelitis took place. A Latex-CRP slide method showed fairly good agreement with CRP assessed by immunoassay. Determination of CRP is a suitable test for following the activity of chronic osteomyelitis.

Human granulocytes contain cationic proteins with chymotrypsin-like activity. These proteases showed a higher relative affinity for alpha1-antichymotrypsin than for alpha1-antitrypsin but the highest affinity for alpha2-macroglobulin. The complexes between cationic protein and alpha1-antichymotrypsin migrate as beta-globulin on agarose gel electrophoresis.

Serum IAP and IS levels were studied in patients with stomach cancer. We found that the serum levels of IAP and IS increased as the disease progressed; their true positive rates as tumor markers were 49.3% and 39.8%, respectively. There was some delicate difference between these glycoproteins and T-cell subpopulation. IAP and IS correlated well with a correlation coefficient of 0.847, their value in clinical oncology are thought to be equivalent. Some difference was found between their substances and ASP, this might indicate the heterogeneous nature of the circulating glycoproteins in varying cancer-bearing status.