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A protease–antiprotease balance is required to maintain the homeostasis of the intrauterine environment in which the fetus develops. Proteases and antiproteases accumulate in meconium exclusively during intrauterine life and are excreted after birth. Proteomic analysis was used to investigate the protein composition in pooled 50 serial meconium portions from 10 neonates. The UniProt, BRENDA and MEROPS databases were the sources of information used to classify the meconium proteases and antiproteases among 946 proteins identified in meconium. A total of 265 enzymatic proteins and 33 protein inhibitors were identified in the meconium. The six main enzyme groups represented in the meconium were oxidoreductases (n = 44), transferases (n = 62), hydrolases (n = 137), lyases (n = 10), isomerases (n = 7) and ligases (n = 5). Six protease families were distinguished: serine (n = 28, 41.2% of all proteases), metallo (n = 23, 33.8%), cysteine (n = 10, 14.7%), aspartic (n = 4, 5.9%), theorine (n = 2, 2.9%) and mixed (n = 1, 1.5%) proteases. The well-characterized meconium-based biomarker panel of proteases and their inhibitors may be a source of important information for use in diagnosing fetal disorders and predicting postnatal health and development. The differences in the composition and function between individual meconium proteases and antiproteases confirm their association with numerous metabolic processes characteristic of the intrauterine environment. [ABSTRACT FROM AUTHOR]

The fruit of Kersen or cherry (Muntingia calabura), a popular shade tree, is not yet promoted as a marketable fruit or a healthy herbal drink. This study aimed to investigate potential health benefits of fruit juice and crude extract of the fruit pulp of M. calabura including their antioxidant potential and inhibitory activities on several enzymes related to metabolic disorders. Fresh juice (FJ) of M.calabura fruit was obtained by juice extractor, whereas the fruit pulp (FP) was dried and macerated with ethanol to obtain the crude extract. Both were evaluated for their inhibitory activities against α-glucosidase, α-amylase, xanthine oxidase, lipase, and protease. Antioxidant activity was evaluated based on their scavenging activity on 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radicals. Both FJ and FP showed good antidiabetic activity by inhibiting α-glucosidase (IC50 162.00 and 84.70 µg/mL, respectively) and α-amylase (IC50 422.34 and 80.46 µg/mL, respectively). In comparison, IC50 of acarbose on α-glucosidase and α-amylase were 130.66 and 152.46 µg/mL, respectively. The FJ and FP extract showed anti-hyperuricemia activity by inhibiting xanthine oxidase (0.02 and 2.78 mg allopurinol equivalent/mL for FJ and FP, respectively). However, weak inhibition activities on protease and lipase were observed for the FJ and FP extract compared to the references used. Similarly, FJ and FP exhibited weak scavenging activity on DPPH, when compared with BHT and ascorbic acid. The study recommends the fruit of M.calabura as a potential source of antidiabetic and anti-hyperuricemia agents. The fruit can be further explored for nutraceutical applications. [ABSTRACT FROM AUTHOR]

Whey acidic protein domain (WAPD) occurs in a variety of proteins in animals and many of WAPD-containing proteins are involved in immunity. In the present study, a novel protein containing three WAPDs was identified from the weather loach, Misgurnus anguillicaudatus , designated as Ma TWD. Ma TWD share high identity with TWDs from fish but low identity with TWDs from other animal phyla. MaTWD transcripts mainly distributed in gills and head kidney responded to bacterial challenge with significant upregulation. In vitro assay with recombinant Ma TWD protein revealed that Ma TWD had antiprotease activity against bacterial proteases. Moreover, Ma TWD exhibited bacterial binding capacity and antimicrobial activity. Most importantly, exogenous Ma TWD protected loach against bacterial infection by reducing loach mortality. We infer that Ma TWD participates in the antibacterial immunity of loach via its antiprotease and antimicrobial activities. • A protein with three whey acidic protein domains (WAPDs) in tandem is identified from weather loach. • MaTWD exhibits antiprotease, antimicrobial and bacterial binding activities. • MaTWD protects loach against bacterial infection. • First functional study of such a WAPD protein in fish. [ABSTRACT FROM AUTHOR]

While GASP‐1 and GASP‐2 proteins are known to regulate myogenesis by inhibiting myostatin, their structural organization suggests a putative role as multivalent protease inhibitors controlling different protease activities. In this study, we show the noncompetitive and competitive antitrypsin activities of the full‐length GASP‐1 and GASP‐2 proteins, respectively, by using a bacterial system production and in vitro enzymatic experiments. The role of the second Kunitz domain in this functional duality is described by assessing the antitrypsin activity of GASP‐1/2 chimeric proteins. Molecular dynamics simulations support the experimental data to rationalize differences in binding modes between trypsin and the GASP‐1 and GASP‐2 second Kunitz domains. A new inhibition mechanism was evidenced for the second Kunitz domain of GASP‐2, in which the conventional cationic residue of trypsin inhibitors was substituted by the strongly interacting glutamine residue. [ABSTRACT FROM AUTHOR]

Neutrophil elastase (NE) is a major inflammatory protease released by neutrophils and is present in the airways of patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, non-CF bronchiectasis, and bronchopulmonary dysplasia. Although NE facilitates leukocyte transmigration to the site of infection and is required for clearance of Gram-negative bacteria, it also activates inflammation when released into the airway milieu in chronic inflammatory airway diseases. NE exposure induces airway remodeling with increased mucin expression and secretion and impaired ciliary motility. NE interrupts epithelial repair by promoting cellular apoptosis and senescence and it activates inflammation directly by increasing cytokine expression and release, and indirectly by triggering extracellular trap release and exosome release, which magnify protease activity and inflammation in the airway. NE inhibits innate immune function by digesting opsonins and opsonin receptors, degrading innate immune proteins such as lactoferrin, and inhibiting macrophage phagocytosis. Importantly, NE-directed therapies have not yet been effective in preventing the pathologic sequelae of NE exposure, but new therapies are being developed that offer both direct antiprotease activity and multifunctional anti-inflammatory properties.

-1 Antitrypsin (AAT) deficiency is the leading genetic cause of emphysema; however, until recently, no genuine animal models of AAT deficiency existed, hampering the development of new therapies. This shortcoming is now addressed by both AAT-null and antisense oligonucleotide mouse models. The goal of this study was to more fully characterize the antisense oligonucleotide model. Both liver AAT mRNA and serum AAT levels were lower in anti-AAT versus control oligonucleotide-treated mice after 6, 12, and 24 wk. Six and twelve weeks of anti-AAT oligonucleotide therapy induced emphysema that was worse in female than male mice: mean linear intercept 73.4 versus 62.5 m (P 0.000003). However, at 24 wk of treatment, control oligonucleotide-treated mice also developed emphysema. After 6 wk of therapy, anti-AAT male and female mice demonstrated a similar reduction serum AAT levels, and there were no sex or treatmentspecific alterations in inflammatory, serine protease, or matrix metalloproteinase mRNAs, with the exception of chymotrypsin-like elastase 1 (Cela1), which was 7- and 9-fold higher in anti-AAT versus control male and female lungs, respectively, and 1.6-fold higher in female versus male anti-AAT-treated lungs (P 0.04). While lung AAT protein levels were reduced in anti-AAT-treated mice, lung AAT mRNA levels were unaffected. These findings are consistent with increased emphysema susceptibility of female patients with AATdeficiency. The anti-AAT oligonucleotide model of AAT deficiency is useful for compartment-specific, in vivo molecular biology, and sex-specific studies of AAT-deficient emphysema, but it should be used with caution in studies longer than 12-wk duration. [ABSTRACT FROM AUTHOR]

Introduction: Bullous lung disease is a common presentation in patients with chronic obstructive pulmonary disease (COPD). The giant pulmonary bulla occupies one third of involved hemithorax and characterized by the existence of centrilobular emphysema in non bullous lung. Sometimes air reabsorbs spontaneously leading to shrinkage and regression of bulla known as autobullectomy. Mechanism of autobullectomy remains unclear. Here, we report a patient with infected bulla who experienced complete resolution after antifungal treatment. Case History: A 63 year old mal with history of twenty pack years of tobacco smoking came to the pulmonary outpatient department with complains of left side chest pain, cough with mild expectoration and low grade fever since five days. Patient also had blood tinged sputum since three days. His chest X-Ray PA view showed a thin walled cavity with air fluid level on the left upper zone with a few calcified parenchymal foci. Patient had symptomatic improvement after two weeks of antifungal treatment. Repeat skiagram chest and CECT chest showed complete resolution of bulla. Discussion: There are two hypotheses which can explain occurrence of bulla: Conclusion: Spontaneous resolution of giant bulla also known as autobullectomy is an ignorant event and follows an infection trigger. The early suspicion and diagnosis by the treating physician can avert the need for a surgical bullectomy in these patients and decline the morbidity and mortality. Our case is of interest not only because of the rarity with which spontaneous regression has been reported in the literature but also because the source of infection was fungal.

The present study investigated the effects of dietary supplementation of yeast nucleotides on the hematology, antioxidant activity, non-specific immunity, expression of intestinal cytokines, and disease resistance in Nile Tilapia (Oreochromis niloticus) . Fish weighing 42.90 ± 0.14 g were randomly divided into four groups. Each group was set in triplicate (15 fish per replicate). Fish were fed on four dietary levels of yeast nucleotides (NTs) supplemented with the basal diet 0% (control), 0.05%, 0.15%, and 0.25% NTs. Significantly higher total serum protein, albumin, total serum globulin, total WBC counts, and lymphocyte and granulocyte contents were recorded in 0.25% NT group as compared to the control. The albumin/globulin ratio (A:G) showed a considerable decrease in the 0.25% NT group. The non-specific immune parameters; serum killing percentage, lysozyme activity, nitric oxide assay, IgM levels, and anti-protease activity, were significantly higher in the 0.25% NT group as compared to the control. Moreover, a 15-day feeding trial demonstrated improved results in terms of serum lysozyme activity, nitric oxide assay, IgM levels, and anti-protease activity than a 30-day feeding trial. A significant increase in the anti-oxidant status of O. niloticus was noticed, as reflected by increased superoxide dismutase and malondialdehyde activity in the serum of 0.25% NT group compared to the control, while glutathione peroxidase displayed a significant increase in all groups as compared to the control. The intestinal cytokines TGF-β, IL-1β, IL-10β, and TNF-α mRNA levels showed a pattern of 0.25% NT > 0.15% NT > 0.05% NT > 0% NT, as relative to the control Ef-1α levels. The relative survival percentages of fish fed on yeast nucleotide-supplemented diets, as analyzed by exposure to Aeromonas sobria, were significantly better than the control group. In conclusion, dietary yeast nucleotide administration at 0.25% improved blood proteins, leukocytes, antioxidant activity, non-specific immunity, cytokine gene expression, and disease resistance of Nile Tilapia. [ABSTRACT FROM AUTHOR]

Introduction: α1-Antitrypsin deficiency is an under-recognized condition with long diagnostic delays between the first symptoms and diagnosis, and there is evidence that patients with suggestive symptoms may see many physicians before an exact diagnosis is made. An increased incidence of serum α1-antitrypsin deficiency has been reported in patients with spontaneous pneumothorax. Aim of the research: To evaluate α1-antitrypsin level in subjects with spontaneous pneumothorax. Material and methods: Thirty-nine patients with the diagnosis of spontaneous pneumothorax and 100 age- and sex-matched control subjects were included in the study. α1-antitrypsin concentrations were determined by nephelometry. Serum qualitative Z antitrypsin variant was analyzed using commercial ELISA kits and α1-antitrypsin phenotyping was carried out by means of isoelectric focusing. Results: Spontaneous pneumothorax occurred in 29 patients for the first time; 10 patients had recurrent pneumothorax. There were no significant differences in age and lung function comparing the patients with and without αα1-antitrypsin deficiency. α1-Antitrypsin level was significantly higher in patients with spontaneous pneumothorax (1.53 ±0.23 g/l) than controls (1.34 ±0.31 g/l) (p = 0.03). Conclusions: Elevated serum α1-antitrypsin level in patients with pneumothorax may show the need to inhibit the activity of proteases that are important for lung damage.

Morbid adherence remains a puzzling disease. This paper suggests that normal and morbidly adherent placentation may be viewed best in terms of trophoblastic stem cells and the mutually exclusive branches of the trophoblastic differentiation pathway-villous trophoblast (VT), interstitial and endovascular nonvillous trophoblast (NVT) at the implantation site, and a positional variation in the chorion. Based on cases of hysterectomies for morbid adherence seen over 30 years at a community hospital, analyzed with routine keratin stains, with actin and trichrome stains as indicated, and with attempts at ultrasonography-pathology correlation, we present selected observations. In true accreta, the site of morbid adherence was to dilated basal plate vessels infiltrated by endovascular NVT, with scant interstitial NVT, and normal myometrium. It appeared that excess blood flow into the placenta was due to excessively deep keratin-positive endovascular NVT that spread-independently of interstitial NVT-in an angiocentric fashion in both accreta and increta. Retroplacental abnormalities were due to myometrial destruction by interstitial NVT in increta, sometimes requiring actin stains for detection; and to an admixture of markedly dilated endometrial glands and vessels in true accreta, best appreciated with keratin stains. Variations of depth and extent in increta may be due to variations in myometrial tone, and in the protease-antiprotease balance. Morbidly adherent fetal membranes are described, and the role of caesarean section scars in incretas is addressed. [ABSTRACT FROM AUTHOR]

This study was aimed to investigate the effects of bromelain on angiogenesis, nitric oxide, and matrix metalloproteinase-3 and -9 in rats exposed to 1200 mV electrical burn injury. Thirty-five, male Wistar albino rats was divided into five groups ( n = 7 each), including control (untreated) group; electrical burn injury (EI) group; electrical burn injury + 21.25 mg/kg BW bromelain group (EIB 1 ); electrical burn injury + 42.50 mg/kg BW bromelain group (EIB 2 ); and electrical burn injury + 85.00 mg/kg BW bromelain group (EIB 3 ). Rat models of electrical burns done by providing electricity in rats that had been anesthetized, a voltage of 1200 mV and strong currents 15 mA for 10 s. The VEFG, NO, and MMP-9 levels were significantly greater in the EI group compared to the untreated group. Out of the 21.25 mg/kg BW, 42.50 mg/kg BW, and 85 mg/kg BW doses of Bromelain extract, only the lowest doses prevented EI-induced increase in NO and MMP-9 level ( P < 0.05). Bromelain at the lowest dose (21.25 mg/kg BW) act as anti-inflammatory and modulate the matrix metalloproteinase-9 in rats exposed to 1200 mV electrical burn injury. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of early peritoneal lavage with ulinastatin on the outcome of a rat model of severe acute pancreatitis (SAP). A total of 80 male Wistar rats were randomly divided into the following groups: Sham-operated (C), SAP model (M), saline lavage (SL), intravenous ulinastatin (IU), early ulinastatin lavage (EUL) and late ulinastatin lavage (LUL). Intraperitoneal lavage or injection were performed immediately subsequent to the establishment of the SAP model in groups SL, IU and EUL and 3 h later in group LUL. Intraperitoneal lavage with or without ulinastatin was performed for 3 h. The survival time of the rats in groups C, M, EUL and LUL was recorded over a 12-h period and the median survival time was calculated. At 3 h after the induction of SAP, histopathological analyses were performed and the biochemical parameters of groups C, M, SL, IU and EUL were assessed. Groups EUL and LUL exhibited an increased median survival time compared with Group M, with the survival time of the rats in group EUL markedly longer than that in the group LUL rats. Group SL, IU and EUL rats were found to have reduced plasma activities of amylase, lipase, aspartate transaminase and alanine transaminase, with the biggest change observed in the group EUL rats. Furthermore, the intervention in groups SL and EUL was more effective at reducing creatinine and urea levels than that in group IU. Rats in group EUL exhibited a greater inhibition of the SAP-induced increase in troponin T levels than rats in groups SL and IU. The pathological severity scores of the pancreas, liver, kidney and lung in group EUL were significantly lower than those in groups M and better than those in groups SL and IU. In conclusion, early intraperitoneal lavage with ulinastatin significantly improves the median survival time and protects multi-organ function in an SAP model. [ABSTRACT FROM AUTHOR]

Background: Although blebs and bullae are frequently found in the apexes of lungs of patients with primary spontaneous pneumothorax (PSP), its pathogens remain unclear. Objectives: To examine the role of proteases [matrix metalloproteinase (MMP)-2, MMP-7 and MMP-9] and antiproteases [tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3 and TIMP-4] in the pathogenesis of PSP. Method: Fifty consecutive PSP patients who received standard surgical care were enrolled in the study. Lung tissues from 20 patients with stage I non-small cell lung cancer were used as a control. Immunohistochemistry (IHC), reverse transcription-polymerase chain reaction (RT-PCR) and gelatin zymography were used to evaluate the expression of MMP and TIMP in the lung tissue of patients with PSP. Results: Overexpression of MMP-2, MMP-7 and MMP-9 was found in the afflicted lung by IHC, zymography and RT-PCR. By IHC, higher expression of MMP-2 and MMP-9 in PSP patients was identified in alveolar macrophages and type II pneumocytes (88 and 92% of patients in macrophages, and 72 and 70% of patients in type II pneumocytes, respectively). MMP-2, MMP-7 and MMP-9 expression in patients was higher in mesothelial cells (66, 76 and 76%). Overexpression of TIMP-2 was detected in the extracellular matrix around bullae and blebs. Expression levels of TIMP-1, TIMP-3 and TIMP-4 were negligible (<10% of cells) in both PSP patients and controls. Conclusions: MMP-2, MMP-9, MMP-7 and TIMP-2 were upregulated in PSP lesions. These results suggest that an imbalance between the expression of proteases and antiproteases may be involved in the pathogeneses of PSP. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1-/- mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection. [ABSTRACT FROM AUTHOR]

Abstract: The present study was designed to test the immunostimulatory efficacy of poly-β hydroxybutyrate–hydroxyvalerate (PHB–HV) extracted from Bacillus thuringiensis B.t.A102 on the immune system of Oreochromis mossambicus. Fish were fed with 0%, 1%, 3% or 5% PHB–HV supplemented feed and were bled at regular intervals of 5 days. The specific immune response was measured in terms of antibody response to sheep red blood cells, the nonspecific immune mechanisms were analysed in terms of serum lysozyme activity, total peroxidases activity and antiprotease activity. The overall functional immunity was tested by experimental challenge with live virulent Aeromonas hydrophila. The results revealed that all the doses of PHB–HV supplementation in feed were effective in stimulating both specific and nonspecific immune mechanisms. The bacterial challenge experiment showed that highest dose of 5% PHB–HV supplementation was more effective than 1% and 3% doses. The study concludes that PHB–HV can be used as a potential immunostimulant in finfish aquaculture. [Copyright &y& Elsevier]

Alpha-1 antitrypsin, a small glycoprotein clade A serpine serine protease inhibitor of neutrophil elastase has been shown to increase in humans following bacterial and viral infection. However, we report here significant reduction of this major inhibitor of elastase in plasma of F. tularensis LVS and SCHU S4 (Type A strain) following pulmonary challenge. Consistent with an imbalance of protease-antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresistivity. These data are suggestive of targeted tissue destruction via unchecked neutrophhil elastase activity in infected animals.

Abstract: The present paper describes the effect of water-soluble fraction of the leaves of the Indian medicinal plant, Tinospora cordifolia (Miers) on the non-specific immunity and disease resistance in Oreochromis mossambicus (Peters). Fish were intraperitoneally injected with 0, 6, 60 or 600 mg kg−1 body weight, of the water soluble fraction. The non-specific humoral (lysozyme, antiprotease and complement) and cellular (production of reactive oxygen and nitrogen species and myeloperoxidase) responses and disease resistance against Aeromonas hydrophila were tested. All the doses of water-soluble fraction tested, significantly enhanced the serum lysozyme, antiprotease and natural haemolytic complement activities on most of the days tested. Similarly, all the doses of water-soluble fraction used, enhanced the cellular myeloperoxidase activity on all the days tested. The enhancement in the ROS and RNI production by peripheral blood leucocytes was observed on almost all the days tested, in most of the treated groups. All the doses of water-soluble fraction when administered as a single or double dose gave protection in terms of reduced percent mortality which is reflected in the increased Relative Percent Survival (RPS) values. The results clearly indicate the immunostimulatory and disease resistance properties of T. cordifolia leaf fraction and so its potential to be used as an immunoprophylactic in finfish aquaculture. [Copyright &y& Elsevier]

Alpha-1-antitrypsin deficiency (AATD) is a rare genetic disorder associated with the development of liver and lung disease. AAT is a 52-kD glycoprotein, produced mainly by hepatocytes and secreted into the blood. Agglomeration of the AAT-protein in hepatocytes can result in liver disease. Exposure to smoke is the major risk factor for the development of lung disease characterised as early chronic obstructive lung disease (COPD). Diagnosis is based on the analysis of the AAT genotype and phenotype. The measurement of the AAT serum level is useful as screening test. Liver biopsy is not necessary to establish the diagnosis. Therapy for AAT-related liver disease is supportive, a specific therapy is not available. AATD is a rare condition (1:5000–10000) and, as a consequence, data and information on diagnosis and treatment are not easily accessible. This chapter provides a comprehensive overview on AATD, covering basic biology, diagnostic and therapeutic approaches. [ABSTRACT FROM AUTHOR]

Abstract: Alpha-1 antitrypsin deficiency is an inherited disease affecting the lung and liver. The typical pulmonary manifestation is chronic obstructive pulmonary disease and emphysema. Severe chronic obstructive pulmonary disease may occur in young adulthood, and terminal respiratory insufficiency causes premature death in many patients. In the liver, alpha-1 antitrypsin deficiency may manifest as benign neonatal hepatitis syndrome; a small percentage of adults develop liver fibrosis, with progression to cirrhosis and hepatocellular carcinoma. The alpha-1 antitrypsin molecule is a serine protease inhibitor that is predominantly produced in the liver. Its most important physiologic functions are the protection of pulmonary tissue from aggressive proteolytic enzymes and regulation of pulmonary immune processes. Diagnosis of alpha-1 antitrypsin deficiency can be established by measurement of the serum alpha-1 antitrypsin concentration or by genetic analysis. Treatment is similar to the usual treatment for patients with chronic obstructive pulmonary disease. A further option is substitution therapy with human alpha-1 antitrypsin. The targets of treatment are the prevention of the accelerated decline of pulmonary function, reduction of lung infections, and improvements in exercise capacity. [Copyright &y& Elsevier]

Chronic lung diseases such as cystic fibrosis and emphysema are characterized by a protease burden, an infective process and a dominant proinflammatory profile. Secretory leucoprotease inhibitor (SLPI) is a prominent innate immune protein of the respiratory tract, possessing serine protease inhibitor activity, antibacterial activity, and anti-inflammatory/immunomodulatory activity. In the course of this review, the authors highlight the findings from a range of studies that illustrate the multiple functions of SLPI and its role in the resolution of the immune response. [ABSTRACT FROM AUTHOR]

Venezuela is a country where indigenous and local communities use more than 1500 species of plants for medicinal purposes. Anticancer drugs derived from plants may act through a direct cytotoxic effect on tumor cells or through other mechanisms such as inhibitory effects on the proteases involved in tumor growth and spread. From our databases of botanical collections and ethnobiological usage, we selected 17 species to conduct an initial survey of cytotoxicity on eight human tumor cell lines (representing lung, breast, colon, and pancreas) and activity against four proteases. Thirteen extracts from 10 species were cytotoxic, at 100 μg/ml or less, on one or more cell lines. Extracts of Jacaranda copaia and Tapirira guianensis were active against several cell lines, whereas extracts of Gnetum nodiflorum, Protium heptaphyllum, Protium unifoliolatum, Costus scaber, and Croton cuneatus were potent and selectively inhibitory for one or two cell lines. Antiprotease activity against one or more enzymes was detected in extracts from 14 plant species, with most activity being directed against leucine aminopeptidase (LAP). The activities observed are discussed in relation to the chemical constituents reportedly isolated from these plants and/or other species of the genus and their traditional uses. [ABSTRACT FROM AUTHOR]

Inflammation is a major component of the vicious cycle characterizing cystic fibrosis pulmonary disease. If untreated, this inflammatory process irreversibly damages the airways, leading to bronchiectasis and ultimately respiratory failure. Antiinflammatory drugs for cystic fibrosis lung disease appear to have beneficial effects on disease parameters. These agents include oral corticosteroids and ibuprofen, as well as azithromycin, which, in addition to its antimicrobial effects, also possesses antiinflammatory properties. Inhaled corticosteroids, colchicine, methotrexate, montelukast, pentoxifylline, nutritional supplements, and protease replacement have not had a significant impact on the disease. Therapy with oral corticosteroids, ibuprofen, and fish oil is limited by adverse effects. Azithromycin appears to be safe and effective, and is thus the most promising antiinflammatory therapy available for patients with cystic fibrosis. Pharmacologic therapy with antiinflammatory agents should be started early in the disease course, before extensive irreversible lung damage has occurred. [ABSTRACT FROM AUTHOR]

Inflammation is an important indicator of tissue injury. In the acute form, there is usually accumulation of fluids and plasma components in the affected tissues. Platelet activation and the appearance in blood of abnormally increased numbers of polymorphonucleocytes, lymphocytes, plasma cells and macrophages usually occur. Infectious disorders such as sepsis, meningitis, respiratory infection, urinary tract infection, viral infection, and bacterial infection usually induce an inflammatory response. Chronic inflammation is often associated with diabetes mellitus, acute myocardial infarction, coronary artery disease, kidney diseases, and certain auto-immune disorders, such as rheumatoid arthritis, organ failures and other disorders with an inflammatory component or etiology. The disorder may occur before inflammation is apparent. Markers of inflammation such as C-reactive protein (CRP) and urinary trypsin inhibitors have changed our appraisal of acute events such as myocardial infarction; the infarct may be a response to acute infection and (or) inflammation. We describe here the pathophysiology of an anti-inflammatory agent termed urinary trypsin inhibitor (uTi). It is an important anti-inflammatory substance that is present in urine, blood and all organs. We also describe the anti-inflammatory agent bikunin, a selective inhibitor of serine proteases. The latter are important in modulating inflammatory events and even shutting them down. [ABSTRACT FROM AUTHOR]

Low-molecular-weight heparins (LMWH) represent depolymerized porcine mucosal heparin derivatives, which are commonly used for the management of thrombotic disorders. Because of their widespread usage, the supplies of the raw material namely unfractionated heparin are nearly exhausted. Porcine mucosal tissue is almost exclusively used for the preparation of these agents. Thus, there is a timely need for the production of heparin like drugs from other sources. Fermentation techniques have been used to produce carbohydrates such as dextran and innulin for therapeutic purposes. Bacterial cell wall polysaccharide mimics the linear hexose units, which constitute heparin. Utilizing Escherichia coli cell membranes produced by fermentation technology, chemical sulfation and enzymatic epimerization, sulfaminoheparosan type of polymer mimicking the structure of heparin has been produced. These semi-synthetic sulfaminoheparosans exhibit biologic actions comparable to that observed with heparin. The sulfaminoheparosan core can also be degraded to obtain low-molecular-weight (LMW) derivatives mimicking LMWHs. Using this technique, a novel LMW sulfaminoheparosan derivative (Q93C/239) was produced by Inalco, Milan, Italy. To compare this heparin analogue, a LMWH, namely tinzaparin, was used to determine the relative anticoagulant, antiprotease, and molecular profile. Additional studies were carried out to determine the susceptibility of this agent to heparinase-I. These comparative studies exhibited both antiprotease and anticoagulant properties similar to those of tinzaparin, However LMW sulfaminoheparosan resisted heparinase-I digestion at low heparinase-I concentrations. These studies demonstrate that the sulfaminoheparosan derived LMW components exhibit similar molecular and anticoagulant profile as tinzaparin and warrant additional preclinical and clinical development to determine their potential usefulness as antithrombotic agents. [ABSTRACT FROM AUTHOR]

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca2++ATPase activity. The smooth muscle plasma membrane possesses a Ca2++ dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA and the tissue inhibitor of metalloprotease-2 (TIMP-2). Since 72 kDa is the molecular mass of MMP-2 and since in our present study the 72 kDa protease in the gelatin containing zymogram is inhibited by matrix metalloprotease inhibitors, EGTA and TIMP-2, it may be suggested that the 72 kDa protease is the MMP-2. In addition to the increasing Ca2++ATPase activity, t-buOOH also enhances the activity of the membrane associated Ca2++ dependent protease that degrades 14C-gelatin. The oxidant triggered protease activity and the Ca2++ATPase activity were found to be prevented by the antioxidant vitamin E, and also by the Ca2++ dependent matrix metalloprotease inhibitors: EGTA and TIMP-2. Adding MMP-2 to the smooth muscle plasma membrane suspension caused an increase in Ca2++ATPase activity and pretreatment with TIMP-2 prevents the increase in Ca2++ATPase activity. Combined treatment of the smooth muscle plasma membrane with low doses of MMP-2 and t-buOOH augments further the Ca2++ATPase activity caused by the respective doses of either t-buOOH or MMP-2. Pretreatment with TIMP-2 prevents the increase in Ca2++ATPase activity elicited by the low doses of MMP-2 and/or t-buOOH. [ABSTRACT FROM AUTHOR]

The use of metal-organic complexes is a potentially fruitful approach for the development of novel enzyme inhibitors. They hold the attractive promise of forming stronger attachments with the target by combining the co-ordination ability of metals with the unique stereoelectronic properties of the ligand. We demonstrated that this approach can be successfully used to inhibit the protease of the human immunodeficiency virus (type 1). Several ligands bearing substituents designed to interact with the catalytic site of the enzyme when complexed to Cu2+ were synthesised. The inhibition pattern of the resulting copper(II) complexes was analysed. We showed that the copper(II) complex of N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide (C1) interacts with the active site of the enzyme leading to competitive inhibition. On the other hand, N2-pyridine-amide ligands and oxazinane carboxamide ligand were found to be poor chelators of the cupric ion under the enzymatic assay conditions. In these cases, the observed inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease. While unchelated metal cations are not likely to be useful agents, metal chelates such as C1 should be considered as promising lead compounds for the development of targeted drugs. [Copyright &y& Elsevier]

Gabexate mesilate is an antiprotease drug, which reduced the severity of pancreatitis and frequency of post-ERCP pancreatitis. In dogs gabexate inhibits sphincter of Oddi motility but no data are available in humans. The aim of this study was to verify by manometry the action of gabexate on human sphincter of Oddi motility. We enrolled 12 patients with idiopathic recurrent pancreatitis (eight males, five females, mean age 46 ± 8 years). Standard preendoscopic sphincter of Oddi manometry was done in basal conditions and during infusion of gabexate 20 mg/min: basal pressure, amplitude and frequency of phasic contractions, and motility index (amplitude per frequency) were calculated before and after gabexate injection. Statistical analysis was performed by using Wilcoxon rank test for paired data. Six patients had a manometric diagnosis of stenosis (basal pressure greater than 40 mm Hg); six had normal findings. Phasic activity was not evaluable in five patients with stenosis. Basal pressure was unaffected by drug infusion, while gabexate caused a significant reduction of phasic activity, both in terms of frequency (4.5 ± 1 vs 3.6 ± 1; P < 0.05) and amplitude (157.4 ± 44 vs 80.0 ± 32; P < 0.05) of contractions. Motility index was reduced on average by 49%. In conclusion, this pilot study confirms, in patients with acute recurrent pancreatitis, the inhibitory action of gabexate on sphincter of Oddi motility already described in dogs. This action needs to be revaluated at therapeutic dosages. On the other hand, prophylactic use of the drug should be avoided during sphincter of Oddi manometry, in order to avoid false negative results. [ABSTRACT FROM AUTHOR]

We have determined effect of the oxidant peroxynitrite (ONOO-) on Ca2+-dependent matrix metalloprotease-2 (MMP-2) activity and the role of the protease on Ca2+ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO--triggered conditions. The smooth muscle plasma membrane possesses a 72-kDa protease activity in a gelatin-containing zymogram. The 72-kDa protease activity has been found to be inhibited by tissue inhibitor of metalloprotease-2 (TIMP-2), indicating that the protease is the matrix metalloprotease-2 (MMP-2). Treatment of the membrane suspension with ONOO- caused stimulation of the MMP-2 activity (as evidenced by 14C-gelatin degradation) and also increased Ca2+ATPase activity. The ONOO--triggered protease activity and the Ca2+ATPase activity were found to be inhibited by the antioxidants: vitamin E, thiourea, and mannitol. Pretreatment with catalase and superoxide dismutase did not significantly alter ONOO--stimulated MMP-2 activity and Ca2+ATPase activity, indicating that peroxide and superoxide are not present in appreciable amount in ONOO-. Under both basal and ONOO- triggered conditions, the MMP-2 activity and the Ca2+ATPase activity were also inhibited by EGTA, 1:10-phenanthroline, and TIMP-2. However, the ONOO--stimulated MMP-2 activity and the Ca2+ATPase activity were found to be insensitive to phenylmethylsulfonylfluoride, Bowman-Birk inhibitor, chymostatin, leupeptin, antipain, N-ethylmaleimide, and pepstatin. These results suggest that ONOO- caused stimulation of MMP-2 activity and that the increased MMP-2 activity subsequently played a pivotal role in stimulating Ca2+ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane. [ABSTRACT FROM AUTHOR]

Neutrophil elastase (NE) is a major inflammatory protease released by neutrophils and is present in the airways of patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, non-CF bronchiectasis, and bronchopulmonary dysplasia. Although NE facilitates leukocyte transmigration to the site of infection and is required for clearance of Gram-negative bacteria, it also activates inflammation when released into the airway milieu in chronic inflammatory airway diseases. NE exposure induces airway remodeling with increased mucin expression and secretion and impaired ciliary motility. NE interrupts epithelial repair by promoting cellular apoptosis and senescence and it activates inflammation directly by increasing cytokine expression and release, and indirectly by triggering extracellular trap release and exosome release, which magnify protease activity and inflammation in the airway. NE inhibits innate immune function by digesting opsonins and opsonin receptors, degrading innate immune proteins such as lactoferrin, and inhibiting macrophage phagocytosis. Importantly, NE-directed therapies have not yet been effective in preventing the pathologic sequelae of NE exposure, but new therapies are being developed that offer both direct antiprotease activity and multifunctional anti-inflammatory properties. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases. [ABSTRACT FROM AUTHOR]

Abstract Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA) protein binds to sialic acid residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host cell membrane, which is mediated by host trypsin-like serine protease. We show data here demonstrating that the protease:antiprotease ratio is increased in the nasal mucosa of asthmatics and that these changes were associated with increased proteolytic activation of influenza. These data suggest that disruption of the protease balance in asthmatics enhances activation and infection of influenza virus.

α -antitrypsin (AAT) deficiency is a genetic disease in which low serum and lung levels of the antiprotease AAT cause a deficiency of the anti-elastase defensive screen of the lower respiratory tract such that neutrophil elastase is free to degrade the connective tissue of the lung, eventually resulting in emphysema. Intravenous AAT infusion therapy restores lung levels of AAT, but is inefficient, costly and a demanding form of therapy. As an alternative, we evaluated aerosol delivery of human plasma AAT (pAAT) and recombinant DNA-produced AAT (rAAT), as a means of providing anti-elastase protection to the lower respiratory tract. In vitro studies demonstrated that both pAAT and rAAT can be aerosolized into droplets suitable for alveolar deposition without loss of antiprotease activity. When administered by aerosol to individuals with AAT deficiency, pAAT and rAAT each significantly raised lung epithelial lining fluid levels of AAT and anti-neutrophil elastase capacity, with the likelihood that twice daily administration of 100 mg of either form would result in normalization of lung anti-elastase defenses at the alveolar surface. Studies in sheep further demonstrated that the aerosolized pAAT and rAAT were each able to pass through alveolar epithelium and gain access to the interstitial compartment of the lung, thus increasing anti-elastase defenses of the lung intersitium. Therapy was safe and well tolerated in all cases. Aerosol therapy with pAAT or rAAT is a safe, feasible, and likely a biochemically efficacious alternative to intravenous AAT augmentation therapy and merits further long-term studies for clinical therapy. [ABSTRACT FROM AUTHOR]

Antiproteases are known to be present in amyloid deposits. We evaluated the possibility of using an anti-serine protease (aprotinin) labelled with technetium-99m (TcA), usually employed as a cortical renal tracer, for the imaging of amyloid deposits. Because of the known high uptake of TcA by the kidneys, we limited our analysis to extra-abdominal amyloid localizations. We report the scintigraphic findings observed in 24 patients with light chain amyloidosis (AL) and one with a hereditary form who were known or suspected to have extra-abdominal involvement. Planar scans obtained 100 min after i.v. TcA administration showed myocardial accumulation in 11 patients, pleuropulmonary accumulation in nine, pericardial accumulation in two and localization in the neck region (thyroid, salivary glands and tongue) in eight. TcA scintigraphy was negative in five patients without clinical or laboratory evidence of extraabdominal involvement, as well as in 12 control group patients with cardiac and renal diseases. These preliminary results indicate TcA to be a low-cost, readily available radiopharmaceutical for imaging of extra-abdominal involvement in AL type amyloidosis. [ABSTRACT FROM AUTHOR]

The saline and alkaline extracts of Fagara zanthoxyloides, Olax subscorpioides and Tetrapleura tetraptera were investigated for antiprotease and membrane stabilizing activities by spectroscopic procedures. Saline extracts of these plants stabilized human red blood cell membrane subjected to hypotonic- and heat-induced lyses. The results revealed that the degree of membrane stabilization was 84% for F. zanthoxyloides while T. tetraptera and O. subscorpioides exhibited 70% and63.2%, respectively. Also, sodium hydroxide extracts of the three plants exhibited antiprotease activity. A 1:5 dilution of F. zanthoxyloides gave 80% inhibition while undiluted extracts of O. subscorpioides and T. tetraptera showed 96% and 73% inhibitions, respectively. [ABSTRACT FROM AUTHOR]