Effect Of Serine Protease Inhibitor Research Articles

Neutrophils use a novel NLRP3 inflammasome-independent mechanism to release bioactive IL-1β in response to lysosomal disruption

Abstract IL-1β is an inflammatory cytokine mainly secreted by myeloid cells in response to infection or sterile tissue damage. Non-canonical secretion of IL-1β from macrophages downstream of activated NLRP3/caspase-1 inflammasomes is the best-characterized model; this is mediated by caspase-1 cleavage of GSDMD allowing N-GSDMD to form pores in the plasma membrane that act as conduits for IL-1β release and inducers of pyroptosis as a lytic cell death. The NLRP3 initiator acts as a sensor of perturbed cellular homeostasis including decreased cytosolic [K+]. In macrophages, this K+ efflux mediated NLRP3 activation is also triggered by agents that disrupt lysosomal integrity. While neutrophils also assemble competent NLRP3 inflammasomes and release bioactive IL-1β via GSDMD-dependent mechanisms, they resist the formation of plasma membrane N-GSDMD pores and progression to pyroptosis. We tested whether lysosome disrupting stimuli in neutrophils would phenocopy macrophage mechanisms to secrete IL-1β in an NLRP3 and GSDMD-dependent manner. Surprisingly, our data indicate that lysosome-disrupting stimuli induce neutrophils to release mature IL-1β and undergo rapid lytic cell death independently of NLRP3 inflammasome assembly and caspase-1 activity with only partial dependence on GSDMD. Ongoing studies testing effects of serine protease inhibitors support a mechanism in which cytosolic accumulation of the multiple serine proteases stored in the lysosome-like azurophilic granules coordinately disrupt canonical NLRP3 inflammasome assembly and directly cleave proIL-1β and GSDMD as part of a novel neutrophil-specific signaling mechanism for lysosomal disruption-induced processing and export of IL-1β. P01-AI141350 R01-EY014362 R21-EY032662

The Effect of Serine Protease Inhibitors on Visceral Pain in Different Rodent Models With an Intestinal Insult.

Background: Serine proteases are believed to play a key role in the origin of abdominal pain in IBD and IBS. We previously demonstrated a reduction of visceral pain in a post-inflammatory IBS rat model after a single intraperitoneal or intracolonic administration of a serine protease inhibitor. The aim of this study was to investigate the efficacy of serine protease inhibition on visceral pain in two different animal models involving a colonic insult based either on acute inflammation or on neonatal irritation. Moreover, protease profiling was explored in the acute colitis model. Methods: An acute 2,4,6-trinitrobenzenesulphonic acid (TNBS) colitis rat model and a chronic neonatal acetic acid mouse model were used in this study. Visceral sensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after intraperitoneal administration of the serine protease inhibitors nafamostat, UAMC-00050 or their vehicles. Colonic samples from acute colitis rats were used to quantify the mRNA expression of a panel of serine proteases and mast cell tryptase by immunohistochemistry. Finally, proteolytic activities in colonic and fecal samples were characterized using fluorogenic substrates. Key Results: We showed a significant and pressure-dependent increase in visceral hypersensitivity in acute colitis and neonatal acetic acid models. UAMC-00050 and nafamostat significantly reduced VMRs in both animal models. In acute colitis rats, the administration of a serine protease inhibitor did not affect the inflammatory parameters. Protease profiling of these acute colitis animals revealed an increased tryptase immunoreactivity and a downregulation of matriptase at the mRNA level after inflammation. The administration of UAMC-00050 resulted in a decreased elastase-like activity in the colon associated with a significantly increased elastase-like activity in fecal samples of acute colitis animals. Conclusion: In conclusion, our results suggest that serine proteases play an important role in visceral hypersensitivity in an acute TNBS colitis model in rats and a neonatal acetic acid model in mice. Moreover, we hypothesize a potential mechanism of action of UAMC-00050 via the alteration of elastase-like proteolytic activity in acute inflammation. Taken together, we provided fundamental evidence for serine protease inhibitors as a promising new therapeutic strategy for abdominal pain in gastrointestinal diseases.

Serine Proteases and Their Inhibitors Affect the Rate of GnRH Neuronal Migration in the Embryonic Chick Model: A Thesis Proposal

Development in an organism is a highly complex task that requires a specific sequence for proper function. One of these tasks is neuronal migration and the extracellular cues that help to guide neurons to their appropriate locations. If neurons do not migrate to their appropriate locations, then there are abnormal neural connections, which can result in neuronal migration disorders (NMD). It has been shown that the inhibition and stimulation of migration may be balanced due to effects of serine protease inhibitors and serine proteases. Serine proteases and their inhibitors can alter the various chemoattraction-signaling cues and thereby modulate neuronal migratory processes. Furthermore, there exists a delicate balance between serine proteases and their inhibitors to modulate migration of neurons within development. Thus, by understanding how the balance of serine protease and serine protease inhibitor activity works, one can further understand how these changes affect neuronal migration in development. Experiments to test in vivo neuronal migration of chick GnRH neurons during embryogenesis were investigated to elucidate the role these proteins play in migration. Protein coated beads were inserted and GnRH levels measured to control. Such experimentation is crucial, as these studies are the first to explore the role of proteases in neural migration in an in vivo model. These findings indicate that the ability to initiate migration lies inherently within the neuron itself such that changes of the extracellular matrix composition are irrelevant in the initial timing. Neurons entering the CNS earlier in trypsin-treated embryos indicates continuous movement may be a cell autonomous event with trypsin-induced proteolysis. In other words, the ability of GnRH neurons to migrate across glial boundaries and through the brain to its target site is independent of further maturation of the neuron or the brain. Taken together, this study can provide knowledge regarding these proteins and their relation to development. This knowledge can potentially lead to therapies that can provide ameliorative care for patients suffering with NMD.

Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

A genetic programming-based approach to identify potential inhibitors of serine protease of Mycobacterium tuberculosis.

Aim: We applied genetic programming approaches to understand the impact of descriptors on inhibitory effects of serine protease inhibitors of Mycobacterium tuberculosis (Mtb) and the discovery of new inhibitors as drug candidates. Materials & methods: The experimental dataset of serine protease inhibitors of Mtb descriptors was optimized by genetic algorithm (GA) along with the correlation-based feature selection (CFS) in order to develop predictive models using machine-learning algorithms. The best model was deployed on a library of 918 phytochemical compounds to screen potential serine protease inhibitors ofMtb. Quality and performance of the predictive models were evaluated using various standard statistical parameters.Result:The best random forest model with CFS-GA screened 126 anti-tubercular agents out of 918 phytochemical compounds. Also, genetic programing symbolic classification method is optimized descriptors and developed an equation for mathematical models. Conclusion: The use of CFS-GA with random forest-enhanced classification accuracy and predicted new serine protease inhibitors of Mtb, which can be used for better drug development against tuberculosis.

The serine protease inhibitors and SERPINE1/2 disrupt prostaglandin E2 production and hyaluronic acid retention in FSH-stimulated pig cumulus-oocyte complexes.

The serine proteases, tissue- and urokinase-type plasminogen activators (PLAT and PLAU) and their inhibitors SERPINE1/2 are regulators of plasminogen to plasmin conversion. They are widely expressed in ovarian tissues, including granulosa and cumulus cells, and their expression is regulated by gonadotropins. The aim of this work was to assess the effect of serine protease inhibitors (aprotinin and AEBSF) and SERPINE1/2 on FSH-induced cumulus cell expansion, the production of prostaglandin E2 (PGE2) and retention of hyaluronic acid (HA) in expanding cumulus. The serine protease activity proved to be essential for the production of PGE2 and also for the retention of HA; the inhibition of plasminogen activators by SERPINE1/2 had the same effect. Collectively, these data indicate that plasmin is required for proper function of expanding cumulus cells in vitro and presumably also in vivo in the pre-ovulatory follicles.

Effect of serine protease inhibitor from squid ovary on gel properties of surimi from Indian mackerel.

Surimi from dark-fleshed fish is prone to degradation of myofibrillar proteins caused by the endogenous proteases, thereby lowering textural property. To tackle the problem, protease inhibitors (egg white, porcine plasma protein, and bovine plasma protein) have been used by the surimi industries. Due to drawbacks of some particular inhibitors (religious constraints, off-white color, off odor, etc.), the novel additives are still in demand. Ovary from squid processing industry, commonly discarded as the waste, can be an alternative natural protease inhibitor, which can improve the gel properties of surimi, especially produced from dark-fleshed fish.

Serine Proteases and their Inhibitors in Human Airway Epithelial Cells: Effects on Influenza Virus Replication and Airway Serine Proteases and their Inhibitors in Human Airway Epithelial Cells: Effects on Influenza Virus Replication and Airway Inflammation

Influenza virus replication and the production of inflammatory cytokines are associated with symptoms, including fever, and exacerbation of bronchial asthma and chronic obstructive pulmonary disease. Proteolytic activation of influenza viruses by serine proteases that are produced by airway epithelial cells is essential for viral entry and replication. Transmembrane protease serine S1 member (TMPRSS) 2, TMPRSS4 and TMPRSS11D have been detected certain cells, including the human alveolar epithelial cell line A549 and the surface epithelial cells of the human nasal mucosa, the trachea, the distal airways, and the lung. Several protease inhibitors, including aprotinin, reduce influenza virus replication. We previously demonstrated the following: (1) TMPRSSs (TMPRSS2, 4, and 11D) are present in primary cultures of human tracheal epithelial cells; (2) serine protease inhibitors, such as camostat and aprotinin, reduce the influenza virus replication and the release of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α into cell supernatants; and (3) camostat reduces the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. These findings suggest that serine proteases expressed by human tracheal epithelial cells induce the proteolytic activation of influenza viruses and that serine protease inhibitors may reduce viral replication and the resultant production of inflammatory cytokines. Thus, serine protease inhibitors are potential candidates for anti-influenza virus drugs Here, we review the expression of serine proteases, the role of serine proteases in influenza virus activation, and the effects of serine protease inhibitors. In this review, we aim to introduce the effects of serine proteases and their inhibitors on influenza virus infection of human airway epithelial cells by discussing the findings of previous studies performed by our group and other research groups. Furthermore, the clinical features and virulence of influenza virus infection are reviewed to clarify the association of virus replication and cytokine release with disease severity.

The serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells

BackgroundSerine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear. MethodsPrimary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2). ResultsCamostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat. ConclusionsThese findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.

A critical assessment of the effect of serine protease inhibitors on porcine fertilization and quality parameters of porcine spermatozoa in vitro

Proteases play an important role during mammalian fertilization. Their function is frequently investigated using specific inhibitors. We analyzed four serine protease inhibitors [4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor from glycine max (STI), Nα-tosyl-l-lysine-chloromethyl ketone hydrochloride (TLCK) and Np-tosyl-l-phenylalanine-chloromethyl ketone (TPCK)] for their in vitro effect on fertilization and sperm quality in pigs. Inhibitor concentrations were chosen based on the reduction of fertilization rate during preliminary dose–response experiments with cryopreserved epididymal spermatozoa. The inhibitor effects on in vitro fertilization (IVF) and sperm parameters (membrane and acrosomal integrity, motility and mitochondrial membrane potential – MMP) were evaluated using diluted fresh semen. AEBSF (100μM), TLCK (100μM) and TPCK (100μM) decreased total fertilization and polyspermy rates by at least 50%. STI (5μM) lowered total fertilization rates but not the level of polyspermy. AEBSF and TPCK reduced fertilization parameters to a similar degree using cryopreserved epididymal spermatozoa (dose–response experiment) or diluted fresh semen. Inhibition by STI was more pronounced using cryopreserved epididymal spermatozoa, whereas TLCK inhibited IVF only with diluted fresh semen. AEBSF and STI had no effect on sperm parameters, and TLCK significantly reduced motility. TPCK diminished MMP and motility and affected membrane and acrosomal integrity in a negative way. In summary, serine protease inhibitors differed in the way they reduce the fertilization rate. These results emphasize the necessity of inhibitor testing before they can be applied in fertilization studies. AEBSF and STI can be used in the future IVF studies without compromising sperm quality.

Protease Inhibitor Reduces Airway Response and Inflammation In Mouse Model Of Cockroach Allergy

RationaleSerine protease(s) are potent inducer of airway resistance and inflammation and the allergic response may be modified by proteases inhibitor. In the present study, effect of serine protease inhibitor in cockroach allergen induced airway resistance and inflammation was determined in mouse model.MethodsMice were immunized with cockroach extract (CE) or Per a 10 and treated with 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) 1 h before challenge. AHR was measured, one day after the last challenge and mice were euthanized to get BALF, blood and lung to evaluate of inflammation and oxidative stress.ResultsAEBSF treatment to mice reduced more than 90% AHR in CE and in Per a 10 challenged mice (p<0.05). EPO, IgE, IL-4, IL-5 and IL-13 levels were significantly (p<0.05) reduced in AEBSF treated mice as compared to untreated group. The treatment lowered the total cell, eosinophil and neutrophil count in BALF to 90, 80 and 83 % in CE and 100, 62 and 90 % in Per a 10 challenged mice (p<0.05), respectively. Lung histology revealed that AEBSF treated mice had reduced inflammation and low mucus production in airways. Furthermore, the treatment had significantly reduced 8-isoprostane and ROS level to 88 and 86 in CE and 82 and 83 % in per a 10 challenged mice in comparison to untreated group (p<0.05).ConclusionsAEBSF reduces allergen induced airway resistance and inflammation indicating that it might be used in “add-on” therapy for respiratory diseases. The therapeutic effect of inhibitor is independent of protease activity of allergen. RationaleSerine protease(s) are potent inducer of airway resistance and inflammation and the allergic response may be modified by proteases inhibitor. In the present study, effect of serine protease inhibitor in cockroach allergen induced airway resistance and inflammation was determined in mouse model. Serine protease(s) are potent inducer of airway resistance and inflammation and the allergic response may be modified by proteases inhibitor. In the present study, effect of serine protease inhibitor in cockroach allergen induced airway resistance and inflammation was determined in mouse model. MethodsMice were immunized with cockroach extract (CE) or Per a 10 and treated with 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) 1 h before challenge. AHR was measured, one day after the last challenge and mice were euthanized to get BALF, blood and lung to evaluate of inflammation and oxidative stress. Mice were immunized with cockroach extract (CE) or Per a 10 and treated with 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) 1 h before challenge. AHR was measured, one day after the last challenge and mice were euthanized to get BALF, blood and lung to evaluate of inflammation and oxidative stress. ResultsAEBSF treatment to mice reduced more than 90% AHR in CE and in Per a 10 challenged mice (p<0.05). EPO, IgE, IL-4, IL-5 and IL-13 levels were significantly (p<0.05) reduced in AEBSF treated mice as compared to untreated group. The treatment lowered the total cell, eosinophil and neutrophil count in BALF to 90, 80 and 83 % in CE and 100, 62 and 90 % in Per a 10 challenged mice (p<0.05), respectively. Lung histology revealed that AEBSF treated mice had reduced inflammation and low mucus production in airways. Furthermore, the treatment had significantly reduced 8-isoprostane and ROS level to 88 and 86 in CE and 82 and 83 % in per a 10 challenged mice in comparison to untreated group (p<0.05). AEBSF treatment to mice reduced more than 90% AHR in CE and in Per a 10 challenged mice (p<0.05). EPO, IgE, IL-4, IL-5 and IL-13 levels were significantly (p<0.05) reduced in AEBSF treated mice as compared to untreated group. The treatment lowered the total cell, eosinophil and neutrophil count in BALF to 90, 80 and 83 % in CE and 100, 62 and 90 % in Per a 10 challenged mice (p<0.05), respectively. Lung histology revealed that AEBSF treated mice had reduced inflammation and low mucus production in airways. Furthermore, the treatment had significantly reduced 8-isoprostane and ROS level to 88 and 86 in CE and 82 and 83 % in per a 10 challenged mice in comparison to untreated group (p<0.05). ConclusionsAEBSF reduces allergen induced airway resistance and inflammation indicating that it might be used in “add-on” therapy for respiratory diseases. The therapeutic effect of inhibitor is independent of protease activity of allergen. AEBSF reduces allergen induced airway resistance and inflammation indicating that it might be used in “add-on” therapy for respiratory diseases. The therapeutic effect of inhibitor is independent of protease activity of allergen.

The effect of serine protease inhibitors on airway inflammation in a chronic allergen-induced asthma mouse model.

Serine protease inhibitors reportedly attenuated airway inflammation and had antioxidant in multiorgan. However, the effects of the serine protease inhibitors nafamostat mesilate (FUT), gabexate mesilate (FOY), and ulinastatin (UTI) on a long-term challenged mouse model of chronic asthma are unclear. BALB/c mice (6 mice/group) were intratracheally inoculated with five doses of Dermatophagoides pteronyssinus (Der p; 50 μL, 1 mg/mL) at one-week intervals. Therapeutic doses of FUT (0.0625 mg/kg), FOY (20 mg/kg), or UTI (10,000 U/kg) were, respectively, injected intraperitoneally into these mice. Control mice received sterile PBS. At 3 days after the last challenge, mice were sacrificed to assess airway hyperresponsiveness (AHR), remodeling, and inflammation; lung histological features; and cytokine expression profiles. Compared with untreated controls, mice treated with FUT, FOY, and UTI had decreased AHR and goblet cell hyperplasia, decreased eosinophil and neutrophil infiltration, decreased Der p-induced IL-4 levels in serum and IL-5, IL-6, IL-13, and IL-17 levels in bronchoalveolar lavage fluid, and inhibited nuclear factor (NF)-κB activity in lung tissues. The serine protease inhibitors FUT, FOY, and UTI have potential therapeutic benefits for treating asthma by downregulating Th2 cytokines and Th17 cell function and inhibiting NF-κB activation in lung tissue.

Effect of a serine protease inhibitor on the progression of chronic renal failure

The number of the chronic renal failure (CRF) patients is increasing explosively. Hypertension, proteinuria, inflammation, fibrosis, and oxidative stress are intertwined in a complicated manner that leads to the progression of CRF. However, the therapeutic strategies to delay its progression are limited. Since serine proteases are involved in many processes that contribute to these risk factors, we investigated the effects of a synthetic serine protease inhibitor, camostat mesilate (CM), on the progression of CRF in 5/6 nephrectomized (Nx) rats. Eighteen male Sprague-Dawley rats were divided into three groups: a sham-operated group (n = 6), a vehicle-treated Nx group (n = 6), and a CM-treated Nx group (n = 6). Following the 9-wk study period, both proteinuria and serum creatinine levels were substantially increased in the vehicle-treated Nx group, and treatment with CM significantly reduced proteinuria and serum creatinine levels. The levels of podocyte-associated proteins in glomeruli, such as nephrin and synaptopodin, were markedly decreased by 5/6 nephrectomy, and this was significantly ameliorated by CM. CM also suppressed the levels of inflammatory and fibrotic marker mRNAs including transforming growth factor-β1, TNF-α, collagen types I, III, and IV, and reduced glomerulosclerosis, glomerular hypertrophy, and interstitial fibrosis in histological studies. Furthermore, CM decreased the expression of NADPH oxidase component mRNAs, as well as reactive oxygen species generation and advanced oxidative protein product levels. Our present results strongly suggest the possibility that CM could be a useful therapeutic agent against the progression of CRF.

Midgut cysteine-proteinase activity in the velvetbean caterpillar (Anticarsia gemmatalis (Hübner))

Proteinase inhibitors are currently targeted as potential insect control agents, but adaptation to proteinase inhibitors is a recognized limitation to such approach requiring the understanding of how phytophagous species can cope with such compounds. The velvetbean caterpillar (Anticarsia gemmatalis) is a key pest of soybean and well-adapted to its host proteinase inhibitors, which is rich in serine-proteinase inhibitors, particularly trypsin-like proteinase inhibitors. As the expression of cysteine proteinases in the midgut of the velvetbean caterpillar is a potential adaptation to circumvent its host defense, we assessed and characterized the digestive cysteine-proteinase activity from velvetbean caterpillars. Significant soluble and membrane-bound proteolytic activity was obtained and was consistent with those of cysteine proteinases based on the substrate and inhibitors used for their characterization. The K m and V max values obtained were 2.35 ± 0.50 mM and 40.89 ± 6.68 nmol min−1 mg−1 for the soluble proteinases and 0.33 ± 0.03 mM and 24.54 ± 0.67 nmol min−1 mg−1 for the membrane-bound proteinases, range of values also consistent with cysteine proteinases. Therefore, the proteolytic activity observed in the velvetbean caterpillar midgut is consistent with that of cysteine proteinases providing preliminary support for the contention of their potential involvement mitigating the negative effects of serine-protease inhibitors in this species.

Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest, Eurygaster integriceps.

In the current study the effects of serine proteinase inhibitors (TLCK, TPCK, SBTI, and a combination of SBTI and TPCK) with concentrations of 1% and 4% of dietary protein in artificial diets were tested against growth of the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), development, and its gut serine proteinase targets. Analysis of variance indicated that protease inhibitors affected nymphal development time, adult weight, and survival. Mean development time of third instar nymphs in control, SBTI (1%), TLCK (1%), and TPCK was 7.18, 9.74, 9.97, and 8.52 days, respectively. The highest mortality (100 % mortality) was observed when a combination of TPCK and SBTI, both at 4% of dietary protein, was used followed by TPCK (4%) that produced 95% mortality. There were significant differences in proteinase activity between treatments and controls when BApNA and SAAPFpNA were used as substrates for trypsin and chymotrypsin, respectively. Reduction of trypsin activity in insects fed with low doses of SBTI (1%), TLCK (1%), and both doses of TPCK (1% and 4%) was 40, 26, 23, and 17%, respectively. Inhibition of chymotrypsin activity was seen in the insects fed on SBTI (1%), TLCK (1%), and TPCK (4%) where inhibition was 14, 9, and 36%, respectively. Maximum inhibition of chymotrypsin activity was observed in the insects fed on diets containing high doses of TPCK (4%). In gel assays, the greatest effects were observed when E. integriceps were fed on high doses of SBTI and TPCK. Therefore, TPCK followed by SBTI proved to be the most effective proteinase inhibitors of E. integriceps.

Inhibition of lung serine proteases in mice: a potentially new approach to control influenza infection

BackgroundHost serine proteases are essential for the influenza virus life cycle because the viral haemagglutinin is synthesized as a precursor which requires proteolytic maturation. Therefore, we studied the activity and expression of serine proteases in lungs from mice infected with influenza and evaluated the effect of serine protease inhibitors on virus replication both in cell culture and in infected mice.ResultsTwo different inbred mouse strains were investigated: DBA/2J as a highly susceptible and C57Bl/6J as a more resistant strain to influenza virus infection. The serine proteases from lung homogenates of mice exhibited pH optima of 10.00. Using the substrate Bz-Val-Gly-Arg-p-nitroanilide or in zymograms, the intensities of proteolysis increased in homogenates from both mouse strains with time post infection (p.i.) with the mouse-adapted influenza virus A/Puerto Rico/8/34 (H1N1; PR8). In zymograms at day 7 p.i., proteolytic bands were stronger and numerous in lung homogenates from DBA/2J than C57Bl/6J mice. Real-time PCR results confirmed differential expression of several lung proteases before and after infecting mice with the H1N1 virus. The most strongly up-regulated proteases were Gzma, Tmprss4, Elane, Ctrl, Gzmc and Gzmb. Pretreatment of mouse and human lung cell lines with the serine protease inhibitors AEBSF or pAB or a cocktail of both prior to infection with the H1N1 or the A/Seal/Massachusetts/1/80 (H7N7; SC35M) virus resulted in a decrease in virus replication. Pretreatment of C57Bl/6J mice with either AEBSF or a cocktail of AEBSF and pAB prior to infection with the H1N1 virus significantly reduced weight loss and led to a faster recovery of treated versus untreated mice while pAB alone exerted a very poor effect. After infection with the H7N7 virus, the most significant reduction of weight loss was obtained upon pretreatment with either the protease inhibitor cocktail or pAB. Furthermore, pretreatment of C57BL/6J mice with AEBSF prior to infection resulted in a significant reduction in the levels of both the H1N1 and H7N7 nucleoproteins in mice lungs and also a significant reduction in the levels of the HA transcript in the lungs of the H1N1- but not the H7N7-infected mice.ConclusionMultiple serine protease activities might be implicated in mediating influenza infection. Blocking influenza A virus infection in cultured lung epithelia and in mice by the used serine protease inhibitors may provide an alternative approach for treatment of influenza infection.

Effect of serine-type protease of Candida spp. isolated from linear gingival erythema of HIV-positive children: critical factors in the colonization

There are several kinds of oral soft tissue lesions that are common manifestations observed in human immunodeficiency virus (HIV)-infected children; for example, linear gingival erythema (LGE) that is a distinctive fiery red band along the margin of the gingivae. The etiology and pathogenesis of LGE are questionable, but a candidal origin has been suggested. Proteases are key virulence attributes produced by a variety of pathogenic fungi, including Candida. The objective of the present study is to identify the protease production in Candida species including, C. albicans (n=5), C. dubliniensis (n=1) and C. tropicalis (n=1), isolated directly from typical LGE lesions observed in six HIV-positive children, and also to test the effect of a serine protease inhibitor on the interaction of Candida spp. and epithelial cells in vitro. The ability of Candida strains to release proteases in the culture supernatant fluids was visualized by gelatin-SDS-PAGE. Gel strips containing 30-fold concentrated supernatant (1.5×10(8) yeasts) were incubated at 37°C for 48 h in 50 mM sodium phosphate buffer, pH 5.5. The concentrated supernatants were also incubated with fibronectin, laminin, immunoglobulin G, bovine serum albumin and human serum albumin. The effect of serine protease inhibitor on the interaction of Candida spp. and epithelial cells (MA 104) was measured after pre-treatment of fungi with the inhibitor (phenylmethylsulphonyl fluoride, PMSF). All the extracellular proteases were completely inhibited by PMSF, identifying these activities as serine-type proteases. Interestingly, a common 62-kDa serine protease was observed in all Candida strains. The culture supernatants, rich in serine protease activities, cleaved several soluble proteinaceous substrates. Additionally, we demonstrated that pre-treatment of C. albicans, C. dubliniensis and C. tropicalis with PMSF diminished the interaction with epithelial cells. Collectively, our results show that Candida spp. isolated from LGE lesions produced and secreted serine proteases and these enzymes may be involved in the initial colonization events.

Effects of Serine Protease Inhibitor and Prostaglandin I 2 on Liver Transplantation From Non–Heart-Beating Rat Donors

Objectives We sought to preserve the microcirculation as a keystone in liver transplantation from a non–heart-beating donor (NHBD). The purpose of this study was to investigate the cytoprotective effects of a serine protease inhibitor, nafamostat mesilate, and prostaglandin I 2 (PGI 2) on livers transplanted from NHBDs. Methods Male Wistar rats were used in five groups of nine rats each. In group 1, livers were retrieved from heart-beating donors (HB group); in group 2, livers were retrieved from NHBDs that had experienced agonal apnea (NHB group); in group 3, livers were retrieved in the same manner as in the NHBD group but were pretreated with nafamostat mesilate (NM), 0.2 mg/kg/h, (NM group); in group 4, livers were retrieved in the same manner as in the NHBD group but were pretreated with prostaglandin (PG) I 2, 33 ng/kg/h for 30 minutes (PG group); and in group 5, livers were retrieved in the same manner as in the NHBD group but were pretreated with NM plus PG, (NM+PG group). Livers were perfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after 6 hours of cold preservation, after which the perfusate and liver tissue were analyzed in one set of experiments. In another set of experiments, livers retrieved and after 1 hour of cold preservation were transplanted according to the Kamada method. Results In the NM+PG group, the values of interleukin-1β, tumor necrosis factor–α, and thromboxane B 2 were significantly lower than those in the NHB group. At histologic analysis, sinusoidal endothelial cells were well preserved in the NM+PG group. The number of survivors at 7 days after liver transplantation in the 5 groups were 9, 0, 1, 1, and 3, respectively. Conclusion The serine protease inhibitor, NM, and PGI 2 supported sinusoidal endothelial cells and preserved microcirculation.

Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes

To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from sea anemone Stichodactyla helianthus (ShPI-I), on the viability and morphology of parasites in culture. Classical inhibitors were selected on the basis of their ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-L: -phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) inhibitors, which are potential Leishmania proteases inhibitors, in all experimental conditions reduced the parasite viability, with regard to time dependence. On the other hand, N-tosyl-lysine chloromethyl ketone (TLCK) did not significantly affect the parasite viability, as it was poor Leishmania enzymes inhibitor. Ultrastructural analysis demonstrated that both Bza and TPCK induced changes in the flagellar pocket region with membrane alteration, including bleb formation. However, TPCK effects were more pronounced than those of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bodies was visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine proteases and the parasite viability. The ultrastructural alterations caused by ShPI-I were more dramatic than those induced by the classical inhibitors. Vesiculation of the flagellar pocket membrane, the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole, and alterations of promastigotes shape resulted.

672. The Effect of Serine Protease Inhibitor 6 (SPI-6) Gene Transfer on a Mouse Model of Sjögren's Syndrome

Top of pageAbstract Sj|[ouml]|gren's syndrome (SS) is an autoimmune disease characterized by a focal and diffuse lymphoid cell infiltration into the salivary and lacrimal glands. This chronic immune cell activation leads to reduced secretory function with symptoms of xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). It has been hypothesized that these sequelae are due to apoptosis of the exocrine epithelial cells. Apoptosis can be induced by cytotoxic T-lymphocytes (CTLs), which contain the enzyme Granzyme B (GrB) in cytoplasmic granules. GrB initiates a caspase-independent apoptosis pathway and is inhibited by serine proteinase inhibitor 6 (SPI-6) in the mouse. In this study, we evaluated the effect of transgenic SPI-6 produced in salivary glands after gene transfer to non-obese diabetic (NOD) mice, which develop exocrine gland infiltrates and are a commonly used disease model for SS. We constructed two serotype 2 rAAV vectors, rAAVSPI-6 and rAAVLacZ (encoding |[beta]|-galactosidase; vector control) using the CMV promoter. rAAVs (1010 particles/gland) were administered directly into both submandibular glands of 8-week old female NOD mice (n=8) via retroductal delivery. Salivary flow rates were determined before vector delivery and at time of sacrifice (16-weeks old). Serum glucose levels and weight were measured weekly and diabetic mice (serum glucose|[ge]|400 mg/dL) were treated with insulin. After sacrifice, submandibular glands were harvested and analyzed for inflammatory infiltrates (focus scores), as well as SPI-6 expression and cytokine profile [mouse interleukins (IL)-2, 4, 6, 12, 18, interferon (IFN)-|[gamma]|, tumor necrosis factor-|[alpha]| and RANTES] in aqueous gland extracts. At 8 weeks, salivary flow rates were not different between mice assigned to the rAAVSPI-6 and rAAVLacZ groups. In contrast, at 16 weeks flow rates were significantly higher in the rAAVSPI-6-treated mice (p=0.018). Submandibular gland IL-10 levels were significantly increased (p=0.009), while IFN-|[gamma]| levels were significantly decreased (p=0.029), in the rAAVSPI-6 mice compared to the rAAVLacZ group. There were no differences in focus scores, serum glucose levels or animal weights between the two treatment groups at 16 weeks. Overall, these results suggest that SPI-6 gene transfer may be beneficial in limiting the autoimmune exocrinopathy of NOD mice.