Omicsynin B4 potently blocks coronavirus infection by inhibiting host proteases cathepsin L and TMPRSS2.

The emergence of SARS-CoV-2 variants represents a major threat to public health and requires identification of novel therapeutic agents to address the unmet medical needs. Small molecules impeding viral entry through inhibition of spike protein priming proteases could have potent antiviral effects against SARS-CoV-2 infection. Omicsynin B4, a pseudo-tetrapeptides identified from Streptomyces sp. 1647, has potent antiviral activity against influenza A viruses in our previous study. Here, we found omicsynin B4 exhibited broad-spectrum anti-coronavirus activity against HCoV-229E, HCoV-OC43 and SARS-CoV-2 prototype and its variants in multiple cell lines. Further investigations revealed omicsynin B4 blocked the viral entry and might be related to the inhibition of host proteases. SARS-CoV-2 spike protein mediated pseudovirus assay supported the inhibitory activity on viral entry of omicsynin B4 with a more potent inhibition of Omicron variant, especially when overexpression of human TMPRSS2. Moreover, omicsynin B4 exhibited superior inhibitory activity in the sub-nanomolar range against CTSL, and a sub-micromolar inhibition against TMPRSS2 in biochemical assays. The molecular docking analysis confirmed that omicsynin B4 fits well in the substrate binding sites and forms a covalent bond to Cys25 and Ser441 in CTSL and TMPRSS2, respectively. In conclusion, we found that omicsynin B4 may serve as a natural protease inhibitor for CTSL and TMPRSS2, blocking various coronavirus S protein-driven entry into cells. These results further highlight the potential of omicsynin B4 as an attractive candidate for broad-spectrum antiviral therapy that could rapidly respond to emerging variants of SARS-CoV-2.

Would the Development of a Multitarget Inhibitor of 3CLpro and TMPRSS2 be Promising in the Fight Against SARS-CoV-2?

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), responsible for generating COVID-19, has spread worldwide and was declared a pandemic by the World Health Organization (WHO) on 11 March 2020, being responsible for various damages to public health, social life, and the economy of countries. Its high infectivity and mutation rates have stimulated researchers and pharmaceutical companies to search for new therapies against this disease. These efforts resulted in several vaccines and the identification of Molnupiravir as an oral treatment for this disease. However, identifying new alternatives and critical information is necessary to fight against this devastating agent. The findings in recent years regarding the structure and biochemistry of SARS-CoV2 are remarkable. In anti-CoV drug discovery, various targets, such as structural, non-structural, and hostrelated proteins are explored. In fact, 3CLpro is the most used among non-structural proteins since this protease cleaves peptide sequences after the glutamine residue, and no human protease has this function. This makes this macromolecule an excellent drug target for discovering new compounds. Another promising target is the transmembrane protease serine 2 (TMPRSS2). Recent studies point to TMPRSS2 as one of the main targets responsible for viral entry related to the cleavage of the S protein. Similar to cathepsins, TMPRSS2 is also responsible for cleaving the spike protein SARS-CoV2, which binds to the ACE2 receptor. Thus, TMPRSS2 is one of the targets that may represent new alternatives in treating SARS-CoV2. In this context, would discovering a multitarget inhibitor be the new strategy in searching for drugs against SARS-CoV2? For many years, new drug discovery was based on the "one drug, one target" premise, where the biological action is related to interactions with only one biological target. However, this paradigm has been overcome as new evidence of multiple mechanisms of action for a single drug. Finally, this review will present a perspective on drug design based on a multitarget strategy against 3CLpro and TMPRSS2. We hope to provide new horizons for researchers worldwide searching for more effective drugs against this devastating agent.

Thermodynamic characterization of a macrocyclic Zika virus NS2B/NS3 protease inhibitor and its acyclic analogs.

Cyclization of small molecules is a widely applied strategy in drug design for ligand optimization to improve affinity, as it eliminates the putative need for structural preorganization of the ligand before binding, or to improve pharmacokinetic properties. In this work, we provide a deeper insight into the binding thermodynamics of a macrocyclic Zika virus NS2B/NS3 protease inhibitor and its linear analogs. Characterization of the thermodynamic binding profiles by isothermal titration calorimetry experiments revealed an unfavorable entropy of the macrocycle compared to the open linear reference ligands. Molecular dynamic simulations and X-ray crystal structure analysis indicated only minor benefits from macrocyclization to fixate a favorable conformation, while linear ligands retained some flexibility even in the protein-bound complex structure, possibly explaining the initially surprising effect of a higher entropic penalty for the macrocyclic ligand.

Kallikrein-related peptidase-8 (KLK8) aggravated hypoxia-induced right ventricular hypertrophy by targeting P38 MAPK/P53 signaling pathway.

Right ventricular (RV) hypertrophy and further heart failure are major co-morbidities, resulting in the premature death of patients with hypoxic pulmonary hypertension (HPH). The regulatory effects of kallikrein-related peptidase (KLK) family members on cardiac function have been extensively studied. However, to the best of the authors' knowledge, the regulatory effects of KLK8 on RV hypertrophy caused by HPH have yet to be reported. The aim of the present study was to assess KLK8 expression in the RV tissue of HPH-modeled rats, and to further explore the effects and underlying mechanism of KLK8 in regulating the hypertrophy of hypoxia-induced H9c2 cardiomyocytes. In HPH model rats, increases in the right ventricle hypertrophy index, the right ventricular systolic pressure, cardiac output, as well as pulmonary artery wall thickness were observed. Western blot analysis revealed that KLK8 expression and MAPK/p53 signaling activity were enhanced in the RVs of rats in an RV HPH rat model. In hypoxia-induced H9c2 cardiomyocytes, KLK8 overexpression promoted cardiomyocyte hypertrophy, whereas KLK8 silencing showed the opposite results. KLK8 overexpression increased the expression levels of ventricular hypertrophy markers, including atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain 7, which were blocked upon addition of the p38 MAPK inhibitor, SB202190. Conversely, KLK8 silencing caused a decrease in the expression levels of the ventricular hypertrophy markers, which were further reduced via inhibition of the p38 MAPK/p53 signaling pathway. Taken together, the results of the present study have shown that KLK8 may subtly regulate RV hypertrophy, and therefore KLK8 may be a promising therapeutic target for treating HPH-induced RV hypertrophy.

Downregulation of matriptase suppresses the PAR­2/PLCγ2/PKC­mediated invasion and migration abilities of MCF­7 breast cancer cells.

Matriptases, members of the type II transmembrane serine protease family, are cell surface proteolytic enzymes that mediate tumor invasion and metastasis. Matriptase is highly expressed in breast cancer and is associated with poor patient outcome. However, the cellular mechanism by which matriptase mediates breast cancer invasion remains unknown. The present study aimed to determine the role of matriptase in the protein kinase C (PKC)­mediated metastasis of MCF­7 human breast cancer cells. Matriptase small interfering RNA­mediated knockdown significantly attenuated the 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced invasiveness and migration of MCF­7 cells, and inhibited the activation of phospholipase C γ2 (PLCγ2)/PKC/MAPK signaling pathways. Matriptase­knockdown also suppressed the expression of MMP­9 and inhibited the activation of NF­κB/activator protein­1 in MCF­7 cells. Additionally, GB83 [an inhibitor of protease­activated receptor­2 (PAR­2)] inhibited PKC­mediated MMP­9 expression and metastatic ability in MCF­7 cells. Furthermore, downregulation of matriptase suppressed TPA­induced MMP­9 expression and invasiveness via PAR­2/PLCγ2/PKC/MAPK activation. These findings shed light on the mechanism underlying the role of matriptase in MCF­7 cell invasion and migration ability, and suggest that matriptase modulation could be a promising therapeutic strategy for preventing breast cancer metastasis.

Reelin restricts dendritic growth of interneurons in the neocortex.

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.

Structural Basis of Covalent Inhibitory Mechanism of TMPRSS2-Related Serine Proteases by Camostat.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.

Cell-free Directed Evolution of a Protease in Microdroplets at Ultrahigh Throughput.

Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these droplets are formed at ultrahigh throughput in microfluidic devices, their perfect monodispersity allows quantitative enzyme assays with a high precision readout. However, despite its potential for high quality cell-free screening experiments, previous demonstrations of enrichment have never been successfully followed up by actual enzyme library selections in monodisperse microfluidic droplets. Here we develop a three-step workflow separating three previously incompatible steps that thus far could not be carried out at once: first droplet-compartmentalized DNA is amplified by rolling circle amplification; only after completion of this step are reagents for in vitro protein expression and, finally, substrate added via picoinjection. The segmented workflow is robust enough to allow the first in vitro evolution in droplets, improving the protease Savinase that is toxic to E. coli for higher activity and identifying a 5-fold faster enzyme.

Potential pharmacological candidates Transmembrane protease, serine 2 inhibitors 2019-ncov treatment / Candidatos potenciales farmacológicos inhibidores de proteasa transmembrana de serina 2 en el tratamiento de 2019-ncov

INTRODUCTION: Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus with characteristic of infecting the respiratory tract, causing severe acute respiratory syndrome. The virus uses the ACE II receptors and the transmembrane protein TMPRSS2 initial step to enter the host cell, this contribution described different types of drug, to perform its inhibition in initial step adhesion. METHODOLOGY: Non-systematic review of articles with the help of preset keywords. RESULTS: In this review we will present drugs that inhibitors of this type of receptor therefore these drugs could be considered potential candidates to mitigate the spread of SARS-CoV-2

INTRODUCCIÓN: La Enfermedad por coronavirus 2019 (COVID-19) causada por el virus SARS-CoV-2, con característica de infectar el tracto respiratorio causando un síndrome respiratorio agudo como paso inicial para ingresar a la célula huésped el virus usa los receptores ACE II y la proteína transmembrana TMPRSS2 para causar la infección, Por lo que se ha descrito diferentes tipos de fármacos para realizar su inhibición en la adhesión del paso inicial. METODOLOGÍA: Revisión no sistemática de artículos con la ayuda de palabras clave preestablecidas. RESULTADOS: En esta revisión presentaremos fármacos que inhiben este tipo de receptor, por lo tanto, estos medicamentos podrían considerarse candidatos potenciales para mitigar la propagación del SARS-CoV-2

Antivirals targeting the polymerase complex of influenza viruses.

Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Encouragingly, several new compounds which inhibit the polymerase of influenza viruses have recently been shown to have enhanced pre-clinical and clinical effectiveness compared to the neuraminidase inhibitors, the mainstay of influenza antiviral therapy over the last two decades. In this review we focus on four compounds which inhibit polymerase function, baloxavir marboxil, favipiravir, pimodivir and AL-794 and discuss their clinical and virological effectiveness, their propensity to select for resistance and their potential for future combination therapy with the most commonly used neuraminidase inhibitor, oseltamivir.

Dust mite-derived Der f 3 activates a pro-inflammatory program in airway epithelial cells via PAR-1 and PAR-2.

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.

Antiviral Activity, Safety, and Pharmacokinetics of AL-794, a Novel Oral Influenza Endonuclease Inhibitor: Results of an Influenza Human Challenge Study.

Background: AL-794 is an orally active prodrug of ALS-033719, which selectively inhibits the endonuclease domain of influenza virus A and B polymerase. Methods: In a phase 1, double-blinded, randomized, placebo-controlled study, healthy subjects were inoculated intranasally with influenza virus (A/Perth/16/2009 H3N2) after confirmation of infection or on day 4. Subjects received 50 mg of AL-794, 150 mg of AL-794, or placebo twice daily for 5 days. Viral load, influenza symptoms, pharmacokinetics, and safety were evaluated. Results: A total of 61 subjects were inoculated. In 42 infected subjects, the mean peak viral load for 50-mg AL-794 recipients, 150-mg AL-794 recipients, and placebo recipients was 3.54, 2.77, and 3.72 log10 50% tissue culture infectious doses (TCID50)/mL, respectively. The mean influenza viral load area under the curve in the corresponding treatment groups was 137, 87.5, and 142 log10 TCID50/mL·h, respectively, and the median time to virus nondetection was 117, 75.3, and 108 hours, respectively. AL-794 was well tolerated, and no viral resistance to ALS-033719 was identified. Conclusion: Following oral administration of AL-794, significant dose-dependent antiviral activity was noted, with a greater decrease in viral load, symptoms, and mucus weight at the 150-mg dose, compared with the 50-mg dose, and no safety concerns for either dose or placebo. Clinical Trials Registration: NCT02588521.

Hepatocyte Growth Factor Activator: A Proteinase Linking Tissue Injury with Repair.

Hepatocyte growth factor (HGF) promotes pleiotropic signaling through its specific receptor tyrosine kinase, MET. As such, it has important roles in the regeneration of injured tissues. Since HGF is produced mainly by mesenchymal cells and MET is expressed in most epithelial, endothelial and somatic stem cells, HGF functions as a typical paracrine growth factor. HGF is secreted as an inactive precursor (proHGF) and requires proteolytic activation to initiate HGF-induced MET signaling. HGF activator (HGFAC) is a serum activator of proHGF and produces robust HGF activities in injured tissues. HGFAC is a coagulation factor XII-like serine endopeptidase that circulates in the plasma as a zymogen (proHGFAC). Thrombin, kallikrein-related peptidase (KLK)-4 or KLK-5 efficiently activates proHGFAC. The activated HGFAC cleaves proHGF at Arg494-Val495, resulting in the formation of the active disulfide-linked heterodimer HGF. Macrophage stimulating protein, a ligand of RON, is also activated by HGFAC in vivo. Although HGFAC functions primarily at the site of damaged tissue, a recent report has suggested that activated HGFAC relays a signal to stem cells in non-injured tissues via proHGF activation in the stem cell niche. This review focuses on current knowledge regarding HGFAC-mediated proHGF activation and its roles in tissue regeneration and repair.

A computational structural analysis of functional attributes of hypodermin A and B proteins: A way forward for vaccine development.

Hypodermosis is a parasitic disease of cattle. The pathogenicity of the disease is attributed to Hypodermin proteins (Hypodermin A, Hypodermin B and Hypodermin C). Studies suggest that Hypodermin proteins may be defined as Serine proteases and collagenases. The structure of both proteases Hypodermin A and Hypodermin B were modeled using the Swiss-model server followed by its validation using Procheck, Errat and Verify-3D. Afterwards, both Hypodermin A and Hypodermin B were docked against collagen in order to study its interaction with respective Hypodermin proteins. The structure of both Hypodermin A and Hypodermin B showed more bent towards hydrophobic nature as more beta sheets were present in them. Both structures were also superimposed to check out similarities and differences present between them. Serine, Aspartic acid, Histidine, Glutamic acid and Lysine are found as interacting residues that are involved in hydrogen bonding with collagen. The interactions are found in the active domain region of Hypodermin proteins. The interacting residues were present in the active region of the hypodermin proteins thus validating the docking studies. This study may help in the drug development against hypodermosis with least side effects.

Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA.

BACKGROUND: Restoration of damaged tissues through the activation of endogenous progenitors is an attractive therapeutic option. A deep evaluation of the intrinsic stem/progenitor cell properties as well as the reciprocal interactions with injured environments is of critical importance. METHODS: Here, we show that bone marrow stromal cell antigen 2 (BST2) allows the isolation of a population of circulating progenitors, the circulating healing (CH) cells, characterized by a distinctive core signature. The bone marrow (BM) origin of BST2pos CH cells has been strengthened by the co-expression of leptin receptor, the hallmark of a subpopulation of BM-skeletal stem cells. RESULTS: BST2pos CH cells retained the capacity to (i) respond to injury signals generated by a bone fracture, (ii) modify the expression of cell motility genes following damage, and (iii) react to hepatocyte growth factor-activator (HGFA), an injury-related stimulus sufficient to induce their transition into GALERT, a state in which cells are functionally activated and participate in tissue repair. CONCLUSIONS: Taken together, these results could pave the way for the identification of new strategies to enhance and potentiate endogenous regenerative mechanisms for future therapies.

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

BACKGROUND: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. CONCLUSIONS/SIGNIFICANCE: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.

Complement C5a inhibition moderates lipid metabolism and reduces tubulointerstitial fibrosis in diabetic nephropathy.

Background: Complement C5 mediates pro-inflammatory responses in many immune-related renal diseases. Given that the C5a level is elevated in diabetes, we investigated whether activation of C5a/C5aR signalling plays a pathogenic role in diabetic nephropathy (DN) and the therapeutic potential of C5a inhibition for renal fibrosis. Methods: Human renal biopsies from patients with DN and control subjects were used for immunohistochemical staining of complement C5 components. Renal function and tubulointerstitial injury were compared between db/m mice, vehicle-treated mice and C5a inhibitor-treated db/db mice. A cell culture model of tubule epithelial cells (HK-2) was used to demonstrate the effect of C5a on the renal fibrotic pathway. Results: Increased levels of C5a, but not of its receptor C5aR, were detected in renal tubules from patients with DN. The intensity of C5a staining was positively correlated with the progression of the disease. In db/db mice, administration of a novel C5a inhibitor, NOX-D21, reduced the serum triglyceride level and attenuated the upregulation of diacylglycerolacyltransferase-1 and sterol-regulatory element binding protein-1 expression and lipid accumulation in diabetic kidney. NOX-D21-treated diabetic mice also had reduced serum blood urea nitrogen and creatinine levels with less glomerular and tubulointerstitial damage. Renal transforming growth factor beta 1 (TGF-ß1), fibronectin and collagen type I expressions were reduced by NOX-D21. In HK-2 cells, C5a stimulated TGF-ß production through the activation of the PI3K/Akt signalling pathway. Conclusions: Blockade of C5a signalling by NOX-D21 moderates altered lipid metabolism in diabetes and improved tubulointerstitial fibrosis by reduction of lipid accumulation and TGF-ß-driven fibrosis in diabetic kidney.

[Effects of complement C5a inhibitor therapy in animal models of non-occlusive mesenteric ischemia]. / Komplement C5a-antagonista-terápia hatása nem okklúzív mesenterialis ischaemia állatmodelljeiben.

INTRODUCTION: Non-occlusive mesenteric ischemia (NOMI) develops without anatomical causes. Early diagnosis is challenging and treatments are of questionable effectiveness. We investigated the role of complement activation in the pathophysiology of NOMI in animal models through the inhibition of complement C5a. MATERIALS AND METHODS: 60-min partial aortic occlusion (PAO; abdominal aorta, proximal to celiac trunk; mean arterial pressure: 30-40 mmHg) was established in Sprague-Dawley rats (n = 28) and 60-min cardiac tamponade in minipigs (n = 19; mean arterial pressure: 40-50 mmHg) to observe short- and long-term circulatory and inflammatory consequences of NOMI. Macro- and microhemodynamics, leukocyte infiltration, plasma levels of inflammatory mediators (endothelin, HMGB-1) were measured. C5a inhibitor (Acetyl-Peptid-A; 4 mg/kg iv) was administered at the 45th min of PAO or tamponade, respectively. RESULTS: Twenty-four hours after PAO systemic inflammatory response increased cardiac output and superior mesenteric artery flow (SMAF). C5a inhibition reduced the elevated cardiac output (203.1 ± 5 vs 269.6 ± 8.1 ml/min/kg) and SMAF and increased ileal microcirculation (833.5 ± 33.8 vs 441.9 ± 22.4 µm/s). In pigs, after the tamponade, C5a inhibition reduced the immediate hemodynamic disturbances, temporarily increased SMAF and permanently the ileal microcirculation. The Acetyl-Peptid-A treatment reduced leukocyte infiltration and plasma levels of inflammatory mediators in both NOMI models. CONCLUSIONS: Complement activation plays central role in the macro- and microcirculatory disturbance during NOMI. C5a inhibition reduces the inflammatory activation and influences the hemodynamic consequences of experimental NOMI.

Protective effect of bacillopeptidase CFR5 from Bacillus subtilis CFR5 on cerulein-induced pancreatitis.

Bacillopeptidase is a serine peptidase, known for its fibrinolytic activity. However, a very little information is known about its in vivo inflammatory and/or anti-inflammatory properties. Thus, to understand whether bacillopeptidase incorporation can regulate pancreatitis or not, the cerulein-induced pancreatitis model was used, and the role of bacillopeptidase on pancreatitis was studied. In this study, 46 kDa protein was purified from Bacillus subtilis and identified as bacillopeptidase CFR5 (BPC) through MS/MS analysis. The nutritional prophylactic group was orally fed with two doses of BPC (100 µg/Kg/BW of rat) 6 h before cerulein administration and analyzed for its effect on intestine and pancreas inflammation, cytokines, and pancreatitis marker gene expression. BPC administration significantly reduced the severity of pancreatitis by decreasing serum amylase, lipase, pancreatic edema and myeloperoxidase activity. The pretreatment with BPC suppressed the pancreatic pro-inflammatory and inflammatory cytokines production including IL-6, IL-1ß, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-13 in both pancreas and serum samples. Moreover, BPC supplementation restored pancreatitis mediated disruption of intestinal barrier integrity by upregulating tight junction proteins (ZO-1, occludin), antimicrobial peptides (DEFB1, CRAMP), MUC-2, TFF3 expression and by enhancing SCFA's production. Pretreatment with BPC suppressed the intestinal inflammation with reduced cytokines production in the colon and ileal region of cerulein-induced pancreatitis. Thus, BPC based pretreatment protocol is a novel intervention to prevent acute pancreatitis.

Matriptase Induction of Metalloproteinase-Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms.

OBJECTIVE: To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. METHODS: Aggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. RESULTS: The addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage. CONCLUSION: Matriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.