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An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4-/-) mice and Mcpt4+/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4-/- mice compared with Mcpt4+/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4-/- mice. Relative to infected Mcpt4+/+ mice, ileal MC accumulation in Mcpt4-/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4+/+ but not Mcpt4-/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4-/- mice, while levels of IL-2, IL-10 and MIP1β (CCL4) were increased over the same period in Mcpt4+/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4-/- mice and type-2 response in Mcpt4+/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4-/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4+/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4-/- mice compared with infected Mcpt4+/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.

Malaria strongly predisposes to bacteremia, which is associated with sequestration of parasitized red blood cells and increased gastrointestinal permeability. The mechanisms underlying this disruption are poorly understood. Here, we evaluated the expression of factors associated with mast cell activation and malaria-associated bacteremia in a rodent model. C57BL/6J mice were infected with Plasmodium yoeliiyoelli 17XNL, and blood and tissues were collected over time to assay for circulating levels of bacterial 16S DNA, IgE, mast cell protease 1 (Mcpt-1) and Mcpt-4, Th1 and Th2 cytokines, and patterns of ileal mastocytosis and intestinal permeability. The anti-inflammatory cytokines (interleukin-4 [IL-4], IL-6, and IL-10) and MCP-1/CCL2 were detected early after P. yoeliiyoelii 17XNL infection. This was followed by the appearance of IL-9 and IL-13, cytokines known for their roles in mast cell activation and growth-enhancing activity as well as IgE production. Later increases in circulating IgE, which can induce mast cell degranulation, as well as Mcpt-1 and Mcpt-4, were observed concurrently with bacteremia and increased intestinal permeability. These results suggest that P. yoeliiyoelii 17XNL infection induces the production of early cytokines that activate mast cells and drive IgE production, followed by elevated IgE, IL-9, and IL-13 that maintain and enhance mast cell activation while disrupting the protease/antiprotease balance in the intestine, contributing to epithelial damage and increased permeability.

As the relationship between the gut microbiome and allergies becomes better understood, targeted strategies to prevent and treat allergies through gut microbiome modulation are being increasingly developed. In the study presented herein, we screened various probiotics for their ability to inhibit mast cell degranulation and identified Lactiplatibacillus plantarum HD02 and MD159 as effective candidates. The two strains significantly attenuated vascular permeability induced by mast cell degranulation in a passive cutaneous anaphylaxis (PCA) model and, in the MC903-induced murine atopic dermatitis (AD) model, demonstrated comparable preventive effects against allergies, reducing blood levels of MCPT-1 (mast cell protease-1) and total IgE. In the house dust mite (HDM)-induced murine AD model, both L. plantarum HD02 and MD159 showed therapeutic effects, with L. plantarum HD02 demonstrating superior efficacy. Nevertheless, L. plantarum MD159 better suppressed transepidermal water loss (TEWL). Furthermore, L. plantarum HD02 and MD159 significantly increased the number of splenic Foxp3 + regulatory T cells, with L. plantarum MD159 having a more pronounced effect. However, only L. plantarum HD02 achieved a reduction in immune cells in the draining lymph nodes. Our findings highlight L. plantarum HD02 and MD159 as promising candidates for the prevention and treatment of allergies, demonstrating significant efficacy in suppressing mast cell degranulation, reducing the number of allergy biomarkers, and modulating immune responses in experimental models of AD. Their distinct mechanisms of action suggest potential complementary roles in addressing allergic diseases, underscoring their therapeutic promise in clinical applications.

Background: Animal models for food allergies serve as crucial tools in understanding allergy mechanisms and assessing the efficacy of potential desensitization methods. The effectiveness of inducing allergies in mice through intragastric lavage sensitization varies. The intraperitoneal method can trigger systemic anaphylaxis, however it lacks anatomical relevance. Hence, a uniform and reliable allergy induction method in mice is required. Tape -stripping can mimic atopic dermatitis (AD), a precursor to lifelong peanut allergies in humans. Furthermore, skin damage triggers the upregulation of skin alarmins and the expansion of small-intestinal mast cells, both implicated in allergy development., Methods: We standardized a skin-based sensitization method in a mouse model of peanut allergy using skin tape-stripping followed by allergen application. We compared this method with intragastric sensitization., Results: Skin-based sensitization led to increased mast cells, goblet cells, and eosinophils in the small intestine, elevated systemic IgE levels, murine mast cell protease-1 (mMCP-1), histamine, and eosinophilic activity in peripheral blood. Moreover, it resulted in a significant hypothermic response, with nearly 30% mortality following an oral challenge one-month post-sensitization., Conclusion: Our research offers a standardized and readily reproducible method for inducing peanut allergy in mice, which could also be adapted for other food allergens., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Harvinder Singh Gill reports financial support was provided by National Institutes of Health. Harvinder Singh Gill reports a relationship with Moonlight Therapeutics that includes: equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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Treatment options are limited for severe asthma, and the need for additional therapies remains great. Previously, we demonstrated that integrin αvβ6-deficient mice are protected from airway hyperresponsiveness, due in part to increased expression of the murine ortholog of human chymase. Here, we determined that chymase protects against cytokine-enhanced bronchoconstriction by cleaving fibronectin to impair tension transmission in airway smooth muscle (ASM). Additionally, we identified a pathway that can be therapeutically targeted to mitigate the effects of airway hyperresponsiveness. Administration of chymase to human bronchial rings abrogated IL-13-enhanced contraction, and this effect was not due to alterations in calcium homeostasis or myosin light chain phosphorylation. Rather, chymase cleaved fibronectin, inhibited ASM adhesion, and attenuated focal adhesion phosphorylation. Disruption of integrin ligation with an RGD-containing peptide abrogated IL-13-enhanced contraction, with no further effect from chymase. We identified α5β1 as the primary fibronectin-binding integrin in ASM, and α5β1-specific blockade inhibited focal adhesion phosphorylation and IL-13-enhanced contraction, with no additional effect from chymase. Delivery of an α5β1 inhibitor into murine airways abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. Finally, α5β1 blockade enhanced the effect of the bronchodilator isoproterenol on airway relaxation. Our data identify the α5β1 integrin as a potential therapeutic target to mitigate the severity of airway contraction in asthma.

Background: Aristolochic acid I (AAI) is an extract from Chinese herbs that causes progressive interstitial nephritis. The aim of this research is to know whether chymases play the crucial role in AAI-induced nephropathy. Methods: The mice were treated with AAI via intraperitoneal injection and the accumulated AAI dosages are 30 mg/kg of body weight for two, four, six, and eight weeks. The animals were sacrificed after another two or four weeks for nephropathy development. Collection of blood, urine, and kidney samples for the further biochemical analysis, hematoxylin-eosin (H and E) and Masson's trichrome stained to detected pathologic, and MMP2 and MMP9 activity assays. Results: After the treatment of AAI, of the mice, their body weights were decreased (P < 0.01), and concentration of creatinine and blood urea nitrogen (BUN) in serum (P < 0.01) and urine collection were increased (P < 0.01). In the renal tissue sections, high amount of inflammatory cells were found by H and E stain, and increased fibrosis in renal interstitial tissue were observed by Masson's trichrome stain. In mice kidney tissue, significantly increased chymase activity after treatment of AAI was found (P < 0.01), but ACE activity did not show significant changes. In ACE KO mice, increased MMP2 and decreased MMP9 activity were found in the AAI-treated mice compared with AAI-untreated control (P < 0.01). Conclusions: Moreover, it was also observed that the deficiency of ACE would accelerate the disease development of AAI-induced nephropathy. These results may help to know more information about the role of AAI-induced chronic kidney disease and can be applied in developing new drug targets for nephropathy. [ABSTRACT FROM AUTHOR]

Rosacea is a chronic inflammatory skin disease whose pathophysiological mechanism is still unclear. However, it is known that mast cell (MC) numbers are increased in the dermis of rosacea patients. MC proteases not only recruit other immune cells, which amplify the inflammatory response, but also cause vasodilation and angiogenesis. MCs are also one of the primary sources of cathelicidin LL-37 (Cath LL-37), an antimicrobial peptide that has been shown to be an enabler of rosacea pathogenesis. Here, we demonstrate that MCs are key mediators of cathelicidin-initiated skin inflammation. After Cath LL-37 injection into the dermis, MC-deficient B6.Cg-Kit(W-sh)/HNihrJaeBsmJ (KitW-sh) mice did not develop rosacea-like features. Conversely, chymase (P

Thymic stromal lymphopoietin (TSLP), mainly expressed by epithelial cells, plays a central role in asthma. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP). Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play key roles in asthma. We evaluated the early proteolytic effects of tryptase and chymase released by HLMCs on TSLP by mass spectrometry. We also investigated whether TSLP and its fragments generated by these enzymes induce angiogenic factor release from HLMs. Mass spectrometry (MS) allowed the identification of TSLP cleavage sites caused by tryptase and chymase. Recombinant human TSLP treated with recombinant tryptase showed the production of 1-97 and 98-132 fragments. Recombinant chymase treatment of TSLP generated two peptides, 1-36 and 37-132. lfTSLP induced the release of VEGF-A, the most potent angiogenic factor, from HLMs. By contrast, the four TSLP fragments generated by tryptase and chymase failed to activate HLMs. Long-term TSLP incubation with furin generated two peptides devoid of activating property on HLMs. These results unveil an intricate interplay between mast cell-derived proteases and TSLP. These findings have potential relevance in understanding novel aspects of asthma pathobiology.

Background: Dengue cases continue to rise and can overwhelm healthcare systems during outbreaks. In dengue, neutrophil mediators, soluble urokinase plasminogen activator receptor (suPAR) and olfactomedin 4, and mast cell mediators, chymase and tryptase, have not been measured longitudinally across the dengue phases. The utility of these proteins as prognostic biomarkers for severe dengue has also not been assessed in an older adult population., Methods: We prospectively enrolled 99 adults with dengue-40 dengue fever, 46 dengue with warning signs and 13 severe dengue, along with 30 controls. Plasma levels of suPAR, olfactomedin 4, chymase and tryptase were measured at the febrile, critical and recovery phases in dengue patients., Results: The suPAR levels were significantly elevated in severe dengue compared to the other dengue severities and controls in the febrile (P < .001), critical (P < .001), and recovery (P = .005) phases. In the febrile phase, suPAR was a prognostic biomarker of severe dengue, with an AUROC of 0.82. Using a cutoff derived from Youden's index (5.4 ng/mL) and an estimated prevalence of severe dengue (16.5%) in our healthcare institution, the sensitivity was 71.4% with a specificity of 87.9% in the febrile phase, and the positive and negative predictive values were 54.7% and 95.8%, respectively. Olfactomedin 4 was elevated in dengue patients but not in proportion to disease severity in the febrile phase (P = .04) There were no significant differences in chymase and tryptase levels between dengue patients and controls., Conclusions: In adult dengue, suPAR may be a reliable prognostic biomarker for severe dengue in the febrile phase., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The angiotensin (Ang)-(1-12)/Ang II pathway contributes to cardiac pathology. However, its involvement in the development of peripheral endothelial dysfunction associated with heart failure (HF) remains unknown. Therefore, this study aimed to characterise the effect of exogenous Ang-(1-12) and its conversion to Ang II on endothelial function using the murine model of HF (Tgαq*44 mice), focusing on the role of chymase and vascular-derived thromboxane A 2 (TXA 2 ). Ex vivo myographic assessments of isolated aorta showed impaired endothelium-dependent vasodilation in late-stage HF in 12-month-old Tgαq*44 mice. However, endothelium-dependent vasodilation was fully preserved in the early stage of HF in 4-month-old Tgαq*44 mice and 4- and 12-month-old FVB control mice. Ang-(1-12) impaired endothelium-dependent vasodilation in 4- and 12-month-old Tgαq*44 mice, that was associated with increased Ang II production. The chymase inhibitor chymostatin did not inhibit this response. Interestingly, TXA 2 production reflected by TXB 2 measurement was upregulated in response to Ang-(1-12) and Ang II in aortic rings isolated from 12-month-old Tgαq*44 mice but not from 4-month-old Tgαq*44 mice or age-matched FVB mice. Furthermore, in vivo magnetic resonance imaging showed that Ang-(1-12) impaired endothelium-dependent vasodilation in the aorta of Tgαq*44 mice and FVB mice. However, this response was inhibited by angiotensin I converting enzyme (ACE) inhibitor; perindopril, angiotensin II receptor type 1 (AT 1 ) antagonist; losartan and TXA 2 receptor (TP) antagonist-picotamide in 12-month-old-Tgαq*44 mice only. In conclusion, the chymase-independent vascular Ang-(1-12)/Ang II pathway and subsequent TXA 2 overactivity contribute to systemic endothelial dysfunction in the late stage of HF in Tgαq*44 mice. Therefore, the vascular TXA 2 receptor represents a pharmacotherapeutic target to improve peripheral endothelial dysfunction in chronic HF., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease-19 (COVID-19). The COVID-19 pandemic has resulted in millions of deaths and widespread socio-economic damage worldwide. Therefore, numerous studies have been conducted to identify effective measures to control the spreading of the virus. Among various potential targets, the 3 chymotrypsin-like protease (3CLpro), also known as Mpro, stands out as the key protease of SARS-CoV-2, playing an essential role in virus replication and assembly, is the most prospective. In this study, we modified the commercial vector, pETM33-Nsp5-Mpro (plasmid # 156475, Addgene, USA), by inserting an autocleavage site (AVLQ) of 3CLpro and 6 × His-tag encoding sequences before and after the Nsp5-Mpro sequence, respectively. This modification enabled the expression of 3CLpro as an authentic N terminal protease (au3CLpro), which was purified to electrophoretic homogeneity by a single-step chromatography using two tandem Glutathione- and Ni-Sepharose columns. The enzyme au3CLpro demonstrated significantly higher activity (3169 RFU/min/μg protein) and catalytic efficiency (K cat /K m of 0.007 μM -1 .s- 1 ) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33-Nsp5-Mpro) with specific activity 889 RFU/min/μg and K cat /K m of 0.0015 μM -1 .s- 1 , respectively. Optimal conditions for au3CLpro activity included a 50 mM Tris-HCl buffer at pH 7, containing 150 mM NaCl and 0.1 mg/ml BSA at 37 °C., Competing Interests: Declaration competing of interest The authors declare that they have no conflict of interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dengue induces a spectrum of severity in humans from the milder dengue fever to severe disease, or dengue hemorrhagic fever (DHF). Chymase is a candidate biomarker that may aid dengue prognosis. This prospective study aimed to identify whether warning signs of severe dengue, including hypovolemia and fluid accumulation, were associated with elevated chymase. Serum chymase levels were quantified prospectively and longitudinally in hospitalized pediatric dengue patients in Sri Lanka. Warning signs were determined based on daily clinical assessments, laboratory tests and ultrasound findings. Chymase was significantly elevated during the acute phase of disease in DHF or Severe dengue, defined by either the 1997 or 2009 WHO diagnosis guidelines, and persisted longer in the most severe patients. Chymase levels were higher in patients with narrow pulse pressure and clinical warning signs such as severe leakage, fluid accumulation, pleural effusion, gall-bladder wall thickening and rapid haematocrit rise concurrent with thrombocytopenia. No association between chymase and liver enlargement was observed. This study confirms that serum chymase levels are associated with DHF/Severe dengue disease in hospitalized pediatric patients. Chymase levels correlate with warning signs of vascular dysfunction highlighting the possible functional role of chymase in vascular leakage during dengue. [ABSTRACT FROM AUTHOR]

Background: Activation of mast cells plays an important role in brain inflammation. CD300a, an inhibitory receptor located on mast cell surfaces, has been reported to reduce the production of pro-inflammatory cytokines and exert protective effects in inflammation-related diseases. Peroxisome proliferator-activated receptor β/δ (PPAR β/δ ), a ligand-activated nuclear receptor, activation upregulates the transcription of CD300a. In this study, we aim to investigate the role of PPAR β/δ in the attenuation of germinal matrix hemorrhage (GMH)-induced mast cell activation via CD300a/SHP1 pathway., Methods: GMH model was induced by intraparenchymal injection of bacterial collagenase into the right hemispheric ganglionic eminence in P7 Sprague Dawley rats. GW0742, a PPAR β/δ agonist, was administered intranasally at 1 h post-ictus. CD300a small interfering RNA (siRNA) and PPAR β/δ siRNA were injected intracerebroventricularly 5 days and 2 days before GMH induction. Behavioral tests, Western blot, immunofluorescence, Toluidine Blue staining, and Nissl staining were applied to assess post-GMH evaluation., Results: Results demonstrated that endogenous protein levels of PPAR β/δ and CD300a were decreased, whereas chymase, tryptase, IL-17A and transforming growth factor β1 (TGF-β1) were elevated after GMH. GMH induced significant short- and long-term neurobehavioral deficits in rat pups. GW0742 decreased mast cell degranulation, improved neurological outcomes, and attenuated ventriculomegaly after GMH. Additionally, GW0742 increased expression of PPAR β/δ , CD300a and phosphorylation of SHP1, decreased phosphorylation of Syk, chymase, tryptase, IL-17A and TGF-β1 levels. PPAR β/δ siRNA and CD300a siRNA abolished the beneficial effects of GW0742., Conclusions: GW0742 inhibited mast cell-induced inflammation and improved neurobehavior after GMH, which is mediated by PPAR β/δ /CD300a/SHP1 pathway. GW0742 may serve as a potential treatment to reduce brain injury for GMH patients., Competing Interests: Declaration of Competing Interest None of the authors have any conflicts of interests related to this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Although no longer a public health emergency of international concern, COVID-19 remains a persistent and critical health concern. The development of effective antiviral drugs could serve as the ultimate piece of the puzzle to curbing this global crisis. 3-chymotrypsin-like protease (3CL pro ), with its substrate specificity mirroring that of the main picornavirus 3C protease and conserved across various coronaviruses, emerges as an ideal candidate for broad-spectrum antiviral drug development. Moreover, it holds the potential as a reliable contingency option to combat emerging SARS-CoV-2 variants. In this light, the approved drugs, promising candidates, and de-novo small molecule therapeutics targeting 3CL pro since the COVID-19 outbreak in 2020 are discussed. Emphasizing the significance of diverse structural characteristics in inhibitors, be they peptidomimetic or nonpeptidic, with a shared mission to minimize the risk of cross-resistance. Moreover, the authors propose an innovative optimization strategy for 3CL pro reversible covalent PROTACs, optimizing pharmacodynamics and pharmacokinetics to better prepare for potential future viral outbreaks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Mast cells have traditionally been associated with allergic inflammatory responses; however, they play important roles in cutaneous innate immunity and wound healing. The Hidradenitis Suppurativa tissue transcriptome is associated with alterations in innate immunity and wound healing-associated pathways; however, the role of mast cells in the disease is unexplored. We demonstrate that mast cell-associated gene expression (using whole tissue RNAseq) is upregulated, and in-silico cellular deconvolution identifies activated mast cells upregulated and resting mast cells downregulated in lesional tissue. Tryptase/Chymase positive mast cells (identified using IHC) localize adjacent to epithelialized tunnels, fibrotic regions of the dermis and at perivascular sites associated with Neutrophil Extracellular Trap formation and TNF-alpha production. Treatment with Spleen Tyrosine Kinase antagonist (Fostamatinib) reduces the expression of mast cell-associated gene transcripts, associated biochemical pathways and the number of tryptase/chymase positive mast cells in lesional hidradenitis suppurativa tissue. This data indicates that although mast cells are not the most abundant cell type in Hidradenitis Suppurativa tissue, the dysregulation of mast cells is paralleled with B cell/plasma cell inflammation, inflammatory epithelialized tunnels and epithelial budding. This provides an explanation as to the mixed inflammatory activation signature seen in HS, the correlation with dysregulated wound healing and potential pathways involved in the development of epithelialized tunnels., (© 2023 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CL pro (also known as the main protease or M pro ) and PL pro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CL pro and PL pro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7- O -glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PL pro and 3CL pro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CL pro and PL pro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

The "Long-COVID syndrome" has posed significant challenges due to a lack of validated therapeutic options. We developed a novel multi-step virtual screening strategy to reliably identify inhibitors against 3-chymotrypsin-like protease of SARS-CoV-2 from abundant flavonoids, which represents a promising source of antiviral and immune-boosting nutrients. We identified 57 interacting residues as contributors to the protein-ligand binding pocket. Their energy interaction profiles constituted the input features for Machine Learning (ML) models. The consensus of 25 classifiers trained using various ML algorithms attained 93.9% accuracy and a 6.4% false-positive-rate. The consensus of 10 regression models for binding energy prediction also achieved a low root-mean-square error of 1.18 kcal/mol. We screened out 120 flavonoid hits first and retained 50 drug-like hits after predefined ADMET filtering to ensure bioavailability and safety profiles. Furthermore, molecular dynamics simulations prioritized nine bioactive flavonoids as promising anti-SARS-CoV-2 agents exhibiting both high structural stability (root-mean-square deviation < 5 Å for 218 ns) and low MM/PBSA binding free energy (<-6 kcal/mol). Among them, KB-2 (PubChem-CID, 14630497) and 9- O -Methylglyceofuran (PubChem-CID, 44257401) displayed excellent binding affinity and desirable pharmacokinetic capabilities. These compounds have great potential to serve as oral nutraceuticals with therapeutic and prophylactic properties as care strategies for patients with long-COVID syndrome.

The orally available chymase inhibitor BAY 1142524 is currently being developed as a first‐in‐class treatment for left‐ventricular dysfunction after myocardial infarction. Results from 3 randomized, single‐center, phase 1 studies in healthy male volunteers examining the safety, tolerability, and pharmacokinetics of BAY 1142524 are summarized. In this first‐in‐human study, single oral doses of 1‐200 mg were administered in fasted state as liquid service formulation or immediate release (IR) tablets. The relative bioavailability and the effect of a high‐fat/high‐calorie meal were investigated at the 5‐mg dose. In a multiple‐dose escalation study, doses of 5‐50 mg twice daily and 100 mg once daily were given for 5 consecutive days. BAY 1142524 was safe and well tolerated and had no effects on heart rate or blood pressure compared with placebo. BAY 1142524 was absorbed with peak concentration 1‐3 hours after administration for IR tablets; it was eliminated from plasma with a terminal half‐life of 6.84‐12.0 hours after administration of liquid service formulation or IR tablets. Plasma exposures appeared to be dose‐linear, with a negligible food effect. There was only low accumulation of BAY 1142524 after multiple dosing. BAY 1142524 exhibited a pharmacokinetic profile allowing for once‐daily dosing. The absence of blood pressure effects after administration of BAY 1142524 supports the combination of this novel anti‐remodeling drug with existing standard of care in patients with left‐ventricular dysfunction after acute myocardial infarction. [ABSTRACT FROM AUTHOR]

We previously reported on the elevated intravitreal activities of tryptase and chymase in association with idiopathic epiretinal membrane (ERM) and idiopathic macular hole (MH). In this present study, we investigated the potential intraocular production of these serine proteases, and measured and compared tryptase and chymase activities in the vitreous body and serum in ERM, MH, proliferative diabetic retinopathy (PDR), and rhegmatogenous retinal detachment (RRD) patients. In addition, nuclear staining with hematoxylin and eosin (H&E) and mast-cell staining with toluidine blue were performed on samples of the vitreous core and bursa premacularis (BPM) of MH. We also performed immunostaining on the above two regions of vitreous samples for MH with anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) antibody, and anti-fibroblast antibody. Moreover, we performed immunostaining with anti-tryptase antibody and anti-chymase antibody on ERMs collected intraoperatively. Tryptase activity in the vitreous body was significantly higher in ERM and MH than in PDR. However, no significant differences were observed in the tryptase activity in the serum among these four diseases. Chymase activity in the vitreous body was significantly higher in MH than in the other three diseases, yet chymase activity in the serum was below detection limit in any of the diseases. Nuclear staining with H&E revealed an abundance of nuclei in the BPM region, but few in the surrounding area. Mast-cell staining with toluidine blue revealed that the BPM showed metachromatic staining. In immunostaining with anti-fibroblasts antibody, anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, and anti-LYVE-1 antibody, the BPM stained more strongly than the vitreous core. Tryptase and chymase-positive cells were also observed in ERM. These findings revealed that the presence of mast cells in the BPM potentially represent the source of these serine proteases. Moreover, the BPM, as a lymphatic tissue, may play an important role in the pathogenesis of macular disease. [ABSTRACT FROM AUTHOR]

Abstract Aims Regular exercise is recommended in postmenopausal women to prevent the development of heart disease, but mechanism underlying the protection is not completely understood. Many studies have suggested that exercise training notably mediated whole body immune and inflammatory functions. Whether exercise training prevents cardiac dysfunction after deprivation of female sex hormones by inhibiting cardiac immune activation is therefore interesting. Main methods Nine-week treadmill running program was introduced in sham-operated and ovariectomized rats. In addition, chronic angiotensin II infusion was further challenged to activate pathological cardiac remodeling. Cardiac remodeling in associated with the density and degranulation of cardiac mast cells was then evaluated. Key findings With exogenous angiotensin II-induced hypertension, cardiac hypertrophy with myocardial fibrosis was shown similarly in both sham-operated controls and ovariectomized rats. Although exercise training did not prevent cardiac hypertrophy, myocardial fibrosis was abolished by exercise. While ovariectomy increased both cardiac mast cell density and degranulation percentage, angiotensin II infusion only enhanced mast cell density. Exercise training could not decrease the density of mast cells, but it did normalize the percentage of degranulation in all groups. Correlation analysis suggested that cardiac mast cell activation is inversely associated with cardiomyocyte hypertrophy due to exercise training but is directly correlated to cardiac hypertrophy by angiotensin II infusion. Significance Exercise training could attenuate cardiac mast cell hyperactivation induced by either deprivation of female sex hormones or excessive angiotensin II. Additionally, cardiac mast cells could be a solution in the distinction between physiological and pathological hypertrophic development. [ABSTRACT FROM AUTHOR]

Adhesion formation that occurred after alkali-induced injury of the cecum was used as a novel adhesion model in rats, and it was compared with that of a common adhesion model after abrading the cecum. Using the novel adhesion model, inhibition of adhesion formation by a chymase inhibitor, Suc-Val-Pro-PheP(OPh)2, and by sodium hyaluronate/carboxymethylcellulose (Seprafilm) was evaluated, and their mechanisms were assessed. The degree of adhesion formation was more severe and more stable in the alkali-induced injury model than in the abrasion-induced injury model. Both the chymase inhibitor and Seprafilm showed significant attenuation of the degree of adhesion 14 days after alkali-induced injury. Chymase activity in the cecum was significantly increased after alkali-induced injury, but it was significantly attenuated by the chymase inhibitor and Seprafilm. Myeloperoxidase and transforming-growth factor (TGF)-β levels were significantly increased after alkali-induced injury, but they were attenuated by both the chymase inhibitor and Seprafilm. At the level of the adhesions, the numbers of both chymase-positive cells and TGF-β-positive cells were significantly increased, but their numbers were reduced by the chymase inhibitor and Seprafilm. In conclusion, a chymase inhibitor attenuated the degree of adhesions to the same degree as Seprafilm in a novel peritoneal adhesion model that was more severe and more stable than the common adhesion model, and not only the chymase inhibitor, but also Seprafilm reduced the chymase increase at the adhesions. [ABSTRACT FROM AUTHOR]

Highlights • The distribution of cardiac mast cells in mice differs from humans and dogs. • This is important since mice are the most widely used genetic model for human pathology. • Loss-of-function mutation in the Mitf gene leads to near-complete lack of cardiac mast cells. • Loss of a single Mitf allele is sufficient for relative mast cell deficiency. Abstract Although mast cell distribution has been described in both human and canine hearts, cardiac mast cells in mice have yet to be categorically localized. We therefore sought to describe mast cell distribution within the mouse heart and characterize their dependence on the Microphthalmia-associated transcription factor (Mitf). Cardiac mast cells were visualized using Toluidine Blue and avidin staining, and their distribution within the heart described. Cardiac mast cells were most prevalent in the epicardium (50%) or myocardium (45%). Less frequently, mast cells were noted in the endocardium (5%). Within the myocardium, 31% of the mast cells had perivascular location. By studying two different Mitf mutant strains, Mitfmi-vga9 and MitfMi-wh , we demonstrated that these mutations led to near-complete deficiency of cardiac mast cells. Accordingly, expression of the mMCP-4 and mMCP-5 genes was lost and chymase enzyme activity was severely reduced. Additionally, hearts from mice heterozygous for these Mitf mutations contained significantly fewer mast cells compared to wild-type mice. Our results demonstrated that the distribution of cardiac mast cells in mice is different from humans and dogs. Cardiac mast cells are dependent on Mitf expression, with loss-of-function mutation in the Mitf gene leading to near-complete lack of cardiac mast cells. Loss of a single Mitf allele is sufficient for relative mast cell deficiency. [ABSTRACT FROM AUTHOR]

Serine proteases constitute the major protein content of mast cell secretory granules. Here we present the extended cleavage specificity of two such proteases from the golden hamster, Mesocricetus auratus. Analysis by phage display technique showed that one of them (HAM1) is a classical chymase with a specificity similar to the human mast cell chymase. However, in contrast to the human chymase, it does not seem to have a particular preference for any of the three aromatic amino acids, Phe, Tyr and Trp, in the P1 position of substrates. HAM1 also efficiently cleaved after Leu similarly to human and many other mast cell chymases. We observed only a 3-fold lower cleavage activity on Leu compared to substrates with P1 aromatic amino acids. Chymotryptic enzymes seem to be characteristic for connective tissue mast cells in mammalian species from opossums to humans, which indicates a very central role of these enzymes in mast cell biology. HAM1 also seems to have the strongest preference for negatively charged amino acids in the P2´position of all mast cell chymases so far characterized. The second hamster chymase, HAM2, is an elastolytic in its activity, similarly to the α-chymases in rats and mice (rMCP-5 and mMCP-5, respectively). The presence of an α-chymase that developed elastase activity thereby seems to be a relatively early modification of the α-chymase within the rodent branch of the mammalian evolutionary tree. [ABSTRACT FROM AUTHOR]

This research paper addresses the hypothesis that mast cells (MCs) contribute to the formation of mammary fibrosis. MCs are important immune regulatory and immune modulatory cells that play major roles in the inflammatory process. Since there is no detailed knowledge, this research study aimed to comparatively investigate the presence, localization, and immunophenotypes of MCs in healthy and mastitic mammary tissues. A total of 264 mammary samples were evaluated for the examination of mast cells and fibrosis. The mean mast cell number in both acute and chronic mastitis samples were very significantly higher than the control group P < 0.001). A 7.9-fold increase in the number of mast cells was found when the chronic mastitis group was compared with the control (healthy) group. Immunohistochemistry revealed presence of all three immune phenotypes in control and mastitic mammary samples (tryptase + (MC T ), chymase + (MC C ) and both chymase and tryptase + (MC TC ). The mean MC T , MC C , and MC TC numbers in the chronic mastitis group were found to be significantly higher than the control ( P < 0.001 for all three phenotypes) but did not differ significantly between control and acute mastitis samples. When the mean numbers of MC T , MC C , and MC TC in the control group and chronic mastitis group were compared, a 10.5, 7.8, and a 4.1-fold increase was observed, respectively. The amount of connective tissue was strongly increased in tissues with chronic mastitis and a 3.01-fold increase was detected compared to the control group. A statistically significant relation was also found between the amount of fibrosis and the increased number of total MCs ( P < 0.001).

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bencosme-Cuevas, Kim, Nguyen, Berry, Li, Adams, Smith, Batool, Swale, Kaufmann, Jones-Hall and Mulenga.)

Despite the remarkable development of highly effective vaccines, including mRNA-based vaccines, within a limited timeframe, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not been entirely eradicated. Thus, it is crucial to identify new effective anti-3CL Pro compounds, pivotal for the replication of SARS-CoV-2. Here, we identified an antcin-B phytosterol-like compound from Taiwanofungus camphoratus that targets 3CL Pro activity. MTT assay and ADMET prediction are employed for assessing potential cytotoxicity. Computational molecular modeling was used to screen various antcins and non-antcins for binding affinity and interaction type with 3CL Pro . Further, these compounds were subjected to study their inhibitory effects on 3CL Pro activity in vitro. Our results indicate that antcin-B has the best binding affinity by contacting residues like Leu141, Asn142, Glu166, and His163 via hydrogen bond and salt bridge and significantly inhibits 3CLPro activity, surpassing the positive control compound (GC376). The 100 ns molecular dynamics simulation studies showed that antcin-B formed consistent, long-lasting water bridges with Glu166 for their inhibitory activity. In summary, antcin-B could be useful to develop therapeutically viable drugs to inhibit SARS-CoV-2 replication alone or in combination with medications specific to other SARS-CoV-2 viral targets., (© 2023. Springer Nature Limited.)

Mast cell (MC)-specific proteases are of particular interest for space biology and medicine due to their biological activity in regulating targets of a specific tissue microenvironment. MC tryptase and chymase obtain the ability to remodel connective tissue through direct and indirect mechanisms. Yet, MC-specific protease expression under space flight conditions has not been adequately investigated. Using immunohistochemical stainings, we analyzed in this study the protease profile of the jejunal, gastric, and hepatic MC populations in three groups of Mongolian gerbils-vivarium control, synchronous experiment, and 12-day orbital flight on the Foton-M3 spacecraft-and in two groups-vivarium control and anti-orthostatic suspension-included in the experiment simulating effects of weightlessness in the ground-based conditions. After a space flight, there was a decreased number of MCs in the studied organs combined with an increased proportion of chymase-positive MCs and MCs with a simultaneous content of tryptase and chymase; the secretion of specific proteases into the extracellular matrix increased. These changes in the expression of proteases were observed both in the mucosal and connective tissue MC subpopulations of the stomach and jejunum. Notably, the relative content of tryptase-positive MCs in the studied organs of the digestive system decreased. Space flight conditions simulated in the synchronous experiment caused no similar significant changes in the protease profile of MC populations. The space flight conditions resulted in an increased chymase expression combined with a decreased total number of protease-positive MCs, apparently due to participating in the processes of extracellular matrix remodeling and regulating the state of the cardiovascular system.

The current study investigated the role of fucoidan from Padina tetrastromatica and Turbinaria conoides against 3-chymotrypsin like protease (3CL pro ) and receptor binding domain (RBD) spike protein of SARS-CoV-2 using an invitro and computational approach. The 3CL pro and RBD genes were successfully cloned in pET28a vector, expressed in BL-21DE3 E. coli rosetta cells and purified by ion exchange affinity and size exclusion chromatography. Fucoidan extracted from both biomass using green approach, subcritical water, was found to inhibit 3CL pro of SARS-CoV-2 with an IC 50 value of up to 0.35 mg mL -1 . However, fucoidan was found to be inactive against the RBD protein. Molecular docking studies demonstrated that fucoidan binds to the active sites of 3CL pro with an affinity of -5.0 kcal mol -1 . In addition, molecular dynamic simulations recorded stabilized interactions of protein-ligand complexes in terms of root mean square deviation, root mean square fluctuation, the radius of gyration, solvent accessible surface area and hydrogen bond interaction. The binding energy of fucoidan with 3CL pro was determined to be -101.821 ± 12.966 kJ mol -1 using Molecular Mechanic/Poisson-Bolt-Boltzmann Surface Area analysis. Fucoidan satisfies the Absorption, Distribution, Metabolism, and Excretion (ADME) properties, including Lipinski's rule of five, which play an essential role in drug design. According to the toxicity parameters, fucoidan does not exhibit skin sensitivity, hepatotoxicity, or AMES toxicity. Therefore, this work reveals that fucoidan from brown macroalgae could act as possible inhibitors in regulating the function of the 3CL pro protein, hence inhibiting viral replication and being effective against COVID-19., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

There is no definitive predictor of dengue severity, and this has led to a very large number of unnecessary hospitalizations worldwide. Although mast cell mediators are believed to a play role in dengue severity, the lack of precise kinetic data demands further research on early predictors. We enrolled 111 patients with confirmed dengue and 85 with “other febrile illness” (OFI) in a hospital-based prospective study in Vietnam. Dengue patients were classified as level 1, 2, or 3 based on the clinical intervention received. Blood samples were collected from each patient every day (pre- and post-defervescence) and after discharge. Plasma chymase, total IgE, and dengue-specific IgE were measured. Dengue-specific IgE levels showed an increasing trend during the course of illness and remained high even at post-discharge, although no significant difference was observed among severity levels. Total IgE showed no such trend. The specific IgE/total IgE ratio (S/T ratio) remained constantly higher in level 3 patients compared to other levels, with a significant difference at some time points. The S/T ratio of acute phase samples (before defervescence) tended to increase with increasing severity (level 1 < 2 < 3), and was significantly higher in level 3 patients than in level 1 and OFI patients. As an early predictor of severity allowing level 3 patients to be distinguished from other dengue patients, the S/T ratio achieved a sensitivity of 75% and specificity of 68%. We describe the kinetic profiles of IgEs, their ratio, and chymase levels at different severity levels. The S/T ratio was found to be associated with dengue severity, suggesting that it could potentially be used as an early predictor of severity. [ABSTRACT FROM AUTHOR]

Background Angiotensin-(1–12) [Ang-(1–12)] is a chymase-dependent source for angiotensin II (Ang II) cardiac activity. The direct contractile effects of Ang-(1–12) in normal and heart failure (HF) remain to be demonstrated. We assessed the hypothesis that Ang-(1–12) may modulate [Ca 2+ ] i regulation and alter cardiomyocyte contractility in normal and HF rats. Methods and results We compared left ventricle (LV) myocyte contractile and calcium transient ([Ca 2+ ] iT ) responses to angiotensin peptides in 16 SD rats with isoproterenol-induced HF and 16 age-matched controls. In normal myocytes, versus baseline, Ang II (10 −6  M) superfusion significantly increased myocyte contractility (dL/dt max : 40%) and [Ca 2+ ] iT (29%). Ang-(1–12) (4 × 10 −6 M) caused similar increases in dL/dt max (34%) and [Ca 2+ ] iT (25%). Compared with normal myocytes, superfusion of Ang II and Ang-(1–12) in myocytes obtained from rats with isoproterenol-induced HF caused similar but significantly attenuated positive inotropic actions with about 42% to 50% less increases in dL/dt max and [Ca 2+ ] iT . Chymostatin abolished Ang-(1–12)-mediated effects in normal and HF myocytes. The presence of an inhibitory cAMP analog, Rp-cAMPS prevented Ang-(1–12)-induced inotropic effects in both normal and HF myocytes. Incubation of HF myocytes with pertussis toxin (PTX) further augmented Ang II-mediated contractility. Conclusions Ang-(1–12) stimulates cardiomyocyte contractile function and [Ca 2+ ] iT in both normal and HF rats through a chymase mediated action. Altered inotropic responses to Ang-(1–12) and Ang II in HF myocytes are mediated through a cAMP-dependent mechanism that is coupled to both stimulatory G and inhibitory PTX-sensitive G proteins. [ABSTRACT FROM AUTHOR]

The chemokine CXCL17 is associated with the innate response in mucosal tissues but is poorly characterized. Similarly, the G protein-coupled receptor GPR35, expressed by monocytes and mast cells, has been implicated in the immune response, although its precise role is ill-defined. A recent manuscript reported that GPR35 was able to signal in response to CXCL17, which we set out to confirm in this study. GPR35 was readily expressed using transfection systems but failed to signal in response to CXCL17 in assays of β-arrestin recruitment, inositol phosphate production, calcium flux, and receptor endocytosis. Similarly, in chemotaxis assays, GPR35 did not confirm sensitivity to a range of CXCL17 concentrations above that observed in the parental cell line. We subsequently employed a real time chemotaxis assay (TAXIScan) to investigate the migratory responses of human monocytes and the monocytic cell line THP-1 to a gradient of CXCL17. Freshly isolated human monocytes displayed no obvious migration to CXCL17. Resting THP-1 cells showed a trend toward directional migration along a CXCL17 gradient, which was significantly enhanced by overnight incubation with PGE2. However, pretreatment of PGE2-treated THP-1 cells with the well-characterized GPR35 antagonist ML145 did not significantly impair their migratory responses to CXCL17 gradient. CXCL17 was susceptible to cleavage with chymase, although this had little effect its ability to recruit THP-1 cells. We therefore conclude that GPR35 is unlikely to be a bona fide receptor for CXCL17 and that THP-1 cells express an as yet unidentified receptor for CXCL17. [ABSTRACT FROM AUTHOR]

A novel chymase inhibitor has been reported to have depressor effect in salt-induced hypertension. Therefore, we examined the hypothesis that chymase inhibitory dried young leaves of Polygonum hydropiper (PPH) or young leaves extract of Polygonum hydropiper (PHE) could reduce salt-induced hypertension. In this study, 8-wk old wild-type mice were allocated into three experiments and experiment I included groups, I- normal water drinking, II- high salt (2% NaCl) water (HSW) drinking, and III- HSW plus PPH (500 mg kg −1 , orally) for 12-wk. Blood pressure (BP) and heart rate (HR) were measured at baseline and weekly up to wk-12. In experiment II, mice were given HSW for 12-wk followed by 8-wk treatment with PPH plus HSW (62.5, 125, 250 and 500 mg kg −1 for groups I, II, III and IV, respectively). BP and HR were measured at baseline and monthly until wk-12, following weekly for 8-wk. Experiment III comprised of four groups of mice for 12-wk HSW and 8-wk treatment with PHE plus HSW (2.5, 5, 10 and 20 mg kg −1 for groups I–IV, respectively). BP and HR were measured at baseline and monthly up to wk-12, following weekly for 8-wk. Significant reduction in BP and HR were observed in mice treated with PPH (500 mg kg −1 ) compared to HSW control. PPH reduced BP and HR dose dependently in hypertensive mice and the higher dose showed maximum reduction. PHE at its maximum dose (20 mg kg −1 ) significantly suppressed BP and HR. Over all, we found that the young leaves of Polygonum hydropiper suppressed salt-induced hypertension. [ABSTRACT FROM AUTHOR]

Human neutrophils express at least four active serine proteases, cathepsin G, N-elastase, proteinase 3 and neutrophil serine protease 4 (NSP4). They have all been extensively studied due to their importance in neutrophil biology and immunity. However, their extended cleavage specificities have never been determined in detail. Here we present a detailed cleavage specificity analysis of human cathepsin G (hCG). The specificity was determined by phage display analysis and the importance of individual amino acids in and around the cleavage site was then validated using novel recombinant substrates. To provide a broader context to this serine protease, a comparison was made to the related mast cell protease, human chymase (HC). hCG showed similar characteristics to HC including both the primary and extended specificities. As expected, Phe, Tyr, Trp and Leu were preferred in the P1 position. In addition, both proteases showed a preference for negatively charged amino acids in the P2´ position of substrates and a preference for aliphatic amino acids both upstream and downstream of the cleavage site. However, overall the catalytic activity of hCG was ~10-fold lower than HC. hCG has previously been reported to have a dual specificity consisting of chymase and tryptase-type activities. In our analysis, tryptase activity against substrates with Lys in P1 cleavage position was indeed only 2-fold less efficient as compared to optimal chymase substrates supporting strong dual-type specificity. We hope the information presented here on extended cleavage specificities of hCG and HC will assist in the search for novel in vivo substrates for these proteases as well as aid in the efforts to better understand the role of hCG in immunity and bacterial defence. [ABSTRACT FROM AUTHOR]