Serine Protease Activity Research Articles

Iripin-1, a new anti-inflammatory tick serpin, inhibits leukocyte recruitment in vivo while altering the levels of chemokines and adhesion molecules.

Serpins are widely distributed and functionally diverse inhibitors of serine proteases. Ticks secrete serpins with anti-coagulation, anti-inflammatory, and immunomodulatory activities via their saliva into the feeding cavity to modulate host's hemostatic and immune reaction initiated by the insertion of tick's mouthparts into skin. The suppression of the host's immune response not only allows ticks to feed on a host for several days but also creates favorable conditions for the transmission of tick-borne pathogens. Herein we present the functional and structural characterization of Iripin-1 (Ixodes ricinus serpin-1), whose expression was detected in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Of 16 selected serine proteases, Iripin-1 inhibited primarily trypsin and further exhibited weaker inhibitory activity against kallikrein, matriptase, and plasmin. In the mouse model of acute peritonitis, Iripin-1 enhanced the production of the anti-inflammatory cytokine IL-10 and chemokines involved in neutrophil and monocyte recruitment, including MCP-1/CCL2, a potent histamine-releasing factor. Despite increased chemokine levels, the migration of neutrophils and monocytes to inflamed peritoneal cavities was significantly attenuated following Iripin-1 administration. Based on the results of in vitro experiments, immune cell recruitment might be inhibited due to Iripin-1-mediated reduction of the expression of chemokine receptors in neutrophils and adhesion molecules in endothelial cells. Decreased activity of serine proteases in the presence of Iripin-1 could further impede cell migration to the site of inflammation. Finally, we determined the tertiary structure of native Iripin-1 at 2.10 Å resolution by employing the X-ray crystallography technique. In conclusion, our data indicate that Iripin-1 facilitates I. ricinus feeding by attenuating the host's inflammatory response at the tick attachment site.

Montane Rattlesnakes in México: Venoms of Crotalus tancitarensis and Related Species within the Crotalus intermedius Group.

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD50) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A2 (PLA2), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA2-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA2s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

Improving the catalytic activity of a detergent-compatible serine protease by rational design.

Serine proteases are among the most important biological additives in various industries such as detergents, leather, animal feed and food. A serine protease gene, Fgapt4, from Fusarium graminearum 2697 was identified, cloned and expressed in Pichia pastoris. The optimal pH and temperature of FgAPT4 were 8.5 and 40°C, respectively. The relative activity was >30% even at 10°C. It had a wide range of pH stability (4.0-12.0) and detergent compatibility. To improve the catalytic activity, a strategy combining molecular docking and evolutionary analysis was adopted. Twelve amino acid residue sites and three loops (A, B and C) were selected as potential hot spots that might play critical roles in the enzyme's functional properties. Twenty-eight mutants targeting changes in individual sites or loops were designed, and mutations with good performance were combined. The best mutant was FgAPT4-M3 (Q70N/D142S/A143S/loop C). The specific activity and catalytic efficiency of FgAPT4-M3 increased by 1.6 (1008.5 vs. 385.9 U/mg) and 2.2-fold (3565.1 vs. 1106.3/s/mM), respectively. Computational analyses showed that the greater flexibility of the substrate pocket may be responsible for the increased catalytic activity. In addition, its application in detergents indicated that FgAPT4-M3 has great potential in washing.

Alpha-1 antitrypsin limits neutrophil extracellular trap disruption of airway epithelial barrier function.

Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT.

Neutrophil serine proteases

The identification and characterization of the four active neutrophil serine proteases (NSPs) have provided a better understanding of their roles in various physiological and pathological processes. The availability of appropriate tools such as substrates, inhibitors, and activity-based probes (ABPs) for studying their activity and functions in cells has become increasingly important. In this paper, the authors provide a comprehensive overview of the current state of knowledge on the tools available for studying NSPs. The substrates, inhibitors, and ABPs that have been developed to date are described, including their strengths and limitations. The authors also discuss the potential implications of these tools for future research on NSPs, including their potential use in the development of new therapeutics for various diseases. Overall, this paper highlights the importance of understanding the activity and functions of NSPs and provides valuable information on the tools available for studying these proteases.

Apoptosis Inducing Factors Involved in the Changes of Flesh Quality in Postmortem Grass Carp (Ctenopharyngodon idella) Muscle.

Alterations of apoptosis have notable influences on flesh quality, but the mechanism is still unclear. Thus, apoptotic behaviors and related triggering mechanisms need to be explored. Fish muscle was prepared and stored at 4 °C for 0, 24, 48, 72, 96, and 120 h for apoptosis analysis. Results showed that positive apoptotic nuclei were positively correlated with drop loss and negatively correlated with shear force and water holding capacity (p < 0.05). Results showed that the triggering apoptotic mechanisms were involved with enhanced transcriptional levels of caspase-2, 3, 7, 8, and 9 through mitochondria and death receptor pathways in the muscle of grass carp. The decreased ATP content, changed cytochrome c redox state, increased protein levels of HSP27 and HSP 90, and enhanced activity of cathepsin (B, L, and D), calpain, and serine proteinase were involved in apoptosis activations. Results indicated that caspases, energy metabolism, cytochrome c redox state, heat shock protein expressions, and protease activities played critical roles in apoptosis alterations in carp muscle during refrigerated storage.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Delayed hair cycle in mnd2 mutant mice lacking HtrA2 serine protease activity

The premature death and degeneration of striatal neurons are typical hallmarks of HtrA2-inactivated motor neuron degeneration 2 (mnd2) mice. Although HtrA2 has been extensively studied in relation to the regulation of apoptosis using mnd2 mice, little is known about the other physiological functions of HtrA2. In this study, we found that the skin color of wild-type (WT) and mnd2 mice was black and pink on postnatal day 32. Using histological and molecular assays (i.e., assessing the activation of MAPK and expression patterns of PCNA), we demonstrated that this differential skin color change is consistent with the delay in the telogen − to − anagen phase of the hair cycle in mnd2 mice. We also examined adipocytes in the subcutaneous skin layer, finding that HtrA2 inactivation leads to the growth retardation of adipocytes, thereby delaying the hair cycle of mnd2 mice. Collectively, these findings show for the first time that HtrA2 plays an essential role in regulating the adipogenesis-associated hair cycle.

Computational modelling predicts impaired barrier function and higher sensitivity to skin inflammation following pH elevation.

Skin surface pH has been identified as a key regulator of the epidermal homeostasis through its action on serine protease activity. These enzymes, like kallikreins (KLK), are responsible for the degradation of corneodesmosomes, the protein structures linking together corneocytes, and are regulated by Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI). KLK activity increases at pH levels higher than physiological. An increase in skin surface pH has been observed in patients suffering from skin diseases characterized by impaired barrier function, like atopic dermatitis. In this work, we introduce an agent-based model of the epidermis to study the impact of a change in skin surface pH on the structural and physiological properties of the epidermis, through the LEKTI-KLK mechanism. We demonstrate that a less acidic pH, compared to the slightly acidic pH observed in healthy skin, is sufficient to significantly affect the water loss at the surface and the amount of irritant permeating through the epidermis. This weakening of the skin barrier function eventually results in a more intense skin inflammation following exposure to an external irritant. This work provides additional evidence that skin surface pH and serine proteases can be therapeutic targets to improve skin barrier integrity.

Genome-wide identification and immune response analysis of serine protease inhibitor genes in the blood clam Tegillarca granosa

Serine protease inhibitors (SPIs) are the main regulators of serine protease activities. In this study, we present a genome-wide identification of SPI genes in T. granosa（TgSPI genes）and their expression characteristics in respond to Vibrio stress. A total of 102 TgSPI genes belonging to eight families, including Serpin, TIL (trypsin inhibitor like cysteine rich domain), Kunitz, Kazal, I84, Pacifastin, WAP (whey acidic protein) and A2M (Alpha-2-macroglobulin) were identified, while no genes belonging to Bowman-Birk, amfpi and Antistasin families were identified. The Kazal family has the most TgSPI genes with 38, and 11 TgSPI genes belong to the mollusc-specific I84 family. The TgSPI genes were found to be randomly distributed on 17 chromosomes with 12 tandem duplicate gene pairs. Expression profiles showed that most TgSPI genes were mainly expressed in immune-related tissues such as hepatopancreas, gill and mantle. In the hepatopancreas, most of TgSPI genes were sensitive to Vibrio stress, 28 and 29 TgSPI genes were up-regulated and down-regulated, respectively. Some up-regulated genes with signal peptides, such as the TgSPIs of I84 family, may act as a mechanism to directly prevent Vibrio from invasion. Six Kazal-type TgSPIs (TgSPI29, 45, 49, 50, 51 and 52) were intracellular proteins and their expression was down-regulated in hemocytes after Vibrio stress. This may have boosted protease activity in hemocytes to the point that more hemoglobin derived peptides were produced and secreted into the hemolymph to exert their anti-Vibrio effects. These findings may provide valuable information for further clarifying the roles of SPIs in the immune defense and will benefit future exploration of the immune function of SPIs in molluscs.

Alkaloid fraction of Mirabilis jalapa Linn. flowers has low cytotoxicity and increases iron absorption through Erythropoietin-Matriptase-2-Hepcidin pathway in iron deficiency Hepatocarcinoma cell model

In this study, we investigated the effects of an alkaloid fraction of Mirabilis jalapa L. flowers in terms of cytotoxicity, Erythropoietin (EPO), hepcidin, and Matriptase-2 (MT-2) expression levels in iron deficiency Hepatocarcinoma (HepG2) cell model. The iron deficiency HepG2 cell model was generated by induction with Deferoxamine (DFO) and was then treated with standard therapy Ferric Ammonium Citrate (FAC) and different alkaloid fraction doses. Subsequently, the type II transmembrane serine proteases (TTSPs) activity and MT-2 expression were measured using a fluorometer and immunocytochemistry methods, while the EPO and hepcidin levels and total iron were examined using an ELISA kit and a colorimetric assay, respectively. The data were then analyzed using ANOVA with a significance level of 95 %. According to the UV–vis Spectrophotometry and HPLC results, the alkaloid fraction of M. jalapa flowers had 6.17- and 4-times higher Betaxanthin levels, respectively, compared to the ethanol extract of M. jalapa flower. Furthermore, LC-MS/MS analysis showed that the most dominant compound is Indicaxanthin. The ethanol extract and alkaloid fraction of M. jalapa flowers were not cytotoxic (IC50 > 30 ppm). Furthermore, the alkaloid fraction containing Indicaxanthin, Miraxanthin-V, and Boeravinone F is capable of increasing EPO levels, membrane and soluble TTSPs activity and MT-2 expression, decreasing hepcidin levels, and increasing intracellular iron levels in iron deficiency HepG2 cell model. In conclusion, the obtained alkaloid fraction of M. jalapa flowers has low cytotoxicity and the later increases iron absorption via EPO-MT2-hepcidin pathway in iron deficiency HepG2 cell model.

Atenativ, a Plasma-Derived Antithrombin Concentrate, Used in Patients with Congenital Antithrombin Deficiency Undergoing Surgery or Parturition: Study Description (NCT04918173)

Background: Antithrombin is an endogenous anticoagulant inhibiting the activity of thrombin, FXa, and other serine proteases. Persons with heterozygous variants are at increased risk for thrombosis and are often insensitive to heparin therapy. Type I homozygous variants are lethal embryologically attesting to antithrombin's indispensability. Current clinical wisdom is to use on-demand anticoagulation with or without antithrombin concentrate for high-risk periods and secondary prophylaxis (prophylaxis after a thrombotic event) for those persons experiencing a thrombus. Two high-risk periods are perioperative and peripartum. Methods: This is a multinational, prospective, open-label, uncontrolled phase 3 study to assess efficacy, safety, and single-dose pharmacokinetics of Atenativ in patients with congenital antithrombin deficiency undergoing surgery or delivery (NCT04918173). Atenativ is a lyophilized, highly pure antithrombin concentrate free of denatured antithrombin prepared from human plasma. The primary objective of the study is to determine the incidence of thrombotic and thromboembolic events after antithrombin replacement therapy in the high risk perioperative and peripartum scenarios. The secondary objectives are to determine single-dose pharmacokinetics (PK), assess coagulation parameters (activated partial thromboplastin time [aPTT], prothrombin time [PT], international normalized ratio [INR] and fibrinogen), and safety and tolerability of Atenativ. We will enroll 18 non-pregnant adult subjects older than 18 and younger than 80 years (4 of which will be 12-16 years of age from the US) to the pharmacokinetics phase and 20 subjects to the treatment phase of the study. Subjects may participate in both phases of the study, but only 1 surgery or delivery per subject is eligible. Salient inclusion criteria are: congenital antithrombin deficiency, defined by plasma activity of antithrombin ≤60%; personal or family history of thrombotic or thromboembolic events; and written informed consent from subject or legal guardian. Salient exclusion criteria are: emergency surgery, precipitous parturition or recent surgery; prior diagnosis of heparin-induced thrombocytopenia; and recent thrombotic or thromboembolic event. Lower extremity venous Doppler examination will be performed for patients entering the treatment phase. Subjects in the PK phase will receive a single dose of 60IU/kg with blood draws at baseline, 20 minutes, 1, 3, and 8 hours, and 2-8, 10, 12, and 14 days after the end of Atenativ infusion. The table illustrates Atenativ dosing schedule for surgical subjects and parturients. Statistics is confined to descriptive analyses without the need for a sample size estimation. Results: Site initiation has begun (8 countries and 15 sites), and subject enrollment has commenced and is planned to continue until Q4 of 2024. Conclusion: This study will endeavor to determine and decrease the incidence of thrombotic and thromboembolic events in subjects with congenital antithrombin deficiency during high-risk periods. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HtrA1 in Gestational Diabetes Mellitus: A Possible Biomarker?

The high-temperature requirement A 1 (HtrA1) is a multidomain secretory protein with serine-protease activity, expressed in many tissues, including placenta, where its expression is higher in the first trimester, suggesting an association of this serine protease in early phases of human placenta development. In this study, we evaluated maternal serum HtrA1 levels in the first and third trimester of gestation. In particular, we evaluated a possible role of HtrA1 as an early marker of gestational diabetes mellitus (GDM) in the first trimester of gestation. We evaluated HtrA1 serum levels in the third trimester (36-40 weeks) in normal pregnancies (n = 20) and GDM pregnancies (n = 20) by using ELISA analysis. Secondly, we performed the same analysis by using the first trimester sera (10-12 weeks) of healthy pregnant women that will develop a normal pregnancy (n = 210) or GDM (n = 28) during pregnancy. We found that HtrA1 serum levels in the third trimester were higher in pregnancies complicated by GDM. Interestingly, higher HtrA1 serum levels were also found in the first trimester in women developing GDM later during the second-third trimester. No significant differences in terms of maternal age and gestational age were found between cases and controls. Women with GDM shown significantly higher pre-pregnancy BMI values compared to controls. Moreover, the probability of GDM occurrence significantly increased with increasing HtrA1 levels and BMI values. The ROC curve showed a good accuracy in predicting GDM, with an AUC of 0.74 (95%CI: 0.64-0.92). These results suggest an important role of HtrA1 as an early predictive marker of GDM in the first trimester of gestation, showing a significative clinical relevance for prevention of this disease.

Pharmacological inhibition of cathepsin C (CatC) as a potential approach for cancer therapeutics

Studies have established that proteolytic enzyme inhibition holds significant promise in cancer prevention and treatment. Cathepsin C (CatC) is conserved lysosomal cysteine dipeptidyl aminopeptidase, which is the key for pro-inflammatory neutrophil serine protease activation and biological functioning. This makes CatC as a promising therapeutic drug target for the management of different cancer types. Considering this, using a wide range of computer aided drug-designing applications, several inhibitors are shortlisted against CatC active pocket, which interact with the enzyme with high affinity and form strong intermolecular interaction network. Compared to control, three molecules ASN_06916232, ASN_06917112 and ASN_06916892 are filtered as best binders of the CatC active pocket with binding energy value of −10.9 kcal/mol, −10.9 kcal/mol and −10.7 kcal/mol, respectively. These compounds interact with several important active side residues of CatC such as Ser233, Cys234, Gly277, Asn380 and His38. Furthermore, the complexes of these compounds with CatC reveal very stable dynamics with average RMSD value less than 3 Å. The binding energy analysis further indicates the compounds to have very stable van der Waals and electrostatic energies. In conclusion, these molecules are promising and require experimental validation to prove them as anti-CatC molecules. Communicated by Ramaswamy H. Sarma

Domain Evolution of Vertebrate Blood Coagulation Cascade Proteins

Vertebrate blood coagulation is controlled by a cascade containing more than 20 proteins. The cascade proteins are found in the blood in their zymogen forms and when the cascade is triggered by tissue damage, zymogens are activated and in turn activate their downstream proteins by serine protease activity. In this study, we examined proteomes of 21 chordates, of which 18 are vertebrates, to reveal the modular evolution of the blood coagulation cascade. Additionally, two Arthropoda species were used to compare domain arrangements of the proteins belonging to the hemolymph clotting and the blood coagulation cascades. Within the vertebrate coagulation protein set, almost half of the studied proteins are shared with jawless vertebrates. Domain similarity analyses revealed that there are multiple possible evolutionary trajectories for each coagulation protein. During the evolution of higher vertebrate clades, gene and genome duplications led to the formation of other coagulation cascade proteins.

Expression of some Serine Protease Encoding Genes and Trypsin-like Protease Activity in Black Tiger Shrimp (Penaeus monodon) Infected with White Spot Syndrome Virus

In this study, trypsin-like protease activity and expression of serine protease encoding genes (PmPPAE1, PmPPAE2 - clip-serine proteases and PmTLP1, PmTLP2 - trypsin-like proteases) in the different tissues of black tiger shrimp were determined. The expression level of PmPPAE1 and PmPPAE2 was the highest in the hemolymph; meanwhile that of PmTLP1 and PmTLP2 was the highest in the hepatopancreas. In the hemolymph of white spot syndrome virus (WSSV)-injected shrimp, the expression of PmPPAE1, PmPPAE2 and PmTLP2 genes was upregulated by 2.32, 2.57 and 4.56-folds, respectively, whereas the expression of PmTLP1 was downregulated by 1.47-folds relative to the control group (injected with phosphate buffered saline). In the hepatopancreas of WSSV-injected shrimp, the expression levels of PmPPAE1, PmPPAE2 increased by 2.24 and 3.99-folds, respectively, and that of PmTLP1 and PmTLP2 decreased by 1.51, 1.29-folds, respectively. Trypsin-like protease activity was found increased 3.25-fold and decreased 4.1-fold, in the hemolymph and hepatopancreas of WSSV-infected shrimp, respectively. These data indicate the role of serine proteases in immune response of the shrimp.

A comprehensive comparison of facial skin hydration based on capacitance and conductance measurements in Chinese women.

The aim of this study was to compare the data of conductance and capacitance measurements of facial skin hydration and to evaluate and discuss the advantages and disadvantages of the different approaches. We measured skin capacitance (Corneometer® CM 825) and skin conductance (Skicon-200EX®) on 30 pre-defined facial sites of 125 Chinese women, resulting in 3750 readings per device. The data were analysed and compared, and continuous colour maps were generated on a 3D avatar for capacitance, conductance, relative difference (Δ%) and correlation (R-value) by interpolating between the individual readings and converting the values to colours. This visualization allows a better interpretation of the results. The complexity of facial skin hydration is revealed by this approach. The similarities and discrepancies in the facial hydration maps are clearly apparent. Due to the superiority of the Skicon in measuring high hydration levels, differences in skin hydration were evident on the forehead compared with the Corneometer maps, which may be related to the more superficial measurement of the Skicon within the stratum corneum. Conversely, a greater understanding of the complexity of facial skin hydration in the nasolabial fold was obvious when using the Corneometer. The best congruence between the instruments was found at two specific but separated facial areas, one around the inner eye region and the other one on a line between the nasolabial sulcus and the oblique, lateral jaw. Interestingly, the data were not normally distributed for both instruments and they had opposite skews. All facial clusters were statistically different from each other (p< 0.001), except the cheek and jaw for the Skicon. Larger than expected percentage coefficients of variance were found for the Corneometer on some facial sites that might be explainable by differences in stratum corneum physiology and biochemistry. Corneometer values of 48 AU and Skicon values of 132 μS were taken as the cutoff for normally hydrated facial skin. Both devices have their advantages and disadvantages suggesting that bio-instrumental measurement of skin hydration is actually more complicated than commonly thought and that the different facial zones and the use of multiple instrumentation have not been adequately considered.

Grass carp SERPINA1 inhibits GCRV infection through degrading CF2.

SERPINA1, a member of the serine protease inhibitor family, plays a role in viral infection and inflammation by regulating the activities of serine and cysteine proteases. To date, there have been no reports on the immune function of SERPINA1 in fishes. In this study, we first cloned the serpina1 gene of grass carp (Ctenopharyngodon idellus) and found that it could respond rapidly to the infection of Grass carp reovirus (GCRV), and overexpression of serpina1 could enhance the antiviral response of CIK cells. A polyclonal antibody of SERPINA1 was prepared, and the protein interacting with SERPINA1 was screened by CoIP/MS in grass carp hepatopancreas tissue. It was found that SERPINA1 interacted with coagulation factor 2 (CF2) and could degrade it in a dose-dependent manner. In addition, overexpression of cf2 contributed to the infection of GCRV in CIK cells, whereas co-expression of serpina1 and cf2 in grass carp reduced the copy number of GCRV in cells. The results showed that grass carp SERPINA1 could inhibit GCRV infection by degrading CF2. This study proposes that SERPINA1 can inhibit viral infection through interaction with the coagulation factor, providing new insights into the molecular mechanism of SERPINA1’s antiviral function.

Dipeptidyl peptidase-1 inhibition in patients hospitalised with COVID-19: a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial

BackgroundNeutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19.MethodsIn a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012.FindingsBetween June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57–0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug.InterpretationBrensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19.FundingSponsored by the University of Dundee and supported through an Investigator Initiated Research award from Insmed, Bridgewater, NJ; STOP-COVID19 trial.

The ketone body β-hydroxybutyrate mitigates ILC2-driven airway inflammation by regulating mast cell function.

Ketone bodies are increasingly understood to have regulatory effects on immune cell function, with β-hydroxybutyrate (BHB) exerting a predominantly anti-inflammatory response. Dietary strategies to increase endogenous ketone body availability such as the ketogenic diet (KD) have recently been shown to alleviate inflammation of the respiratory tract. However, the role of BHB has not been addressed. Here, we observe that BHB suppresses group 2 innate lymphoid cell (ILC2)-mediated airway inflammation. Central to this are mast cells, which support ILC2 proliferation through interleukin-2 (IL-2). Suppression of the mast cell/IL-2 axis by BHB attenuates ILC2 proliferation and the ensuing type 2 cytokine response and immunopathology. Mechanistically, BHB directly inhibits mast cell function in part through GPR109A activation. Similar effects are achieved with either the KD or 1,3-butanediol. Our data reveal the protective role of BHB in ILC2-driven airway inflammation, which underscores the potential therapeutic value of ketone body supplementation for the management of asthma.