Endogenous Serine Protease Inhibitor Research Articles

Therapeutic potential of elafin in airway inflammatory disease

Elafin, which is derived from trappin-2 or pre-elafin by proteolysis, is an endogenous serine protease inhibitor with a low molecular weight. Its inhibitory activity is dependent on anchoring to the extracellular matrix by forming covalent bonds with its distinctive N-terminal domain via tissue transglutaminases. In addition to inhibiting proteases, it also exhibits anti-inflammatory, antibiotic, antifungal, antiviral and immunomodulatory functions. Elafin plays an important role in inflammatory disease and is a promising candidate for the anti-inflammatory treatment of respiratory diseases. This review will discuss the therapeutic potential of elafin in airway inflammation and provide evidence and suggestions for the future treatment of airway inflammatory diseases. In addition, the therapeutic potential of elafin in lung cancer is also discussed.

Native and activated antithrombin inhibits TMPRSS2 activity and SARS-CoV-2 infection.

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad‐spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS‐CoV, MERS‐CoV, hCoV‐229E, SARS‐CoV‐2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS‐CoV‐2. Thus, AT is an endogenous inhibitor of SARS‐CoV‐2 that may be involved in COVID‐19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti‐TMPRSS2 and anti‐SARS‐CoV‐2 activity of AT, suggesting that repurposing of native and activated AT for COVID‐19 treatment should be explored.

Antithrombin-III mitigates thrombin-mediated endothelial cell contraction and sickle red blood cell adhesion in microscale flow.

SummaryIndividuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin‐III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient‐derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin‐activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule‐1 (VCAM‐1) mediated by thrombin. Our findings suggest that, by attenuating thrombin‐mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.

Evaluation of Elafin Immunohistochemical Expression as Marker of Cervical Cancer Severity

Introduction: The main risk factor for the development of cervical cancer (CC) is persistent infection by human papillomavirus (HPV) oncogenic types. In order to persist, HPV exhibits a plethora of immune evasion mechanisms. PI3/Elafin (Peptidase Inhibitor 3) is an endogenous serine protease inhibitor involved in epithelial protection against pathogens. PI3/Elafin’s role in CC is still poorly understood. Materials and Methods: In the present study, we addressed PI3/Elafin protein detection in 123 CC samples by immunohistochemistry and mRNA expression in several datasets available at Gene Expression Omnibus and The Cancer Genome Atlas platforms. Results: We observed that PI3/Elafin is consistently downregulated in CC samples when compared to normal tissue. Most of PI3/Elafin-positive samples exhibited this protein at the plasma membrane. Besides, high PI3/Elafin expression at the cellular membrane was more frequent in in situ stages I + II than in invasive cervical tumor stages III + IV. This indicates that PI3/Elafin expression is gradually lost during the CC progression. Of note, advanced stages of CC were more frequently associated with a more intense PI3/Elafin reaction in the nuclei and cytoplasm. Conclusion: Our results suggest that PI3/Elafin levels and subcellular localization may be used as a biomarker for CC severity.

Predictions of novel Schistosoma mansoni - human protein interactions consistent with experimental data

Infection by the human blood fluke, Schistosoma mansoni involves a variety of cross-species protein- protein interactions. The pathogen expresses a diverse arsenal of proteins that facilitate the breach of physical and biochemical barriers present in skin evasion of the immune system, and digestion of human plasma proteins including albumin and hemoglobin, allowing schistosomes to reside in the host for years. However, only a small number of specific interactions between S. mansoni and human proteins have been identified. We present and apply a protocol that generates testable predictions of S. mansoni-human protein interactions. In this study, we have preliminary predictions of novel interactions between schistosome and human proteins relevant to infection and the ability of the parasite to evade the immune system. We applied a computational whole-genome comparative approach to predict potential S. mansoni-human protein interactions based on similarity to known protein complexes. We first predict S. mansoni -human protein interactions based on similarity to known protein complexes. Putative interactions were then scored and assessed using several contextual filters, including the use of annotation automatically derived from literature using a simple natural language processing methodology. Next, in vitro experiments were carried out between schistosome and host proteins to validate several prospective predictions. Our method predicted 7 out of the 10 previously known cross-species interactions involved in pathogenesis between S. mansoni and its human host. Interestingly, two novel putative interactions involving Schistosoma proteins, the cercarial elastase SmCE, and the adult tegument surface protein Sm29, were also predicted and experimentally characterized. Preliminary data suggest that elafin, a host endogenous serine protease inhibitor, may be a novel substrate for SmCE. Additionally, CD59, an inhibitor of the membrane attack complex, could interact with Sm29. Furthermore, the application framework provides an integrated methodology for investigation of host-pathogen interactions and an extensive source of orthogonal data for experimental analysis. We have made the predictions available for community perusal.

Functional attribution of LdISP, an endogenous serine protease inhibitor from Leishmania donovani in promoting infection

Leishmaniasis, a parasitic disease caused by unicellular eukaryotic protozoa of the genus Leishmania, affects more than 12 million people worldwide. Events of leishmaniasis are based on the infection of the mammalian host, precisely macrophages, where both host and parasite derived proteases and endogenous inhibitors are significant. Pathogen derived protease inhibitors have generated considerable interest as they often act as an agent promoting infection and parasitic survivability. An endogenous serine protease inhibitor from Indian strain of Leishmania donovani was previously identified by our group and named as LdISP. It has been found to inhibit neutrophil elastase (NE), responsible for natural inflammation process. However, LdISP's role in progression of infection or the proteomics based structural exposition has not been explored. The present study is aimed to localize and validate the potential role of LdISP in infectivity. We found that LdISP localized endogenously and treatment of infected host cells with LdISP curbs ROS and NO production. Additionally, in silico studies are carried out to predict the putative amino acid residues of LdISP involved in the inhibition process. Taken together, our results demonstrate that LdISP eventually exerts a pronounced role in L. donovani infection.

Identification and characterization of a Leishmania donovani serine protease inhibitor: Possible role in regulation of host serine proteases.

This study aims to identify, purify, and characterize an endogenous serine protease inhibitor from an Indian strain of Leishmania donovani, which causes the fatal visceral leishmaniasis. (i) Reverse zymography was used to identify the serine protease inhibitor by inhibiting the gelatinolytic activity of serine protease. (ii) Purification was performed by combining heat treatment, ultracentrifugation, and affinity and gel permeation chromatography. (iii) Spectrophotometric assays were conducted to quantify and compare the inhibitory activity of the L. donovani serine protease inhibitor (LdISP). (iv) Further, the protein was identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS). An endogenous inhibitor with an apparent molecular weight of 21.8 kDa, which is acidic in nature, having a pI of 5.9 was identified. The Ki value of the inhibitor for trypsin was determined to be in the nanomolar range. The protein has the following features: (i) ecotin-like nature, (ii) cross-organism functionality, that is, an inhibitory effect on the serine proteases of higher organisms other than its own, and (iii) homology with other such proteins from a different species of Leishmania on conducting protein mass fingerprinting after MALDI ToF MS. The inhibitor shows varying and entirely contrasting efficacies toward serine proteases of its own as well as of higher organisms. This indicates that it accelerates disease progression and drives parasite survival as it inhibits the activities of the host serine proteases.

Elafin: a double agent in breast and ovarian cancer.

Elafin: a double agent in breast and ovarian cancer.

Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein.

Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.

Elafin is downregulated during breast and ovarian tumorigenesis but its residual expression predicts recurrence.

IntroductionElafin is an endogenous serine protease inhibitor. The majority of breast cancer cell lines lack elafin expression compared to human mammary epithelial cells. In this study, we hypothesized that elafin is downregulated during breast and ovarian tumorigenesis.MethodsWe examined elafin expression by immunohistochemistry (IHC) in specimens of normal breast tissue (n = 24), ductal carcinoma in situ (DCIS) (n = 54), and invasive breast cancer (n = 793). IHC analysis of elafin expression was also performed in normal fallopian tube tissue (n = 20), ovarian cystadenomas (n = 9), borderline ovarian tumors (n = 21), and invasive ovarian carcinomas (n = 216). To understand the significance of elafin in luminal breast cancer cell lines, wild-type or M25G elafin (lacking the protease inhibitory function) were exogenously expressed in MCF-7 and T47D cells.ResultsElafin expression was downregulated in 24% of DCIS and 83% of invasive breast tumors when compared to elafin expression in the normal mammary epithelium. However, the presence of elafin-positive cells in invasive breast tumors, even at low frequency, correlated with poor recurrence-free survival (RFS), reduced overall survival (OS), and clinicopathological markers of aggressive tumor behavior. Elafin-positive cells were an especially strong and independent prognostic marker of reduced RFS in IHC-defined luminal A-like tumors. Elafin was also downregulated in 33% of ovarian cystadenomas, 43% of borderline ovarian tumors, and 86% of invasive ovarian carcinomas when compared to elafin expression in the normal fallopian tube. In ovarian tumors, elafin-positive cells were correlated with reduced RFS, OS and disease-specific survival (DSS) only in stage I/II patients and not in stage III/IV patients. Notably, exogenous expression of elafin or elafin M25G in the luminal breast cancer cell lines MCF-7 and T47D significantly decreased cell proliferation in a protease inhibitory domain-independent manner.ConclusionsElafin predicts poor outcome in breast and ovarian cancer patients and delineates a subset of endocrine receptor-positive breast cancer patients susceptible to recurrence who could benefit from more aggressive intervention. Our in vitro results suggest that elafin arrests luminal breast cancer cells, perhaps suggesting a role in tumor dormancy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0497-4) contains supplementary material, which is available to authorized users.

Novel Role of the Serine Protease Inhibitor Elafin in Gluten-Related Disorders

Elafin, an endogenous serine protease inhibitor, modulates colonic inflammation. We investigated the role of elafin in celiac disease (CD) using human small intestinal tissues and in vitro assays of gliadin deamidation. We also investigated the potential beneficial effects of elafin in a mouse model of gluten sensitivity. Epithelial elafin expression in the small intestine of patients with active CD, treated CD, and controls without CD was determined by immunofluorescence. Interaction of elafin with human tissue transglutaminase-2 (TG-2) was investigated in vitro. The 33-mer peptide, a highly immunogenic gliadin peptide, was incubated with TG-2 and elafin at different concentrations. The degree of deamidation of the 33-mer peptide was analyzed by liquid chromatography-mass spectrometry. Elafin was delivered to the intestine of gluten-sensitive mice using a recombinant Lactococcus lactis vector. Small intestinal barrier function, inflammation, proteolytic activity, and zonula occludens-1 (ZO-1) expression were assessed. Elafin expression in the small intestinal epithelium was lower in patients with active CD compared with control patients. In vitro, elafin significantly slowed the kinetics of the deamidation of the 33-mer peptide to its more immunogenic form. Treatment of gluten-sensitive mice with elafin delivered by the L. lactis vector normalized inflammation, improved permeability, and maintained ZO-1 expression. The decreased elafin expression in the small intestine of patients with active CD, the reduction of 33-mer peptide deamidation by elafin, coupled to the barrier enhancing and anti-inflammatory effects observed in gluten-sensitive mice, suggest that this molecule may have pathophysiological and therapeutic importance in gluten-related disorders.

In Vivo Anti-HIV Activity of the Heparin-Activated Serine Protease Inhibitor Antithrombin III Encapsulated in Lymph-Targeting Immunoliposomes

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log10 reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention.

A Prospective Comparison Between 1.5t Magnetic Resonance and 3t Magnetic Resonance in Ileo-Colonic Crohn's Disease: A Single Center Experience

Studies using animal models suggest that proteolytic balance at the surface of inflamed mucosa is broken towards an increased proteolytic activity. This increase could be explained by an important release of active proteases, a decreased expression of endogenous protease inhibitors, or both. We have investigated here the proteolytic activity released by fresh colonic tissue biopsies of IBD patients (Crohn's disease: CD, and Ulcerative Colitis: UC) and non-IBD patients, and we have investigated in those tissues, the expression of Elafin, an endogenous serine protease inhibitor, possibly released by many cell types. This molecule is considered as an “alarm” antiprotease released by immune or epithelial cells, and that is able to inhibit neutrophil proteases such as elastase and Proteinase-3. Methods: Non-IBD patients (Control), CD and UC colonic biopsies were harvested and freshly incubated for 1-h at 37 degrees, in culture media (HBSS). Arginin cleavage specific activity was measured in biopsy supernatants using a chromogenic substrate (tosyl-GPR-pNa). Other biopsies were paraffin-embedded and used for the detection of elafin mRNA using sense and antisense RNA riboprobes (200pb) against elafin mRNA. Results: A significant increased proteolytic activity was detected in culture supernatants from CD, and UC biopsies compared to control patients supernatants. In non-inflamed biopsies, we detected a strong expression of elafin mRNA in epithelium while only few cells in the sub-mucosa expressed this mRNA. In both UC and CD biopsies, elafin mRNA was detected in the sub-mucosa in a zone that correlated with infiltrated immune cells. However in the intestinal epithelium itself, elafin mRNA expression was clearly reduced. Conclusion:Our results showed that in IBD patients, proteolytic balance of the mucosa is disrupted towards an increased proteolytic activity. Elafin mRNA was constitutively expressed by epithelial cells in non-IBD patient tissues, but in IBD patient tissues, a specific down-regulation of elafin expression was observed in the epithelium. This study showed that the intestinal protease-antiprotease balance is altered in inflammatory bowel disease (IBD) and proposes that deficient expression of elafin by the epithelium could participate to explain this unbalance.

Differential effects of periopathogens on host protease inhibitors SLPI, elafin, SCCA1, and SCCA2

Objective: Secretory leukocyte peptidase inhibitors (SLPI), elafin, squamous cell carcinoma antigen 1 and 2 (SCCA1 and SCCA2) are specific endogenous serine protease inhibitors expressed by epithelial cells that prevent tissue damage from excessive proteolytic enzyme activity due to inflammation. To determine the effects of various periopathogens on these protease inhibitors, we utilized human gingival epithelial cells (GECs) challenged with cell-free bacteria supernatants of various periopathogens Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum.Design: The gene expression and secretion of SLPI, elafin, SCCA1, and SCCA2 were determined using real-time PCR and ELISA, respectively. The direct effects of periopathogens and P. gingivalis gingipain mutants on these inhibitors were examined in vitro by Western Blot. The effect on the innate immune response of GECs was measured by expression of antimicrobial peptides: human beta-defenisin-2 and chemokine (C-C motif) ligand 20 (CCL20).Results: We found that SLPI, SCCA2, elafin, hBD2, and CCL20 gene expression levels were significantly induced (p<0.001) in response to P. gingivalis, whose virulence factors include cysteine proteases, but not in response to stimulation by other bacteria. P. gingivalis reduced the secretion of SLPI and elafin significantly in GECs, and degraded recombinant SLPI, elafin, SCCA1, and SCCA2. Differential degradation patterns of SLPI, elafin, SCCA1, and SCCA2 were observed with different bacteria as well as P. gingivalis mutants associated with the loss of specific gingipains secreted by P. gingivalis. In addition, pretreatment of GECs with SLPI, SCCA1, or SCCA2 partially blocked hBD2 and CCL20 mRNA expression in response to P. gingivalis, suggesting a protective effect.Conclusion: Our results suggest that different periopathogens affect the host protease inhibitors in a different manner, suggesting host susceptibility may differ in the presence of these pathogens. The balance between cellular protease inhibitors and their degradation may be an important factor in susceptibility to periodontal infection.

Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] : Cholesterol (DOPS : Chol) liposomes for inhalation. Incubation of rSLPI with cathepsin L leads to complete loss of activity while encapsulation of rSLPI in DOPS : Chol liposomes retained 92.6% of its activity after challenge with cathepsin L. rSLPI-loaded liposomes were aerosolized efficiently using a standard nebulizer with a minimal loss of activity and stability. This formulation was biocompatible and encapsulation did not appear to diminish access to intracellular sites of action in in vitro cell culture studies. Liposome encapsulation of rSLPI therefore improves stability and potentially reduces the level and frequency of dosing required for therapeutic effect after inhalation.

Differential Regulation of Elafin in Normal and Tumor-Derived Mammary Epithelial Cells Is Mediated by CCAAT/Enhancer Binding Protein β

CCAAT/enhancer binding protein beta (C/EBP beta) is a transcription factor implicated in the control of development, differentiation, and proliferation of mammary epithelial cells. However, it remains unclear how C/EBP beta is involved in tumor suppression through its interaction with specific downstream genes in breast cancer. Tumor cells overexpress serine proteases, which play crucial roles in tumor invasion and metastasis. Elafin is an endogenous serine protease inhibitor and is transcriptionally down-regulated in most tumor cell lines. In this study, we show that C/EBP beta is differentially expressed in normal versus tumor cell lines and normal adjacent versus tumor tissues obtained from breast cancer patients. We identified elafin as a downstream effector of C/EBP beta and show that elafin is also differentially regulated between normal and tumor cells. The mechanism by which C/EBP beta regulates elafin expression is through its direct interaction with the elafin promoter. There are three C/EBP beta binding sites involved in the elafin promoter activity, and the overexpression of C/EBP beta transactivates the elafin gene through these sites in tumor cells. RNA interference studies in normal cells further evidenced the requirement of the C/EBP beta for the elafin expression and negative feedback loop between C/EBP beta and elafin. We suggest that elafin is a novel substrate of C/EBP beta, and alterations in C/EBP beta isoforms result in their differential binding to the elafin promoter, leading to the altered expression of the elafin between normal and tumor cells.

Longitudinal studies of inter-alpha inhibitor proteins in severely septic patients: A potential clinical marker and mediator of severe sepsis*

To determine the clinical relevance and prognostic significance of serial measurement of inter-alpha inhibitor proteins (IalphaIp) in severely septic patients. A laboratory-based study of serial plasma samples over the first 5 days of severe sepsis from a prospective clinical trial. Small business and academic medical center research laboratories. Two hundred sixty-six patients with severe sepsis from a multiple-center phase III clinical trial. None. Inter-alpha inhibitor proteins serve as endogenous serine protease inhibitors in human plasma. The levels of IalphaIp were markedly reduced to a mean value of 290+/-15 microg/mL at the onset of severe sepsis compared with normal plasma levels (617+/-197 microg/mL). Failure of IalphaIp levels to recover over the first 5 days of sepsis was associated with an unfavorable outcome (p<.001). IalphaIp levels were inversely correlated with interleukin-6 levels at study entry and over the first 5 days of management of severe sepsis. IalphaIp levels were significantly lower in women, with increased age, in the presence of multiple organ failure and in patients with intra-abdominal sources of sepsis. Inter-alpha inhibitor proteins are markedly reduced in severe sepsis, and failure of recovery of IalphaIp levels over the course of sepsis is associated with an unfavorable outcome.

INTER-ALPHA INHIBITORS IN ADULT AND NEONATAL SEPSIS: A POTENTIAL APPLICATION AS AN INTEGRATED CLINICAL MARKER AND THERAPEUTIC AGENT

A consistent finding in the complex pathophysiology of sepsis is evidence of excess systemic protease activity. An array of endogenous protease inhibitors counterbalances these actions. Many of these inhibitors are rapidly consumed in sepsis, often leading to a failure to appropriately regulate protease activity. A complex polypeptide family termed as Inter-alpha inhibitor proteins (IAIP) is one of the endogenous serine protease inhibitors found abundantly in human blood. IAIP have been implicated to play a role in systemic inflammation/sepsis. Studies of the IAIP levels in adult patients and infants with clinically proven sepsis indicated that IAIP decreased significantly and the plasma IAIP levels inversely correlated with the disease severity and mortality. In a longitudinal study of more than 250 severe septic patients, failure of recovery of IAIP levels over the course of sepsis was associated with a poor outcome suggesting IAIP levels are potentially useful as a clinical marker in sepsis. The rapid IAIP depletion might lead to uncontrolled proteolytic activity and exacerbates the systemic inflammation. In a preclinical study to investigate the efficacy of IAIP replacement therapy, administration of purified IAIP significantly improved outcome in experimental sepsis models. The increased survival rates were observed even when IAIP were administered at delayed timepoints in a polymicrobial sepsis rat model and in a LPS challenge neonatal mouse model. IAIP administration appeared to have immunomodulatory effects as TNF-alpha was significantly attenuated and IL-10 was augmented by IAIP treatment. Further studies in genetically altered mice pups (IL-10−/−) suggested that the beneficial effects of IAIP might be independent of the augmentation of antiinflammatory cytokines. Taken together, IAIP are potentially useful in the clinical management of sepsis as an integrated predictive marker and effective therapeutic replacement agent in reducing morbidity and mortality of this deadly disease.

Cysteine protease inhibition attenuates acute right heart failure

Acute right ventricular pressure overload (RVPO) causes RV failure, morbidity, and mortality in patients, and currently has no direct treatment. We previously showed that 90 min RVPO without ischemia causes RV free wall (RVFW) dysfunction (RVD) that persists following relief of RVPO. We tested the hypothesis that cysteine protease (CP) activation contributes to, and therefore that CP inhibition would attenuate, RVD during RVPO. Methods Under open-chest, anesthetized conditions, 32 pigs were subjected to 4 hrs RVPO by constriction of the pulmonary artery. Prior to initiation of RVPO, pigs were randomized to treatment with MDL-28170 (INH), a membrane permeable CP inhibitor, or inactive vehicle (VEH). Investigators were blinded to treatment assignment until all analyses were completed. Results Neither INH nor VEH had any hemodynamic effects prior to RVPO. Beginning RV systolic pressure was similar in both groups, but INH significantly attenuated the decline in RV stroke work vs VEH during the ensuing 4 hrs (32±3% vs 49±5% decline, p<0.05). INH significantly attenuated degradation of the endogenous serine protease inhibitor α-1-antichymotrypsin by 51% (p<0.05) that otherwise occurs in RVFW during RVPO, but did not affect degradation of troponin, desmin, or spectrin, substrates of the cysteine protease calpain. Conclusions The CP inhibitor MDL-28170 attenuates RV dysfunction during sustained RVPO. The mechanism for the salutary effect may involve indirect or cross-class inhibition of endogenous serine proteases. Supported by Department of Veterans Affairs and NHLBI.

Inter-Alpha-Inhibitor Proteins Are Endogenous Furin Inhibitors and Provide Protection against Experimental Anthrax Intoxication

Inter-alpha-inhibitor protein (IalphaIp) functions as an endogenous serine protease inhibitor in human plasma, and IalphaIp levels diminish rapidly during acute inflammatory states. One potential target for IalphaIp is furin, a cell-associated serine endopeptidase essential for the activation of protective antigen and the formation of anthrax lethal toxin (LT). IalphaIp blocks furin activity in vitro and provides significant protection against cytotoxicity for murine peritoneal macrophages exposed to up to 500 ng/ml LT. A monoclonal antibody (MAb), 69.31, that specifically blocks the enzymatic activity of IalphaIp eliminates its protective effect against LT-induced cytotoxicity. IalphaIp (30 mg/kg of body weight) administered to BALB/c mice 1 hour prior to an intravenous LT challenge resulted in 71% survival after 7 days compared with no survivors among the control animals (P < 0.001). We conclude that human IalphaIp may be an effective preventative or therapeutic agent against anthrax intoxication.