Aprotinin (II): Inhalational Administration for the Treatment of COVID-19 and Other Viral Conditions.

Aprotinin is a broad-spectrum inhibitor of human proteases that has been approved for the treatment of bleeding in single coronary artery bypass surgery because of its potent antifibrinolytic actions. Following the outbreak of the COVID-19 pandemic, there was an urgent need to find new antiviral drugs. Aprotinin is a good candidate for therapeutic repositioning as a broad-spectrum antiviral drug and for treating the symptomatic processes that characterise viral respiratory diseases, including COVID-19. This is due to its strong pharmacological ability to inhibit a plethora of host proteases used by respiratory viruses in their infective mechanisms. The proteases allow the cleavage and conformational change of proteins that make up their viral capsid, and thus enable them to anchor themselves by recognition of their target in the epithelial cell. In addition, the activation of these proteases initiates the inflammatory process that triggers the infection. The attraction of the drug is not only its pharmacodynamic characteristics but also the possibility of administration by the inhalation route, avoiding unwanted systemic effects. This, together with the low cost of treatment (≈2 Euro/dose), makes it a good candidate to reach countries with lower economic means. In this article, we will discuss the pharmacodynamic, pharmacokinetic, and toxicological characteristics of aprotinin administered by the inhalation route; analyse the main advances in our knowledge of this medication; and the future directions that should be taken in research in order to reposition this medication in therapeutics.

Aprotinin-Drug against Respiratory Diseases.

Aprotinin (APR) was discovered in 1930. APR is an effective pan-protease inhibitor, a typical "magic shotgun". Until 2007, APR was widely used as an antithrombotic and anti-inflammatory drug in cardiac and noncardiac surgeries for reduction of bleeding and thus limiting the need for blood transfusion. The ability of APR to inhibit proteolytic activation of some viruses leads to its use as an antiviral drug for the prevention and treatment of acute respiratory virus infections. However, due to incompetent interpretation of several clinical trials followed by incredible controversy in the literature, the usage of APR was nearly stopped for a decade worldwide. In 2015-2020, after re-analysis of these clinical trials' data the restrictions in APR usage were lifted worldwide. This review discusses antiviral mechanisms of APR action and summarizes current knowledge and prospective regarding the use of APR treatment for diseases caused by RNA-containing viruses, including influenza and SARS-CoV-2 viruses, or as a part of combination antiviral treatment.

Aprotinin does not Impair Vascular Function in Patients Undergoing Coronary Artery Bypass Graft Surgery.

Bleeding is a major complication in coronary artery bypass graft surgery. Antifibrinolytic agents like serine protease inhibitor aprotinin can decrease postoperative bleeding and complications of cardiac surgery. However, the effects of aprotinin on vascular function are not completely elucidated. We compared the ex vivo vascular function of left internal mammary arteries from patients undergoing coronary artery bypass graft surgery with and without intraoperative application of aprotinin using a Mulvany Myograph. Human internal mammary arteries were treated with aprotinin ex vivo and tested for changes in vascular function. We analyzed the impact of aprotinin on vascular function in rat aortic rings. Finally, impact of aprotinin on expression and activity of endothelial nitric oxide synthase was tested in human endothelial cells. Intraoperative application of aprotinin did not impair ex vivo vascular function of internal mammary arteries of patients undergoing coronary artery bypass graft surgery. Endothelium-dependent and -independent relaxations were not different in patients with or without aprotinin after nitric oxide synthase blockade. A maximum vasorelaxation of 94.5%±11.4vs. 96.1%±5.5% indicated a similar vascular smooth muscle function in both patient groups (n=13 each). Long-term application of aprotinin under physiological condition preserved vascular function of the rat aorta. In vitro application of increasing concentrations of aprotinin on human endothelial cells resulted in a similar expression and activity of endothelial nitric oxide synthase. In conclusion, intraoperative and ex vivo application of aprotinin does not impair the endothelial function in human internal mammary arteries and experimental models.

Bovine pancreatic trypsin inhibitor and soybean Kunitz trypsin inhibitor: Differential effects on proteases and larval development of the soybean pest Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Pest management is challenged with resistant herbivores and problems regarding human health and environmental issues. Indeed, the greatest challenge to modern agriculture is to protect crops from pests and still maintain environmental quality. This study aimed to analyze by in silico, in vitro, and in vivo approaches to the feasibility of using the inhibitory protein extracted from mammals - Bovine Pancreatic Trypsin Inhibitor (BPTI) as a potential inhibitor of digestive trypsins from the pest Anticarsia gemmatalis and comparing the results with the host-plant inhibitor - Soybean Kunitz Trypsin Inhibitor (SKTI). BPTI and SKTI interacts with A. gemmatalis trypsin-like enzyme competitively, through hydrogen and hydrophobic bonds. A. gemmatalis larvae exposed to BPTI did not show two common adaptative mechanisms i.e., proteolytic degradation and overproduction of proteases, presenting highly reduced trypsin-like activity. On the other hand, SKTI-fed larvae did not show reduced trypsin-like activity, presenting overproduction of proteases and SKTI digestion. In addition, the larval survival was reduced by BPTI similarly to SKTI, and additionally caused a decrease in pupal weight. The non-plant protease inhibitor BPTI presents intriguing element to compose biopesticide formulations to help decrease the use of conventional refractory pesticides into integrated pest management programs.

Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

BACKGROUND: Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK). OBJECTIVE: We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC. METHODS: A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract. RESULTS: No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC. CONCLUSION: Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.

Experimental nephrotic syndrome leads to proteolytic activation of the epithelial Na+ channel in the mouse kidney.

Proteolytic activation of the renal epithelial Na+ channel (ENaC) involves cleavage events in its α- and γ-subunits and is thought to mediate Na+ retention in nephrotic syndrome (NS). However, the detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α-ENaC and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the NH2-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed Na+ retention and increased expression of fragments of α-ENaC and γ-ENaC cleaved at both the proximal cleavage site and, more prominently, the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented Na+ retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α-ENaC and γ-ENaC was similarly found in healthy mice treated with a low-salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and, more prominently, distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.NEW & NOTEWORTHY This study demonstrates that murine experimental nephrotic syndrome leads to aprotinin-sensitive proteolytic activation of the epithelial Na+ channel at both the α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.

Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo.

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

Synergistic Effect of ß-alanine and Aprotinin on Mesenteric Ischemia.

BACKGROUND: Acute mesenteric ischemia arises through sudden interruption of mesenteric blood flow, mostly due to an occlusion of the superior mesenteric artery and is associated with a high mortality of approximately 50% to 90%. In previous studies, the single application of ß-alanine or aprotinin caused an ameliorated intestinal damage but without any systemic effects. METHODS: To analyze the combined effect of ß-alanine and aprotinin on acute ischemia and reperfusion of the small intestine, a model with anesthetized rats was used. Ischemia and reperfusion were initiated by occluding and reopening the superior mesenteric artery. After 120 min of ischemia and 180 min of reperfusion, the intestine was analyzed for tissue damage, the activity of the saccharase, and accumulation of granulocytes. In addition, systemic and metabolic as well as inflammatory parameters were measured in blood at certain points in time. RESULTS: The combination of ß-alanine and aprotinin resulted in a clearly stabilized mean arterial blood pressure and blood glucose level during the reperfusion period. Furthermore, the combined administration resulted in significantly reduced tissue damage parameters, cytokine and cell-free hemoglobin concentrations in blood plasma. In addition, the damage to the small intestine was significantly attenuated, so that the animals ultimately survived the entire test period because of the administration of both substances. CONCLUSIONS: Overall, the simultaneous application of both substances leads to a synergistic protection without the occurrence of undesirable side effects. The combined usage of ß-alanine and aprotinin can be seen as a promising approach to inhibit the onset of acute mesenteric ischemia.

Methylene blue may have a role in the treatment of COVID-19.

In this paper, we raise the hypothesis that Methylene Blue may be a treatment option for Corona Virus Disease of 2019 specially when combined with Non Steroid Anti-Inflammatory Drugs. In previous publications including ours, the role of kininogen system has been postulated. A correlation between clinical findings of the disease and this mechanism has been drawn to denote a pivotal role of kininogen-kallikrein system in pathophysiology of the disease. Therein the possible role of Icatibant, Ecallantide and Aprotinin in the treatment of this disease has been raised. Here we want to emphasize on an important post-receptor mechanism of bradykinin that is Nitric Oxide. We came to this aim because we found out how access to these novel treatment nominees may be expensive and unaffordable. For this reason we are focusing on possible role of an old albeit "mysterious" drug namely Methylene Blue. This medication may abort effects of Bradykinin by inhibition of Nitric Oxide synthase inhibitor and promote oxygen saturation while it is inexpensive and ubiquitously accessible. Clinical studies cannot be over emphasized.

Aprotinin Inhibits SARS-CoV-2 Replication.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.

In vitro effect of a novel protease inhibitor cocktail on Toxoplasma gondii tachyzoites.

Toxoplasmosis is a zoonotic disease and a global food and water-borne infection. The disease is caused by the parasite Toxoplasma gondii, which is a highly successful and remarkable pathogen because of its ability to infect almost any nucleated cell in warm-blooded animals. The present study was done to demonstrate the effect of protease inhibitors cocktail (PIC), which inhibit both cysteine and serine proteases, on in vitro cultured T. gondii tachyzoites on HepG2 cell line. This was achieved by assessing its effect on the invasion of the host cells and the intracellular development of T.gondii tachyzoites through measuring their number and viability after their incubation with PIC. Based on the results of the study, it was evident that the inhibitory action of the PIC was effective when applied to tachyzoites before their cultivation on HepG2 cells. Pre-treatment of T.gondii tachyzoites with PIC resulted in failure of the invasion of most of the tachyzoites and decreased the intracellular multiplication and viability of the tachyzoites that succeeded in the initial invasion process. Ultrastructural studies showed morphological alteration in tachyzoites and disruption in their organelles. This effect was irreversible till the complete lysis of cell monolayer in cultures. It can be concluded that PIC, at in vitro levels, could prevent invasion and intracellular multiplication of Toxoplasma tachyzoites. In addition, it is cost effective compared to individual protease inhibitors. It also had the benefit of combined therapy as it lowered the concentration of each protease inhibitor used in the cocktail. Other in vivo experiments are required to validate the cocktail efficacy against toxoplasmosis. Further studies may be needed to establish the exact mechanism by which the PIC exerts its effect on Toxoplasma tachyzoites behavior and its secretory pathway.

Amblyomin-X, a recombinant Kunitz-type inhibitor, regulates cell adhesion and migration of human tumor cells.

In a tumor microenvironment, endothelial cell migration and angiogenesis allow cancer to spread to other organs causing metastasis.  Indeed, a number of molecules that are involved in cytoskeleton re-organization and intracellular signaling have been investigated for their effects on tumor cell growth and metastasis. Alongside that, Amblyomin-X, a recombinant Kunitz-type protein, has been shown to reduce metastasis and tumor growth in in vivo experiments. In the present report, we provide a mechanistic insight to these antitumor effects, this is,  Amblyomin-X modulates Rho-GTPases and uPAR signaling, and reduces the release of MMPs, leading to disruption of the actin cytoskeleton and decreased cell migration of tumor cell lines. Altogether, our data support a role for Amblyomin-X as a novel potential antitumor drug. ABBREVIATIONS: Amb-X: Amblyomin-X; ECGF: endotelial cell growth factor; ECM: extracellular matrix; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cell; LRP1: low-density lipoprotein receptor-related protein; MMP: matrix metalloproteinase; HPI-4: hedgehog pathway inhibitor 4; PAI-1: plasminogen activator inhibitor 1; PMA: phorbol 12-myristate-13-acetate; TFPI: tissue factor pathway inhibitor; uPA: urokinase plasminogen activator; uPAR: uPA receptor.

A nanometer-sized protease inhibitor for precise cancer diagnosis and treatment.

Inhibition of pro-cancer proteases is a potent anticancer strategy. However, protease inhibitors are mostly developed in the forms of small molecules or peptides, which normally suffer from insufficient metabolic stability. The fast clearance significantly impairs the antitumor effects of these inhibitors. In this study, we report a nanometer-sized inhibitor of a pro-cancer protease, suppressor of tumorigenicity 14 (st14), which has been reported as a potent prognostic marker for multiple cancers. This st14 inhibitor was fabricated by conjugating a recombinant st14 inhibitor (KD1) with carbon quantum dots (CQDs). CQD-KD1 not only demonstrated high potency of inhibiting st14 activity in biochemical experiments, but also remarkably suppressed the invasion of breast cancer cells. In contrast to the original recombinant KD1, CQD-KD1 demonstrated a prolonged retention time in plasma and at the tumor site because of the reduced renal clearance. Consistently, CQD-KD1 demonstrated enhanced efficacies of suppressing tumor growth and cancer metastases in vivo. In addition, CQD-KD1 precisely imaged tumor tissues in cancer-grafted mice by specifically targeting the over-expressed st14 on the tumor cell surface, which indicates CQD-KD1 as a potent probe for the fluorescence guided surgery of tumor resection. In conclusion, this study demonstrates that CQD-KD1 is a highly potent diagnostic and therapeutic agent for cancer treatments.

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

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

S-Nitrosylation of CHIP Enhances F508Del-CFTR Maturation.

S-nitrosothiols (SNOs) are endogenous signaling molecules that have numerous beneficial effects on the airway via cyclic guanosine monophosphate-dependent and -independent processes. Healthy human airways contain SNOs, but SNO levels are lower in the airways of patients with cystic fibrosis (CF). In this study, we examined the interaction between SNOs and the molecular cochaperone C-terminus Hsc70 interacting protein (CHIP), which is an E3 ubiquitin ligase that targets improperly folded CF transmembrane conductance regulator (CFTR) for subsequent degradation. Both CFBE41o- cells expressing either wild-type or F508del-CFTR and primary human bronchial epithelial cells express CHIP. Confocal microscopy and IP studies showed the cellular colocalization of CFTR and CHIP, and showed that S-nitrosoglutathione inhibits the CHIP-CFTR interaction. SNOs significantly reduced both the expression and activity of CHIP, leading to higher levels of both the mature and immature forms of F508del-CFTR. In fact, SNO inhibition of the function and expression of CHIP not only improved the maturation of CFTR but also increased CFTR's stability at the cell membrane. S-nitrosoglutathione-treated cells also had more S-nitrosylated CHIP and less ubiquitinated CFTR than cells that were not treated, suggesting that the S-nitrosylation of CHIP prevents the ubiquitination of CFTR by inhibiting CHIP's E3 ubiquitin ligase function. Furthermore, the exogenous SNOs S-nitrosoglutathione diethyl ester and S-nitro-N-acetylcysteine increased the expression of CFTR at the cell surface. After CHIP knockdown with siRNA duplexes specific for CHIP, F508del-CFTR expression increased at the cell surface. We conclude that SNOs effectively reduce CHIP-mediated degradation of CFTR, resulting in increased F508del-CFTR expression on airway epithelial cell surfaces. Together, these findings indicate that S-nitrosylation of CHIP is a novel mechanism of CFTR correction, and we anticipate that these insights will allow different SNOs to be optimized as agents for CF therapy.

ClTI, a Kunitz trypsin inhibitor purified from Cassia leiandra Benth. seeds, exerts a candidicidal effect on Candida albicans by inducing oxidative stress and necrosis.

Cassia leiandra is an Amazonian plant species that is used popularly for the treatment of mycoses. Recently, a protease inhibitor, named ClTI, with insecticidal activity against Aedes aegypti, was purified from the mature seeds of C. leiandra. In this work, we show that ClTI has antifungal activity against Candida species and describe its mode of action towards Candida albicans. This study is relevant because the nosocomial infections caused by Candida species are a global public health problem that, together with the growing resistance to current drugs, has increased the urgency of the search for new antifungal compounds. ClTI inhibited the growth of Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida krusei. However, ClTI was more potent against C. albicans. The candidicidal mode of action of ClTI on C. albicans involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+ -ATPase), induction of oxidative stress, and DNA damage. ClTI also exhibited antibiofilm activity and non-cytotoxicity to mammalian cells. These results indicate that ClTI is a promising candidate for the future development of a new, natural, and safe agent for the treatment of infections caused by C. albicans.

Anti-neoplastic effects of aprotinin on human breast cancer cell lines: In vitro study.

BACKGROUND: Aprotinin is a nonspecific serine protease inhibitor, which can inhibit plasminogen-plasmin system and matrix metalloproteinases. Aprotinin has been investigated as an antitumor agent. However, its antineoplastic effects on breast cancer (BC) have not been investigated yet. OBJECTIVES: The objective of this study was to assess the inhibitory effects of aprotinin on human BC cell lines. We assessed the effects of aprotinin on local invasion and survival of human BC cell lines MDA-MB-231, SK-BR-3 and MCF-7 in vitro. MATERIAL AND METHODS: CHEMICON cell invasion assay kit was used to assess local invasion, and (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) assay was used to determine the antiproliferative activity of aprotinin. Human dermal fibroblast (HDF-1) cell line was used as control normal cells. RESULTS: Cancer cell lines showed more invasion characteristics compared to HDF-1. Aprotinin significantly decreased the invasiveness of MDA-MB-231 in concentrations of 1 trypsin inhibitor unit (TIU)/mL, 1.3 TIU/mL and 1.7 TIU/mL in comparison with the untreated group (analysis of variance (ANOVA) p < 0.001). Treatment of SK-BR-3 with 1.3 TIU/mL aprotinin caused no significant reduction in invasiveness (p = 0.06). Treatment with different concentrations of aprotinin significantly decreased the surviving fraction and inhibited the growth of all cell lines tested in this study (analysis of variance (ANOVA) p < 0.001). Compared to cancer cell lines, normal HDF-1 cell line showed less sensitivity to antiproliferative effects of aprotinin, both in low and high doses. CONCLUSIONS: Aprotinin significantly inhibited the growth of human breast cancer cell lines MDA-MB-231, SK-BR-3 and MCF-7, and normal fibroblast cell line HDF-1. The growth inhibitory effect was more dominant in cancer cell lines. Inhibition of local invasion by aprotinin was significant only in the case of MDA-MB-231. Future molecular studies could shed further lights on mechanisms underlying antineoplastic effects of aprotinin and its potential therapeutic effects.

Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro. RESULTS: The human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells. CONCLUSIONS: We designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its γ-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Three-Dimensional Hydrogel-Based Culture to Study the Effects of Toxicants on Ovarian Follicles.

Various toxicants, such as drugs and their metabolites, can cause potential ovarian toxicity. As the functional units of the ovary, ovarian follicles are susceptible to this type of damage at all developmental stages. Studying the effects of toxicants on ovarian follicles is an important task. Three-dimensional (3D) hydrogels, such as fibrin alginate interpenetrating networks (FA-IPNs), can support ovarian follicle culture in vitro for extended periods of time and serve as a suitable tool for studying ovotoxicity. Growing follicles encapsulated in the FA-IPN can proteolytically degrade the fibrin component in the FA-IPN. The degradation of fibrin mirrors the follicle growth and serves as a surrogate reporter for follicle health. The speed of fibrin degradation can be further controlled by aprotinin, a small molecule that inhibits plasmin-driven proteolytic degradation, which further expands the application of the described system. In this chapter, we describe methods to (1) isolate and encapsulate mouse ovarian follicles in FA-IPN, (2) follow follicle growth and development in vitro, and (3) evaluate the effects of toxicants on folliculogenesis using fibrin degradation.