Serum Protease Inhibitors Research Articles

Interaction of Synthetic Pyrethroid Insecticide Deltamethrin with Human Alpha-2-Macroglobulin: Spectroscopic and Molecular Docking Studies.

Deltamethrin (DLM) is a commercial insecticide of the synthetic pyrethroid family that is used to control disease-causing insects and vectors. When humans are exposed to the fumes or aerosols of DLM, it enters the body via cuticular absorption and reacts with proteins and other biomolecules. Alpha-2-macroglobulin (α2M) is a serum proteinase inhibitor that also carries out receptor- mediated endocytosis of extracellular substances. This study was done to decipher the structural and functional alterations of α2M by DLM. Various spectroscopic techniques, including UV absorption and fluorescence spectroscopy, binding studies, and molecular docking, were used to characterize the interaction of DLM with α2M. The affinity constant was calculated from the Stern-Volmer equation using fluorescence data. The UV-Vis and fluorescence spectral studies indicated the formation of a complex between α2M and DLM. Thermodynamically, the interaction was found to be spontaneous with ΔG = -4.23 kcal/mol. CD spectra suggested a change in the secondary structure of the protein from β to α helical content with increasing concentration of DLM. The molecular docking study by Autodock Vina established the interaction of DLM with Glu-926, Ala-1103, Ala-1108, Val-1116, Asn-1159, Glu-1220, Leu-1261, Thr-1272, Ile-1390, Pro-1391, Lys-1393, Val-1396, Lys-1397, Thr-1408, Glu-1409, Val-1410, Ser-1411, Ser-1412, and Asn-1413 with an improved docking score of -6.191 kcal/mol. The binding was carried out in the vicinity of the receptor-binding domain at the C-terminal of α2M. The decrease in the functional activity and structural changes of protein after binding with DLM has a significant effect on human α2M. The information may be useful for exploring the role of DLM in a clinical chemistry laboratory.

The Efficacy of Ezetimibe Combined with Simvastatin in Patients with Coronary Heart Disease and Its Effect on Serum Retinol Binding Protein 4, Thioredoxin Interaction Protein and Visceral Adipose Tissue Serine Protease Inhibitor

To observe the efficacy of ezetimibe combined with simvastatin with coronary heart disease patients and its effect on retinol binding protein 4, thioredoxin interaction protein and visceral adipose tissue serine protease inhibitor. 120 patients with coronary heart disease admitted in our hospital from May 2019 to May 2020 were divided into the control group and the observation group. Changes in serum retinol binding protein 4, thioredoxin interaction protein and visceral adipose tissue serine protease inhibitor and blood lipids were recorded. Changes in the Seattle angina scale in the observation group, 18 patients were effective and the total effective rate were 93.33 %; 27 patients showed effect in the control group, 21 patients were effective and the total effective rate was 80.00 %; the total effective rate in the observation group was higher than that in the control group, with statistical significance (p<0.05). Compared with those before treatment, visceral adipose tissue serine protease inhibitor and high density lipoprotein cholesterol increased, retinol binding protein 4, thioredoxin interaction protein, triglyceride, total cholesterol and low density lipoprotein cholesterol decreased in the two groups and visceral adipose tissue serine protease inhibitor and high density lipoprotein cholesterol were higher and retinol binding protein 4, thioredoxin interaction protein, triglyceride, total cholesterol and low density lipoprotein cholesterol were lower in the observation group than those in the control group after 3 mo of treatment (p<0.05). The ezetimibe combined with simvastatin can regulate blood lipid, decrease the expression of retinol binding protein 4, thioredoxin interaction protein and visceral adipose tissue serine protease inhibitor in serum and improve the onset of angina pectoris in patients with coronary heart disease.

Secretory leukocyte protease inhibitor as a marker of proinflammatory response in children with community-acquired pneumonia

The study of the secretory leukocyte protease inhibitor (SLPI) level in children with pneumonia is important because it may indicate protease-antiprotease imbalance and predict local regulation of proteolytic activity at different degrees of pneumonia in children. The aim of the study was to assess the level of SLPI as a marker of proinflammatory response in the serum of young children, depending on the severity of pneumonia, including the presence or absence of lesions of the hepatobiliary system. The study investigated the SLPI as a marker of the inflammatory response in children with community-acquired pneumonia, depending on the severity of the disease, lesions of the hepatobiliary system, age and sex. The level of SLPI in the serum was determined by enzyme-linked immunosorbent assay using a diagnostic test system from “IMMUNOTECH” (France). “IBM SPSS Statistica” Version 12 (20) was used for statistical processing of the results. It was found that the course of community-acquired pneumonia is accompanied by an increase in the serum level of SLPI in children in parallel with the severity of the disease. The development of a lesion from the GBS reduces the level of a protease inhibitor in the blood serum of children with pneumonia, which indicates a decrease in the body's defense systems. In children 1-12 months, the level of SLPI is higher than in children 13-36 months, which indicates more significant protective capabilities of the body of children in the first year of life. It has been shown that an increase in the level of SLPI correlates with an increase in the level of inflammatory cytokines, which indicates a unidirectional change in the formation of local immunity in response to damage to the lung tissue. At the same time, a negative correlation was found between the level of SLPI and the activities of AST, GGT and CRP (rxy= -0.054; rxy= -0.215; rxy= -0.215, respectively), which indicates a violation of liver function during the development of an infectious-inflammatory process in the body. Studies of the nature of the effects of cytokines and protease inhibitors (protective factors) on the development and maintenance of inflammatory process in the lungs in young children with community-acquired pneumonia with lesions of the hepatobiliary system may be the basis for predicting its course.

Knockdown of Alpha-1 Antitrypsin with antisense oligonucleotide does not exacerbate smoke induced lung injury

Alpha-1 Antitrypsin (AAT) is a serum protease inhibitor that regulates increased lung protease production induced by cigarette smoking. Mutations in the Serpina1 gene cause AAT to form hepatoxic polymers, which can lead to reduced availability for the protein’s primary function and severe liver disease. An AAT antisense oligonucleotide (ASO) was previously identified to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knockdown of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups each for the smoking and smoke-flu injury models. The ASO and control (No-ASO) were injected subcutaneously starting with smoking or four days prior to influenza infection and then injected weekly at 50 mg/kg body weight. ASO treatment during a 3-month smoke exposure significantly decreased the serum and lung AAT expression, resulting in increased Cela1 expression and elastase activity. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly worsened by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure, but did not observe an ASO treatment effect. Similarly, with the smoke-flu model, differences were only observed between smoke-flu and room air controls, but not as a result of ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment, while sufficient to protect from liver injury, may not be robust enough to lead to lung injury. The results also suggest that previously described AAT ASO treatment for AAT mutation related liver disease may attenuate hepatic injury without being detrimental to the lungs. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.

Serum proteome assessment in nonalcoholic fatty liver disease in children: a preliminary study

ABSTRACT Objectives Nonalcoholic fatty disease (NAFLD) affects 3–10% of the pediatric population, making it the most common chronic liver disease among children. The aim of the study is to identify potential biomarkers enabling the diagnosis of NAFLD and monitoring the course of the disease. Methods Proteome analysis was performed in a group of 30 patients (19 boys and 11 girls) in total, of whom 16 children had previously diagnosed NAFLD based on the abdominal ultrasound after excluding other diseases of this organ. Results A total of 297 proteins have been identified. Thirty-seven proteins (responsible for inflammation, stress response, and regulation of this process) differentiating both experimental groups were identified. Up-regulated proteins included afamin, retinol-binding protein-4, complement components, and hemopexin; while serum protease inhibitors, clusterin, immunoglobulin chains, and vitamin D binding protein were found in the down-regulated group. The correlation between selected proteins and indicators of noninvasive assessment of liver fibrosis (APRI, FIB-4) as well as differences between the serum proteome of patients with normal weight, overweight, and obesity were also assessed. Conclusion The plasma protein profile is significantly altered in nonalcoholic liver disease in children and may prove to be a valuable source of biomarkers to evaluate the extent of liver disease.

Distinct anti-inflammatory properties of alpha1-antitrypsin and corticosteroids reveal unique underlying mechanisms of action

Alpha1-antitrypsin (AAT) is a serum protease inhibitor that rises during inflammation and healthy pregnancies. Plasma-derived AAT, indicated for genetic AAT deficiency, is presently being explored for additional medical indications. Unlike corticosteroids, some anti-inflammatory activities of AAT involve NF-κB–dependent outcomes, e.g., induction of IL-1R antagonist. AAT activities were compared to dexamethasone (DEX), using various in-vitro cells assays, animal studies, and NF-κB–p65 localization and activity studies. Results demonstrate a cytokine shift towards resolution in AAT-treated cells, as opposed to pan-suppression in DEX-treated cells. AAT enhanced, while DEX suppressed LPS-induced IL-1Ra production and re-epithelialization. When drugs were combined, AAT allowed the immunosuppressive DEX activities, while DEX at medium to high levels antagonized beneficial AAT effects. Interestingly, lower levels of DEX maintained the immunosuppressive effect, while allowing upregulation of IL-1Ra. Therefore, AAT may represent a distinct endogenous anti-inflammatory, resolution-promoting agent that may improve tissue well-being while preventing undesired corticostroids side effects.

Increasing Neutrophil Elastase and Decreasing Its Inhibitor, Alpha 1-Antitrypsin, in Patients with Non-Alcoholic Fatty Liver Disease

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in modern human life. Serum protease and protease inhibitor concentrations can be potentially considered the important risk factors for NAFLD, together with the main risk factors of this disorder, such as diabetes, obesity, and dyslipidemia. Objectives: The present study aimed to investigate the plasma level of a neutral serine protease family called neutrophil elastase (NE), as well as its inhibitor, alpha1-antitrypsin (A1AT), in NAFLD patients compared to normal subjects. Additionally, the most common deficient variants of A1AT (S and Z) were determined in both patient and healthy groups. Methods: The study included 54 consecutive unrelated NAFLD patients and 120 matched healthy subjects as controls over two years. Plasma concentrations of A1AT and neutrophil elastase activity were determined for all patients and controls using the enzyme immunoassay and alpha1-proteinase inhibitor-immunoglobulin A complex, respectively. The A1AT variants of PiZ (rs28929474) and PiS (rs17580) were analyzed by the polymerase chain reaction. Results: The NAFLD patient group had more plasma elastase activity than the healthy control group (1.7 (0.5) vs. 1.3 (0.5) (U/mL), P < 0.01). Furthermore, the plasma A1AT level was significantly lower in NAFLD patients than in controls (216.1 (171.3) vs. 244.6 (152.9) (mg/dL), P = 0.01). The heterozygous carriages of PiS and PiZ variants were not statistically different between NAFLD patients and controls. Conclusions: Higher elastase activity and lower A1AT concentration in plasma can be considered the potential prognostic and diagnostic biomarkers in patients with NAFLD.

Towards simultaneous quantification of protease inhibitors and inflammatory biomarkers in serum for people living with HIV

Detecting small and big molecules together: simultaneous quantification of protease inhibitor (DRV) and inflammatory biomarker in serum by Arrayed Imaging Reflectometry (AIR).

THE VALUE OF THE TUMOR NECROSIS FACTOR-ALPHA AND VASPIN RATIO IN THE DEVELOPMENT OF INSULIN RESISTANCE

Diabetes mellitus type 2 is a chronic disease that results in metabolic disorders in the whole organism, primarily due to insulin resistance and relative insufficiency of insulin. The fatty tissue is no longer considered only as a passive depositary for the accumulation of energy and participation in the regulation of thermogenesis. In the last decades, it has been noted that this is an active endocrine organ that in addition to regulation of fat mass and participating in homeostasis, also releases a large number of bioactive mediators called adipokines. Adipokines are influenced by an autocrine or paracrine method on a variety of metabolic processes. Increased body weight is a factor in the development of different metabolic disorders. Excess fatty tissue can directly affect one of the main metabolic links — activity and insulin sensitivity. Increased body weight is a factor in the development of a carbohydrate metabolism disorder. Vaspin is an adipocytokine, a representative of widely spread serpins (serine protease inhibitors). The elevated serum protease inhibitor profile was noted in overweight and insulin-resistant mice. It is suggested that vaspin is an insulin sensitizer with anti-inflammatory action and can take part as a compensatory mechanism in response to reduced insulin sensitivity. Therefore, the activation of vaspin may decrease insulin resistance. Evaluation of protease which can be inhibited by vaspin may increase the development of new approaches of compensation based on etiology of impaired metabolic and glucose tolerance. The tumor necrosis factor-α (TNF-α) is a cytokine that is involved in systemic inflammation. The elevated levels of TNF-α are considered as one of the causative factors in insulin resistance, which is associated with obesity in the pathogenesis of DM-2. This review article provides an analysis of mostly foreign scientific articles about the importance of adipokines, particularly TNF-α and vaspin, in the development of insulin resistance and the possibility of further consideration of these markers as an indicator of the severity of the impaired carbohydrate metabolism.

Antimicrobial peptides, disease severity and exacerbations in bronchiectasis

RationaleRecently a frequent exacerbator phenotype has been described in bronchiectasis, but the underlying biological mechanisms are unknown. Antimicrobial peptides (AMPs) are important in host defence against microbes but can be...

Protein modeling to assess the pathogenicity of rare variants of SERPINA1 in patients suspected of having Alpha 1 Antitrypsin Deficiency

BackgroundAlpha 1 Antitrypsin (AAT) is a key serum proteinase inhibitor encoded by SERPINA1. Sequence variants of the gene can cause Alpha 1 Antitrypsin Deficiency (AATD), a condition associated with lung and liver disease. The majority of AATD cases are caused by the ‘Z’ and ‘S’ variants – single-nucleotide variations (SNVs) that result in amino acid substitutions of E342K and E264V. However, SERPINA1 is highly polymorphic, with numerous potentially clinically relevant variants reported. Novel variants continue to be discovered, and without reports of pathogenicity, it can be difficult for clinicians to determine the best course of treatment.MethodsWe assessed the utility of next-generation sequencing (NGS) and predictive computational analysis to guide the diagnosis of patients suspected of having AATD. Blood samples on serum separator cards were submitted to the DNA1 Advanced Screening Program (Biocerna LLC, Fulton, Maryland, USA) by physicians whose patients were suspected of having AATD. Laboratory analyses included quantification of serum AAT levels, qualitative analysis by isoelectric focusing, and targeted genotyping and NGS of the SERPINA1 gene. Molecular modeling software UCSF Chimera (University College of San Francisco, CA) was used to visualize the positions of amino acid changes as a result of rare/novel SNVs. Predictive software was used to assess the potential pathogenicity of these variants; methods included a support vector machine (SVM) program, PolyPhen-2 (Harvard University, Cambridge, MA), and FoldX (Centre for Genomic Regulation, Barcelona, Spain).ResultsSamples from 23 patients were analyzed; 21 rare/novel sequence variants were identified by NGS, including splice variants (n = 2), base pair deletions (n = 1), stop codon insertions (n = 2), and SNVs (n = 16). Computational modeling of protein structures caused by the novel SNVs showed that 8 were probably deleterious, and two were possibly deleterious. For the majority of probably/possibly deleterious SNVs (I50N, P289S, M385T, M221T, D341V, V210E, P369H, V333M and A142D), the mechanism is probably via disruption of the packed hydrophobic core of AAT. Several deleterious variants occurred in combination with more common deficiency alleles, resulting in very low AAT levels.ConclusionsNGS and computational modeling are useful tools that can facilitate earlier, more precise diagnosis, and consideration for AAT therapy in AATD.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter total A2M levels, which can potentially alter levels of a variety of growth factors such as IL-6, TGF-beta, basic FGF, and PDGF. Alterations in levels of these cytokines and proteolytic degradation of small peptide hormones may have profound effect on host-cancer interaction.

Mutational Analysis of Prostate-Specific Antigen Defines the Intrinsic Proteolytic Activity of the proPSA Zymogen.

Prostate-specific antigen (PSA) is an important prostate cancer biomarker. It is also a protease expressed at high concentrations by the normal and malignant prostate. PSA is secreted as a zymogen (proPSA) with an inhibitory prodomain that must be removed for full activity. ProPSA variants, assumed to be inactive, are found in the blood of prostate cancer patients, and are indicative of poor clinical outcome. Despite the abundance of clinical reports, our understanding of PSA's enzymology is limited, in part due to a lack of appropriate experimental systems. We sought to develop a series of PSA-derived mutants that would help to enhance our understanding of the gene. Sixteen rPSA variants were generated and characterized by a variety of biochemical methods. The wildtype cDNA (WT) provided the template for generating a panel of recombinants. These included variants that abolished removal of the prodomain (R24A), disabled its enzymatic activity (S213A), and/or facilitated a cell-based conversion to the active conformation (FR). The purified variants' proteolytic activity was examined using a fluorogenic substrate, known PSA-cleavable proteins, and physiologically relevant inhibitors. Upon demonstrating our successful generation and purification of the PSA variants, we characterized proPSA activity, describing cleavage of synthetic and biologic substrates, but not serum protease inhibitors. This finding was exploited in the development of a self-activating mutant (PSA_QY) that exhibited the greatest enzymatic activity of all the variants. The system described herein will prove useful for varied applications. ProPSA is partially functional with relatively high activity compared to the mature enzyme. In demonstrating the zymogen's intrinsic activity, we suggest that the proPSA in prostate cancer patient serum is not inert. This may have implications for our understanding of the disease. Prostate 76:1203-1217, 2016. © 2016 Wiley Periodicals, Inc.

Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2% of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54% recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

Protein profiles in mucosal and systemic compartments in response to Vibrio cholerae in a mouse pulmonary infection model

We have recently shown that a mouse lung infection model resulting in acute pneumonia could be used for evaluating the protective immunity induced by mucosal vaccines against Vibrio cholerae. In order to gain insight and better understanding of the pathogenicity of V. cholerae infection, we identified and compared proteins induced by V. cholerae in nasal washes, bronchoalveolar lavages (BAL), and sera. Intranasal administration of V. cholerae increased the concentration of total proteins in nasal washes and BAL fluids, but not in sera. LTQ-Orbitrap hybrid Fourier transform mass spectrometry showed that cytoskeletal proteins, protease inhibitors and anti-inflammatory mediators were present in nasal washes from uninfected mice. The distinctly expressed proteins in nasal washes in response to V. cholerae mainly consisted of protease inhibitors, anti-inflammatory proteins, and anti-microbial proteins. A number of protease inhibitors and anti-inflammatory proteins were selectively expressed in BAL fluids from V. cholerae-infected mice, while cytoskeletal proteins and heat shock proteins were mainly observed in BAL fluids from uninfected mice. A large number of serum complements, protease inhibitors, and acute phase proteins were expressed in V. cholerae-infected mice. Collectively, these results suggest that intranasal administration of V. cholerae leading to acute pneumonia elicited alterations of protein profiles associated with immune homeostasis and host protection in both the mucosal and systemic compartments.

α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes.

Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α2-macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α2M and markedly lowers the concentration of α2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind α2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α2M—(and IgM-) binding domains of PfEMP1, which would be independent of the host receptor specificity of clinically important PfEMP1 antigens.

Immune protective effect of human alpha-1-antitrypsin gene during β cell transplantation in diabetic mice.

Type 1 diabetes (T1D) is a chronic autoimmune disease in which β cells are destroyed. Islet transplantation is the most promising therapeutic treatment for T1D patients. However, allograft rejection and autoimmune reaction have been recognized as primary causes of graft loss after transplantation. Alpha-1-antitrypsin (AAT) is an important serine protease inhibitor in serum. AAT is characterized by anti-inflammation, anti-apoptosis, and induction-specific immunological tolerance. In this study, we successfully established NIT-hAAT cell lines, which are murine islet β cell lines with stable expression of human AAT (hAAT) gene. These NIT-hAAT cells were transplanted under the left kidney capsule of BALB/c diabetic mice. Interestingly, the sustained expression of hAAT in vivo can block the inflammatory cell infiltration and reduce the production of proinflammatory cytokines to effectively prevent nonspecific inflammation. Results showed that hAAT can inhibit the proliferation of lymphocytes, shift the balance between Th17 and Treg, and suppress the maturation of dendritic cells. Therefore, hAAT can serve as a beneficial immunomodulator that limits immune rejection to prolong islet allograft survival and achieve long-term successful transplant outcomes.

Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens.

Abstract 4939: Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo

Abstract Prostate-Specific Antigen (PSA) is a 237 amino acid, 33 kDa, extracellular serine protease expressed in prostate epithelial tissue. In the normal prostate, high concentrations of PSA are stored in the prostatic ductal network. However, cancer leads to the disruption of normal tissue architecture and subsequent leakage of PSA into the tissue interstitium and circulation. Enzymatically active PSA is only present in prostate tissue and at other sites of prostate cancer growth. In circulation, active PSA immediately forms complexes with the serum protease inhibitor alpha-1-antichymotrypsin (ACT), while the inactive forms remains “free”. Traditional assays relied on testing for the presence of total PSA (mostly inactive and complexed) and do not provide information regarding the amount of enzymatically active PSA, a more biologically relevant tumor biomarker. To fill this gap, we developed a novel near infrared (NIR) fluorescent agent, designed to detect active PSA with no detection of inactive or complexed PSA. This agent, PSA750 FAST, contains a PSA-cleavable peptide sequence labeled with NIR fluorophores (ex/em 750/770 nm) and coupled to a pharmacokinetic modifier designed to improve its plasma availability. In its native state, the agent is nearly completely optically quenched (&amp;gt; 95%); and only becomes fluorescent upon enzymatic cleavage with active PSA, yielding a 300-1800 fold increase in signal as compared to the signal obtained using inactive or complexed PSA. Proteolytic cleavage was also selective for PSA over a large panel of enzymes, including Kallikrein 1, Cathepsin B, MMP-9, MMP-12, MMP-13, uPA, chymotrypsin and thrombin. We hypothesized that the enzymatic activity of PSA could be non-invasively and quantitatively monitored in 3D using Fluorescence Molecular Tomography (FMT), a powerful near-infrared imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals. LNCaP (PSA positive) and PC3 (PSA negative) cells were implanted in the chest area of male nude mice. Real time imaging was performed after systemic administration of PSA750 FAST when tumors reached the desired size. Our results demonstrated a significantly higher fluorescent signal in LNCaP tumors as compared the adjoining muscle (34.1 +/- 3.8 nM versus 7.2 +/- 0.6 nM, p=0.0043). Likewise, fluorescence concentration in tumors was significantly higher in LNCaP as compared to PC3 tumors (34.1 +/- 3.8 nM vs 17.14 +/- 3.48 nM, p=0.0191; 12.99 +/- 3.7 vs 2.8 +/- 0.8 pmoles, p=0.0233). This is the first report that demonstrates the feasibility of non-invasive, real-time, molecular imaging of active PSA in vivo in a relevant tumor model. These findings demonstrate the effectiveness of this agent in conjunction with optical imaging as a functional platform for molecular imaging in the field of prostate cancer. Citation Format: Jeffrey D. Peterson, Guojie Ho, Jeffrey Morin, Jeannine Delaney, Wael Yared, Milind Rajopadhye, Sylvie C. Kossodo. Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4939. doi:10.1158/1538-7445.AM2014-4939

Abstract 699: Mesenchymal stem cells (MSC) as cell-based vectors for PSA-activated proaerolysin to sites of prostate cancer

Abstract Mesenchymal stem cells (MSC) traffic to sites of inflammation, such as those commonly found at sites of prostate and other cancers. Using multi-parameter flow cytometry, our group has shown that MSCs represent 0.01-1.1% of the total cells present at sites of primary prostate cancer (PCa). Allogeneic MSCs have been safely administered to &amp;gt;1000 patients in over 350 clinical trials worldwide for a variety of diseases. Coupled with their ability to evade the immune system, this suggests that allogeneic MSCs can be used as cell-based delivery vectors for anti-cancer agents. Towards this goal, a method has been developed to load MSCs with PLGA microparticles (MP) encapsulating PSA-activated proaerolysin. Proaerolysin is a highly potent (low pM) bacterial pore-forming toxin that selectively kills PSA-expressing cells in a proliferation-independent manner, which is critical due to the low proliferative index of PCa. Proaerolysin has been modified using site-directed mutagenesis to replace the wildtype activation domain with a PSA cleavage sequence. Though prodrug delivery will be enriched at cancer sites using MSC ‘Trojan horses’, entrapment in non-malignant tissue, such as the lung, following systemic administration is expected and must be addressed; here, using a prodrug strategy. Importantly, enzymatically active PSA is only present in the prostate and at sites of PCa, including metastases. Circulating PSA is inactive due to covalent binding to serum protease inhibitors. Therefore, toxicity to non-target tissues will be minimized through both selective delivery and prodrug activation. The prodrug is released from the MPs in a controlled manner over at least a 1 week period in vitro. Hemolysis assays in the presence of MP-conditioned supernatant demonstrated that MP fabrication does not neutralize drug toxicity. Furthermore, incubation of LNCaP (PSA+) and PC3 (PSA-) cells with MP-conditioned supernatant demonstrates selective toxicity to PSA-expressing cells at low nM concentrations. MP internalization by MSCs has been confirmed using both flow cytometry and confocal microscopy. Chitosan-modification of the MPs permitted increased loading of MSCs (100 ug/mL of MPs). For long-term development, preclinical studies such as these have raised the question of tumor homing efficiency in humans. Therefore, an ongoing FDA-approved first-in-man pre-prostatectomy clinical trial to quantify the number of systemically-delivered allogeneic MSCs that traffic to sites of primary PCa has been initiated. BEAMing (digital PCR) technology will be used to accurately quantify donor MSCs based on differential SNP profiles (detection threshold: ≥0.01%). Data from this trial will be used to determine the amount of prodrug-loaded MPs necessary to deliver per MSC to achieve a therapeutic effect. This data will subsequently be used in ongoing animal studies to model clinical relevance in efficacy studies prior to further translation. Citation Format: W. Nathaniel Brennen, Oren Levy, Sudhir Ranganath, Michael Schweizer, Marc Rosen, Sandrine Billet, Neil Bhowmick, Samuel Denmeade, Jeffrey Karp, John Isaacs. Mesenchymal stem cells (MSC) as cell-based vectors for PSA-activated proaerolysin to sites of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 699. doi:10.1158/1538-7445.AM2014-699