Serum Inhibitor Research Articles

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway

Background/Aims: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. Methods: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. Results: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. Conclusion: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.

Stirring-controlled solidified floating solid-liquid drop microextraction as a new solid phase-enhanced liquid-phase microextraction method by exploiting magnetic carbon nanotube-nickel hybrid

A specific technique is introduced to overcome limitations of classical solidification of floating organic drop microextraction, such as tedious and time-consuming centrifuge step and using disperser solvent, by facile and efficient participation of solid and liquid phases. In this proposed method of stirring-controlled solidified floating solid-liquid drop microextraction (SC-SF-SLDME), magnetic carbon nanotube-nickel hybrid (MNi-CNT) as a solid part of the extractors are dispersed ultrasonically in sample solution, and the procedure followed by dispersion of liquid phase (1-undecanol) through high-rate stirring and easily recollection of MNi-CNT in organic solvent droplets through hydrophobic force. With the reduction in speed of stirring, one solid-liquid drop is formed on top of the solution. MNi-CNT acts as both extractor and the coalescence helper between organic droplets for a facile recollection. MNi-CNT was prepared by spray pyrolysis of nickel oleate/toluene mixture at 1000 °C. Four tyrosine kinase inhibitors were selected as model analytes and the effecting parameters were investigated. The results confirmed that magnetic nanoadsorbent has an important role in the procedure and complete collection of dispersed solvent is not achieved in the absence of the solid phase. Also, short extraction time exhibited success of the proposed method and effect of dispersed solid/liquid phases. The limits of quantification (LOQs) for imatinib, sunitinib, erlotinib, and nilotinib were determined to be as low as 0.7, 1.7, 0.6, and 1.0 μg L−1, respectively. The intra-day precisions (RSDs) were lower than 4.5%. Method performance was investigated by determination of mentioned tyrosine kinase inhibitors (TKIs) in human serum and cerebrospinal fluid samples with good recoveries in the range of 93–98%.

Properdin: a tightly regulated critical inflammatory modulator.

SummaryThe complement alternative pathway is a powerful arm of the innate immune system that enhances diverse inflammatory responses in the human host. Key to the effects of the alternative pathway is properdin, a serum glycoprotein that can both initiate and positively regulate alternative pathway activity. Properdin is produced by many different leukocyte subsets and circulates as cyclic oligomers of monomeric subunits. While the formation of non‐physiological aggregates in purified properdin preparations and the presence of potential properdin inhibitors in serum have complicated studies of its function, properdin has, regardless, emerged as a key player in various inflammatory disease models. Here, we review basic properdin biology, emphasizing the major hurdles that have complicated the interpretation of results from properdin‐centered studies. In addition, we elaborate on an emerging role for properdin in thromboinflammation and discuss the potential utility of properdin inhibitors as long‐term therapeutic options to treat diseases marked by increased formation of platelet/granulocyte aggregates. Finally, we describe the interplay between properdin and the alternative pathway negative regulator, Factor H, and how aiming to understand these interactions can provide scientists with the most effective ways to manipulate alternative pathway activation in complex systems.

Inhibiting ability of benzathine penicillin G towards group A Streptococcus β -hemolyticus in 21 and 28 days after a single intramuscular injection

Background The effectiveness of a single intramuscularbenzathine penicillin G as a secondary prevention of acute rheu-matic fever after 28 days is still controversial. The latest studyshowed that serum penicillin concentration was below protectivelevel in 4 weeks after a single intramuscular injection.Objective This study was a preliminary study to compare the in-hibiting ability of benzathine penicillin G towards group A Strepto-coccus β -hemolyticus in 21 and 28 days after a single IM injectionby using Mueller-Hinton broth method.Methods The inhibiting ability was measured by the serum in-hibitory concentration and serum bactericidal concentration aftera single intramuscular injection of benzathine penicillin G 1.2 mil-lion units.Results The subjects were 28 patients, with mean age of 15.5years (SD 3.4). The numbers of male and female were equal. Meanserum inhibitory concentration in 21 days after a single injectionwas 926.4 (SD 3080.1) and after 28 days was 1540.9 (SD 4275.1),which was not significantly different (p=0.0662). Mean serum bac-tericidal concentration in 21 days after IM injection was 1579.6(SD 4265.7) and in 28 days was 2417 (SD 6849.4) and it was notstatistically different (p=0.2276).Conclusion This study concluded that there was no significantdifference in the inhibiting ability of benzathine penicillin G to-wards group A Streptococcus β -hemolyticus between 21 and 28days after a single intramuscular injection

Local Inhibition of Complement Improves Mesenchymal Stem Cell Viability and Function After Administration.

The results of recent clinical trials using mesenchymal stem cells (MSCs) have been unsatisfactory, indicating that current MSC-based therapies need to be improved. We and others have previously demonstrated that MSCs activate complement by unknown mechanisms after infusion, leading to damaged MSCs. In the study reported here, we found that incorporation of N-glycolylneuraminic acid onto MSCs during in vitro culture was a factor in the activation of complement by MSCs. In addition, we developed a way to "paint" heparin onto MSCs. This novel method improved the viability of MSCs and enhanced their function after infusion by directly inhibiting complement and by recruiting factor H, another potent complement inhibitor in serum, onto the surface of the MSCs. These data suggest that cell-surface engineering of MSCs with heparin to locally inhibit complement activation on MSCs might be a straightforward and effective method for improving the outcome of current MSC-based therapies.

α2-adrenoreceptor modulated FAK pathway induced by dexmedetomidine attenuates pulmonary microvascular hyper-permeability following kidney injury.

Renal ischemia-reperfusion (rI/R) could cause remote acute lung injury (ALI) and combination of these two organ injuries can remarkably increase the mortality. This study aims to determine whether dexmedetomidine, an α2-adrenoreceptor agonist sedative, can ameliorate pulmonary microvascular hyper-permeability following rI/R injury and explore the underlying mechanisms. In vivo, C57BL/6J mice received dexmedetomidine (25μg/kg, i.p.) in the absence or presence of α2-adrenergic antagonist atipamezole (250μg/kg, i.p.) or focal adhesion kinase (FAK) inhibitor (30mg/kg, i.p.) before bilateral renal pedicle clamping for 45 minutes followed by 24 hours reperfusion. The lung histopathological changes and the permeability of pulmonary microvascular were assessed respectively. In vitro, the cultured C57BL/6J mice pulmonary microvascular endothelial cells (PMVECs) were treated with serum from mice with rI/R with or without dexmedetomidine and atipamezole. Trans-endothelial permeability and phospho-tyrosine397FAK, F-actin, VE-cadherin and ZO-1 in monolayer PMVECs were measured respectively in the presence or absence of rI/R serum, dexmedetomidine and FAK inhibitor. In vivo, dexmedetomidine remarkably attenuated lung injury and pulmonary microvascular hyper-permeability caused by rI/R injury, which was abolished by atipamezole or FAK inhibitor co-administration. In vitro, the permeability of PMVECs monolayer following exposure to serum from rI/R mice was increased significantly, and decreased by dexmedetomidine. Dexmedetomidine increased phospho-tyrosine397FAK in a time- and dose-dependent manner, which was correlated with the changes in trans-endothelial permeability. Our data indicated that dexmedetomidine is able to ameliorate remote pulmonary microvascular hyper-permeability induced by rI/R, at least in part, via FAK modulation.

Phytanic acid activates NADPH oxidase through transactivation of epidermal growth factor receptor in vascular smooth muscle cells.

BackgroundPhytanic acid (PA) has been implicated in development of cancer and its defective metabolism is known to cause life-threatening conditions, such as Refsum disease, in children. To explore molecular mechanisms of phytanic acid-induced cellular pathology, we investigated its effect on NADPH oxidase (NOX) and epidermal growth factor receptor (EGFR) in rat aortic smooth muscle cells (RASMC).MethodsSmooth muscle cells were isolated from rat aortae using enzymic digestion with collagenase and elastase. Cultured RASMC were treated with varying concentrations (0.5-10 μg/ml) of phytanic acid in the presence/absence of fetal bovine serum (FBS) and/or EGFR inhibitor, AG1478. Following treatment with experimental agents, NOX activity was assayed in RASMC cultures by luminescence method. Gene expression of NOX-1 and p47phox was assessed using RT-PCR. NOX-1, p47phox and, total EGFR protein and its phosphorylated form were measured by Western blotting.ResultsTreatment of RASMC with supraphysiological concentrations (>2.5 μg/ml) of PA significantly (p < 0.01) increased the NOX activity. PA also significantly increased gene/protein expression of NOX-1 and p47phox whereas p22phox and p67phox remained unaffected. Interestingly, PA (2.5-10 μg/ml) markedly (2–3 folds) increased the total and phosphorylated EGFR. Treatment of cells with EGFR inhibitor, AG1478, significantly blocked the PA-induced enhancement of NOX activity.ConclusionsOur findings that PA transactivates EGFR and induces NOX activity in vascular smooth muscle cells provide new insights into molecular mechanisms of PA’s role in cancer and Refsum disease.

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.

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Abstract 18834: IF1 And ApoA-I Serum Concentrations, but Not HDL-cholesterol, Predict Mortality in Coronary Artery Disease Patients: The Genes Study

Introduction: High-density lipoprotein cholesterol (HDL-C) may not be the best clinical measure of HDL for assessing cardiovascular risk. Serum inhibitor factor 1 (IF1), an endogenous inhibitor of the ecto-F1-ATPase apoA-I receptor, has been identified as an independent determinant of HDL-C associated with low risk of coronary artery disease (CAD). Objective: To determine the relationship between serum IF1 concentration and mortality in stable CAD patients in comparison with HDL-C and apoA-I Methods: Serum IF1 was measured in 577 coronary male patients (enrollment age 45-74 years; enrollment period, 2001-2004) from the GENES (Genetique et ENvironement en Europe du Sud) study. Vital status was assessed yearly through December 31, 2013. Results: During a median follow up of 11 years, mortality rate was 29.5%. Cardiovascular mortality accounted for the majority (62.5%) of deaths. Serum IF1 (median value: 0.42 mg/mL) was positively correlated with HDL-C and apoA-I, and negatively with triglycerides and the severity of CAD (Gensini, Duke-Jeopardy scores). Mortality was lower the higher the quartile score for IF1 and apoA-I, but not for HDL-C. Inverse associations of IF1 and apoA-I with mortality remained significant, even after multivariate adjustments for classical cardiovascular risk factors - age, smoking, diabetes, dyslipidemia, waist circumference, physical activity and CRP. Furthermore, associations of the highest IF1 quartile with mortality reduction remained significant after further adjustment for clinical parameters proven to be predictive of complications in CAD patients, such as ankle-brachial index, heart rate and CAD duration. Interestingly, when levels of both IF1 and apoA-I were high, mortality was much lower than when only one of these was high. Conclusions: IF1 is a new biological marker strongly predictive of mortality in CAD patients. Among patients with normal or elevated apoA-I levels, IF1 measurement may help to identify those who are at low risk of complications and those who are not. IF1 measurement could be use in a panel of other emerging biomarkers that have been proven to be better than HDL-C level, for the better assessment of cardiovascular risk or in the setting of pharmacotherapy.

P43: Endogenous inhibitors of cysteine proteases cystatin C and cystatin SN in biological fluids of patients with intraocular melanoma as possible biomarkers and therapy targets

Cystatins are natural endogenous inhibitors of cysteine proteases universally involved into development of different tumors. Tumor growth and metastazing include increased consumption of these inhibitors, followed by decreased level of cystatins and dysregulation of proteases/protease inhibitors system. However, the biological role of individual cystatins is still not clear. The most known cystatin C was shown to relate to some types of tumor development. Cystatins include a group of intracellular cystatins (belonging to type 1) and extracellular cystatins (type 2), among them cystatins C, D, E/M, F, G, S, SN and SA, which functions were not studied enough. It was suggested that cystatin SN was responsible in regulation of tumor growth locally. To investigate cystatin C and cystatin SN concentrations in biological fluids of patients with intraocular melanoma as tumor biomarkers and possible therapy targets. The patients with melanoma chorioidea (57 patients; among them woman 36, men 21; aged from 28 to 80, of middle age of 56.6 ± 2.4 years) were under investigation. In all cases the pathological process involved one eye. The control group consisted of 37 healthy persons (volunteers), medical personnel in clinic and students, aged from 20 to 49 years; the middle age 31 ± 4.1 years); 7 patients with age-related cataract aged from 57 to 80 (middle age 71 ± 2.6 years; man 3, woman 4). The biological fluids studied: tears, intraocular fluid (obtained during operation) and blood serum. In all cases investigation was made according to informed agreement of patients and control group members. Cystatins concentration was measured by ELISA kits: for Cystatin C (BioVendor, Chechia) and for cystatin SN with help of Human Cystatin SN (CST1) Elisa Kit Cusabio, China. Statistical analysis was made by non-parametric statistic test of Kruskal–Wallis, for correlations – Spearman test. The difference between groups studied was considered significant p < 0.05. Increased serum level of cystatin C was revealed in patients with melanoma chorioides (1023.5 ± 78.9 ng/ml, p = 0.019) compared with the control (809.9 ± 146.8 ng/ml). In tears of patients with melanoma chorioidea, cystatin C concentration (441.7 ± 14.5 ng/ml) had a tendency to increase as compared to the data obtained in tears of the control group (287.5 ± 20.01 ng/ml) as well as the cystatin C level of intraocular fluid of these patients vs the control group (p < 0.1). Cystatin SN concentration in serum of patients with melanoma chorioidea (1.45 ± 0.30 ng/ml) was lower vs the control group (3.12 ± 0.32 ng/ml, p = 0.0038), as well as Cystatin SN level in intraocular fluid (1.43 ± 0.10 ng/ml) vs the control (2.60 ± 0.60 ng/ml, p < 0.05). There was no difference in cystatin SN concentration in tears of patients and control group. In serum of control (healthy) group, cystatin C concentration is significantly higher than cystatin SN level in serum, tears and intraocular fluid. The reverse correlation was revealed between the level of these inhibitors in serum, that is suggested their possible interaction. In melanoma patients the reciprocal changes in cystatin C and cystatin SN were shown: increased cystatin C and decreased cystatin SN level in all biological fluids studied. On the basis of cystatins distribution in biological fluids of patients one can suggest their involvement in pathological process as system reaction of organism on tumor development.

Serum and fecal canine α1-proteinase inhibitor concentrations reflect the severity of intestinal crypt abscesses and/or lacteal dilation in dogs

Gastrointestinal (GI) protein loss, due to lymphangiectasia or chronic inflammation, can be challenging to diagnose. This study evaluated the diagnostic accuracy of serum and fecal canine α1-proteinase inhibitor (cα1PI) concentrations to detect crypt abscesses and/or lacteal dilation in dogs. Serum and fecal cα1PI concentrations were measured in 120 dogs undergoing GI tissue biopsies, and were compared between dogs with and without crypt abscesses/lacteal dilation. Sensitivity and specificity were calculated for dichotomous outcomes. Serial serum cα1PI concentrations were also evaluated in 12 healthy corticosteroid-treated dogs.Serum cα1PI and albumin concentrations were significantly lower in dogs with crypt abscesses and/or lacteal dilation than in those without (both P < 0.001), and more severe lesions were associated with lower serum cα1PI concentrations, higher 3 days-mean fecal cα1PI concentrations, and lower serum/fecal cα1PI ratios. Serum and fecal cα1PI, and their ratios, distinguished dogs with moderate or severe GI crypt abscesses/lacteal dilation from dogs with only mild or none such lesions with moderate sensitivity (56–92%) and specificity (67–81%). Serum cα1PI concentrations increased during corticosteroid administration. We conclude that serum and fecal α1PI concentrations reflect the severity of intestinal crypt abscesses/lacteal dilation in dogs. Due to its specificity for the GI tract, measurement of fecal cα1PI appears to be superior to serum cα1PI for diagnosing GI protein loss in dogs. In addition, the serum/fecal cα1PI ratio has an improved accuracy in hypoalbuminemic dogs, but serum cα1PI concentrations should be carefully interpreted in corticosteroid-treated dogs.

Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3–7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1+ and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase− spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.

Abstract 266: Screening and Validation of Small Molecule Inhibitors of Serum and Glucocorticoid Regulated Kinase-1 as Novel Therapy for Cardiac Arrhythmia Disorders

Alterations in sodium flux (INa) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. Recent data demonstrates a critical role for the serum and glucocorticoid regulated kinase-1 (SGK1) by modulation of INa in the heart, by regulating the voltage-gated sodium channel NaV1.5. To better understand and pharmacologically probe the significance of SGK1 in cardiac dysrhythmias, we have used computer aided drug discovery (CADD) to identify small molecule inhibitors of SGK1. Expression of a constitutively active form of SGK1 (SGK1-CA) increased INa (1.7 fold, p &lt;0.005) in a stable line of HEK cells expressing NaV1.5. Conversely, expression of a dominant negative form (SGK-DN) decreased NaV1.5 channel activity (2.8 fold, p &lt;0.005). We examined the effects of SGK1 inhibition in a LQT model, by quantifying the ability of SGK1 inhibition to rescue the 2:1 AV block phenotype of the potassium channel zebrafish mutant, breakdance (bkd). Morpholino injection or expression of SGK1-DN significantly rescued the 2:1 AV block phenotype as compared to controls (p &lt; 0.05). Using CADD partnered with iterative empirical screens we identified several hit chemical scaffolds. Our lead compound inhibits the phosphorylation of the SGK1 target gene, GSK3-β in a dose dependent manner in cardiomyocytes (CMs) expressing SGK1-CA at the lowest effective concentration of 0.5μM. There was no significant inhibition of AKT dependent phosphorylation of GSK3-β up to a concentration of 50μM, demonstrating specificity of the inhibitor for SGK1. Incubation of bkd zebrafish mutants with the inhibitor rescued the 2:1 AV block in a dose dependent manner (60% rescue with 45μM, p &lt; 0.05). Acute application of the inhibitor dramatically inhibited INa with either expression of SGK1-CA (90.8% reduction, p &lt;0.05 ) or with RFP only (77.5% reduction p &lt; 0.005). The half-time of inhibition was 200s with resulting current densities that were not statistically different than those observed with genetic inhibition by expression of SGK1-DN. We conclude SGK1 activity regulates INa and speculate that structure activity relationship (SAR) derivatives of our lead compound might have a role in treatment of human cardiac arrhythmias.

Melanoma inhibitory activity in Brazilian patients with cutaneous melanoma.

BACKGROUND:Melanoma inhibitory activity is a protein secreted by melanoma cells and has been used as a tumor marker. Increased Melanoma inhibitory activity serum levels are related to metastatic disease or tumor recurrence. Currently there are no studies on Melanoma inhibitory activity and cutaneous melanoma involving Brazilian patients.OBJECTIVE:To evaluate the performance and feasibility of measuring Melanoma inhibitory activity levels in Brazilian patients with cutaneous melanoma.METHODS:Blood was obtained from ten patients with proved metastatic cutaneous melanoma (Group 1), 15 patients resected for cutaneous melanoma without metastasis (Group 2) and 5 healthy donors (Group 3). Melanoma inhibitory activity was measured using a commercially available ELISA kit.RESULTS:There was a statistically significant difference of Melanoma inhibitory activity levels between patients with and without metastasis (p=0.002), and between patients with metastasis and healthy donors (p=0.002). There was no difference between patients without metastasis and healthy donors (p=0.443).CONCLUSION:Melanoma inhibitory activity is a tumor marker for cutaneous melanoma and the Melanoma inhibitory activity-ELISA test can be easily performed. Patients with metastasis have increased Melanoma inhibitory activity serum levels when compared to patients without metastasis and healthy donors.

EFFECT OF SILVER NANOPARTICLES IN EXPERIMENTAL PERITONITIS

In our research we have investigated the effect of intraperitoneal introduction of silver nanoparticles solution (linear particle size of 10–20 nm) into intact animals and in the simulation of experimental peritonitis. We have evaluated the indices of nonspecific proteinases and their inhibitors in blood serum and peritoneum lavage. The intraperitoneal introduction of silver nanoparticles solution to intact animals leads to the minimal reaction of proteinase-inhibitor systems components in blood serum and peritoneum lavage. When modeling inflammatory process in peritoneum cavity by intraperitoneal injection of 10% filtered fecal suspension of rats, application of silver nanoparticles solution is accompanied by decrease in extent of activation of proteinases and preservation of inhibitory potential, both at systemic and local level, which can indicate antiinflammatory effects of nanosilver.

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.

Manipulating adenovirus hexon hypervariable loops dictates immune neutralisation and coagulation factor X-dependent cell interaction in vitro and in vivo.

Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.

Antipapain activity in the serum of patients with breast cancer.

Complement information about the share the role of antipapain activity in serum people with breast cancer. We measured the activity of cysteine peptidase inhibitors in the sera of 150 patients with breast cancer. Patients were divided into four groups depending on the cancer type and treatment method. We also analysed the control group. The activity of cysteine peptidase inhibitors was defined as a 'defensiveness' marker. The activity of cysteine peptidase inhibitors was measured against papain using the colorimetric method and the BANA substrate. The highest activity of enzymes was found in the group of patients with BC and hereditary predisposition to it, and the lowest activity was found in patients after surgical treatment. The activity of cysteine peptidase inhibitors in serum was measured against papain. We found that the activity levels were correlated with the cancer stage and treatment method. The lowest activity was found in patients after surgical treatment; the highest in women with active cancer and a hereditary predisposition to it.

From Naive to Primed Pluripotency: In Vitro Conversion of Mouse Embryonic Stem Cells in Epiblast Stem Cells.

Mouse embryonic stem cells (ESCs) derive from the inner cell mass (ICM) of a blastocyst at E3.5 while mouse epiblast stem cells (EpiSCs) derive from the late epiblast of a post-implantation embryo at E5.5-E7.5. Both cells are able to differentiate into derivatives of the three germs layers but only ESCs are able to produce chimeras when they are introduced into a blastocyst. To support the naive state of pluripotency, ESC culture requires Leukemia inhibitory factor (Lif) and either serum or inhibitors of Erk and Gsk3 pathways (2i) while the primed pluripotency of EpiSCs is maintained using Activin A and Fibroblast Growth Factor 2 (FGF2). It is possible to obtain EpiSCs in vitro starting from ESCs but also to induce ESCs starting from EpiSCs even if this second process is very difficult and inefficient. In this protocol we describe how we perform the process of conversion from ESCs to EpiSCs.