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Humans have turned to natural products for medicinal therapeutics for millennia. In recent years, the ribosomally synthesized and post-translationally modified peptide (RiPP) class of natural products have garnered a lot of attention due to their ability for heterologous expression, relative ease of mutation, and vast biological activates. Microviridins are a family of RiPPs that contain a tricyclic cage-like structure through the formation of two lactone rings and one lactam ring catalyzed by two ATP-grasp ligase family enzymes. Microviridins are potent inhibitors of serine proteases and mutational studies of a small number of residues indicates the potential of peptide engineering protease inhibition. The screening of large libraries of microviridin mutants could allow for targeted protease inhibition. Here we describe work toward the yeast surface display of microviridin B. It is demonstrated that Microviridin B retains its elastase inhibitory activity with an nM IC50 when the leader region of the precursor peptide is attached, opening the door to the surface display of the peptide for library screening. An assay was developed screen microviridin B libraries to determine the specificity of APT grasp ligase MdnC. The proof of concept showed promising results, however there is indication of incomplete peptide modification by MdnC. An investigation into MdnC expression in yeast shows a 2µ origin and constitutive GPD promoter results in higher MdnC expression compared to a CEN origin and Gal1 promoter, even on galactose media.

Background & Aims: Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response. Methods: The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion. Results: In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion. Conclusions: Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response. Impact and implications: Our results indicate that neutrop

One cluster of the extrapulmonary manifestations in chronic obstructive pulmonary disease (COPD) is related to the brain, which includes anxiety, depression and cognitive impairment. Brain-related comorbidities are related to worsening of symptoms and increased mortality in COPD patients. In this study, a murine model of COPD was used to examine the effects of emphysema and repetitive pulmonary inflammatory events on systemic inflammatory outcomes and brain function. In addition, the effect of a dietary intervention on brain-related parameters was assessed. Adult male C57Bl/6J mice were exposed to elastase or vehicle intratracheally (i.t.) once a week on three consecutive weeks. Two weeks after the final administration, mice were i.t. exposed to lipopolysaccharide (LPS) or vehicle for three times with a 10 day interval. A dietary intervention enriched with omega-3 PUFAs, prebiotic fibers, tryptophan and vitamin D was administered from the first LPS exposure onward. Behavior and cognitive function, the degree of emphysema and both pulmonary and systemic inflammation as well as blood-brain barrier (BBB) integrity and neuroinflammation in the brain were assessed. A lower score in the cognitive test was observed in elastase-exposed mice. Mice exposed to elastase plus LPS showed less locomotion in the behavior test. The enriched diet seemed to reduce anxiety-like behavior over time and cognitive impairments associated with the presented COPD model, without affecting locomotion. In addition, the enriched diet restored the disbalance in splenic T-helper 1 (Th1) and Th2 cells. There was a trend toward recovering elastase plus LPS-induced decreased expression of occludin in brain microvessels, a measure of BBB integrity, as well as improving expression levels of kynurenine pathway markers in the brain by the enriched diet. The findings of this study demonstrate brain-associated comorbidities - including cognitive and behavioral impairments - in this murine model for COPD.

[ES] El déficit de alfa-1-antitripsina (DAAT) es una enfermedad rara monogénica, que provoca en los pacientes un desarrollo temprano de enfermedad pulmonar obstructiva crónica (EPOC) y/o de daño hepático (cirrosis hepática y hepatocarcinoma). Estos pacientes se caracterizan por tener niveles séricos reducidos de la proteína alfa-1-antitripsina (AAT), lo que supone que tienen bajas concentraciones en los alveolos pulmonares, donde desarrolla su función protectora como inhibidora de proteasas. Debido a un exceso de actividad proteasa, las paredes alveolares son dañadas por la elastasa generada por los neutrófilos lo que conduce al desarrollo de enfisema. En el hígado, la acumulación de polímeros de AAT en el retículo endoplasmático, está asociado con daño mitocondrial y aumento de las vías de señalización de apoptosis, que llevan a la fibrosis y cirrosis hepáticas. Esta enfermedad se produce debido a mutaciones en el gen de la SERPINA1, que codifica para la AAT y son responsables de la reducción de su concentración en circulación. Pese al gran número de mutaciones existentes en este gen, la gran parte de la población presenta estos 3 alelos: M, alelo normal; los alelos patológicos S y Z, siendo el último el que afecta con mayor gravedad a los pacientes. Estudios previos realizados por nuestro grupo de investigación han demostrado que las proteínas totales de los pacientes con DAAT se encuentran más oxidadas que las de los controles sanos, ya que estos pacientes están expuestos a una situación de estrés oxidativo crónico. Como la exposición al estrés oxidativo reduce la afinidad de la AAT por la elastasa del neutrófilo, es importante conocer el grado de oxidación de la AAT (OxyAAT) para conocer el grado real de protección del paciente frente al daño generado por la elastasa del neutrófilo. Por lo tanto, el objetivo del trabajo es estudiar las posibles diferencias de OxyAAT circulante entre pacientes con DAAT e individuos control. La medida se realizó mediante ensayos e, [EN] Alpha-1 antitrypsin deficiency (AATD) is a rare monogenic hereditary disease, that increases the risk to suffer from early chronic obstructive pulmonary disease (COPD) or liver disease (hepatic cirrhosis and hepatocellular carcinoma) among patients. These patients are characterized for having lower alpha-1 antitrypsin (AAT) serum concentrations than non-affected people. This leads to lower alveoli activity of AAT, where it performs its protective function against the neutrophil elastase by inhibiting its protease activity. Due to an excess of protease activity, alveoli walls are damaged by neutrophil elastase and leads to the development of emphysema. In the liver, the accumulation of AAT polymers in endoplasmic reticulum vesicles are found in association with mitochondrial damage and an increase of apoptotic signaling pathways, which lead to fibrosis and hepatic cirrhosis. This disease is produced by mutations in the gene SERPINA1 that encodes for AAT, which are responsible for its circulation level decrease. Although there are several variants of this locus, the most common alleles are: M allele, normal AAT; the pathologic alleles S and Z, being Z the one that affects patients with higher severity. Previous works of our research group have shown that there is higher total protein oxidation in AATD patients than in healthy controls due to a chronic exposure to oxidative stress. Since oxidative stress reduces AAT affinity for the neutrophil elastase, it is important to know the oxidation state of AAT (OxyAAT) to understand the real degree of protection of the patient to the elastase damage. Thus, the objective of this work is to study the possible differences of circulatory OxyAAT between AATD patients and control individuals. The measure was performed through enzymatic assays determining elastase inhibitory capacity (EIC) and trypsin inhibitory capacity (TIC) of serum samples by spectrophotometry. The percentage of OxyAAT was calculated through the EIC/TIC rat

[ES] El déficit de alfa-1-antitripsina (DAAT) es una enfermedad rara monogénica, que provoca en los pacientes un desarrollo temprano de enfermedad pulmonar obstructiva crónica (EPOC) y/o de daño hepático (cirrosis hepática y hepatocarcinoma). Estos pacientes se caracterizan por tener niveles séricos reducidos de la proteína alfa-1-antitripsina (AAT), lo que supone que tienen bajas concentraciones en los alveolos pulmonares, donde desarrolla su función protectora como inhibidora de proteasas. Debido a un exceso de actividad proteasa, las paredes alveolares son dañadas por la elastasa generada por los neutrófilos lo que conduce al desarrollo de enfisema. En el hígado, la acumulación de polímeros de AAT en el retículo endoplasmático, está asociado con daño mitocondrial y aumento de las vías de señalización de apoptosis, que llevan a la fibrosis y cirrosis hepáticas. Esta enfermedad se produce debido a mutaciones en el gen de la SERPINA1, que codifica para la AAT y son responsables de la reducción de su concentración en circulación. Pese al gran número de mutaciones existentes en este gen, la gran parte de la población presenta estos 3 alelos: M, alelo normal; los alelos patológicos S y Z, siendo el último el que afecta con mayor gravedad a los pacientes. Estudios previos realizados por nuestro grupo de investigación han demostrado que las proteínas totales de los pacientes con DAAT se encuentran más oxidadas que las de los controles sanos, ya que estos pacientes están expuestos a una situación de estrés oxidativo crónico. Como la exposición al estrés oxidativo reduce la afinidad de la AAT por la elastasa del neutrófilo, es importante conocer el grado de oxidación de la AAT (OxyAAT) para conocer el grado real de protección del paciente frente al daño generado por la elastasa del neutrófilo. Por lo tanto, el objetivo del trabajo es estudiar las posibles diferencias de OxyAAT circulante entre pacientes con DAAT e individuos control. La medida se realizó mediante ensayos e, [EN] Alpha-1 antitrypsin deficiency (AATD) is a rare monogenic hereditary disease, that increases the risk to suffer from early chronic obstructive pulmonary disease (COPD) or liver disease (hepatic cirrhosis and hepatocellular carcinoma) among patients. These patients are characterized for having lower alpha-1 antitrypsin (AAT) serum concentrations than non-affected people. This leads to lower alveoli activity of AAT, where it performs its protective function against the neutrophil elastase by inhibiting its protease activity. Due to an excess of protease activity, alveoli walls are damaged by neutrophil elastase and leads to the development of emphysema. In the liver, the accumulation of AAT polymers in endoplasmic reticulum vesicles are found in association with mitochondrial damage and an increase of apoptotic signaling pathways, which lead to fibrosis and hepatic cirrhosis. This disease is produced by mutations in the gene SERPINA1 that encodes for AAT, which are responsible for its circulation level decrease. Although there are several variants of this locus, the most common alleles are: M allele, normal AAT; the pathologic alleles S and Z, being Z the one that affects patients with higher severity. Previous works of our research group have shown that there is higher total protein oxidation in AATD patients than in healthy controls due to a chronic exposure to oxidative stress. Since oxidative stress reduces AAT affinity for the neutrophil elastase, it is important to know the oxidation state of AAT (OxyAAT) to understand the real degree of protection of the patient to the elastase damage. Thus, the objective of this work is to study the possible differences of circulatory OxyAAT between AATD patients and control individuals. The measure was performed through enzymatic assays determining elastase inhibitory capacity (EIC) and trypsin inhibitory capacity (TIC) of serum samples by spectrophotometry. The percentage of OxyAAT was calculated through the EIC/TIC rat

[ES] El déficit de alfa-1-antitripsina (DAAT) es una enfermedad rara monogénica, que provoca en los pacientes un desarrollo temprano de enfermedad pulmonar obstructiva crónica (EPOC) y/o de daño hepático (cirrosis hepática y hepatocarcinoma). Estos pacientes se caracterizan por tener niveles séricos reducidos de la proteína alfa-1-antitripsina (AAT), lo que supone que tienen bajas concentraciones en los alveolos pulmonares, donde desarrolla su función protectora como inhibidora de proteasas. Debido a un exceso de actividad proteasa, las paredes alveolares son dañadas por la elastasa generada por los neutrófilos lo que conduce al desarrollo de enfisema. En el hígado, la acumulación de polímeros de AAT en el retículo endoplasmático, está asociado con daño mitocondrial y aumento de las vías de señalización de apoptosis, que llevan a la fibrosis y cirrosis hepáticas. Esta enfermedad se produce debido a mutaciones en el gen de la SERPINA1, que codifica para la AAT y son responsables de la reducción de su concentración en circulación. Pese al gran número de mutaciones existentes en este gen, la gran parte de la población presenta estos 3 alelos: M, alelo normal; los alelos patológicos S y Z, siendo el último el que afecta con mayor gravedad a los pacientes. Estudios previos realizados por nuestro grupo de investigación han demostrado que las proteínas totales de los pacientes con DAAT se encuentran más oxidadas que las de los controles sanos, ya que estos pacientes están expuestos a una situación de estrés oxidativo crónico. Como la exposición al estrés oxidativo reduce la afinidad de la AAT por la elastasa del neutrófilo, es importante conocer el grado de oxidación de la AAT (OxyAAT) para conocer el grado real de protección del paciente frente al daño generado por la elastasa del neutrófilo. Por lo tanto, el objetivo del trabajo es estudiar las posibles diferencias de OxyAAT circulante entre pacientes con DAAT e individuos control. La medida se realizó mediante ensayos e, [EN] Alpha-1 antitrypsin deficiency (AATD) is a rare monogenic hereditary disease, that increases the risk to suffer from early chronic obstructive pulmonary disease (COPD) or liver disease (hepatic cirrhosis and hepatocellular carcinoma) among patients. These patients are characterized for having lower alpha-1 antitrypsin (AAT) serum concentrations than non-affected people. This leads to lower alveoli activity of AAT, where it performs its protective function against the neutrophil elastase by inhibiting its protease activity. Due to an excess of protease activity, alveoli walls are damaged by neutrophil elastase and leads to the development of emphysema. In the liver, the accumulation of AAT polymers in endoplasmic reticulum vesicles are found in association with mitochondrial damage and an increase of apoptotic signaling pathways, which lead to fibrosis and hepatic cirrhosis. This disease is produced by mutations in the gene SERPINA1 that encodes for AAT, which are responsible for its circulation level decrease. Although there are several variants of this locus, the most common alleles are: M allele, normal AAT; the pathologic alleles S and Z, being Z the one that affects patients with higher severity. Previous works of our research group have shown that there is higher total protein oxidation in AATD patients than in healthy controls due to a chronic exposure to oxidative stress. Since oxidative stress reduces AAT affinity for the neutrophil elastase, it is important to know the oxidation state of AAT (OxyAAT) to understand the real degree of protection of the patient to the elastase damage. Thus, the objective of this work is to study the possible differences of circulatory OxyAAT between AATD patients and control individuals. The measure was performed through enzymatic assays determining elastase inhibitory capacity (EIC) and trypsin inhibitory capacity (TIC) of serum samples by spectrophotometry. The percentage of OxyAAT was calculated through the EIC/TIC rat

Elastic fibers containing elastin play an important role in tendon functionality, but the knowledge on presence and function of elastin during tendon healing is limited. The aim of this study was to investigate elastin content and distribution in intact and healing Achilles tendons and to understand how elastin influence the viscoelastic properties of tendons. The right Achilles tendon was completely transected in 81 Sprague-Dawley rats. Elastin content was quantified in intact and healing tendons (7, 14, and 28 days post-surgery) and elastin distribution was visualized by immunohistochemistry at 14 days post-surgery. Degradation of elastin by elastase incubation was used to study the role of elastin on viscoelastic properties. Mechanical testing was either performed as a cyclic test (20x 10 N) or as a creep test. We found significantly higher levels of elastin in healing tendons at all time-points compared to intact tendons (4% in healing tendons 28 days post-surgery vs 2% in intact tendons). The elastin was more widely distributed throughout the extracellular matrix in the healing tendons in contrast to the intact tendon where the distribution was not so pronounced. Elastase incubation reduced the elastin levels by approximately 30% and led to a 40%-50% reduction in creep. This reduction was seen in both intact and healing tendons. Our results show that healing tendons contain more elastin and is more compliable than intact tendons. The role of elastin in tendon healing and tissue compliance indicates a protective role of elastic fibers to prevent re-injuries during early tendon healing. Plain Language Summary Tendons transfer high loads from muscles to bones during locomotion. They are primarily made by the protein collagen, a protein that provide strength to the tissues. Besides collagen, tendons also contain other building blocks such as, for example, elastic fibers. Elastic fibers contain elastin and elastin is important for the extensibility of the tendon. Wh, Funding Agency:Östergotland Country council LIO-796831

Proteases and reactive oxygen species (ROS) have long been implicated in playing key roles in host tissue injury at sites of inflammation dominated by macrophage activations and/or neutrophil infiltrations. Imbalances between proteases/antiproteases and ROS/antioxidants are recognized to contribute to amplification of inflammatory-based host tissue injury. This has been especially well-documented in such respiratory tract diseases as chronic obstructive pulmonary disease, cystic fibrosis, and acute respiratory distress syndrome. Inflammation-related protease/ROS disequilibria are further confounded by recognition that proteases can increase ROS by several different mechanisms and that ROS can inactivate proteases. The major human antiprotease, alpha-1 antitrypsin (AAT), is dramatically inactivated by ROS. AAT deficiency is the most prevalent genetic predisposing factor leading to emphysema, a condition treated by replacement infusions of plasma-derived AAT (hAAT) at a cost of up to $200,000 per year per patient. An updated method for production of a plant-made recombinant AAT (prAAT) engineered for enhanced oxidation resistance compared to hAAT is presented. Plant-made recombinant AAT shows comparable antiprotease activity to hAAT, and retains full activity under oxidative conditions that would deactivate hAAT. Additionally, we show that prAAT has similar effectiveness in preventing neutrophil elastase-induced cell death in an in vitro human bronchial epithelial cell culture model. We conclude that prAAT is potentially a "biobetter" AAT product that could be made available to individuals with a wide spectrum of inflammatory disorders characterized by overly aggressive neutrophilic infiltrations.

The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.

Доведено, що нирки мають надзвичайно високий рівень неспецифічного імунітету за рахунок високої активності лізоциму і підвищеного рівня активності нейтрофілів. Адреналіновий стрес активує неспецифічний імунітет, а лінкоміцин його пригнічує., Доказано, что почки имеют исключительно высокий уровень неспецифического иммунитета за счет высокой активности лизоцима и повышенного уровня активности нейтрофилов. Адреналиновый стресс активирует неспецифический иммунитет, а линкомицин его угнетает., It has been proved that kidneys have extremely high level of non-specific immunity due to high activity of lysocim and high activity level of neutrophils. Adrenal stress activates non-specific immunity, but lincomycin depresses it.

Uvod. Cilj studije je bio da se ispita uloga antineutrofilnih citoplazmatskih antitela (ANCA) i aktivnosti serumske DNaze I kod idiopatskog sistemskog lupus erythematosusa (SLE) i lupus-like sindroma (LLS) indukovanog propiltiouracilom (PTU). Metod. Poređena su 142 pacijenta sa idiopatskim SLE i 17 pacijenata sa LLS indukovanim PTU. Ispitan je ANCA profil (mijeloperoksidaza-MPO, proteinaza 3-PR3, elastaza-El, laktoferin-Lf, katepsin G, BPI-engl. bactericidal/permeability increasing protein), serološki parametri (anti-dsDNK, anti-extractable nuclear antigens (ENA), anti-nukleozomalna, antihistonska, anti-C1q, anti-kardiolipinska antitela, komponente komplementa, krioglobulini) i aktivnost serumske DNaze I kod pacijenata sa SLE i LLS indukovanim PTU. Kontrolne grupe su formirane od zdravih osoba i od pacijenata lečenih PTU ali bez LLS. Klinički i serološki je praćeno 25 ANCA pozitivnih SLE i 12 LLS pacijenata tokom 1.8±0.5 i 4.1±2.0 godina. Rezultati. ANCA su nađena kod 28% pacijenata sa SLE i kod 100% pacijenata sa LLS izazvanim PTU. Pacijenti sa LLS izazvanim PTU znatno češće su imali kutani vaskulitis, a znatno ređe artritis i zahvaćenost bubrega (p<0.01) u odnosu na pacijente sa SLE. ANCA pozitivni SLE pacijenti su češće imali autoimunski hepatitis (p<0.05) i neurološke/psihijatrijske manifestacije (p<0.01), a značajno ređe su imali kutane manifestacije (p<0.05) u odnosu na ANCA negativne pacijente sa SLE. Učestalost hematoloških, nefroloških i artikularnih manifestacija, kao i polioserozitisa, nije se razlikovala kod ANCA pozitivnih i ANCA negativnih pacijenata sa SLE. ANCA su kod pacijenata sa SLE bila monospecifična (77.5%) i to najčešće anti-Lf (52.5%), anti-MPO (47.5%) ili anti-El (15%). Anti-Lf kod pacijenata sa SLE nisu bila udružena sa prisustvom anti-MPO (p<0.01) i anti-El (p<0.05). Kod pacijenata sa LLS indukovanim PTU nađena su polispecifična ANCA (82.4%) i to najčešće anti-MPO (100%), anti-El (82.4%) i anti-PR3 (52.9%)...

Transformation of circulating leukocytes from a dormant into an activated state with changing rheological properties leads to a major shift of their behavior in the microcirculation. Low levels of pseudopod formation or expression of adhesion molecules facilitate relatively free passage through microvessels while activated leukocytes with pseudopods and enhanced levels of adhesion membrane proteins become trapped in microvessels, attach to the endothelium and migrate into the tissue. The transformation of leukocytes into an activated state is seen in many diseases. While mechanisms for activation due to infections, tissue trauma, as well as non-physiological biochemical or biophysical exposures are well recognized, the mechanisms for activation in many diseases have not been conclusively liked to these traditional mechanisms and remain unknown. We summarize our recent evidence suggesting a major and surprising role of digestive enzymes in the small intestine as root causes for leukocyte activation and microvascular disturbances. During normal digestion of food digestive enzymes are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. When permeability of this barrier increases, these powerful degrading enzymes leak into the wall of the intestine and into the systemic circulation. Leakage of digestive enzymes occurs for example in physiological shock and multi-organ failure. Entry of digestive enzymes into the wall of the small intestine leads to degradation of the intestinal tissue in an autodigestion process. The digestive enzymes and tissue/food fragments generate not only activate leukocytes but also cause numerous cell dysfunctions. For example, proteolytic destruction of membrane receptors, plasma proteins and other biomolecules occurs. We conclude that escape of digestive enzymes from the intestinal track serves as a major source of cell dysfunction, morbidity and even mortality, including abnormal leukocyte activation seen in rhe

Transformation of circulating leukocytes from a dormant into an activated state with changing rheological properties leads to a major shift of their behavior in the microcirculation. Low levels of pseudopod formation or expression of adhesion molecules facilitate relatively free passage through microvessels while activated leukocytes with pseudopods and enhanced levels of adhesion membrane proteins become trapped in microvessels, attach to the endothelium and migrate into the tissue. The transformation of leukocytes into an activated state is seen in many diseases. While mechanisms for activation due to infections, tissue trauma, as well as non-physiological biochemical or biophysical exposures are well recognized, the mechanisms for activation in many diseases have not been conclusively liked to these traditional mechanisms and remain unknown. We summarize our recent evidence suggesting a major and surprising role of digestive enzymes in the small intestine as root causes for leukocyte activation and microvascular disturbances. During normal digestion of food digestive enzymes are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. When permeability of this barrier increases, these powerful degrading enzymes leak into the wall of the intestine and into the systemic circulation. Leakage of digestive enzymes occurs for example in physiological shock and multi-organ failure. Entry of digestive enzymes into the wall of the small intestine leads to degradation of the intestinal tissue in an autodigestion process. The digestive enzymes and tissue/food fragments generate not only activate leukocytes but also cause numerous cell dysfunctions. For example, proteolytic destruction of membrane receptors, plasma proteins and other biomolecules occurs. We conclude that escape of digestive enzymes from the intestinal track serves as a major source of cell dysfunction, morbidity and even mortality, including abnormal leukocyte activation seen in rhe

Beta2-integrins are important in leukocyte trafficking and function, and are regulated through the binding of cytoplasmic proteins, such as kindlin-3, to their intracellular domain. Here, we investigate the involvement of beta2-integrins in the regulation of metabolic disease using mice where the kindlin-3 binding site in the beta2-integrin cytoplasmic tail has been mutated (TTT/AAA-beta2-integrin knock-in (KI) mice), leading to expressed but dysfunctional beta2-integrins and significant neutrophilia in vivo. Beta2-integrin KI mice fed on a high fat diet showed normal weight gain, and normal accumulation of macrophages and lymphocytes in white adipose tissue (WAT) and liver, but increased neutrophil numbers especially in WAT. In addition, beta2-integrin KI mice fed on a high fat diet showed significantly increased peripheral insulin resistance in response to high-fat feeding. However, this was associated with improved glucose disposal following glucose load. Interestingly, beta2-integrin KI neutrophils produced more elastase in vitro, in response to stimulation. Beta2-integrin KI mice displayed variability of tissue inflammatory status, with liver and WAT exhibiting little or no difference in inflammation compared to high fat fed controls, whereas skeletal muscle demonstrated a raised inflammatory profile in association with higher elastase levels and diminished signalling through the IRS1-PKB pathway. In conclusion, although expression of dysfunctional beta2-integrins increased neutrophil production and infiltration into tissue, skeletal muscle was the most affected tissue exhibiting evidence of higher neutrophil activity and insulin resistance. Thus, beta2-integrins modulate glucose homeostasis during high fat feeding predominantly through actions on skeletal muscle to affect metabolic phenotype in vivo.

Background: During continuous venovenous haemofiltration (CVVH), regional anticoagulation with citrate may be superior to heparin in terms of biocompatibility, since heparin as opposed to citrate may activate complement (reflected by circulating C5a) and induce neutrophil degranulation in the filter and myeloperoxidase (MPO) release from endothelium. Methods. No anticoagulation (n = 13), unfractionated heparin (n = 8) and trisodium citrate (n = 17) regimens during CVVH were compared. Blood samples were collected pre- and postfilter; C5a, elastase and MPO were determined by ELISA. Additionally, C5a was also measured in the ultrafiltrate. Results: In the heparin group, there was C5a production across the filter which most decreased over time as compared to other groups (P = 0.007). There was also net production of elastase and MPO across the filter during heparin anticoagulation (P = 0.049 or lower), while production was minimal and absent in the no anticoagulation and citrate group, respectively. During heparin anticoagulation, plasma concentrations of MPO at the inlet increased in the first 10 minutes of CVVH (P = 0.024). Conclusion: Citrate confers less filter-induced, potentially harmful complement activation and neutrophil degranulation and less endothelial activation than heparin when used for anticoagulation during continuous venovenous haemofiltration in critically ill patients.

In a previous study we were able to accurately fit experimental data on arterial tissues at supra-physiological loads using a material model that accounts for softening/damage only in the portion of the model associated with the collagen fibers (Weisbecker et al., 2012). Naturally, this result leads to the hypothesis that the softening behavior is related only to the collagen fibers, and not to the matrix material. In this study we test this hypothesis by conducting uniaxial extension tests on elastase and collagenase treated tissues and on untreated control specimens from the media of human thoracic aortas. We relate structural changes in the tissue after enzyme treatment to changes in the corresponding mechanical behavior. Collagenase treated tissue does not exhibit any softening behavior under quasi-static cyclic loading, a result supporting our hypothesis. Conversely, elastase treated tissue exhibits continuous softening under the same loading conditions, indicating that the integrity of the tissue is destroyed upon removal of the elastin. Finally, we fit isotropic and anisotropic constitutive models to the mechanical response of the collagenase treated arterial tissue, while our anisotropic model better approximates the response of collagenase treated arterial tissues, we show that an isotropic matrix model is sufficient to accurately reproduce the mechanical response of untreated control specimens, consistent with current practice in the literature., QC 20131001

Background: The aim of this manuscript was to characterize airway ceramide profiles in a rodent model of elastase-induced emphysema and to examine the effect of pharmacological intervention directed towards ceramide metabolism.Methods: Adult mice were anesthetized and treated with an intratracheal instillation of elastase. Lung function was measured, broncho-alveolar lavage fluid collected and histological and morphometrical analysis of lung tissue performed within 3 weeks after elastase injection, with and without sphingomyelinase inhibitors or serine palmitoyltransferase inhibitor. Ceramides in broncho-alveolar lavage (BAL) fluid were quantified by tandem mass spectrometry.Results: BAL fluid showed a transient increase in total protein and IgM, and activated macrophages and neutrophils. Ceramides were transiently upregulated at day 2 after elastase treatment. Histology showed persistent patchy alveolar destruction at day 2 after elastase installation. Acid and neutral sphingomyelinase inhibitors had no effect on BAL ceramide levels, lung function or histology. Addition of a serine palmitoyltransferase inhibitor ameliorated lung function changes and reduced ceramides in BAL.Conclusions: Ceramides were increased during the acute inflammatory phase of elastase-induced lung injury. Since addition of a serine palmitoyltransferase inhibitor diminished the rise in ceramides and ameliorated lung function, ceramides likely contributed to the early phase of alveolar destruction and are a potential therapeutic target in the elastase model of lung emphysema.

Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions.

The present study was carried out to analyze the effects of erythromycin on functions of polymorphonuclear leukocytes (PMN) and immunocompetent cells in mice. Administration of erythromycin (EM) reduced phagocytic activity as determined by chemiluminescence, although chemotaxsis and elastase production of PMN were not affected. These were consistent with results obtained from PMN of EM-treated patients with chronic lower respiratory infections. However, serum of EM-treated mice exhibited suppressive activity to mitogenic activity of recombinant interleukin 1 (rIL-1), when serum was prepared from mice 2h after intravenous injection of heat-killed Pseudomonas aeruginosa. Inhibition of IL-1 activity by this serum was neutralized by anti-mouse α＿1-antitrypsin (α＿1-AT) antiserum to a significant extent. Furthermore, mouse splenocytes cultured with several concentrations of EM produced higher levels of IL-2 upon stimulation with concanavaline A (Con A). IL-2 production by splenocytes increased in proportion to increasing doses of EM (2-5 μg/ml), but EM suppressed IL-2 production at doses higher than 10 μg/ml. These results were consistent with the results of Con A-induced blastogenesis of splenocytes. Additionally, IL-1 production by PMN and alveolar macrophage was also enhanced by EM in the dose range of 2-5 μg/ml. To analyze the kinetics of production of IL-1 suppressive factor in serum, supernatant fluids from cultures of mouse hepatocytes with EM were tested for α＿1-AT activity and IL-1 inhibitory activity. Upon stimulation with IL-1, cultured hepatocytes produced α＿1-AT in the presence of EM (5-10 μg/ml) higher than did hepatocytes cultured without EM. Furthermore, supernatant fluids of hepatocytes cultured with EM exhibited suppressive activity to IL-1. These results suggest that EM can exert regulatory functions in host defense mechamsms.

The present study was carried out to analyze functions of polymorphonuclear leukocyte (PMN) in the peripheral blood of patients with chronic infections in the lower respiratory tract after long-term treatment with erythromycin (EM). The number of total leukocytes was significantly higher in patients than in healthy controls before treatment, and it returned to normal levels after a 3-month treatment. Phagocytic activity of PMN was determined by measuring the intensity of luminol-mediated chemiluminescence. EM treatment reduced chemiluminescence of PMN in patients to a significant extent, in proportion to the improvement of clinical symptoms. Intracellular production of elastase, which was recognized as one of the predisposing factors in tissue damage of the respiratory tract, did not increase in PMN of patients throughout observation, but extracellular release of elastase from PMN upon stimulation with interleukin 1 (IL-1) was much reduced by EM treatment. Furthermore, serum obtained from EM-treated patients exhibited significanty higher levels of anti-elastase activity as compared with serum of healthy controls. The anti-elastase activity in EM-treated patients was completely neutralized by monoclonal antibody to human α＿1-antitrypsin. These results suggest that EM can modify PMN functions and also increase the anti-elastase activity in serum.

In this work, the effect of aqueous and methanolic extracts of the plants species Critoniella acuminata, Salvia rubescens, Phenax rugosus (Poir.) Wedd and Tabebuia chrysanta G. on the enzymes elastase and myeloperoxidase, involved in degranulation leukocyte process, was evaluated, identifying the potential direct inhibitory effect on the enzyme and/or inhibition of the desgranulation of polymorphonuclear neutrophils. Extracts of Critoniella acuminata andSalvia rubescens presented effects on the degranulation process and the inhibition of the enzyme elastase and myeloperoxidase; the extracts of Phenax rugosus do not showed significant effect. Tabebuia chrysanta methanolic extract only showed effect on inhibition of elastase activity., Se evaluó el efecto de extractos metanólicos y acuosos de las especies vegetales Critoniella acuminata, Salvia rubescens, Phenax rugosus (Poir.) Wedd y Tabebuia chrysanta G. Nicholson sobre las enzimas elastasa y mieloperoxidasa, relacionadas con el proceso de desgranulación leucocitaria, y se determinó el potencial efecto inhibitorio directo sobre la enzima o la inhibición de la desgranulación de los neutrófilos polimorfonucleares. Los extractos de Critoniella acuminata y Salvia rubescens presentaron efectos sobre el proceso de desgranulación y la actividad de las enzimas mieloperoxidasa y elastasa; en el caso de los extractos de Phenax rugosus, estos no mostraron un efecto significativo sobre ninguna de las enzimas. De la especieTabebuia chrysanta solamente el extracto metanólico mostró efecto sobre la inhibición de la actividad elastasa.

Albumin is the most abundant protein found in healing wounds. Traditional and chromatographic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressings is adsorption and accumulation of proteins like albumin at the solid-liquid interface of the biological fluid and wound dressing fiber. To better understand the effect of fiber charge and molecular modifications in cellulose-containing fibers on the binding of serum albumin as observed in protease sequestrant dressings, albumin binding to modified cotton fibers was compared with traditional and chromatographic isotherms. Modified cotton including carboxymethylated, citrate-crosslinked, dialdehyde and phosphorylated cotton, which sequester elastase and collagenase, were compared for their albumin binding isotherms. Albumin isotherms on citrate-cellulose, cross-linked cotton demonstrated a two-fold increased binding affinity over untreated cotton. A comparison of albumin binding between traditional, solution isotherms and chromatographic isotherms on modified cellulose yielded similar equilibrium constants. Application of the binding affinity of albumin obtained in the in vitro protein isotherm to the in vivo wound dressing uptake of the protein is discussed. The chromatographic approach to assessment of albumin isotherms on modified cellulose offers a more rapid approach to evaluating protein binding on modified cellulose over traditional solution approaches.

Målet med studien var att undersöka om parodontala parametrar och elastas i gingivalvätska (GCF) skilde sig hos individer med alfa-1-antrypsin-brist (AAT-brist) jämfört med individer med normal AAT-halt. I studien ingick 30 individer, varav 20 med allvarlig AAT-brist, fenotyp PiZZ. Tio individer med AAT- brist led av kronisk obstruktiv lungsjukdom (KOL) (grupp 1) och 10 var symptomfria (grupp 2). Tio individer med normal AAT-halt, fenotyp PiMM (grupp 3), utgjorde kontrollgrupp och rekryterades från en allmäntandvårdsklinik. Undersökningen bestod av insamling av GCF, gingivalindex (GI), plackindex (PlI), fickdjupsmätning (PPD) och röntgen. GCF samlades in med hjälp av pappers-strips (Periopaper®). AAT i plasma mättes med nefelometri och AAT i GCF mättes med ELISA. Elastasaktivitet och proteinmängd i GCF bestämdes med spektrofotometri. Medelvärden för GI, PlI, PPD och röntgenmätningar visade inga statistiskt signifikanta skillnader mellan grupperna. AAT i plasma och GCF visade mycket låga värden i grupp 1 och 2 utan några signifikanta skillnader mellan grupperna men en signifikant skillnad i jämförelse med grupp 3. Elastas i gingivalvätska visade inga skillnader mellan de tre grupperna. Sammanfattningsvis visade varken parodontala värden eller elastas i GCF några skillnader hos individer med AAT- brist, fenotyp PiZZ, jämfört med individer med normal AAT-halt, fenotyp PiMM, i detta material., The aim of this study was to investigate if periodontal parameters and elastase in gingival crevicular fluid (GCF) are different in alpha-1-antitrypsin deficient (AATD) subjects compared to subjects with normal AAT level. Thirty subjects were included, 20 of whom with severe AATD, phenotype PiZZ. Ten AATD subjects suffered from chronic obstructive pulmonary disease (COPD, group 1) and 10 were asymptomatic (group 2). Ten control subjects, phenotype PiMM, (group 3) were recruited from a public dental clinic. The examination comprised of sampling of GCF, Gingival Index (GI), Plaque Index (PlI), probing pocket depth (PPD) and radiography. GCF was collected with paper strips (Periopaper®). Plasma AAT concentration was measured by nephelometry and AAT in GCF with ELISA. Elastase activity and protein in GCF were determined by spectrophotometry. The mean values for GI, PlI, PPD and the radiological measurements did not show any statistically significant differences between the groups. AAT in plasma and GCF demonstrated very low values in groups 1 and 2 with no significant difference between these groups but a statistical difference in comparison with group 3. Elastase in GCF did not show any difference between the three groups. In conclusion, neither the periodontal parameters nor the elastase in GCF were different in AATD subjects, phenotype PiZZ, when compared to subjects with normal AAT level, phenotype PiMM, in this material.

Målet med studien var att undersöka om parodontala parametrar och elastas i gingivalvätska (GCF) skilde sig hos individer med alfa-1-antrypsin-brist (AAT-brist) jämfört med individer med normal AAT-halt. I studien ingick 30 individer, varav 20 med allvarlig AAT-brist, fenotyp PiZZ. Tio individer med AAT- brist led av kronisk obstruktiv lungsjukdom (KOL) (grupp 1) och 10 var symptomfria (grupp 2). Tio individer med normal AAT-halt, fenotyp PiMM (grupp 3), utgjorde kontrollgrupp och rekryterades från en allmäntandvårdsklinik. Undersökningen bestod av insamling av GCF, gingivalindex (GI), plackindex (PlI), fickdjupsmätning (PPD) och röntgen. GCF samlades in med hjälp av pappers-strips (Periopaper®). AAT i plasma mättes med nefelometri och AAT i GCF mättes med ELISA. Elastasaktivitet och proteinmängd i GCF bestämdes med spektrofotometri. Medelvärden för GI, PlI, PPD och röntgenmätningar visade inga statistiskt signifikanta skillnader mellan grupperna. AAT i plasma och GCF visade mycket låga värden i grupp 1 och 2 utan några signifikanta skillnader mellan grupperna men en signifikant skillnad i jämförelse med grupp 3. Elastas i gingivalvätska visade inga skillnader mellan de tre grupperna. Sammanfattningsvis visade varken parodontala värden eller elastas i GCF några skillnader hos individer med AAT- brist, fenotyp PiZZ, jämfört med individer med normal AAT-halt, fenotyp PiMM, i detta material., The aim of this study was to investigate if periodontal parameters and elastase in gingival crevicular fluid (GCF) are different in alpha-1-antitrypsin deficient (AATD) subjects compared to subjects with normal AAT level. Thirty subjects were included, 20 of whom with severe AATD, phenotype PiZZ. Ten AATD subjects suffered from chronic obstructive pulmonary disease (COPD, group 1) and 10 were asymptomatic (group 2). Ten control subjects, phenotype PiMM, (group 3) were recruited from a public dental clinic. The examination comprised of sampling of GCF, Gingival Index (GI), Plaque Index (PlI), probing pocket depth (PPD) and radiography. GCF was collected with paper strips (Periopaper®). Plasma AAT concentration was measured by nephelometry and AAT in GCF with ELISA. Elastase activity and protein in GCF were determined by spectrophotometry. The mean values for GI, PlI, PPD and the radiological measurements did not show any statistically significant differences between the groups. AAT in plasma and GCF demonstrated very low values in groups 1 and 2 with no significant difference between these groups but a statistical difference in comparison with group 3. Elastase in GCF did not show any difference between the three groups. In conclusion, neither the periodontal parameters nor the elastase in GCF were different in AATD subjects, phenotype PiZZ, when compared to subjects with normal AAT level, phenotype PiMM, in this material.

Although the adverse health effects of nanoparticles have been proposed and are being clarified, their aggravating effects on pre-existing pathological conditions have not been fully investigated. In this review, we provide insights into the immunotoxicity of both airborne and engineered nanoparticles as an exacerbating factor on hypersusceptible subjects, especially those with immune-mediated pulmonary inflammation, using our in vivo experimental model. First, we exhibit the effects of nanoparticles on pulmonary inflammation induced by bacterial endotoxin (lipopolysaccharide: LPS) as a disease model in innate immunity, and demonstrate that nanoparticles instilled through both an intratracheal tube and an inhalation system can exacerbate the lung inflammation. Second, we introduce the effects of nanoparticles on allergic pulmonary inflammation as a disease model in adaptive immunity, and show that repetitive pulmonary exposure to nanoparticles has aggravating effects on allergic inflammation, including adjuvant effects on Th2-milieu. Third, we show that very small nanoparticle exposure exacerbates emphysematous pulmonary inflammation, which is concomitant with enhanced lung expression of proinflammatory molecules (including those that are innate immunity related). Taken together, nanoparticle exposure may synergistically facilitate pathological pulmonary inflammation via both innate and adaptive immunological impairment.

Although the adverse health effects of nanoparticles have been proposed and are being clarified, their aggravating effects on pre-existing pathological conditions have not been fully investigated. In this review, we provide insights into the immunotoxicity of both airborne and engineered nanoparticles as an exacerbating factor on hypersusceptible subjects, especially those with immune-mediated pulmonary inflammation, using our in vivo experimental model. First, we exhibit the effects of nanoparticles on pulmonary inflammation induced by bacterial endotoxin (lipopolysaccharide: LPS) as a disease model in innate immunity, and demonstrate that nanoparticles instilled through both an intratracheal tube and an inhalation system can exacerbate the lung inflammation. Second, we introduce the effects of nanoparticles on allergic pulmonary inflammation as a disease model in adaptive immunity, and show that repetitive pulmonary exposure to nanoparticles has aggravating effects on allergic inflammation, including adjuvant effects on Th2-milieu. Third, we show that very small nanoparticle exposure exacerbates emphysematous pulmonary inflammation, which is concomitant with enhanced lung expression of proinflammatory molecules (including those that are innate immunity related). Taken together, nanoparticle exposure may synergistically facilitate pathological pulmonary inflammation via both innate and adaptive immunological impairment.

[EN] Tissue injury during surgery as well as anaesthesia can lead to modulation of neutrophil function. The aim of this study was to assess how two types of anaesthesia, i.e. combination of ketamine/midazolam/propofol (Group I) and ketamine/propofol (Group II) influenced neutrophil function during and 24 h after operation in rabbits. Blood samples were taken prior to anaesthesia, at 30 and 60 min, and 24 h after starting the experiment. At these time points, degranulation and free radical generation were assessed in both groups. After 30 min of anaesthesia, we observed a decrease in elastase release from 50.96±0.71% to 26.52±4.85% in Group I and from 51.00±0.7% to 41.00±5.48% in Group II, respectively. In subsequent measurements, the elastase level increased to values lower than before starting anaesthesia. The myeloperoxidase (MPO) release decreased significantly after 30 min of anaesthesia in Group I, then increased to the value similar to the pre-anaesthetic level after 60 min. MPO level in Group II also decreased after 30 min but to a lesser degree, and then after 1 h MPO slightly exceeded the pre-anaesthetic level. In both groups, 24 h after the start anaesthesia the values obtained decreased, but remained at a level higher than at the time before anaesthesia. We observed a decrease in alkaline phosphatase (ALKP) levels in both studied groups after 30 min of anaesthesia (from 24.77±5.9% to 15.7±2.1% and from 23.6±1.14% to 10.6±0.89%, in Groups I and II, respectively), with a subsequent increase after 60 min. In Group I, we observed a significant (P<0.01) decrease in NO production 30 min after the start of anaesthesia. After increasing to the pre-anaesthetic level, the NO level was almost constant in both subsequent measurements. In Group II, NO level decreased 30 min after the onset of anaesthesia and then increased, reaching a slightly higher level than at the start of anaesthesia. The superoxide generation by neutrophils from rabbits of both groups decreased

The lysosomal enzyme elastase was earlier shown to cleave the complement molecule C3. During somepreliminary experiments on the interactions of certain pathogenic bacteria with the innate defencemechanisms, we observed C3 cleavage, in the presence of elastase, to fragments not previouslydescribed. To elucidate this proteolytic reaction, the present study was conducted. Degradation of C3in mixtures with elastase or cathepsin G was detected by an immunoblot procedure using anti-C3c andanti-C3d antibodies after separating the proteins by SDS-PAGE. Certain C3 fragments were analysedfor amino acid sequence. The results revealed the existence of a cleavage site for elastase at the positionalanine1350 ⁄ lysine1351 of the C3 molecule, which has not been previously described. The fragmentresulted from this cleavage has a size of about 39 kDa and it contains a part or the whole of C3d. Thiscleavage was distinct from the one previously described at position 987 ⁄ 988, which gives a 34 kDaC3d-containing fragment.

[EN] Tissue injury during surgery as well as anaesthesia can lead to modulation of neutrophil function. The aim of this study was to assess how two types of anaesthesia, i.e. combination of ketamine/midazolam/propofol (Group I) and ketamine/propofol (Group II) influenced neutrophil function during and 24 h after operation in rabbits. Blood samples were taken prior to anaesthesia, at 30 and 60 min, and 24 h after starting the experiment. At these time points, degranulation and free radical generation were assessed in both groups. After 30 min of anaesthesia, we observed a decrease in elastase release from 50.96±0.71% to 26.52±4.85% in Group I and from 51.00±0.7% to 41.00±5.48% in Group II, respectively. In subsequent measurements, the elastase level increased to values lower than before starting anaesthesia. The myeloperoxidase (MPO) release decreased significantly after 30 min of anaesthesia in Group I, then increased to the value similar to the pre-anaesthetic level after 60 min. MPO level in Group II also decreased after 30 min but to a lesser degree, and then after 1 h MPO slightly exceeded the pre-anaesthetic level. In both groups, 24 h after the start anaesthesia the values obtained decreased, but remained at a level higher than at the time before anaesthesia. We observed a decrease in alkaline phosphatase (ALKP) levels in both studied groups after 30 min of anaesthesia (from 24.77±5.9% to 15.7±2.1% and from 23.6±1.14% to 10.6±0.89%, in Groups I and II, respectively), with a subsequent increase after 60 min. In Group I, we observed a significant (P<0.01) decrease in NO production 30 min after the start of anaesthesia. After increasing to the pre-anaesthetic level, the NO level was almost constant in both subsequent measurements. In Group II, NO level decreased 30 min after the onset of anaesthesia and then increased, reaching a slightly higher level than at the start of anaesthesia. The superoxide generation by neutrophils from rabbits of both groups decreased

[EN] Tissue injury during surgery as well as anaesthesia can lead to modulation of neutrophil function. The aim of this study was to assess how two types of anaesthesia, i.e. combination of ketamine/midazolam/propofol (Group I) and ketamine/propofol (Group II) influenced neutrophil function during and 24 h after operation in rabbits. Blood samples were taken prior to anaesthesia, at 30 and 60 min, and 24 h after starting the experiment. At these time points, degranulation and free radical generation were assessed in both groups. After 30 min of anaesthesia, we observed a decrease in elastase release from 50.96±0.71% to 26.52±4.85% in Group I and from 51.00±0.7% to 41.00±5.48% in Group II, respectively. In subsequent measurements, the elastase level increased to values lower than before starting anaesthesia. The myeloperoxidase (MPO) release decreased significantly after 30 min of anaesthesia in Group I, then increased to the value similar to the pre-anaesthetic level after 60 min. MPO level in Group II also decreased after 30 min but to a lesser degree, and then after 1 h MPO slightly exceeded the pre-anaesthetic level. In both groups, 24 h after the start anaesthesia the values obtained decreased, but remained at a level higher than at the time before anaesthesia. We observed a decrease in alkaline phosphatase (ALKP) levels in both studied groups after 30 min of anaesthesia (from 24.77±5.9% to 15.7±2.1% and from 23.6±1.14% to 10.6±0.89%, in Groups I and II, respectively), with a subsequent increase after 60 min. In Group I, we observed a significant (P<0.01) decrease in NO production 30 min after the start of anaesthesia. After increasing to the pre-anaesthetic level, the NO level was almost constant in both subsequent measurements. In Group II, NO level decreased 30 min after the onset of anaesthesia and then increased, reaching a slightly higher level than at the start of anaesthesia. The superoxide generation by neutrophils from rabbits of both groups decreased

[EN] Tissue injury during surgery as well as anaesthesia can lead to modulation of neutrophil function. The aim of this study was to assess how two types of anaesthesia, i.e. combination of ketamine/midazolam/propofol (Group I) and ketamine/propofol (Group II) influenced neutrophil function during and 24 h after operation in rabbits. Blood samples were taken prior to anaesthesia, at 30 and 60 min, and 24 h after starting the experiment. At these time points, degranulation and free radical generation were assessed in both groups. After 30 min of anaesthesia, we observed a decrease in elastase release from 50.96±0.71% to 26.52±4.85% in Group I and from 51.00±0.7% to 41.00±5.48% in Group II, respectively. In subsequent measurements, the elastase level increased to values lower than before starting anaesthesia. The myeloperoxidase (MPO) release decreased significantly after 30 min of anaesthesia in Group I, then increased to the value similar to the pre-anaesthetic level after 60 min. MPO level in Group II also decreased after 30 min but to a lesser degree, and then after 1 h MPO slightly exceeded the pre-anaesthetic level. In both groups, 24 h after the start anaesthesia the values obtained decreased, but remained at a level higher than at the time before anaesthesia. We observed a decrease in alkaline phosphatase (ALKP) levels in both studied groups after 30 min of anaesthesia (from 24.77±5.9% to 15.7±2.1% and from 23.6±1.14% to 10.6±0.89%, in Groups I and II, respectively), with a subsequent increase after 60 min. In Group I, we observed a significant (P<0.01) decrease in NO production 30 min after the start of anaesthesia. After increasing to the pre-anaesthetic level, the NO level was almost constant in both subsequent measurements. In Group II, NO level decreased 30 min after the onset of anaesthesia and then increased, reaching a slightly higher level than at the start of anaesthesia. The superoxide generation by neutrophils from rabbits of both groups decreased

[EN] Tissue injury during surgery as well as anaesthesia can lead to modulation of neutrophil function. The aim of this study was to assess how two types of anaesthesia, i.e. combination of ketamine/midazolam/propofol (Group I) and ketamine/propofol (Group II) influenced neutrophil function during and 24 h after operation in rabbits. Blood samples were taken prior to anaesthesia, at 30 and 60 min, and 24 h after starting the experiment. At these time points, degranulation and free radical generation were assessed in both groups. After 30 min of anaesthesia, we observed a decrease in elastase release from 50.96±0.71% to 26.52±4.85% in Group I and from 51.00±0.7% to 41.00±5.48% in Group II, respectively. In subsequent measurements, the elastase level increased to values lower than before starting anaesthesia. The myeloperoxidase (MPO) release decreased significantly after 30 min of anaesthesia in Group I, then increased to the value similar to the pre-anaesthetic level after 60 min. MPO level in Group II also decreased after 30 min but to a lesser degree, and then after 1 h MPO slightly exceeded the pre-anaesthetic level. In both groups, 24 h after the start anaesthesia the values obtained decreased, but remained at a level higher than at the time before anaesthesia. We observed a decrease in alkaline phosphatase (ALKP) levels in both studied groups after 30 min of anaesthesia (from 24.77±5.9% to 15.7±2.1% and from 23.6±1.14% to 10.6±0.89%, in Groups I and II, respectively), with a subsequent increase after 60 min. In Group I, we observed a significant (P<0.01) decrease in NO production 30 min after the start of anaesthesia. After increasing to the pre-anaesthetic level, the NO level was almost constant in both subsequent measurements. In Group II, NO level decreased 30 min after the onset of anaesthesia and then increased, reaching a slightly higher level than at the start of anaesthesia. The superoxide generation by neutrophils from rabbits of both groups decreased

[EN] Tissue injury during surgery as well as anaesthesia can lead to modulation of neutrophil function. The aim of this study was to assess how two types of anaesthesia, i.e. combination of ketamine/midazolam/propofol (Group I) and ketamine/propofol (Group II) influenced neutrophil function during and 24 h after operation in rabbits. Blood samples were taken prior to anaesthesia, at 30 and 60 min, and 24 h after starting the experiment. At these time points, degranulation and free radical generation were assessed in both groups. After 30 min of anaesthesia, we observed a decrease in elastase release from 50.96±0.71% to 26.52±4.85% in Group I and from 51.00±0.7% to 41.00±5.48% in Group II, respectively. In subsequent measurements, the elastase level increased to values lower than before starting anaesthesia. The myeloperoxidase (MPO) release decreased significantly after 30 min of anaesthesia in Group I, then increased to the value similar to the pre-anaesthetic level after 60 min. MPO level in Group II also decreased after 30 min but to a lesser degree, and then after 1 h MPO slightly exceeded the pre-anaesthetic level. In both groups, 24 h after the start anaesthesia the values obtained decreased, but remained at a level higher than at the time before anaesthesia. We observed a decrease in alkaline phosphatase (ALKP) levels in both studied groups after 30 min of anaesthesia (from 24.77±5.9% to 15.7±2.1% and from 23.6±1.14% to 10.6±0.89%, in Groups I and II, respectively), with a subsequent increase after 60 min. In Group I, we observed a significant (P<0.01) decrease in NO production 30 min after the start of anaesthesia. After increasing to the pre-anaesthetic level, the NO level was almost constant in both subsequent measurements. In Group II, NO level decreased 30 min after the onset of anaesthesia and then increased, reaching a slightly higher level than at the start of anaesthesia. The superoxide generation by neutrophils from rabbits of both groups decreased

Several species in the genus Arnica have been used in traditional medicine to treat inflammatory-related disorders. Extracts of twelve Arnica species and two species closely related to Arnica (Layia hieracioides and Madia sativa) were investigated for inhibition of human neutrophil elastase release and inhibition of transcription factor NF-κB. Statistical analyses reveal significant differences in inhibitory capacities between extracts. Sesquiterpene lactones of the helenanolide type, of which some are known inhibitors of human neutrophil elastase release and NF-κB, are present in large amounts in the very active extracts of A. montana and A. chamissonis. Furthermore, A. longifolia, which has previously not been investigated, shows a high activity similar to that of A. montana and A. chamissonis in both bioassays. Sesquiterpene lactones of the xanthalongin type are present in large amounts in A. longifolia and other active extracts and would be interesting to evaluate further.

In the present study, hypertrophic and metaplastic changes of goblet cells were induced in rat nasal epithelium by intranasal instillation of endotoxin or elastase. A significant increase in the amount of intraepithelial mucosubstance was observed after 24 hours during 3 days of instillation. The elastase-induced mucus production was not inhibited in neutrophil-depleted rats, but the endotoxin-induced change was significantly inhibited. Intranasal instillation of the neutrophil elastase inhibitor ONO-5046 partially inhibited the endotoxin-induced mucus production. Epithelial mucus secretion was evaluated by the temporary decrease in the amount of intraepithelial mucosubstance. The endotoxin-induced mucus secretion peaked 3 to 6 hours after intranasal instillation, coinciding with the peak of the intraepithelial neutrophil infiltration. The elastase-induced mucus secretion peaked 1 to 3 hours after intranasal instillation; intraepithelial neutrophil infiltration was not induced by elastase. These results indicate that neutrophil elastase is an important mediator of the intraepithelial mucus synthesis and secretion induced by endotoxin

The objective of this study was to quantify and compare the effects of sow¿s milk and 2 milk replacer diets (containing clotting or nonclotting protein sources) on exocrine pancreatic secretion, plasma cholecystokinin, and immunoreactive cationic trypsin in pigs. In addition, the relationship between exocrine pancreatic secretion and growth in milk-fed pigs was studied. In a changeover experiment, 9 chronically catheterized pigs of 6.6 ± 0.19 kg of BW were studied for 3 wk. Pigs were assigned to each of 3 diets. Exocrine pancreatic secretion was measured from the third to the seventh day on each diet. The protein content and trypsin activity of the pancreatic juice were measured. Blood samples were taken at 10 min before and after milk ingestion and were analyzed for cholecystokinin and immunoreactive cationic trypsin. Pancreatic protein and trypsin secretion did not differ between pigs fed sow¿s milk and those fed milk replacer, but the volume secreted was less for the pigs fed sow¿s milk (0.75 vs. 1.03 mL·kg¿1·h¿1; P <0.01). A postprandial response to milk intake was not observed. The 2 milk replacer diets did not affect exocrine pancreatic secretion differently. The average exocrine pancreatic secretion (volume, 0.94 mL·kg¿1·h¿1; protein, 4.28 mg·kg¿1·h¿1; trypsin, 1.65 U·kg¿1·h¿1) was intermediate between literature values for suckling and weaned pigs. Plasma cholecystokinin was elevated (18 pmol·L¿1) and showed low correlations with the pancreatic secretion traits. Plasma immunoreactive cationic trypsin was not significantly related to any of the pancreatic secretion traits and should therefore not be used as an indicator for exocrine pancreatic function in milk-fed pigs. Exocrine pancreatic secretion varied substantially among individual pigs (protein, 0.22 to 13.98 mg·kg¿1·h¿1). Pancreatic protein and trypsin secretion showed a positive, nonlinear relationship with performance traits. It was concluded that neither specific sow¿s milk ingredients nor the prote

Uncontrolled cell proliferation is one of the hallmarks of cancer and the transition from the G1 to S phase is the most commonly reported cell cycle abnormality in tumors. It has been shown that the oncogenic activity of G1 cyclin E (CCNE) can be amplified by generating hyperactive low molecular weight forms (LMW) through elastase-mediated proteolytic processing. Neutrophil elastase (NE) and proteinase 3 (PR3) are 2 proteases that are aberrantly expressed in breast cancer cells and seem to be involved in cell proliferation. In this study, we evaluated the effect of the expression of these 2 proteases in addition to 2 potential intracellular targets of NE (CCNE1 and CCNE2) on clinical outcome in a population of 205 primary breast cancer patients. By univariate analysis, CCNE1, CCNE2, estrogen receptor and grade significantly predicted relapse free interval (RFI). NE and PR3 did not achieve statistical significance. In a multivariate analysis, elevated CCNE2 [hazard ratio (HR) 2.10, p = 0.008] predicted shorter RFI. In subgroup analyses of the tamoxifen-only treated patients, high CCNE1 levels predicted treatment resistance, while high levels of CCNE2 were associated with poor RFI in untreated patients. Investigation of the relationship between CCNE1, CCNE2 and NE did not show any impact on RFI. To conclude, this study was the first to evaluate these markers at the mRNA level by RT-PCR in a series of primary breast cancer patients, and our results confirmed the impact of high CCNE levels on clinical outcome in systemically untreated and of CCNE1 in tamoxifen-only treated early breast cancer patients., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Uncontrolled cell proliferation is one of the hallmarks of cancer and the transition from the G1 to S phase is the most commonly reported cell cycle abnormality in tumors. It has been shown that the oncogenic activity of G1 cyclin E (CCNE) can be amplified by generating hyperactive low molecular weight forms (LMW) through elastase-mediated proteolytic processing. Neutrophil elastase (NE) and proteinase 3 (PR3) are 2 proteases that are aberrantly expressed in breast cancer cells and seem to be involved in cell proliferation. In this study, we evaluated the effect of the expression of these 2 proteases in addition to 2 potential intracellular targets of NE (CCNE1 and CCNE2) on clinical outcome in a population of 205 primary breast cancer patients. By univariate analysis, CCNE1, CCNE2, estrogen receptor and grade significantly predicted relapse free interval (RFI). NE and PR3 did not achieve statistical significance. In a multivariate analysis, elevated CCNE2 [hazard ratio (HR) 2.10, p = 0.008] predicted shorter RFI. In subgroup analyses of the tamoxifen-only treated patients, high CCNE1 levels predicted treatment resistance, while high levels of CCNE2 were associated with poor RFI in untreated patients. Investigation of the relationship between CCNE1, CCNE2 and NE did not show any impact on RFI. To conclude, this study was the first to evaluate these markers at the mRNA level by RT-PCR in a series of primary breast cancer patients, and our results confirmed the impact of high CCNE levels on clinical outcome in systemically untreated and of CCNE1 in tamoxifen-only treated early breast cancer patients., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes. (c) 2006 Elsevier B.V. All rights reserved.

A series of 1,4-bis(alkoxycarbonyl)azetidin-2-ones, designed as potential suicide-inhibitors of serine proteases, has been synthesized and evaluated against porcine pancreatic elastase (PPE). The most active compound (K(i)similar to10 muM; reversible inhibitor) was equipped with phenethyloxycarbonyl and benzyloxycarbonyl side-chains at positions N1 and C4, respectively, with the (S)-configuration. H-1 NMR spectroscopic analysis of the reaction mixtures showed that the ester function is preferentially hydrolyzed, in both chemical and enzyme-catalyzed reactions, with regard to the azetidinone and urethane functions. Considering the three potentially sensitive carbonyl functions and the two stereoisomers, ab initio calculations were performed to determine the energetic barriers required to reach the transition state structures of hydrolysis in a model of the enzyme pocket. (C) 2002 Elsevier Science Ltd. All rights reserved.

A series of monocyclic azetidinones were prepared, bearing, at position C-3, an acetylamino or a bromo substituent, at position N-1, a carboxymethyl group protected as p-nitrobenzyl ester (PNB) and alpha-functionalized with a potential leaving group (LG). These structures were designed as potential suicide-inhibitors of enzymes containing a serine nucleophile in their active site. The beta-lactam ring of these molecules was found to be stable in phosphate buffer (pH 7.5), but the PNB ester was rapidly cleaved. This constitutes a practical method of in situ deprotection. Depending on the nature of the LG group on the carboxymethyl chain, substitution of this group (LG = F) or decarboxylation (LG = SO2Ph) was observed under hydrolytic conditions. The 1,3-disubstituted azetidinones were inactive against beta-lactamases of classes A, B, C, and D. Three compounds behaved as weak reversible inhibitors of porcine pancreatic elastase (PPE). (C) 1999 Elsevier Science Ltd. All rights reserved.

Increased levels of solubile intercellular adhesion molecole-1, neutrophils and elastase in the lung of preterm infants with bronchopulmonary displasia.

Ischaemia is a common clinical event leading to local and remote injury. Evidence indicates that tissue damage is largely caused by activated neutrophils which accumulate when the tissue is perfused. If the area of ischaemic tissue is large, neutrophils also sequester in the lungs, inducing non-cardiogenic pulmonary oedema. Ischaemia-reperfusion injury is initiated by production of reactive oxygen species which initially appear responsible for the generation of chemotactic activity for neutrophils. Later, once adherent to endothelium, neutrophils mediate damage by secretion of additional reactive oxygen species as well as proteolytic enzymes, in particular elastase. Therapeutic options for limiting ischaemia-reperfusion injury include inhibition of oxygen radical formation, pharmacological prevention of neutrophil activation and chemotaxis, and also use of monoclonal antibodies which prevent neutrophil-endothelial adhesion, a prerequisite for injury.

To date, using high-resolution X-ray crystallography, we have determined the structures of 10 small molecule ligands or inhibitors to the serine protease, porcine pancreatic elastase. These complexes reveal details of binding or steps on the catalytic pathway (Michaelis complex, acyl intermediate, transition state intermediate) that now can be used to design novel, more specific inhibitors or intermediates. Keywords: Enzyme inhibitors.

Předkládaná práce se věnuje problematice aneurysmat břišní aorty (AAA) s přihlédnutím k možnosti využití mechanických zkoušek tkání aortálních výdutí ke zlepšení úrovně jejich konstitutivních modelů. Úvodní část se zabývá úvodem do problematiky, popisu struktury stěny zdravé aorty, jejím hlavním komponentám a degenerativním změnám, které vedou až ke vzniku AAA. Na tento úvod navazuje stručný exkurz do konstitutivního modelování, který se úzce zaměřuje na popis modelů používaných pro popis mechanického chování měkkých tkání. Teoretická část je poté doplněna užším výběrem konstitutivních modelů používaných pro modelování stěny aorty a intraluminálního trombu, společně s publikovanými výsledky, které jsou v závěru této části zhodnoceny a diskutovány. Stěžejní část práce je věnována zkouškám mechanických vlastností arteriálních tkání. Nejprve je představena metodika testování společně s popisem úprav, kterými prošlo vybavení laboratoře, společně s testovacím zařízením. Dále je pozornost zaměřena na výsledky mechanických zkoušek intraluminálního trombu, kde jsou prezentovány výsledky jak z jednoosých tahových zkoušek, tak i z ekvibiaxiálního testování a je zde vyšetřován vliv vzdálenosti ILT od krevního řečiště na mechanické vlastnosti trombu. Další oblast, které je věnována pozornost, je vyšetření vlivu elastázy na změnu mechanických vlastností prasečí aorty. V tomto případě jsou vzorky prasečích aort experimentálně testovány pouze pomocí dvouosých zkoušek a je zde analyzována doba působení elastázy na změnu mechanických vlastností tak, aby výsledná tkáň měla podobnou deformačně-napěťovou odezvu jako tkáň aneurysmatická. Na závěr jsou shrnuty dosažené výsledky experimentálního měření, limitace a také navrženy další možné směry výzkumu., This paper deals with the problem of abdominal aortic aneurysms (AAA), taking into account the possibility of using mechanical tests of aortic tissues for improvement of level of their constitutive models. First part of thesis deals with the introduction into the problem, description of the structure of the wall of the healthy aorta, its main components and the degenerative changes which lead to formation of AAA. This is followed by a brief excursion into constitutive modeling, which focuses closely on the description of the models used to describe the mechanical behavior of soft tissues. The theoretical part is then supplemented by a narrower selection of constitutive models used for modeling aortic wall and intraluminal thrombus, together with published results, which are reviewed and discussed at the end of this section. The main part of this thesis is devoted to tests of mechanical properties of arterial tissues. First, the methodology is presented together with the description of the customizations of the laboratory equipments together with the test rig. In addition, attention is focused on the results of mechanical tests of intraluminal thrombus, where the results of both uniaxial tensile tests and equbiaxial testing are presented. Also the influence of distance ILT from the lumen on the mechanical properties of the thrombus is examined. Another area of interest is the investigation of the effect of elastase on the chnage of mechanical properties of pig aorta. In this case, porcine aortas are experimentally tested only by biaxial testing, and the time of elastase action to alter the mechanical properties is analyzed so that the resulting tissue has a similar stress-strain response as aneurysmal tissue. Finally, the results of experimental measurements, limitations and other possible ways of research are summarized.

Production and release of proteases is among the strategies by which pathogenic bacteria increase their virulence. Moreover, these bacteria are also known to produce a cocktail of enzymes to degrade and inhibit antimicrobial peptides. Cyclotides, on the other hand, are circular plant mini-proteins having a unique cyclic topology (termed as the cyclic cystine knot, CCK) which endows them with exceptional stability. Their ultra-stable framework has attracted interest in drug design as a scaffold to otherwise susceptible drugs including antibacterial peptides. We have determined the stability of three peptides with the CCK motif, i.e., the cyclotides cycloviolacin O2, kalata B1 and MCoTI-II in elastase and aureolysin and also in the supernatant cultures of P. aeruginosa and S. aureus. The cyclotides have resisted enzymatic degradation both from the specific proteases and the bacterial cultures, with MCoTI-II being the least stable of the three. The cyclotides find application in drug engineering as their stable framework can be used as a scaffold or as a template for unstable drugs. The findings of the current study further support this idea; the cyclotide structure can be utilized as a novel strategy in enhancing the stability of antibacterial peptides.

Předkládaná práce se věnuje problematice aneurysmat břišní aorty (AAA) s přihlédnutím k možnosti využití mechanických zkoušek tkání aortálních výdutí ke zlepšení úrovně jejich konstitutivních modelů. Úvodní část se zabývá úvodem do problematiky, popisu struktury stěny zdravé aorty, jejím hlavním komponentám a degenerativním změnám, které vedou až ke vzniku AAA. Na tento úvod navazuje stručný exkurz do konstitutivního modelování, který se úzce zaměřuje na popis modelů používaných pro popis mechanického chování měkkých tkání. Teoretická část je poté doplněna užším výběrem konstitutivních modelů používaných pro modelování stěny aorty a intraluminálního trombu, společně s publikovanými výsledky, které jsou v závěru této části zhodnoceny a diskutovány. Stěžejní část práce je věnována zkouškám mechanických vlastností arteriálních tkání. Nejprve je představena metodika testování společně s popisem úprav, kterými prošlo vybavení laboratoře, společně s testovacím zařízením. Dále je pozornost zaměřena na výsledky mechanických zkoušek intraluminálního trombu, kde jsou prezentovány výsledky jak z jednoosých tahových zkoušek, tak i z ekvibiaxiálního testování a je zde vyšetřován vliv vzdálenosti ILT od krevního řečiště na mechanické vlastnosti trombu. Další oblast, které je věnována pozornost, je vyšetření vlivu elastázy na změnu mechanických vlastností prasečí aorty. V tomto případě jsou vzorky prasečích aort experimentálně testovány pouze pomocí dvouosých zkoušek a je zde analyzována doba působení elastázy na změnu mechanických vlastností tak, aby výsledná tkáň měla podobnou deformačně-napěťovou odezvu jako tkáň aneurysmatická. Na závěr jsou shrnuty dosažené výsledky experimentálního měření, limitace a také navrženy další možné směry výzkumu., This paper deals with the problem of abdominal aortic aneurysms (AAA), taking into account the possibility of using mechanical tests of aortic tissues for improvement of level of their constitutive models. First part of thesis deals with the introduction into the problem, description of the structure of the wall of the healthy aorta, its main components and the degenerative changes which lead to formation of AAA. This is followed by a brief excursion into constitutive modeling, which focuses closely on the description of the models used to describe the mechanical behavior of soft tissues. The theoretical part is then supplemented by a narrower selection of constitutive models used for modeling aortic wall and intraluminal thrombus, together with published results, which are reviewed and discussed at the end of this section. The main part of this thesis is devoted to tests of mechanical properties of arterial tissues. First, the methodology is presented together with the description of the customizations of the laboratory equipments together with the test rig. In addition, attention is focused on the results of mechanical tests of intraluminal thrombus, where the results of both uniaxial tensile tests and equbiaxial testing are presented. Also the influence of distance ILT from the lumen on the mechanical properties of the thrombus is examined. Another area of interest is the investigation of the effect of elastase on the chnage of mechanical properties of pig aorta. In this case, porcine aortas are experimentally tested only by biaxial testing, and the time of elastase action to alter the mechanical properties is analyzed so that the resulting tissue has a similar stress-strain response as aneurysmal tissue. Finally, the results of experimental measurements, limitations and other possible ways of research are summarized.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2018., Tissue infiltration and elevated peripheral circulation of granulocytic myeloid-derived cells is associated with poor outcomes in prostate cancer and other malignancies. Although myeloid-derived cells have the ability to suppress T-cell function, little is known about the direct impact of these innate cells on prostate tumor growth. Here, we report that granulocytic myeloid-derived suppressor cells (MDSC) are the predominant tumor-infiltrating cells in prostate cancer xenografts established in athymic nude mice. MDSCs significantly increase in number in the peripheral circulation as a function of xenograft growth and are successfully depleted in vivo by Gr-1 antibody treatment. Importantly, MDSC depletion significantly decreases xenograft growth. We hypothesized that MDSCs might exert their pro-tumorigenic actions in part through neutrophil elastase (NE). Indeed, we demonstrate that NE is expressed by infiltrating immune cells and enzymatically active in prostate cancer xenografts and tumors of prostate-specific Ptennull mice. Importantly, treatment with sivelestat, a small-molecule inhibitor specific for NE, significantly decreases xenograft and Pten-null tumor growth. Mechanistically, NE activates MAPK signaling and induces MAPK-dependent transcription of the proliferative gene cFOS in prostate cancer cells. Functionally, NE stimulates proliferation, migration, and invasion of prostate cancer cells in vitro. Immunohistochemistry on human prostate cancer biopsies reveals expression of NE by infiltrating CD33+ MDSCs, which predict poor patient prognosis. Interestingly, reduced expression of SERPINB1, an endogenous NE inhibitor, is also correlated with diminished survival in several cancers. Accordingly, examining gene expression datasets of human prostate cancer, we find reduced SERPINB1 in metastatic and locally advanced disease compared to normal tissue, with low expression predicting reduced progression-free survival. Moreover, SERPINB1 is down-regulated in Pten

Ninety children, aged from 2 month to 3 years, with thymomegaly and 25 aged-matched controls were studied. Most children with thymomegaly had disturbances of psychomotor development. Depending on their types, the cohort of children was stratified into 4 subgroups: 1(st) - 36 patients (40%) with schizotypal signs; 2(nd) - 30 hyperactive children (33%); 3(rd) - 19 children with hyperthymia signs (21%); 4(th) - 5 normal children (6%). The deviations of locomotion and psychiatric development were correlated with the extent of thymus enlargement and activation of innate and adaptive immunity.