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Objectives: Alpha-1-Antitrypsin (AAT) is one of the major plasmatic protease inhibitors. In the last decade, an association between Alpha-1-Antitrypsin Deficiency (AATD) and Abdominal Aortic Aneurysms (AAA) has been hypothesized. Multiple factors may be involved in AAA's etiopathogenesis, and an underlying structural defect of the extracellular matrix (ECM) is always present. AATD could be a reasonable risk factor for AAA because it is related to protease/antiprotease imbalance and enhanced ECM degradation of the vessel wall. Methods: We performed genotyping of 138 patients hospitalized in the Vascular Surgery Division of the ASST-Spedali Civili di Brescia, Italy, for non-traumatic rupture of AAA. The second purpose was to observe the distribution of main non-genetic risk factors for AAA between patients with and without AATD. Results: Out of 138 patients, 22 were found with AATD: 16 MS, 1 SS, 3 MZ, and 2 with a new rare AAT variant. When compared to the general Italian population, our cohort's frequency of deficient S allele was significantly higher (7.8 vs 2.2% respectively, P0.05). Although we found no differences in age, gender, hypertension, diabetes, and smoke habits between AAA patients with and without AATD, hyperlipidemia was significantly less frequent in patients with AATD (46.4 vs 12.5% respectively, P

Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory diseases. However, their therapeutical exploitation is slowed down by severe adverse manifestations that can hardly be foreseen, mainly due to incomplete knowledge of how HDIs impact the delicate balance of inflammatory mediators. In this work, we characterized the effects of the HDI trichostatin A (TSA) on the expression of TNFAIP3, which is a crucial inhibitor of the classical NF-kB pathway and an LPS-induced negative feedback regulator. The accumulation of TNFAIP3 mRNA after LPS stimulation showed biphasic behavior, with one wave within the first hour of stimulation and a second wave several hours later, which were both reduced by TSA. By using inhibition and knockdown approaches, we identified two temporally and mechanistically distinct modes of action. The first wave of TNAIP3 accumulation was directly blunted by the histone deacetylase (HDAC) blockade. By contrast, the second wave was decreased mainly because of the lack of endogenous TNF-α induction, which, in turn, depended on the intact HDAC activity. In both cases, class I HDACs appeared to play a nonredundant role, with HDAC3 required, but not sufficient, for TNF-α and TNFAIP3 induction. In addition to TNFAIP3, TNF-α is known to induce many response genes that orchestrate the inflammatory cascade. Thus, suppression of TNF-α may represent a general mechanism through which HDIs regulate a selected set of target genes.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001—is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide—a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

The inflammatory and IFN pathways of innate immunity play a key role in both resistance and pathogenesis of Coronavirus Disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-Associated Molecular Patterns (SAMPs) remain to be completely defined. Here we identify single-stranded RNA (ssRNA) fragments from SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and functions, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identify TLR7/8 as crucial cellular sensors of ssRNAs encoded by SARS-CoV-2 involved in host resistance and disease pathogenesis of COVID-19.

Orientador: José de Assis Fonseca Faria Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: O sódio é um elemento essencial na alimentação, contudo, o seu consumo em excesso poderá ter efeitos nocivos para a saúde, tais como o aumento da pressão sanguínea e a redução da absorção de cálcio. Devido a estes efeitos, a redução do teor de sódio nos alimentos tem sido uma das prioridades nas campanhas realizadas por órgãos governamentais, nomeadamente no setor de laticínios onde o queijo é considerado um gênero alimentício com elevados níveis deste elemento. Dessa forma, o presente trabalho objetivou avaliar o efeito da substituição parcial de NaCl por KCl e MgCl2 nos parâmetros de qualidade do queijo cottage probiótico durante 28 dias de estocagem refrigerada e verificar a viabilidade da cultura starter e das culturas probióticas utilizadas para o processamento do queijo. Além disso, objetivou-se avaliar a aceitação sensorial para identificar direções para a melhoria de atributos sensoriais do produto perante consumidores. Com base nos resultados obtidos observou-se que o ideal de gosto salgado para o queijo cottage probiótico com NaCl foi de 1,12% (p/p), e as concentrações equivalentes em salinidade para todos os sais hipossódicos avaliados mantiveram-se as mesmas do NaCl (1,12% p/p). As contagens de Lactobacillus acidophilus e Bifidobacterium animalis subsp. lactis variaram de 7,74 a 6,20 e 8,58 a 7,93 log (UFC. g-1), respectivamente no primeiro e no último dia de estocagem, valores esses suficientes para benefícios à saúde humana. Porém, as contagens da cultura starter permaneceram baixas com valores inferiores a 3,0 log UFC. g-1 para todas as formulações. Com relação aos ácidos orgânicos, o ácido lático foi o mais encontrado em todas as formulações, seguido pelo ácido cítrico e ácido acético. Quanto a atividade proteolítica, os resultados demonstraram que a formulação controle (100% p/p NaCl sem probióticos) foi a que apresentou menor atividade durante 28 dias de estocagem refrigerada, diferindo estatisticamente das demais formulações. Todos os queijos desse estudo podem ser classificados como de baixo teor de gordura, porque o teor de gordura variou de 4.33 a 4.69 %, e muito alta umidade (74.60 a 75.59 %), segundo a legislação brasileira. A adição de culturas probióticas e a redução de sódio não provocaram mudanças significativas no perfil de ácidos graxos em todos os queijos formulados. Os resultados demonstraram que todas as formulações de queijo cottage apresentaram potencial probiótico, desde que consumidas numa quantidade adequada (2 porções diárias de 50 g), uma vez que as concentrações dos probióticos permaneceram dentro dos padrões estipulados pela legislação brasileira. Entre as formulações elaboradas com sais hipossódicos, a formulação mais aceita foi aquela com redução de sódio de 25% (p/p) de NaCl e substituição por KCl (12,5% p/p) e MgCl2 (12,5% p/p), e o atributo que mais afetou a aceitação dessas formulações foi a textura das amostras. Dessa forma, o queijo cottage probiótico demonstrou ser uma excelente matriz para inserção de sais-hipossódicos, pois não modifica o poder de salga em relação ao NaCl Abstract: Sodium intake is essential for the welfare of human being, however, its overconsumption may lead to unhealthy effects, namely increased blood pressure and lower calcium absorption. Due to the aforementioned effects, sodium content in food is one of the main focuses of attention in campaigns by government agencies, especially in the dairy sector where cheese is perceived as being a food with high sodium content. Thus, this study aimed to evaluate the effect of partial replacement of NaCl by KCl and MgCl2 on the parameters of quality probiotic cottage cheese during 28 days of refrigerated storage and to determine the viability of starter culture and probiotic cultures used in cheese processing. Furthermore, the objective was to evaluate the sensory acceptance to identify directions for improving the sensory attributes of the product before consumers.Using magnitude estimation method, the salty flavor in probiotic cottage was generated using 1.12% (w/w) NaCl, and equivalent concentrations in salinity for all low-sodium salts, was the same as for NaCl (1.12% w/w). The counts of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis ranged from 7.74 to 6.20 and from 8.58 to 7.93 log (CFU. g-1), respectively in the first and last day of storage, enough for benefits to consumers. However, the starter culture counts remained at values below 3.0 log CFU. g-1 for all formulations. Lactic acid was the organic acid found in greater quantities in all formulations, followed by citric acid and acetic acid. The proteolytic activity results showed that the control formulation (100% w/w NaCl without probiotics) showed the lowest activity during 28 days of refrigerated storage, differing from the other formulations. All cheeses of this study can be classified as low-fat, because the fat content ranged 4.33 to 4.69% and very high humidity (74.6 to 75.59%), according to Brazilian regulation. The addition of probiotic cultures and reducing sodium did not cause significant changes in the fatty acid profile of all tested cheeses.The results showed that all the formulations showed probiotic potential if consumed in appropriate portion (2 portions of 50 g daily), since the concentration of probiotics remained within the Brazilian standards. Among formulations prepared with low-sodium salt the best one was that with a reduction of 25%(w/w) sodium substitution of KCl and NaCl (12.5% w/w) and MgCl2 (12.5% w/w) and the attribute that most affected the acceptance was the texture of the samples. Thus, the probiotic cottage cheese proved to be an excellent matrix for the insertion of low-sodium salts, because it does not change the power of brining in relation to NaCl Mestrado Tecnologia de Alimentos Mestra em Tecnologia de Alimentos

The presence of Alpha1-Antitrypsin (AAT) polymers, known to promote a sustained pro-inflammatory activity, has been previously demonstrated in bronchial biopsies of subjects with Z-AAT deficiency (AATD) suggesting a possible role in the development of COPD through a small airway disease impairment. The study aimed to assess the presence of small airways dysfunction and the potential correlation with the presence of Z-AAT polymers obtained by Exhaled Breath Condensate (EBC) collection in PiZZ subjects, as compared with matched healthy PiMM subjects. We enrolled 19 asymptomatic, never smoker subjects: 9 PiZZ and 10 PiMM as controls, without obstructive ventilatory defect (i.e., normal FEV1/VC% ratio). All subjects underwent complete pulmonary function tests (PFT). EBC was collected in all subjects. ELISA test was applied to search for Z-AAT polymers. The PiZZ subjects showed normal lung volumes and DLCO values. However, in comparison with PiMM subjects, the single breath test N2 wash-out revealed significant differences regarding the phase III slope (1.45±0.35 N2/L vs. 0.96±0.40 N2/L) (p

Introduction: The presence of Alpha1-Antitrypsin (AAT) polymers that are pro-inflammatory has been previously demonstrated in bronchial biopsies of subjects with Z-AAT deficiency (Respir Res, 2014 Sep 14;15:112), suggesting a possible role in the development of COPD. Aim: The aim of the study was to assess the presence of small airways dysfunction and the potential correlation with the presence of Z-AAT polymers obtained by Exhaled Breath Condensate (EBC) collection in PiZZ subjects, comparing with matched healthy PiMM subjects. Methods: We enrolled 17 asymptomatic non-smoker subjects: 9 PiZZ and 8 PiMM as controls, without obstructive ventilatory defect (i.e.: normal FEV1/VC ratio). All subjects underwent complete pulmonary function tests (PFT). EBC was collected in PiZZ patients. ELISA test was applied to search for Z-AAT polymers. Results: The PiZZ subjects showed normal lung volumes and DLCO values. However, In comparison with PiMM subjects, the single breath test N2 wash-out revealed significant differences regarding the phase III slope (1.5±0.4 N2/L vs 1.1±0.3 N2/L) and the closing volume/vital capacity ratio (14.3±4.5 % vs 9.5±5.3 %) (p Conclusions: PiZZ subjects showed small airways dysfunction when compared to PiMM subjects. Surprisingly, Z-AAT polymers were found only in 4 PiZZ subjects; this is probably due to the sample methodology. New studies are needed to evaluate the correlation between bronchial Z-AAT polymers and small airway impairment in subjects with Z-AAT deficiency.

Dendritic cells (DCs) constitute a complex network of cell subsets with common functions but also with many divergent aspects. All dendritic cell subsets share the ability to prime T cell response and to undergo a complex trafficking program related to their stage of maturation and function. For these reasons, dendritic cells are implicated in a large variety of both protective and detrimental immune responses, including a crucial role in promoting anti-tumor responses. Although cDC1s are the most potent subset in tumor antigen cross-presentation, they are not sufficient to induce full-strength anti-tumor cytotoxic T cell response and need close interaction and cooperativity with the other dendritic cell subsets, namely cDC2s and pDCs. This review will take into consideration different aspects of DC biology, including the functional role of dendritic cell subsets in both fostering and suppressing tumor growth, the mechanisms underlying their recruitment into the tumor microenvironment, as well as the prognostic value and the potentiality of dendritic cell therapeutic targeting. Understanding the specificity of dendritic cell subsets will allow to gain insights on role of these cells in pathological conditions and to design new selective promising therapeutic approaches.

Dendritic cells (DCs) are professional antigen-presenting cells responsible for the activation of specific T-cell responses and for the development of immune tolerance. Immature DCs reside in peripheral tissues and specialize in antigen capture, whereas mature DCs reside mostly in the secondary lymphoid organs where they act as antigen-presenting cells. The correct localization of DCs is strictly regulated by a large variety of chemotactic and nonchemotactic signals that include bacterial products, DAMPs (danger-associated molecular patterns), complement proteins, lipids, and chemokines. These signals function both individually and in concert, generating a complex regulatory network. This network is regulated at multiple levels through different strategies, such as synergistic interactions, proteolytic processing, and the actions of atypical chemokine receptors. Understanding this complex scenario will help to clarify the role of DCs in different pathological conditions, such as autoimmune diseases and cancers and will uncover new molecular targets for therapeutic interventions.

Persistent and excessive cytokine production is a hallmark of autoimmune diseases and may play a role in disease pathogenesis and amplification. Therefore, cytokine neutralization is a useful therapeutic strategy to treat immune-mediated conditions. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression in diverse biological processes. Altered miRNA levels are observed in most autoimmune diseases and are recognized to influence autoimmunity through different mechanisms. Here, we review the impact of altered miRNA levels on the expression of cytokines that play a relevant pathogenic role in autoimmunity, namely primary pro-inflammatory cytokines, the IL-17/IL-23 axis, type I interferons and IL-10. Regulation can be either “direct” on the target cytokine, or “indirect,” meaning that one given miRNA post-transcriptionally regulates the expression of a protein that in turn influences the level of the cytokine. In addition, miRNAs associated with extracellular vesicles can regulate cytokine production in neighboring cells, either post-transcriptionally or via the stimulation of innate immune RNA-sensors, such as Toll-like receptors. Because of their tremendous potential as physiological and pathological regulators, miRNAs are in the limelight as promising future biopharmaceuticals. Thus, these studies may lead in the near future to the design and testing of therapeutic miRNAs as next generation drugs to target pathogenic cytokines in autoimmunity.

Orientador: Marcelo Cristianini Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: O açaí (Euterpe oleracea Mart.) é uma fruta altamente perecível que necessita de tratamentos que prolonguem sua vida de prateleira, inativando as enzimas naturalmente presentes e micro-organismos deteriorantes e patogênicos. O processamento de alimentos utilizando uma tecnologia emergente não térmica (alta pressão) surge como uma alternativa aos processos tradicionais de pasteurização, pelas vantagens que apresenta ao minimizar perdas sensoriais e nutricionais. Neste contexto, este trabalho objetivou avaliar o efeito da tecnologia de alta pressão isostática (API) na inativação das principais enzimas presentes na polpa do açaí (polifenoloxidase e peroxidase), bem como na inativação de um micro-organismo deteriorante (Lactobacillus fructivorans), um esporulado (Alicyclobacillus acidoterrestris) e uma cepa de Escherichia coli permitindo estabelecer parâmetros de processo adequados para garantir a segurança microbiológica e preservando as propriedades funcionais e sensoriais do fruto. Foram utilizadas pressões de 400, 500 e 600 MPa, tempos de 5 e 15 min e temperaturas de 25 e 65 °C. As análises realizadas foram: atividade enzimática, cor, microbiológicas, sensorial, antocianinas, compostos fenólicos totais, atividade antioxidante e reológicas. Os resultados demostraram que que a enzima peroxidase é termorresistente e barorresistente em polpa de açaí nas condições estudadas, contrariamente à polifenoloxidase, que apresentou maior inativação (71,7 %) na condição de 600 MPa/15 min/65 °C. O processamento por API foi comparado a um processo térmico convencional (85 ºC/1 min), onde foi observada inativação parcial das duas enzimas, mas com alteração da cor do produto (?E = 4,22) perceptível ao olho humano. Para Escherichia coli, a API permitiu a inativação de 6,1 ciclos log em todas as condições estudadas. Para Alicyclobacillus acidoterrestris, foi determinado um tratamento a 600 MPa/65 °C (5, 10, 15, 20 e 25 min) resultando em um valor de 4 reduções decimais de 13,5 min. Foram então selecionadas duas melhores condições 600 MPa/5 min/25 °C e 600 MPa/5 min/65 °C, que juntamente com a amostra controle e a pasteurizada foram submetidas à avaliação durante a estocagem a 5 °C. Para o grupo de micro-organismos (aeróbios mesófilos, aeróbios psicotróficos e bactérias lácticas), verificou-se que após o processamento por API (600 MPa/5 min/65 °C) as contagens permaneceram em valores abaixo de 2 log UFC/mL durante os 28 dias. Em nenhuma das amostras de polpa foi detectada a presença de Salmonella sp, o que indica que o produto permaneceu de acordo com os padrões legais vigentes. Para as amostras processadas por API e a pasteurizada, os valores de contagem de micro-organismos permaneceram abaixo do limite máximo permitido pela legislação durante os 28 dias. Os resultados da análise sensorial mostraram que o tratamento por API influenciou na percepção da textura das amostras de açaí (textura mais consistente) e durante as análises reológicas pôde-se perceber que as mesmas apresentaram um aumento de viscosidade aparente. Em polpas de açaí pressurizadas, a extração das antocianinas aumentou em média 37%, em relação a amostra controle, para ambos os processos de API. Em oposição, o tratamento térmico reduziu o teor desse pigmento em 16,3% em comparação com a amostra in natura. Para fenólicos totais, ambos os processos a 600 MPa tanto a 25°C como a 65°C quando comparados a amostra in natura, aumentaram a extração em 11,44% e 10,25%, respectivamente. A API também foi capaz de manter a atividade antioxidante para as polpas processadas a 600 MPa por 5 min a 25 e 65°C. Diante dos resultados, conclui-se que o processamento de API se mostrou como uma alternativa para tratamentos térmicos de pasteurização, gerando um produto seguro microbiologicamente, com preservação de suas características nutricionais e sensoriais Abstract: Açaí (Euterpe oleracea Mart.) is a highly perishable fruit which needs treatments that prolong its shelf life, inactivating enzymes naturally present and spoilage and pathogenic microorganisms. Food processing using an emerging non-thermal technology (high pressure) could be an alternative to conventional pasteurization processes for thermosensitive products, by the advantages of minimizing sensorial and nutritional losses. In this context, this study aimed to evaluate the effect of high isostatic pressure technology (HIP) on the inactivation of the main enzymes present in açaí pulp (polyphenol oxidase and peroxidase), as well as inactivation of a spoilage microorganism (Lactobacillus fructivorans), a strain of Escherichia coli and a sporulated microorganism (Alicyclobacillus acidoterrestris), allowing to establish adequate process parameters to ensure the microbiological safety of the product and preserve the functional and sensory properties of fruit. Pressures of 400, 500 and 600 MPa, times of 5 and 15 minutes and temperatures of 25 and 65°C were used. The analyzes were: enzymatic activity, color, microbiological, sensorial, anthocyanins, total phenolic compounds, antioxidant activity and rheological. The results showed that peroxidase enzyme is heat resistant and baroresistente in açaí pulp under the conditions studied, in opposition, polyphenoloxidase, which presented greater inactivation (71.7%) in the condition of 600 MPa / 15 min / 65 ° C. The HIP process was compared to a conventional thermal process (85 °C/1min), where partial inactivation of the two enzymes was observed, but with a change in the color (?E = 4.22) visible to the human eye. For Escherichia coli, the HIP allowed the inactivation of 6.1 log cycles in all conditions studied. For Alicyclobacillus acidoterrestris, a treatment at 600 MPa/65°C (5, 10, 15, 20 and 25 min) was determined resulting in a value of 4 decimal reductions of 13.5 min. Two better conditions were selected, 600 MPa/5 min/25 °C and 600 MPa/5 min/65 °C and together with the control and pasteurized samples, were evaluated during storage at 5 °C. For the group of microorganisms (mesophilic aerobes, psychotrophic aerobes and lactic acid bacteria), it was found that after processing by HIP (600 MPa / 5min / 65°C) the counts remained below 2 log CFU/mL during 28 days. No presence of Salmonella sp was detected in any sample, which indicates that the product remained in accordance with current legal standards. For both, the API and pasteurized samples, the microorganism count values remained below the maximum allowable limit required by the legislation for 28 days. The results of the sensory analysis showed that API process influenced the texture perception of açaí samples (more consistent texture) and during rheological analysis it was observed that samples processed by HIP showed an increase in viscosity. In pressurized açaí pulps, the extraction of anthocyanins increased 37%, compared to control sample, for both HIP processes. In contrast, the heat treatment reduced the pigment content by 16.3% compared to the in natura sample. For total phenolics, both processes at 600 MPa at 25°C and 65°C when compared to the in natura sample, increased the extraction by 11.44% and 10.25%, respectively. The API was also able to maintain the antioxidant activity for the pulps processed at 600 MPa for 5 min at 25 and 65 ° C. In view of the results, it was concluded that the HIP processing was shown as an alternative for thermal treatments of pasteurization, generating a safe microbiological product, with preservation of its nutritional and sensorial characteristics Doutorado Tecnologia de Alimentos Doutora em Tecnologia de Alimentos FAPESP 2015/01570-0 CAPES

The tumor microenvironment consists of both malignant and non-malignant cells and a plethora of soluble mediators. Different types of tumors have specific tumor microenvironments characterized by distinct chemokines and chemotactic factors that influence leukocyte recruitment. The immune cell infiltrate continuously interacts with stroma cells and influence tumor growth. Emerging evidence suggests that the regulation of the composition and the metabolic state of tumor-associated leukocytes may represent a new promising intervention strategy. Here we summarize the current knowledge on the role of tumor-associated immune cells in tumor growth and dissemination, with a specific focus on the nature of the chemotactic factors responsible for their accumulation and activation in tumors.

Alpha1-antitrypsin (alpha(1)AT) deficiency is a hereditary disorder associated with reduced alpha(1)AT serum level, predisposing adults to pulmonary emphysema. Among the known mutations of the alpha(1)AT gene (SERPINA1) causing alpha(1)AT deficiency, a few alleles, particularly the Z allele, may also predispose adults to liver disease. We have characterized a new defective alpha(1)AT allele (c.745G>C) coding for a mutant alpha(1)AT (Gly225Arg), named P(brescia). The P(brescia) alpha(1)AT allele was first identified in combination with the rare defective M(wurzburg) allele in an 11-year-old boy showing significantly reduced serum alpha(1)AT level. Subsequently, the P(brescia) allele was found in the heterozygous state with the normal M or the defective Z allele in nine and three adults respectively. In cellular models of the disease, we show that the P(brescia) mutant is retained in the endoplasmic reticulum as ordered polymers and is secreted more slowly than the normal M alpha(1)AT. This behaviour recapitulates the abnormal cellular handling and fate of the Z alpha(1)AT and suggests that the mutation present in the P(brescia) alpha(1)AT causes a conformational change of the protein which, by favouring polymer formation, is etiologic to both severe alpha(1)AT deficiency in the plasma and toxic protein-overload in the liver.

In the liver, CCl4 induces cell necrosis followed by regeneration. Cell injury is caused by free radical damage and may be due, at least in part, to oxidative stress and the subsequent formation of reactive oxygen intermediates (ROIs). In a rat model of acute CCl4-induced hepatic injury, we examined the expression of genes involved in cellular response to different kinds of stress, including oxidative stress (hsp 70 family, heme oxygenase), in free radical detoxification (Mn superoxide dismutase and Cu/ Zn superoxide dismutase), in iron homeostasis (H and L ferritin subunits) and in the cell cycle (c-fos, c-jun, histone H3). As an experimental approach, we first analysed the pattern of protein synthesised by liver slices in vitro. Then we studied the mechanisms regulating the expression of different genes, by analysing both mRNA steady state levels and transcription rates. Activation of the specific heat shock transcription factor (HSF) by CCl4 was also investigated. We observed that different members of the hsp70 family (hsp70, hsc73, grp78) are activated by different kinetics and are regulated mainly at the transcriptional level. Induction of the hsp70 gene occurs rapidly and transiently and is preceded by the activation of HSF DNA-binding activity. We demonstrated an increase in the steady-state levels of mRNAs for heme oxygenase, Mn and Cu/Zn superoxide dismutases and H and L ferritin subunits. However, different kinetics and regulatory mechanisms occurred with different genes. We showed that induction of c-fos and c-jun protooncogenes is the earliest event after CCl4 administration, whereas histone H3 expression peaked at 24-48 h. The results of this study are interpreted as evidence that activation of specific stress response genes is primarily related to the defence against the rapidly occurring cell damage, but may also be related to subsequent processes of tissue inflammation and cell proliferation.

Summary HIV‐1 matrix protein p17 activates a variety of cell responses which play a critical role in viral replication and infection. Its activity depends on the expression of p17 receptors (p17R) on the surface of target cells. Whether p17 also plays a role in stimulating human monocytes, a major HIV‐1 reservoir, is not known. Here we show that human monocytes constitutively express p17Rs and that p17 selectively triggers these cells to produce MCP‐1. The effect of p17 on MCP‐1 expression was observed at the transcriptional level and was primarily dependent on the activation of the transcription factor AP‐1. p17 increased the binding activity of AP‐1 complexes in a time‐ and dose‐dependent manner. Deletion of the AP‐1 binding sites in the MCP‐1 promoter resulted in the lack of p17‐induced MCP‐1 transcription. In particular, the P3 binding site located between −69 and −63 position seems to be essential to MCP‐1 mRNA induction in p17‐treated monocytes. An ever increasing amount of evidences shows a tight link between biologically dysregulated monocytes, AP‐1 activation, MCP‐1 release and HIV‐1 pathogenesis. Overall our results suggest that p17 may play a critical role in the monocyte‐mediated inflammatory processes, which are suspected to be major precipitating events in AIDS‐defining diseases.

Numerous animal studies simulating liver injury have demonstrated that interleukin-6 (IL-6) exerts a protective effect. This study was designed to further analyze the molecular mechanisms underlying the protective role of IL-6 in a rat model of liver ischemia/reperfusion injury. We show that IL-6: (i) at high doses reduces cell damage which occurs in ischemic-reperfused liver, while at low doses displays only a limited protective capacity, (ii) anticipates and enhances hepatocyte compensatory proliferation seen in ischemic-reperfused liver also at a low, more pharmacologically acceptable dose, (iii) sustains the acute phase response which is dampened in ischemic-reperfused liver, (iv) strengthens the heat shock-stress response shown by ischemic-reperfused liver and (v) overcomes the dysfunctions of the unfolding protein response found in ischemic-reperfused liver. We also show that IL-6-enhanced STAT3 activation probably plays a crucial role in the potentiation of the different protective pathways activated in ischemic-reperfused liver. Our data confirm that IL-6 is a potential therapeutic in liver injury of different etiologies and reveal novel mechanisms by which IL-6 sustains liver function after ischemia/reperfusion injury.

Background Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown. We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients. Methods Experiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of

Myc is a transcriptional activator whose deregulated expression not only promotes proliferation but also induces or sensitizes cells to apoptosis. Here we demonstrate that c-myc plays a role in triggering apoptosis in CEM T leukaemia cells exposed to progressive medium exhaustion. Indeed starved cells undergo apoptosis in the presence of constitutively elevated c-myc expression and the phorbol ester, phorbol 12-miristate 13-acetate (PMA), which rescues cells from apoptosis, induces complete c-myc down-regulation. We also investigate the hypothesis that ornithine decarboxylase (ODC), a transcriptional target of c-myc, is a down-stream mediator of c-myc driven apoptosis. We demonstrate that PMA induces in starved cells an earlier and larger decrease in ODC expression (mRNA and activity) and intracellular polyamine content, compared to untreated starved cells. Moreover we show that alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzymatic activity, effectively reduces, while exogenous added polyamines enhance apoptosis in starved cells. All these data indicate that ODC and polyamines may act as facilitating factors in triggering apoptosis induced by growth/survival factors withdrawal.

Background: Cigarette smoke extract (CSE), a model for studying the effects of tobacco smoke in vivo and in vitro, induces cell oxidative stress and affects the antioxidative glutathione system. We evaluated the impact of CSE on airway epithelial cells and the possible cytoprotective effect of the mucolitic drug S-carboximethilcysteine lysine salt (S-CMC-Lys). Methods: Reduced glutathione (GSH) and reactive oxygen species (ROS) intracellular levels were evaluated by fluorimetry in human bronchial epithelial cells (16-HBE) and the expression and activity of enzymes of the GSH metabolic pathway were investigated by RT-PCR, Western blot and colorimetric assays. Results: CSE significantly increased cell mortality in a time and dose dependent manner, via an apoptosis-independent pathway. Short-term (3 hours) CSE exposure induced an increase in ROS levels and a GSH intracellular concentration drop. In parallel, the expression of glutathione peroxidases 2 and 3, glutathione reductase and glutamate-cysteine-ligase was increased. S-CMC-Lys was effective in counteracting these effects. Conclusion: CSE affects ROS levels, GSH concentration and GSH enzymes pathway. These effects can be to some extent reversed by S-CMC-Lys, that could represent a therapeutic tool to counteract CSE induced oxidative cellular injuries.

Angiogenesis plays a crucial role in tumor growth and progression. Low expression of mineralocorticoid receptor (MR) in several malignant tumors correlates with disease recurrence and overall survival. Previous studies have shown that MR expression is decreased in colorectal cancer (CRC). Here we hypothesize that decreased MR expression can contribute to angiogenesis and poor patient survival in colorectal malignancies. In a cohort of CRC patients, we analyzed tumor MR expression, its correlation with tumor microvascular density and its impact on survival. Subsequently, we interrogated the role of MR in angiogenesis in an in vitro model, based on the colon cancer cell line HCT116, ingenierized to re-express a physiologically controlled MR. In CRC, decreased MR expression was associated with increased microvascular density and poor patient survival. In pchMR transfected HCT116, aldosterone or natural serum steroids largely inhibited mRNA expression levels of both VEGFA and its receptor 2/KDR. In CRC, MR activation may significantly decrease angiogenesis by directly inhibiting dysregulated VEGFA and hypoxia-induced VEGFA mRNA expression. In addition, MR activation attenuates the expression of the VEGF receptor 2/KDR, possibly dampening the activation of a VEGFA/KDR dependent signaling pathway important for the survival of tumor cells under hypoxic conditions.

Major hepatic resection in cirrhotic patients is associated with impaired liver regeneration and failure, leading to high peri-operative mortality. In this work, the causes of defective regeneration in cirrhotic liver and the utility of IL-6 treatment were investigated in an experimental model combining cirrhosis and partial hepatectomy in the rat. Relative to normal controls, decompensated cirrhotic animals showed decreased survival, while compensated cirrhotic animals showed similar survival but reduced hepatic DNA synthesis and newly regenerated liver mass amount. Defective liver regeneration was associated with a decrease in STAT3 and NF-kB activation, consistent with an increased accumulation of their respective inhibitors PIAS3 and IkBalpha, and with a decreased induction of Bcl-xL. Treatment with recombinant IL-6 enhanced survival of decompensated cirrhotic animals, while it did not affect survival of compensated cirrhotic animals but sustained liver regeneration, by restoring STAT3 and NF-kB activation and Bcl-xL induction to the levels found in normal controls. The pro-growth effects exerted by IL-6 treatment in cirrhotic liver were attained also at low, pharmacologically acceptable doses. In conclusion, our results suggest that IL-6 treatment may be therapeutic in major resection of cirrhotic liver.

In the liver IL-6 displays growth-inducing and pro-survival activities. We studied the pro-proliferative and protective mechanisms of IL-6 treatment in a model of liver cirrhosis in wild type rat, investigating the theoretical basis for a potential pharmacologic role of IL-6 in cirrhosis.We analyzed IL-6 receptors levels in cirrhotic liver. We also studied the activation of signaling pathways downstream IL-6 receptors by analyzing the DNA-binding activity of transcription factors STAT3, AP-1, HNF-1 and NF-kappaB and the phosphorylation status of AKT and eNOS. We also analyzed hepato-cell proliferation, by determining BrdU incorporation into DNA, and liver mass expansion.We show that liver cells from cirrhotic animals have increased expression of the IL-6 receptor alpha/gp80. In addition, we show that in cirrhosis the main molecular pathways downstream the receptors are intact and that IL-6 activates STAT3, AP-1 and NF-kappaB transcription factors, induces AKT and eNOS phosphorylation and increases hepato-cell proliferation and liver mass expansion in a dose-dependent manner.Our data demonstrate that the theoretical basis exists for the therapeutic employment of IL-6 in liver cirrhosis.

We analysed the expression of the hsp70 gene, the phosphorylation status of different members of the mitogen-activated protein kinase (MAPK) family, the behaviour of the Akt-GSK3 pathway, as well as the DNA-binding activity of several transcription factors, potential targets of these kinases, in the brain of rats exposed to a fever-like increase in body temperature. Two different brain regions, the cerebellum and the hippocampus, were studied. Hyperthermia caused HSF activation and the induction of hsp70 mRNA and protein to a greater extent in the cerebellum than in the hippocampus. In the cerebellum, ERK1/2 and p38 MAPK phosphorylation were increased by hyperthermia and returned to basal levels during the recovery from heat stress, whereas JNK3 phosphorylation decreased and recovered to above control levels within 60 min of recovery. JNK1 phosphorylation was never modified. In the hippocampus, ERK phosphorylation did not increase but rather decreased, whereas the behaviour of p38 MAPK and JNK was similar to that observed in the cerebellum. Akt phosphorylation increased after hyperthermia and was accompanied by an increased phosphorylation of two substrates, GSK3 and FKHRL1, in both brain areas, with a major effect in the cerebellum. DNA-binding activities of AP-1, NF-kappaB, and MEF2 were activated by heat shock in the cerebellum, whereas only MEF2 was activated in the hippocampus. Our data indicate that a physiologically relevant increase in body temperature induces brain injury and survival response to it as demonstrated by induction of hsp70 gene expression and activation of specific signalling pathways. Reprogramming of gene expression, by the specific transcription factors activated, probably plays a central role in cell adaptation and survival to heat stress. The hippocampus shows less responsiveness to hyperthermia than the cerebellum.

A Sample-Saving Preparation to Extract DNA-Binding Proteins from Cardiac and Vascular Tissues

Two groups of prolactinoma cell lines were identified. One group (responder) expresses both D(2) dopamine receptors and an autocrine loop mediated by nerve growth factor (NGF) and one group (nonresponder) lacks both D(2) receptors and NGF production. D(2) receptor expression in these cell lines is dependent on NGF. Indeed, NGF inactivation in responder cells decreases D(2) receptor density, while NGF treatment induces D(2) receptor expression in nonresponders. Here we show that inactivation of p75(NGFR), but not of trkA, resulted in D(2) receptor loss in responder cells and prevented D(2) receptor expression induced by NGF in the nonresponder. Analysis of nuclear factor-kappaB (NF-kappaB) nuclear accumulation and binding to corresponding DNA consensus sequences indicated that in NGF-secreting responder cells, but not in nonresponders, NF-kappaB is constitutively activated. Moreover, NGF treatment of nonresponder cells induced both nuclear translocation and DNA binding activity of NF-kappaB complexes containing p50, p65/RelA, and cRel subunits, an effect prevented by anti-p75(NGFR) antibodies. Disruption of NF-kappaB nuclear translocation by SN50 remarkably impaired D(2) receptor expression in responder cells and prevented D(2) gene expression induced by NGF in nonresponders. These data indicate that in prolactinoma cells the effect of NGF on D(2) receptor expression is mediated by p75(NGFR) in a trkA-independent way and that NGF stimulation of p75(NGFR) activates NF-kappaB, which is required for D(2) gene expression. We thus suggest that NF-kappaB is a key transcriptional regulator of the D(2) gene and that this mechanism may not be confined to pituitary tumors, but could also extend to other dopaminergic systems.

Laura Pini,1 Valentina Pinelli,2 Denise Modina,1 Michela Bezzi,3 Laura Tiberio,4 Claudio Tantucci1 1Unit of Respiratory Medicine, Department of Clinical and Experimental Sciences, University of Brescia, 2Department of Respiratory Medicine, Spedali Civili di Brescia, 3Department Bronchoscopy, Spedali Civili di Brescia, 4Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy Background: The contribution to airflow obstruction by the remodeling of the peripheral airways in chronic obstructive pulmonary disease (COPD) patients has been well documented, but less is known about the role played by the large airways. Few studies have investigated the presence of histopathological changes due to remodeling in the large airways of COPD patients. Objectives: The aim of this study was to verify the presence of airway remodeling in the central airways of COPD patients, quantifying the airway smooth muscle (ASM) area and the extracellular matrix (ECM) protein deposition, both in the subepithelial region and in the ASM, and to verify the possible contribution to airflow obstruction by the above mentioned histopathological changes. Methods: Biopsies of segmental bronchi spurs were performed in COPD patients and control smoker subjects and immunostained for collagen type I, versican, decorin, biglycan, and alpha-smooth muscle actin. ECM protein deposition was measured at both subepithelial, and ASM layers. Results: The staining for collagen I and versican was greater in the subepithelial layer of COPD patients than in control subjects. An inverse correlation was found between collagen I in the subepithelial layer and both forced expiratory volume in 1 second and ratio between forced expiratory volume in 1 second and forced vital capacity. A statistically significant increase of the ASM area was observed in the central airways of COPD patients versus controls. Conclusion: These findings indicate that airway remodeling also affects the large airways in COPD patients who have greater deposition of ECM proteins in the subepithelial layer and a larger smooth muscle area than control smoker subjects. These changes may contribute to chronic airflow obstruction in COPD patients. Keywords: COPD, central airways, remodeling, airway smooth muscle, proteoglycans

In-vivo heat-shock induced heat shock factor (HSF) DNA-binding activity and accumulation of heat shock protein (hsp)70 mRNA in newborn and adult rat cerebellum was studied. We identified a high basal level of c-Jun N-terminal kinase (JNK) and p38 MAP kinase phosphorylation in the cerebellum, independently of age. Hyperthermia increased JNK1, decreased JNK2 but did not modify JNK3 phosphorylation in the newborn cerebellum, whereas decreased the phosphorylation of both JNK1 and JNK3 in adult rats. During recovery from hyperthermia, JNK2 phosphorylation returned to control level in the newborn, JNK1 appeared hyperphosphorylated only in the newborn, and JNK3 in all animals. JNK2 never appeared phosphorylated in the adult cerebellum. Hyperthermia increased p38 MAP kinase phosphorylation in the cerebellum, with different trends in newborn and adult rats during recovery. Heat shock increased extracellular signal-regulated kinase phosphorylation concomitant to tyrosine kinase receptor activation (epidermal growth factor-receptor in the newborn and insulin-like growth factor-receptor in the adult cerebellum). The behavior of stress kinases may underlie a different age-related vulnerability to heat stress of the cerebellum.

Background: In cultured cells of various origin, ethanol induces the synthesis of 70 kDa family heat shock proteins (hsp70 family), which play a role in the protection of protein traffic and secretion, as well as in cytoskeleton organization. To assess whether ethanol also can induce such genes in vivo, we studied the behavior of hsp70, hsc73, and grp78 messenger ribonucleic acids (mRNAs) and related proteins in the liver and brain of rats acutely treated with ethanol. Methods: Overnight fasted Sprague-Dawley rats (220-250 g) were acutely treated with a low (2 g/kg body weight) or a high (5 g/kg body weight) dose of ethanol as a 30% solution in saline or an equal volume of saline (controls) by gastric intubation. Animals were killed at various times after treatments (3-72 hr). Messenger RNA levels for different members of hsp70 family (hsp70; 73 kDa heat shock cognate, or hsc73; and 78 kDa glucose-regulating protein, or grp78) were determined by Northern blot analysis and hybridization with specific complementary deoxyribonuclcic acid (cDNA) probes. The amounts of related proteins were assayed by Western blot analysis with specific antibodies. Autoradiograms and fluorograms were subjected to densitometric scanning. Results: Ethanol (2 g/kg) caused a slight increase in hsc73 and grp78 mRNA levels only in the liver, without enhancing the amount of proteins. Ethanol (5 g/kg) increased the level of hsc73 and grp78 mRNAs and related proteins in the liver. In the brain, the amount of hsc73 mRNA was enhanced, but this did not change hsc73 protein. In addition, we observed an increase in cerebral grp78 transcript and related protein. Hsp70 gene was not induced in the examined tissues by either dose of ethanol. Conclusions: Hepatic and cerebral hsc73 and grp78 genes are responsive to ethanol in vivo, and their activation may signal the cell's effort to counteract the harmful action of ethanol.

We investigated the intracellular distribution of heat shock factors 1 and 2 (HSF1, HSF2) in rat spinal cord by immunoblotting and immunohistochemistry using selective policlonal antibodies. Our results showed that both HSF1 and HSF2 were expressed in spinal cord cells (both neurons and glia) but at different intensity and cell localization. HSF1 was unusually distributed in the perinuclear compartment of selected neurons of the gray matter while astrocytes, oligodendrocytes and ependymal cells were predominantly stained in the nucleus. HSF2 was expressed at lower levels than HSF1 and was scattered in both nucleus and cytoplasm of the motoneurons of the ventral horns while glial cells again showed a nuclear positivity. This study suggested that the different ability of neurons vs. glial cells to react against adverse conditions might well be correlated with the different constitutive localization of HSFs.