Expressão de citocinas durante a gestação de porcos

Pig production in Argentina is constantly growing. Reproductive management is essential to achieve optimal birth rates that translate into greater profitability and investment efficiency. Prenatal losses limit pig production. Alterations in the migration of the embryos, elongation, immunological recognition of pregnancy by the mother and embryonic competition for the implantation site can arise in this process. In a successful pregnancy, the dialogue established between the conceptus and the endometrium involves, among others, the immune system and its main molecules called cytokines. Cytokines are a group of low molecular weight proteins that mediate complex interactions between different cell types. Numerous studies describe the role of various cytokines that are involved in the regulation of the inflammatory process characteristic of the fetus/maternal interface throughout the normal pig pregnancy. This review describes the main cytokines that act during pig pregnancy, both in the early gestation period and in the late pregnancy period.La producción porcina en Argentina se encuentra en constante crecimiento. El manejo reproductivo es fundamental para alcanzar índices óptimos de nacimientos que se traduzcan en una mayor rentabilidad y eficiencia de la inversión ya que las pérdidas prenatales limitan la producción porcina. En la gestación pueden ocurrir alteraciones en la migración de los embriones, su elongación, el reconocimiento inmunológico de la preñez por la madre y la competencia embrionaria por el lugar de implantación. Para que la gestación sea exitosa, el diálogo que se establece entre el conceptus y el endometrio involucra, entre otros, al sistema inmunológico y sus moléculas llamadas citoquinas. Las citoquinas son un grupo de proteínas de bajo peso molecular que actúan mediando interacciones complejas entre distintos tipos celulares. Numerosos estudios describen el rol de diversas citoquinas que se encuentran involucradas en la regulación del proceso inflamatorio característico de la interfase feto/materna a lo largo de la gestación porcina normal. En esta revisión se describen las principales citoquinas que actúan durante la gestación porcina tanto en el período de gestación temprana como en el período de gestación tardía.A produção suína na Argentina está em constante crescimento. O manejo reprodutivo é essencial para atingir índices ótimos que se traduzam em maior rentabilidade e eficiência do investimento, uma vez que as perdas pré-natais limitam a produção de suínos. Na patogênese do processo de gestação intervêm alterações na migração dos embriões, seu alongamento, o reconhecimento imunológico da gravidez pela mãe e a competição embrionária pelo local de implantação. Para que a gravidez seja bem sucedida, o diálogo estabelecido entre o concepto e o endométrio envolve, entre outros, o sistema imunológico e suas moléculas chamadas citocinas. As citocinas são um grupo de proteínas de baixo peso molecular que medeiam interações complexas entre diferentes tipos de células. Numerosos estudos descrevem o papel de várias citocinas que estão envolvidas na regulação do processo inflamatório característico da interface feto/materno durante a gestação suína. Nesta revisão, são descritas as principais citocinas que atuam durante a gestação de suínos, tanto no início da gestação quanto no final da gestação. de várias citocinas que estão envolvidas na regulação do process

IFN-γ and IL-10: seric and placental profile during pig gestation Seric and placental cytokines in pig gestation

Abstract Concentration of interferon-gamma and interleukin-10 in maternal serum and in maternal and fetal porcine placental extracts from different gestation periods was determined. Crossbred pigs’ placental samples of 17, 30, 60, 70, and 114 days gestation and non-pregnant uteri were used. Interferon-gamma concentration was increased at the placental interface at 17 days, in maternal and fetal placenta, and decreased significantly in the remaining gestation periods. Interferon-gamma showed a peak in serum at 60 days. Regarding interleukin-10, placental tissue concentrations were unaltered, there were no significant differences with non-gestating uteri samples. In serum interleukin-10 increased at 17, 60, and 114 days gestation. At 17 days there are uterus structural and molecular changes that allow the embryos implantation and placenta development. The presence of interferon-gamma found at this moment in the interface would favor that placental growth. Moreover, its significant increase in serum at 60 days, would generate a proinflammatory cytokine pattern that facility the placental remodeling characteristic of this moment of porcine gestation. On the other hand, a significant interleukin-10 increase in serum at 17, 60 and 114 days could indicate its immunoregulatory role at a systemic level during pig gestation

Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry

Abstract: Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloridesensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern–Volmer constant (KCl ) for chloride in water solution was 115.0 ± 2.8 M1 , whereas the intracellular KCl was 17.8 ± 0.8 M1 , for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 lM) and glibenclamide (100 lM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way

The chloride anion acts as a second messenger in mammalian cells modifying the expression of specific genes

Abstract: Background/Aims: Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTRdependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-Clconcentration, regulating in turn the expression of specific gene

CFTR modulates RPS27 gene expression using chloride anion as signaling effector

Abstract: In Cystic Fibrosis (CF), the impairment of the CFTR channel activity leads to a variety of alterations, including differential gene expression. However, the CFTR signaling mechanisms remain unclear. Recently, culturing IB3-1 CF cells under different intracellular Clconcentrations ([Cl- ]i), we observed several Cl- -dependent genes and further characterized one of them as RPS27. Thus, we hypothesized that Cl- might act as a signaling effector for CFTR signaling. Here, to test this idea, we study RPS27 expression in T84 cells modulating the CFTR activity by using CFTR inhibitors. First, we observed that incubation of T84 cells with increasing concentrations of the CFTR inhibitors CFTR(inh)-172 or GlyH-101 determined a progressive increase in the relative [Cl- ]i (using the Cl- fluorescent probe SPQ). The [Cl- ]i rise was concomitant with a dose-dependent down-regulation of RPS27. These results imply that CFTR inhibition produce Cl- accumulation and that RPS27 expression can be modulated by CFTR inhibition. Therefore, Cl- behaves as a signaling effector for CFTR in the modulation of RPS27 expression. In addition, the IL-1β receptor antagonist IL1RN or the JNK inhibitor SP600125, both restored the down-regulation of RPS27 induced by CFTRinh-172, implying a role of autocrine IL-1β and JNK signaling downstream of Cl- in RPS27 modulation

N-acetyl cysteine reverts the proinflammatory state induced by cigarette smoke extract in lung Calu-3 cells

Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are lethal pulmonary diseases. Cigarette consumption is the main cause for development of COPD, while CF is produced by mutations in the CFTR gene. Although these diseases have a different etiology, both share a CFTR activity impairment and proinflammatory state even under sterile conditions. The aim of this work was to study the extent of the protective effect of the antioxidant N-acetylcysteine (NAC) over the proinflammatory state (IL-6 and IL-8), oxidative stress (reactive oxygen species, ROS), and CFTR levels, caused by Cigarette Smoke Extract (CSE) in Calu-3 airway epithelial cells. CSE treatment (100 µg/ml during 24 h) decreased CFTR mRNA expression and activity, and increased the release of IL-6 and IL-8. The effect on these cytokines was inhibited by N-acetyl cysteine (NAC, 5 mM) or the NF-kB inhibitor, IKK-2 (10 µM). CSE treatment also increased cellular and mitochondrial ROS levels. The cellular ROS levels were normalized to control values by NAC treatment, although significant effects on mitochondrial ROS levels were observed only at short times (5´) and effects on CFTR levels were not observed. In addition, CSE reduced the mitochondrial NADH-cytochrome c oxidoreductase (mCx I-III) activity, an effect that was not reverted by NAC. The reduced CFTR expression and the mitochondrial damage induced by CSE could not be normalized by NAC treatment, evidencing the need for a more specific reagent. In conclusion, CSE causes a sterile proinflammatory state and mitochondrial damage in Calu-3 cells that was partially recovered by NAC treatment. Keywords: Cigarette smoke extract, Mitochondria, CFTR, ROS, COPD, Cystic fibrosi

Histamine H4 receptor agonism induces antitumor effects in human T-cell lymphoma

Abstract: The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 µM) in combination with histamine (10 µM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses

Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1β. We have previously shown that IL-1β, at low doses (∼30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signaling. However, at higher doses (>2.5 ng/ml, ∼150 pM), IL-1β inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1β in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1β (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-κB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1β blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ∼50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1β, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels

Epidermal growth factor receptor activity upregulates lactate dehydrogenase a expression, lactate dehydrogenase activity, and lactate secretion in cultured ib3-1 cystic fibrosis lung epithelial cells

Abstract: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It has been postulated that reduced HCO3− transport through CFTR may lead to a decreased airway surface liquid pH. In contrast, others have reported no changes in the extracellular pH (pHe). We have recently reported that in carcinoma Caco-2/pRS26 cells (transfected with short hairpin RNA for CFTR) or CF lung epithelial IB3-1 cells, the mutation in CFTR decreased mitochondrial complex I activity and increased lactic acid production, owing to an autocrine IL-1β loop. The secreted lactate accounted for the reduced pHe, because oxamate fully restored the pHe. These effects were attributed to the IL-1β autocrine loop and the downstream signaling kinases c-Src and JNK. Here we show that the pHe of IB3-1 cells can be restored to normal values (∼7.4) by incubation with the epidermal growth factor receptor (EGFR, HER1, ErbB1) inhibitors AG1478 and PD168393. PD168393 fully restored the pHe values of IB3-1 cells, suggesting that the reduced pHe is mainly due to increased EGFR activity and lactate. Also, in IB3-1 cells, lactate dehydrogenase A mRNA, protein expression, and activity are downregulated when EGFR is inhibited. Thus, a constitutive EGFR activation seems to be responsible for the reduced pHe in IB3-1 cells