Can the addition of Interleukin-13 affect the cryosurvival of bovine embryos?

In this study, we investigated the impact of incorporating Interleukin-13 (IL-13) into the embryonic culture medium and its influence on cryotolerance and cellular viability of vitrified bovine embryos. Two distinct time points for IL-13 supplementation were explored: during the final hours of culture prior to cryopreservation and during the period of recultivation following cryopreservation and warming. Cryosurvival rates, total cell count, and cell viability were assessed using the TUNEL technique to determine the apoptotic percentage. Re-expansion and hatching rates did not show differences among all groups (P > 0.05), and the total cell number was comparable between the treated and control groups (P > 0.05). However, the group that received IL-13 before vitrification exhibited a higher apoptotic percentage (P < 0.05). This suggests that the anti-inflammatory effect of IL-13 may have impacted the embryo's defense capacity against the stress induced by cryopreservation, leading to an increased percentage of apoptosis, although it did not influence the developmental resumption capability.

The in vitro anti-inflammatory activity of N-antipyrine-3,4-dichloromaleimide derivatives is due to an immunomodulatory effect on cytokines environment.

BACKGROUND AND AIM: Inflammation is the immune response to a harmful stimulus, and its purpose is to destroy foreign agents so that the affected site can be repair. When uncontrolled or unresolved, inflammation can lead to significant tissue damage. Many classes of compounds are used today as anti-inflammatory drugs. However, there is an ongoing demand for new, more effective molecules with higher safety margins. In this regard, the anti-inflammatory effect of six synthetic compounds of N-antipyrine-3,4-dichloromaleimide was evaluated. METHODS: RAW 264.7 cells were used to evaluate the cytotoxicity and the anti-inflammatory activity, by measuring the effect of these molecules on nitric oxide, IL-1ß, IL-6, MCP-1 (CCL2), TNF-α, INF-γ, IL-4, and IL-13 levels, as well as under NF-κB activation. RESULTS: Some of the tested compounds showed significant cytotoxicity (CC50 < 100 µM). Subsequently, the potential of nitric oxide (NO) inhibition as screening for potential anti-inflammatory action was evaluated. Three of the compounds tested showed a promising profile (1, 3, and 5). When the effect of these compounds was evaluated on the production of IL-1ß, IL-6, MCP-1 (CCL2), TNF-α, and INF-γ, only N-antipyrine-3,4-dichloromaleimide (1) and N-antipyrine-3-chloro-4-(3,4-dichloroaniline) maleimide (3) showed significant inhibition profiles. These two compounds were also able to increase the production of cytokines known for having an anti-inflammatory profile (IL-4 and IL-13) and inhibit the phosphorylation of the p-p65 NF-κB subunit significantly. CONCLUSION: In conclusion, these two compounds present a significant and unusual anti-inflammatory mechanism (increasing the production of anti-inflammatory mediators). They are therefore considered promising prototypes for the development of new anti-inflammatory drugs with immunomodulatory characteristics.

Aberrantly low STAT3 and STAT5 responses are associated with poor outcome and an inflammatory gene expression signature in pediatric acute myeloid leukemia.

The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions.

Blockage of Eosinopoiesis by IL-17A Is Prevented by Cytokine and Lipid Mediators of Allergic Inflammation.

Interleukin- (IL-) 17A, a pleiotropic mediator of inflammation and autoimmunity, potently stimulates bone-marrow neutrophil production. To explore IL-17A effects on eosinopoiesis, we cultured bone-marrow from wild-type mice, or mutants lacking inducible nitric oxide synthase (iNOS-/-), CD95 (lpr), IL-17RA, or IL-4, with IL-5, alone or associated with IL-17A. Synergisms between IL-17A-activated, NO-dependent, and NO-independent mechanisms and antagonisms between IL-17A and proallergic factors were further examined. While IL-17A (0.1-10 ng/mL) had no IL-5-independent effect on eosinopoiesis, it dose-dependently suppressed IL-5-induced eosinophil differentiation, by acting during the initial 24 hours. Its effectiveness was abolished by caspase inhibitor, zVAD-fmk. The effect of IL-17A (0.1-1 ng/mL) was sensitive to the iNOS-selective inhibitor aminoguanidine and undetectable in iNOS-/- bone-marrow. By contrast, a higher IL-17A concentration (10 ng/mL) retained significant suppressive effect in both conditions, unmasking a high-end iNOS-independent mechanism. Lower IL-17A concentrations synergized with NO donor nitroprusside. Eosinopoiesis suppression by IL-17A was (a) undetectable in bone-marrow lacking IL-17RA or CD95 and (b) actively prevented by LTD4, LTC4, IL-13, and eotaxin. Sensitivity to IL-17A was increased in bone-marrow lacking IL-4; adding IL-4 to the cultures restored IL-5 responses to control levels. Therefore, effects of both IL-17A and proallergic factors are transduced by the iNOS-CD95 pathway in isolated bone-marrow.

TGF-ß-mediated sustained ERK1/2 activity promotes the inhibition of intracellular growth of Mycobacterium avium in epithelioid cells surrogates.

Transforming growth factor beta (TGF-ß) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-ß production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-ß and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-ß produced by ECs are sufficient to induce a self-sustaining autocrine TGF-ß signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-ß to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-ß receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-ß produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-ß receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-ß production. In summary, our findings indicate that the amplitude of TGF-ß signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.

Keratinocytes treated with peptidoglycan from Staphylococcus aureus produce vascular endothelial growth factor, and its expression is amplified by the subsequent production of interleukin-13.

BACKGROUND: Vascular endothelial growth factor (VEGF)-transgenic mice develop psoriasiform plaques that resemble human psoriasis, demonstrating that VEGF is an important factor in the development of psoriasis. In human keratinocytes, VEGF is regulated by human cathelicidin LL37, and the expression of this peptide, as well as interleukin-13 receptor-alpha1 (IL-13Ralpha1) and VEGF, is increased in psoriatic skin. Peptidoglycan (PGN) stimulates innate immunity, inducing cytokines and antimicrobial peptides (cathelicidins), but PGN can also activate psoriatic T-helper-1 (Th1) lymphocytes. As neither the bacterial component that induces LL37 and VEGF expression nor the function of IL-13 in keratinocytes has been clarified, the role of PGN in the induction of these molecules was evaluated in this work. METHODS: HaCaT keratinocytes were treated with PGN from Staphylococcus aureus, recombinant VEGF (rVEGF), and recombinant IL-13 (rIL-13). The mRNA expression of LL37, VEGF, and IL-13 was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), and VEGF and IL-13 production was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Keratinocytes treated with PGN show increased expression of LL37 at 24 h, VEGF at 36 h, and IL-13 at 72 h post-treatment. Anti-Toll-like receptor 2 (anti-TLR2) antibody blocked the production of these molecules. LL37 and VEGF expression in keratinocytes treated with PGN was dose dependent. rVEGF induced IL-13 production and rIL-13 induced VEGF production at 36 h. The anti-Flt-1 antibody blocked IL-13 expression. CONCLUSIONS: These results suggest that PGN from S. aureus can induce LL37 and VEGF expression in keratinocytes, and that this VEGF production can be amplified by subsequent IL-13 overproduction.

The monocyte-derived chemokine is released in the bronchoalveolar lavage fluid of steady-state asthmatics.

Infiltration of the airways by T helper type 2 (Th2) lymphocytes is a well-recognized feature of bronchial asthma. Monocyte-derived chemokine (MDC) is a potent attractant which activates Th2 lymphocytes via the chemokine receptor CCR4. We have investigated both leukocyte recruitment and MDC release into the airways of asthmatic patients. Differential cell counts in bronchoalveolar lavage (BAL) fluid showed that numbers of lymphocytes and eosinophils were elevated in asthmatics compared with normal subjects (median, 6.1 vs. 1.0 x 10(3)/ml, P < 0.005 and 1.4 vs. 0.24 x 10(3)/ml, P = 0.001, respectively). By enzyme-linked immunosorbent assay it was demonstrated that MDC concentrations were significantly elevated in BAL fluid from asthmatics compared with normals (medians 282 pg/ml, range 190-780 pg/ml vs. median 29 pg/ml range 17-82 pg/ml, P < 0.001). Interestingly, there was a significant correlation between MDC levels and the bronchoconstrictive response to methacholine [PC20 forced expiratory volume (FEV)1, r = -0.78, P = 0.001], suggesting that MDC may be involved in the severity of the disease. By immunohistochemistry, MDC was localized predominantly to the bronchial epithelium in bronchial biopsies derived from stable asthmatics. Moreover, primary human airway epithelial cells were found to release MDC upon cytokine stimulation. These findings suggest that MDC may play a major role in the pathogenesis of bronchial asthma.

Antinociceptive effects of interleukin-4, -10, and -13 on the writhing response in mice and zymosan-induced knee joint incapacitation in rats.

The antinociceptive effects of interleukin (IL)-4, -10, and -13 were investigated in two different experimental pain models. Our results showed that pretreatment (30 min) with IL-4 (1-5 ng/animal), IL-10 (0.4-10 ng/animal), or IL-13 (0.4-2.5 ng/animal) inhibited the writhing response induced by the i.p. administration of acetic acid (53-89%) or zymosan (63-74%) in mice, and the knee joint incapacitation induced by i.a. injection of zymosan (49-66%) in rats. Neither of the cytokines affected the pain elicited in mice using the hot-plate test. This analgesic effect of IL-4, -10, and -13 was not reversed by the combined pretreatment with the opioid receptor antagonist naloxone. IL-4, -10, or -13 significantly inhibited the release of both tumor necrosis factor (TNF)-alpha (60, 53, and 100%, respectively) and IL-1beta (80, 100, and 100%, respectively) by mice peritoneal macrophages obtained after local (i.p.) injection of zymosan. Antisera against IL-4, -10, and -13 potentiated both the zymosan-induced writhing response and the articular incapacitation. Our results demonstrate that IL-4, -10, and -13 display analgesic activity that is probably not due to endogenous opioid release. This analgesic effect could be related to a peripheral mechanism, probably via the inhibition of the release of the pro-inflammatory cytokines TNF-alpha and IL-1beta by resident peritoneal macrophages.

Citoquinas: la internet biológica ¿futura terapia en el asma bronquial? / Cytokines: the biological internet, the future therapy of bronchial asthma?

Las citoquinas son polipéptidos producidos por variadas células nucleadas que actúan como intercomunicadores celulares. Participan en funciones de defensa y reparación del adño del organismo y restablecimiento de la homeostasis. En los últimos años y gracias al desarrollo de la biología molecular, ha sido posible identificar y producir en el laboratorio numerosas citoquinas disponibles en el tratamiento de diversas enfermedades. En el asma bronquial existe un desbalance de algunas citoquinas con predominio de la producción de las interleuquinas (ILs) dependientes de los linfocitos tipo Th-2, como IL-4 e IL-5, las cuales inducen la producción de IgE y la eosinofilia, respectivamente. Actualmente están en marcha estudios clínicos tendientes a bloquear o impedir la acción de la IL-4 e IL-5 mediante anticuerpos monoclonales anti-IL o mediante la acción inhibidora sobre estas citoquinas que ejerce la IL-12. En esta revisión bibliográfica se analiza el estado actual de esta nueva futura terapia del asma

IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control of Toxoplasma gondii replication in human fibroblasts activated with IFN-gamma.

The ability of up-regulatory [recombinant (r) IFN-gamma, rIFN-beta and rTNF-alpha] and down-regulatory (rIL-4, rIL-10 and rIL-13) cytokines to control the expression of indoleamine 2,3-dioxygenase (INDO) and anti-Toxoplasma activity in the human fibrosarcoma cell line 2C4 was evaluated. Activation of fibroblasts with rIFN-gamma, rIFN-beta and rTNF-alpha resulted in augmentation of INDO expression and activity leading to 40.0, 25.0 and 27.0 % inhibition of tachyzoite growth, respectively. An additive effect was observed when host cells were incubated with rIFN-gamma plus rTNF-alpha. With regard to the down-regulatory cytokines we observed that IL-4 as well as IL-13, but not IL-10, induced significant inhibition of IFN-gamma-induced control of parasite replication, INDO mRNA expression and tryptophan catabolism. Similarly, IL-4 but not IL-10 inhibited the cell surface expression of HLA-DR and CD2 induced by IFN-gamma. Consistent with these findings we were able to detect by reverse transcription-PCR the expression of mRNA for different chains of IL-4 and IL-13 receptors (IL-4Ralpha, IL-13Ralpha1 and IL-13Ralpha2) but not for IL-10 receptor in the 2C4 and other human lung fibroblast cell lines (LL24 and MRC5). Together our results indicate that IL-4 and IL-13, but not IL-10, are implicated in the negative regulation of IFN-gamma-induced anti-Toxoplasma activity in human cells from fibroblast lineage.