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As recently described, the administration of extremely low doses (pg/kg) of CCL4 (Macrophage inflammatory protein 1β, MIP-1β) can induce antinociceptive effects in mice (Garcia-Dominguez et al., 2019b). We describe here that hydrodynamic delivery of a plasmid containing CCL4 cDNA provokes a biphasic response consisting in an initial thermal hyperalgesic reaction for 8 days followed by analgesia at days 10–12, being both responses blocked after the administration of the CCR5 antagonist DAPTA. Both the luminiscence evoked in liver after the administration of a plasmid containing CCL4 and luciferase cDNAs and the hepatic concentration of CCL4 measured by ELISA were maximal 4 days after plasmid administration and markedly diminished at day 10. A dose–effect curve including a wide dose range of exogenous CCL4 revealed thermal analgesia after the administration of 10–100 pg/kg whereas 1000 times higher doses (30–100 ng/kg) induced, instead, thermal hyperalgesia inhibited by DAPTA. This hyperalgesia was absent in mice with reduced white blood cells after cyclophosphamide treatment, thus supporting the involvement of circulating leukocytes. A multiarray bioluminescent assay revealed increased plasma levels of IL-1α, CCL2, CXCL1, CXCL13, IL-16 and TIMP-1 in mice treated with 100 ng/kg of CCL4. The hyperalgesic response evoked by CCL4 was prevented by IL-1R, CXCR2 or CCR2 antagonists or by the neutralization of CXCL13 or IL-16, but not TIMP-1, with selective antibodies. The administration of the anti-IL-16 antibody was the unique treatment able to convert hyperalgesia evoked by 100 ng/kg of CCL4 in an analgesic effect. The ability of IL-16 to evoke hypernociception was confirmed by studying the response to its exogenous administration (10–30 ng/kg). In summary, the present results demonstrate that CCL4 induces a dual modulation of nociception and describe some mechanisms involved in the hyperalgesic response evoked by this chemokine.

Matrix metalloproteinases can modulate the inflammatory response through processing of cyto- and chemokines. Among them, MMP-14 is a non-dispensable collagenase responsible for the activation of other enzymes, triggering a proteolytic cascade. To identify the role of MMP-14 during the pro-inflammatory response, wildtype and Mmp14 −/− mice were challenged with lipopolysaccharide. MMP-14 levels decreased after endotoxemia. Mutant animals showed 100% mortality, compared to 50% in wildtype mice. The increased mortality was related to a more severe lung injury, an impaired lung MMP-2 activation, and increased levels of the alarmin S100A9. There were no differences in the expression of other mediators including Il6, Cxcl2, Tgfb, Il10, or S100a8. A similar result was observed in lung explants of both genotypes cultured in presence of lipopolysaccharide. In this ex vivo model, exogenous activated MMP-2 ameliorated the observed increase in alarmins. Samples from septic patients showed a decrease in serum MMP-14 and activated MMP-2 compared to non-septic critically ill patients. These results demonstrate that the MMP-14-MMP-2 axis is downregulated during sepsis, leading to a proinflammatory response involving S100A9 and a more severe lung injury. This anti-inflammatory role of MMP-14 could have a therapeutic value in sepsis. • MMP-14 levels decrease in lungs from endotoxemic mice and serum from septic patients. • Mmp14 −/− mice show increased lung injury and mortality following endotoxemia. • Absence of Mmp14 decreases activated MMP-2 and increases S100A9 levels in lung tissue. • MMP-14 ameliorates inflammation by promoting S100A9 cleavage by activated MMP-2.

MT1‐MMP ( MMP14 ) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14 −/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1‐MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1‐MMP induces a senescent phenotype characterized by up‐regulation of p16 INK 4a and p21 CIP 1/ WAF 1 , increased activity of senescence‐associated β‐galactosidase, generation of a senescence‐associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14 −/− mice with all‐ trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.

Autophagy has emerged as a key regulator of the inflammatory response. To examine the role of autophagy in the development of organ dysfunction during endotoxemia, wild-type and autophagy-deficient (Atg4b-null) mice were challenged with lipopolysaccharide. Animals lacking Atg4b showed increased mortality after endotoxemia. Among the different organs studied, only the lungs showed significant differences between genotypes, with increased damage in mutant animals. Autophagy was activated in lungs from wild-type, LPS-treated mice. Similarly, human bronchial cells show an increased autophagy when exposed to serum from septic patients. We found an increased inflammatory response (increased neutrophilic infiltration, higher levels of Il6, Il12p40, and Cxcl2) in the lungs from knockout mice and identified perinuclear sequestration of the anti-inflammatory transcription factor ATF3 as the putative mechanism responsible for the differences between genotypes. Finally, induction of autophagy by starvation before LPS exposure resulted in a dampened pulmonary response to LPS in wild-type, but not knockout, mice. Similar results were found in human bronchial cells exposed to LPS. Our results demonstrate the central role of autophagy in the regulation of the lung response to endotoxemia and sepsis and its potential modulation by nutrition.Endotoxemia and sepsis trigger autophagy in lung tissue. Defective autophagy increases mortality and lung inflammation after endotoxemia. Impairment of autophagy results is perinuclear ATF3 sequestration. Starvation ameliorates lung injury by an autophagy-dependent mechanism.

Toll-like receptors (TLRs) have raised an extraordinary interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs drive an inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, TLR4, and TLR9 in gastric cancer. For this purpose, an immunohistochemical study on cancer specimens from 106 patients with gastric cancer was performed using tissue arrays and specific antibodies against TLR3, TLR4, and TLR9. The results indicate that gastric carcinomas samples show high expression of TLR3, TLR4, and TLR9 by cancer cells. The expression of TLR3 by cancer cells was significantly associated with a poor overall survival in patients with resectable tumors. Moreover, in patients with resectable tumors and lymph node invasion, a high TLR3 expression defines a population with even worse prognosis. Therefore, TLR3 may have clinical interest as indicator of tumor aggressiveness and as a prognostic indicator in gastric cancer.

Toll-like receptors (TLRs) have garnered an extraordinary amount of interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs induce type I interferons and other cytokines, which drive an inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, 4, and 9 in prostate cancer.The expression levels of TLR3, TLR4, and TLR9 were analyzed on tumors from 133 patients with prostate cancer. The analyses were performed by immunohistochemistry on tissue arrays and real time-PCR.Cancerous cells showed high expression levels of TLRs compared with controls. Samples of carcinomas with recurrence exhibited a significant increase in the mRNA levels of TLR3, TLR4, and TLR9. In addition, the tumors that showed high TLR3 or TLR9 expression levels were significantly associated with higher probability of biochemical recurrence.TLR expression is associated with prostate cancer with recurrence and the role of TLR receptors in the biology of malignancy merits study. Therapeutic strategies to boost or block TLRs may be of interest.

The use of live bioreactors for the expression of human genes in the mammary gland of transgenic animals is one of the most cost-effective ways for the production of valuable recombinant therapeutic proteins. Among the transgenic species used so far, rabbits are good candidates for the expression of tens to hundreds of grams of complex proteins in the milk during lactation. The lactating mammary gland of rabbits has proven to be effective in the processing of complex proteins. In this work, the potential use of rabbits as bioreactors is discussed based on our results and the published data.

An understanding of the expression of transgenes in the mammary gland during gestation and lactation is crucial for the use of transgenic mammals as bioreactors. Here we describe the temporal pattern of expression of the endogenous rabbit WAP gene and human erythropoietin (hEPO) transgenes under the control of rabbit WAP promoter and 3′ flanking sequences. The endogenous rabbit WAP gene was expressed throughout gestation including the day of mating, as well as during lactation in transgenic rabbits bearing a minigene construct. In non-pregnant cycling females, WAP expression was found independent of transgenic status; however, WAP expression was not detected in non-cycling females. The significance of this new finding is not clear at present. hEPO mRNA was detected in mammary gland biopsies from pregnant transgenic rabbits only on day 28 of gestation. During lactation, transcripts were present in mammary gland biopsy samples taken on days 0, 7, 14 and 21. A sharp decline in the levels ...

Critically ill patients frequently develop neuropsychological disturbances including acute delirium or memory impairment. The need for mechanical ventilation is a risk factor for these adverse events, but a mechanism that links lung stretch and brain injury has not been identified.To identify the mechanisms that lead to brain dysfunction during mechanical ventilation.Brains from mechanically ventilated mice were harvested, and signals of apoptosis and alterations in the Akt survival pathway were studied. These measurements were repeated in vagotomized or haloperidol-treated mice, and in animals intracerebroventricularly injected with selective dopamine-receptor blockers. Hippocampal slices were cultured and treated with micromolar concentrations of dopamine, with or without dopamine receptor blockers. Last, levels of dysbindin, a regulator of the membrane availability of dopamine receptors, were assessed in the experimental model and in brain samples from ventilated patients.Mechanical ventilation triggers hippocampal apoptosis as a result of type 2 dopamine receptor activation in response to vagal signaling. Activation of these receptors blocks the Akt/GSK3β prosurvival pathway and activates the apoptotic cascade, as demonstrated in vivo and in vitro. Vagotomy, systemic haloperidol, or intracerebroventricular raclopride (a type 2 dopamine receptor blocker) ameliorated this effect. Moreover, ventilation induced a concomitant change in the expression of dysbindin-1C. These results were confirmed in brain samples from ventilated patients.These results prove the existence of a pathogenic mechanism of lung stretch-induced hippocampal apoptosis that could explain the neurological changes in ventilated patients and may help to identify novel therapeutic approaches.

Background Mechanical ventilation can promote lung injury by triggering a pro-inflammatory response. Macrolides may exert some immunomodulatory effects and have shown significant benefits over other antibiotics in ventilated patients. We hypothesized that macrolides could decrease ventilator-induced lung injury. Methods Adult mice were treated with vehicle, clarithromycin or levofloxacin, and randomized to receive mechanical ventilation with low (12 cmH2O, PEEP 2 cmH2O) or high (20 cmH2O, ZEEP) inspiratory pressures for 150 minutes. Histological lung injury, neutrophil infiltration, inflammatory mediators (NFκB activation, Cxcl2, IL-10) and levels of adhesion molecules (E-selectin, ICAM) and proteases (MMP-9 and MMP-2) were analyzed. Results There were no differences among groups after low-pressure ventilation. Clarithromycin significantly decreased lung injury score and neutrophil count, compared to vehicle or levofloxacin, after high-pressure ventilation. Cxcl2 expression and MMP-2 and MMP-9 levels increased and IL-10 decreased after injurious ventilation, with no significant differences among treatment groups. Both clarithromycin and levofloxacin dampened the increase in NFκB activation observed in non-treated animals submitted to injurious ventilation. E-selectin levels increased after high pressure ventilation in vehicle- and levofloxacin-treated mice, but not in those receiving clarithromycin. Conclusions Clarithromycin ameliorates ventilator-induced lung injury and decreases neutrophil recruitment into the alveolar spaces. This could explain the advantages of macrolides in patients with acute lung injury and mechanical ventilation.

Excessive lung stretch triggers lung inflammation by activation of the NF-κB pathway. This route can be modulated by autophagy, an intracellular proteolytic system. Our objective was to study the impact of the absence of autophagy in a model of ventilator-induced lung injury. Mice lacking Autophagin-1/ATG4B ( Atg4b −/−), a critical protease in the autophagic pathway, and their wild-type counterparts were studied in baseline conditions and after mechanical ventilation. Lung injury, markers of autophagy, and activation of the inflammatory response were evaluated after ventilation. Mechanical ventilation increased autophagy and induced lung injury in wild-type mice. Atg4b −/− animals showed a decreased lung injury after ventilation, with less neutrophilic infiltration than their wild-type counterparts. As expected, autophagy was absent in mutant animals, resulting in the accumulation of p62 and ubiquitinated proteins. Activation of the canonical NF-κB pathway was present in ventilated wild-type, but not Atg4b-deficient, animals. Moreover, these mutant mice showed an accumulation of ubiquitinated IκB. High-pressure ventilation partially restored the autophagic response in Atg4b −/− mice and abolished the differences between genotypes. In conclusion, impairment of autophagy results in an ameliorated inflammatory response to mechanical ventilation and decreases lung injury. The accumulation of ubiquitinated IκB may be responsible for this effect.

The tissue specificity of the expression of the hepatitis B surface antigen (HBsAg) in transgenic mice was studied. Northern blot hybridization and electron microscopy of various tissues revealed the higher HBsAg transcription levels confined to the liver and kidney, and to a lesser extent to the spleen and intestines. In electron microscopic studies we found HBsAg particles in the serum of transgenic animals to be positive by ELISA to HBsAg. The expression of the transgene was localized in situ in the ovary, liver, kidney, and spleen of transgenic mice, employing a polyclonal antiserum directed against the HBsAg and conjugated with protein A-gold. Surprisingly, F1 transgenic females from the CB-104 line, expressing HBsAg in the serum, showed impaired fertility, although the ovarian function was not diminished. Normal off-spring were obtained after cross embryo transfer between transgenic (Tg) females to non-transgenic (Ntg) recipients or from Ntg embryos transferred to Tg recipients. We speculate that this phenomenon could be related to a disruption on a gene(s) somehow involved in the reproductive performance of the transgenic females.

Matrix metalloproteinase-8, released mainly from neutrophils, is a critical regulator of the inflammatory response by its ability to cleave multiple mediators. Herein, we report the results of a model of endotoxemia after intraperitoneal LPS injection in mice lacking MMP-8 and their wildtype counterparts. Control, saline-treated animals showed no differences between genotypes. However, there was an increased lung inflammatory response, with a prominent neutrophilic infiltration in mutant animals after LPS treatment. Using a proteomic approach, we identify alarmins S100A8 and S100A9 as two of the main differences between genotypes. Mice lacking MMP-8 showed a significant increase in these two molecules in lung homogenates, but not in spleen and serum. Mice lacking MMP-8 also showed an increase in MIP-1α levels and a marked activation of the non-canonical NF-κB pathway, with no differences in CXC-chemokines such as MIP-2 or LIX. These results show that MMP-8 can modulate the levels of S100A8 and S100A9 and its absence promotes the lung inflammatory response during endotoxemia.

This study was designed to determine the possibility of using F1 crossbreed cattle (Holstein × Zebu) as donors of oocytes for in vitro fertilization (IVF) and for pronuclear gene microinjection into in vitro-produced embryos. In the first part of the experiment oocytes from Bostaurus (Holstein), Bosindicus (Zebu) and F1 crossbred Bostaurus × Bosindicus (Holstein × Zebu) genotypes were inseminated with Bostaurus (Holstein) semen and were allocated for in vitro embryo production using conventional IVF procedures. No differences were observed on the in vitro maturation (IVM) rates between breeds (Holstein × Holstein:85%, Zebu × Holstein:84% and Zebu × Holstein × Holstein:88%). Holstein cows yielded the highest number of cumulus oocyte complexes (6.8 per ovary) for in vitro maturation, differing (P