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Regulatory T (Treg) cells induce immunologic tolerance by suppressing effector functions of conventional lymphocytes in the periphery. On the other hand, immune silencing is mediated by recognition of phosphatidylserine (PS) on apoptotic cells by phagocytes. Here we describe expression of the PS‐binding protein Annexin V (ANXA5) in CD4(+) CD25(hi) Treg cells at the mRNA and protein levels. CD4(+) ANXA5(+) T cells constitute about 0·1%–0·6% of peripheral blood CD3(+) T cells, exhibit co‐expression of several Treg markers, such as Forkhead box P3, programmed cell death protein‐1, cytotoxic T‐lymphocyte antigen‐4 and CD38. In vitro, ANXA5(+) Treg cells showed enhanced adhesion to PS(+) endothelial cells. Stimulated by anti‐CD3 and PS(+) syngeneic antigen‐presenting cells CD4(+) ANXA5(+) T cells expanded in the absence of exogenous interleukin‐2. CD4(+) ANXA5(+) T cells suppressed CD4(+) ANXA5(−) T‐cell proliferation and mammalian target of rapamycin phosphorylation, partially dependent on cell contact. CD4(+) ANXA5(+) T‐cell‐mediated suppression was allo‐specific and accompanied by an increased production of anti‐inflammatory mediators. In vivo, using a model of delayed type hypersensitivity, murine CD4(+) ANXA5(+) T cells inhibited T helper type 1 responses. In conclusion, we report for the first time expression of ANXA5 on a subset of Treg cells that might bridge classical regulatory Treg function with immune silencing.

Background Animal models of sciatic nerve injury are commonly used to study neuropathic pain as well as axon regeneration. Inflammation/immune response at the site of nerve lesion is known to be an essential trigger of the pathological changes that have a critical impact on nerve repair and regeneration; moreover, the damage to peripheral nerve can cause a loss of sensory function and produces a persistent neuropathic pain. N-Acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, of which is N-palmitoylethanolamide (PEA) that arouses great attention owing to its anti-inflammatory, analgesic, and neuroprotective activities. The modulation of specific amidases for NAEs (and in particular NAE-hydrolyzing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels. Here, we investigated, in a mice model of sciatic nerve crush, the effect of 2-pentadecyl-2-oxazoline (PEA-OXA) the oxazoline of PEA that reportedly modulates activity of NAAA. Methods In this experimental model, the mice, following the sciatic nerve crush, were treated daily with PEA-OXA at a dose of 10 mg\kg for 14 days. Therefore, we evaluated the effects of PEA-OXA on the degree of injury, on the inhibition of neuropathic pain, and on the inflammatory process, as in the improvement of reparative processes and therefore in the restoration of locomotor function. Results Our results showed that PEA-OXA (10 mg/kg) treatment, daily, for 14 days after sciatic nerve crush, have an anti-inflammatory and neuroprotective effect and moreover have an analgesic protective effect on hypersensitivity, and improve the functional recovery after nerve crush. Conclusions Therefore, treatment with PEA-OXA as a whole has shown a protective effect, which makes it a powerful candidate for the treatment of peripheral nerve injury and neuropathic pain.

Inflammatory bowel disease is characterised by intricate immune cell interactions with tissue cells and such cross-talks can become deregulated. The formyl peptide receptor 1 (Fpr1) is expressed by both immune and stromal cells including epithelial cells. We evaluated the development of the physiopathology of the DNBS induced colitis in Fpr1 KO mice on the C57BL/6 genetic background compared to C57BL/6 genetic background animals. We have assessed both macroscopic and histological markers of the diseased, together with the immunohistochemical and molecular changes. DNBS-treated Fpr1 KO mice showed a i) reduction in weight loss, ii) lower extent of colon injury and iii) an increase in MPO activity. Molecular analyses indicated that in absence of Fpr1 there was reduced NF-κB translocation into the nucleus, cytokines levels, FOXP3 and GATA3, CD4, CD8 and CD45 expression as well as a dysregulation of TGF-β signalling. In addition, the colon of DNBS-injected Fpr1 KO mice displayed a lower degree of expression of Bax and higher expression of Bcl-2 compared correspondent WT mice. Finally, intravital microscopy investigation of the microcirculation post-DNBS instillation revealed a lower degree of neutrophil-endothelial cell rolling and adhesion - mediated by P-selectin and ICAM-1 - in Fpr1 KO mice. All the main outcome in the study have a P-value, statistical significance of evidence, less than 0.05. We provide evidence for an important pathogenic role of mouse Fpr1 in experimental colitis, an outcome effected through modulation of immune cell recruitment together with a modulation of local cellular activation and survival.

Current pharmacological management of Parkinson disease (PD) does not provide for disease modification, but addresses only symptomatic features. Here, we explore a new approach to neuroprotection based on the use of 2-pentadecyl-2-oxazoline (PEA-OXA), the oxazoline derivative of the fatty acid amide signaling molecule palmitoylethanolamide (PEA), in an experimental model of PD. Daily oral treatment with PEA-OXA (10 mg/kg) significantly reduced behavioral impairments and neuronal cell degeneration of the dopaminergic tract induced by four intraperitoneal injections of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on 8-week-old male C57 mice. Moreover, PEA-OXA treatment prevented dopamine depletion, increased tyrosine hydroxylase and dopamine transporter activities, and decreased α-synuclein aggregation in neurons. PEA-OXA treatment also diminished nuclear factor-κB traslocation, cyclooxygenase-2, and inducible nitric oxide synthase expression and through upregulation of the nuclear factor E2-related factor 2 pathway, induced activation of Mn-superoxide dismutase and heme oxygenase-1. Further, PEA-OXA modulated microglia and astrocyte activation and preserved microtubule-associated protein-2 alterations. In conclusion, pharmacological activation of nuclear factor E2-related factor 2 pathways with PEA-OXA may be effective in the future therapy of PD.

Cilj istraživanja: Istražiti postoji li povećan izražaj gena za p62, Beclin 1, LC3A, Atg5 i Ripk3f u tumorskom tkivu u odnosu na zdravo tkivo mokraćnog mjehura miša. Materijali i metode: Eksperimentalno istraživanje provedeno je u Laboratoriju za istraživanje raka Medicinskog fakulteta u Splitu. Miševi C57BL/6J divljeg tipa (wt) stari 6 do 8 tjedana podijeljeni su u 3 eksperimentalne skupine, netretiranu kontrolnu skupinu, ispitivanu skupinu podvrgnutu akutnom tretmanu i ispitivanu skupinu podvrgnutu kroničnom tretmanu. Ispitivane skupine izložene su kemikaliji N-butil-N-(4-hidroksibutil) nitrozaminu (BBN-u) u vodi za piće sa svrhom indukcije tumora mokraćnog mjehura. Akutna skupina miševa bila je izložena 0,05% BBN-u tijekom 2 tjedna, dok je skupina kroničnih miševa BBN-u (0,05%) bila izložena 12 tjedana. Životinje su žrtvovane 2 tjedna (skupina akutnih) i 20 tjedana (skupina kroničnih) nakon početka tretmana. Iz uzoraka je izolirana mRNA, nakon čega je prevedena u cDNA koja je služila kao predložak za qRT-PCR. Kao standard za genski izražaj korišten je gen za aktin. Rezultati: Izražaj gena za p62, Beclin 1, LC3A i Atg5 nije promijenjen u akutnoj skupini u odnosu na kontrolnu netretiranu skupinu. Izražaj gena za Ripk3f statistički je značajno povećan u akutnoj skupini u odnosu na kontrolnu (p, Objectives: To determine expression of autophagy related genes p62, Beclin 1, LC3A and Atg5, as well as gene expression of necroptosis related gene Ripk3f, in bladder carcinoma compared to healthy bladder tissue of mice. Materials and methods: Experimental research was conducted at Laboratory for cancer research at University of Split School of Medicine. Wild type C57BL/6J mice 6-8 weeks old were divided into 3 groups: untreated control group, experimental group that undergone acute treatment and experimental group that undergone chronic treatment. Experimental groups were exposed to a chemical carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) administered in drinking water, in order to induce bladder carcinoma. Acute experimental group was exposed to BBN (0.05%) during 2 weeks, while the chronic experimental group was exposed to BBN (0.05%) during 12 weeks. Mice were sacrificed 2 and 20 weeks after commencement of treatment. RNA was isolated from tissue samples and then converted into cDNA by reverse transcription. cDNA was used as a template for qRT-PCR reaction. Actin gene was used as a housekeeping control. Results: Expression of p62, Beclin 1, LC3A and Atg5 was not changed in acute experimental group compared to nontreated control group. Ripk3f expression was significantly increased in both acute and chronic experimental groups compared to nontreated control group (p

Background: Decreased dipeptidylpeptidase IV (DPPIV) activity within the human nasal mucosa has previously been shown to contribute to the severity of chronic inflammatory rhinosinusitis. Objective: To investigate and correlate the role of DPPIV activity with regard to bronchial inflammation. Methods: DPPIV/CD26 activity/concentration was investigated in the bronchial tissue of human subjects suffering from chronic bronchial inflammation. In addition, the effect of a recombinant Aspergillus fumigatus DPPIV (fuDPPIV) was investigated on histamine-induced bronchoconstriction in anesthetized rabbits. Results and Conclusions: DPPIV/CD26 was present in submucosal seromucous glands, in leukocytes and to a very low degree in endothelial cells of human bronchi. DPPIV activity was correlated with tissue CD26 content measured by immunoassay. As previously reported for the nasal mucosa, DPPIV/CD26 activity was inversely correlated with the degree of airway inflammation. Systemic pretreatment with recombinant fuDPPIV markedly reduced the increase in histamine-induced airway resistance in rabbits. In conclusion, DPPIV activity modulates lower airway tone by degrading unknown peptidic substrates released by histamine in response to an allergen. Contrasting with our observations in the nose, this modulation is apparently not mediated via a neurokinin (NK1) receptor.

Inflammation is fundamentally a protective cellular response aimed at removing injurious stimuli and initiating the healing process. However, when prolonged, it can override the bounds of physiological control and becomes destructive. Inflammation is a key element in the pathobiology of chronic pain, neurodegenerative diseases, stroke, spinal cord injury, and neuropsychiatric disorders. Glia, key players in such nervous system disorders, are not only capable of expressing a pro-inflammatory phenotype but respond also to inflammatory signals released from cells of immune origin such as mast cells. Chronic inflammatory processes may be counteracted by a program of resolution that includes the production of lipid mediators endowed with the capacity to switch off inflammation. These naturally occurring lipid signaling molecules include the N-acylethanolamines, N-arachidonoylethanolamine (an endocannabinoid), and its congener N-palmitoylethanolamine (palmitoylethanolamide or PEA). PEA may play a role in maintaining cellular homeostasis when faced with external stressors provoking, for example, inflammation. PEA is efficacious in mast cell-mediated models of neurogenic inflammation and neuropathic pain and is neuroprotective in models of stroke, spinal cord injury, traumatic brain injury, and Parkinson disease. PEA in micronized/ultramicronized form shows superior oral efficacy in inflammatory pain models when compared to naive PEA. Intriguingly, while PEA has no antioxidant effects per se, its co-ultramicronization with the flavonoid luteolin is more efficacious than either molecule alone. Inhibiting or modulating the enzymatic breakdown of PEA represents a complementary therapeutic approach to treat neuroinflammation. This review is intended to discuss the role of mast cells and glia in neuroinflammation and strategies to modulate their activation based on leveraging natural mechanisms with the capacity for self-defense against inflammation.

CD8 T cells are recognized key players in control of persistent virus infections, but increasing evidence suggests that assistance from other immune mediators is also needed. Here, we investigated whether specific antibody responses contribute to control of lymphocytic choriomeningitis virus (LCMV), a prototypic mouse model of systemic persistent infection. Mice expressing transgenic B cell receptors of LCMV-unrelated specificity, and mice unable to produce soluble immunoglobulin M (IgM) exhibited protracted viremia or failed to resolve LCMV. Virus control depended on immunoglobulin class switch, but neither on complement cascades nor on Fc receptor γ chain or Fc γ receptor IIB. Cessation of viremia concurred with the emergence of viral envelope-specific antibodies, rather than with neutralizing serum activity, and even early nonneutralizing IgM impeded viral persistence. This important role for virus-specific antibodies may be similarly underappreciated in other primarily T cell–controlled infections such as HIV and hepatitis C virus, and we suggest this contribution of antibodies be given consideration in future strategies for vaccination and immunotherapy., Author Summary Persistent viruses such as hepatitis C virus (HCV) or HIV can defeat the body's defense system and cause devastating epidemics worldwide. Recent attempts at vaccinating against HIV have relied on the induction of specific antiviral killer T lymphocytes but have failed to confer protection on the host. Better knowledge about how a successful defense should operate is therefore essential for developing and refining new vaccines. Here, we have used a prototypic mouse model to investigate basic defense mechanisms required to eliminate persisting viruses. Experiments in several genetically engineered mouse models show that contrary to common belief, not only antiviral killer T cells, but also antibodies (produced by B cells), are needed to prevent a virus from persisting in its host. These findings suggest that induction of antibodies, along with antiviral killer T lymphocytes, should be envisaged when devising new strategies for vaccinating against HIV or HCV., Andreas Bergthaler and colleagues reveal that T cell control of systemic persistent viral infections requires support by antibodies produced by B cells, which may have important implications for future vaccination strategies against HIV and hepatitis C.

We previously reported that reducing the expression of the gap junction protein connexin (Cx)43 in mice restricts intimal thickening formation after acute vascular injury by limiting the inflammatory response and the proliferation and migration of smooth muscle cells (SMCs) toward the damaged site. SMC populations isolated from porcine coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening, suggesting that they participate in the restenotic process. Here, we further investigate the relationship between connexin expression and SMC phenotypes using porcine coronary artery SMCs. Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was downregulated and Cx43 was markedly upregulated in stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. In R-SMCs, Cx43 expression was increased and Cx40 was absent. We confirmed that S-SMCs treated with platelet-derived growth factor-BB acquire an R phenotype. This was accompanied by an upregulation of Cx43 and a loss of Cx40. Importantly, platelet-derived growth factor-BB–induced S-to-R phenotypic change was prevented by a reduction of Cx43 expression with antisense, ie, S-SMCs retained their typical elongated appearance and the expression of α-smooth muscle actin, a well-known SMC differentiation marker, whereas the expression of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in S-SMCs prevents platelet-derived growth factor-BB–induced S-to-R modulation. This suggests that Cx43 may be an additional target for local delivery strategies aimed at reducing restenosis.

International audience; Chronic granulomatous disease (CGD), a genetic disorder characterized by the absence of a functional phagocyte NADPH oxidase, is a severe immune deficiency. However, non-infectious hyperinflammation is a second hallmark of the disease. In CGD mouse models, sterile hyperinflammation can be induced by A. fumigatus cell wall preparations. In this study, we used subcutaneous injection of microbial cell walls and cell wall components to identify causes of CGD hyperinflammation and to characterize its histological features. Sterile cell wall preparations from fungi (A. fumigatus, C. albicans, S. cerevisiae), but not from bacteria (S. aureus, P. aeruginosa, E. coli), caused prolonged and severe skin inflammation in CGD mice. To identify fungal cell wall elements responsible for this process, we investigated microbial cell wall-derived monosubstances. Injection of beta(1-3)(1-6)-glucan induced severe hyperinflammation in CGD mice, while other fungal cell components [mannan, (1-3) beta-glucan] or bacterial cell wall components (lipopolysaccharide, lipoteichoic acid) caused no or only moderate inflammation. beta-glucan-induced hyperinflammation was predominantly due to a defect in termination of inflammation, as in the initial stage (2 days), the severity of inflammation and the extent of cell death were comparable in wild-type and CGD mice. At later stages (7 days), beta(1-3)(1-6)-glucan-induced inflammation had subsided in wild-type mice. In contrast, CGD mice showed persistent severe inflammation with central necrosis, containing abundant apoptotic and necrotic cells. In summary, branched fungal beta-glucan induces a severe inflammatory reaction in the absence of phagocyte NADPH oxidase. As opposed to the commonly perceived notion that reactive oxygen species are the cause of cell death, our results demonstrate that tissue necrosis can be caused by the absence of a superoxide-producing enzyme.

Neurogenesis is nearly completed after birth, whereas gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. Little is known about the effects of hypoxic-ischemic (HI) injury on these critical postnatal processes. Here we explored the glial reactions to mild HI injury of the neonatal rat cerebral cortex at P3. We show that the HI lesion results in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. The morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggest an accelerated transformation of radial glia into astrocytes. In addition, BrdU/GFAP immunostaining revealed a significant increase of double-labelled cells indicating an acute proliferation of astrocytes after HI. This enhanced proliferative activity of astrocytes persisted for several weeks. We found an elevated number and increased mitotic activity of both NG2-positive oligodendrocyte progenitors and RIP-positive oligodendrocytes after injury. These findings imply that glial responses are central to cortical tissue remodelling following neonatal ischemia and represent a potential target for therapeutic approaches.

BACKGROUND & AIMS: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor and a regulator of insulin sensitivity in peripheral tissues. In the liver, PTEN deletion increases insulin sensitivity, but induces steatosis, steatohepatitis, and hepatocellular carcinoma. Here, we investigated the pathophysiologic mechanisms regulating PTEN expression in the liver and the development of steatosis. METHODS: PTEN expression was evaluated in the liver of rats and human beings having metabolic syndrome. Signaling pathways regulating PTEN expression and lipid accumulation in hepatocytes were examined in vitro. RESULTS: PTEN expression is down-regulated in the liver of rats having steatosis and high plasma levels of fatty acids, as well as in steatotic human livers. Unsaturated fatty acids inhibited PTEN expression in HepG2 cells via activation of a signaling complex formed by the mammalian target of rapamycin (mTOR) and nuclear factor-kappaB (NF-kappaB). Down-regulation of PTEN expression induced steatosis by affecting import, esterification, and extracellular release of fatty acids. CONCLUSIONS: Hepatic steatosis can be mediated by alterations of PTEN expression in hepatocytes exposed to high levels of unsaturated fatty acids. Furthermore, our data revealed interaction between mTOR and NF-kappaB, suggesting cross-talk between these 2 pathways.

Com o objetivo de padronizar normas técnicas para obtenção de modelo animal com tumor de Walker 256 e de estabelecer o número de células tumorais necessárias para que esse tumor tenha grande porcentagem de pega e longevidade, possibilitando o desenvolvimento de pesquisas em várias áreas da saúde, foi realizado trabalho em duas etapas. Na primeira foram utilizados 120 ratos para treinamento e definição da técnica. Na segunda etapa foram utilizados 84 ratos, sendo estes separados em 7 grupos (G) de 12 animais cada. O tumor, na forma ascítica, foi inoculado no tecido celular subcutâneo do dorso dos ratos com os seguintes números de células: GI, 1 x 10(7); GII, 5 x 10(6); GIII, 2,5 x 10(6); GIV, 1 x 10(6); GV, 5 x 10(5); GVI, 3 x 10(5) e GVII, 2 x 10(5). Foram avaliadas a porcentagem de pega e a longevidade nos grupos. Os animais dos GI, GII, GIII e GIV obtiveram 100% de desenvolvimento tumoral, porém baixa longevidade. Os dos GV e GVI obtiveram desenvolvimento tumoral em frequência maior que 90% e longevidade satisfatória. Os do GVII não apresentaram desenvolvimento tumoral. Concluiu-se que todos os procedimentos devem ser exaustivamente treinados e que o número de células tumorais viáveis para inoculação, em tecido celular subcutâneo de ratos, deve estar na faixa entre 5 x 10(5) e 3 x 10(5). The aim of this work was standardize technical norms to obtain a model of Walker 256 tumor in animals and get the tumorous cells number needed to increase the tumorous join percentage and longevity, it makes possible the research development in several health areas. The work was realized in two stages. In first were used 120 rats to crew’s training and technicals definitions. In second stage were used 84 rats, these separated in 7 groups (G) with 12 animals each one. The tumor, in ascitic form was inoculated on subcutaneous cellular tissue on dorsal of rats with the follow number of cells : GI, 1 x 10(7); GII, 5 x 10(6); GIII, 2,5 x 10(6); GIV, 1 x 10(6); GV, 5 x 10(5); GVI, 3 x 10(5) e GVII, 2 x 10(5). Were evaluate the join percentage and longevity in groups. The animals of GI, GII, GIII e GIV obtained 100% of tumorous development, however low longevity. The two GV and GVI had tumorous development often high that 90% and satisfactory longevity. The GVII didn’t show tumorous development . The conclusion was made all procedures must be exhaustivelly trained and the number of tumorous cells passable to inoculation, in subcutaneous cellular tissue of rats, must be in the range of 5x10(5) and 3x10(5) .

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development.