RESUMO
Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin-muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic deletion of interleukin-1 (IL-1) using IL-1αß knockout (KO) mice and perioperative IL-1 receptor type 1 (IL-1R1) blockade with the drug anakinra, but not treatment with the glucocorticoid prednisolone, prevented these changes. Anakinra treatment also reversed the established sensitization phenotype when initiated 8 days after incision. Furthermore, with the generation of an IL-1ß floxed(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1ß, suggesting that both peripheral and central inflammatory mechanisms contribute to the transferred disease phenotype. These results indicate that persistent CRPS is often contributed to by autoantibodies and highlight a potential therapeutic use for clinically licensed antagonists, such as anakinra, to prevent or treat CRPS via blocking IL-1 actions.
Assuntos
Autoanticorpos/imunologia , Síndromes da Dor Regional Complexa/imunologia , Imunoglobulina G/imunologia , Interleucina-1alfa/imunologia , Interleucina-1beta/imunologia , Adulto , Animais , Autoanticorpos/administração & dosagem , Autoanticorpos/sangue , Síndromes da Dor Regional Complexa/sangue , Síndromes da Dor Regional Complexa/diagnóstico , Síndromes da Dor Regional Complexa/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/sangue , Proteína Antagonista do Receptor de Interleucina 1/administração & dosagem , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Extremidade Inferior/lesões , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Microglia/patologia , Pessoa de Meia-Idade , Medição da Dor , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Receptores Tipo I de Interleucina-1/imunologia , Receptores Tipo I de Interleucina-1/metabolismo , Corno Dorsal da Medula Espinal/imunologia , Corno Dorsal da Medula Espinal/patologiaRESUMO
Pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) is a multifunctional neuropeptide, which may play a role in cardioprotection. However, little is known about the presence of PACAP-38 in heart failure (HF) patients. The aim of our study was to measure the alterations of PACAP-38 like immunoreactivity (LI) in acute (n = 13) and chronic HF (n = 33) and to examine potential correlations between PACAP-38 and HF predictors (cytokines, NT-proBNP). Tissue PACAP-38 LI and PAC1 receptor levels were also investigated in heart tissue samples of patients with HF. Significantly higher plasma PACAP-38 LI was detected in patients with acute HF, while in chronic HF patients, a lower level of immunoreactivity was observed compared to healthy controls (n = 13). Strong negative correlation was identified between plasma PACAP-38 and NT-proBNP levels in chronic HF, as opposed to the positive connection seen in the acute HF group. Plasma IL-1 ß, IL-2 and IL-4 levels were significantly lower in chronic HF, and IL-10 was significantly higher in patients with acute HF. PACAP-38 levels of myocardial tissues were lower in all end-stage HF patients and lower PAC1 receptor levels were detected in the primary dilated cardiomyopathy group compared to the controls. We conclude that PACAP-38 and PAC1 expression correlates with some biomarkers of acute and chronic HF; therefore, further studies are necessary to explore whether PACAP could be a suitable prognostic biomarker in HF patients.
Assuntos
Insuficiência Cardíaca , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Humanos , Miocárdio/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismoRESUMO
Psoriasis is a chronic inflammatory disease with unmet medical needs. To clarify potential therapeutic targets, different animal models have been developed. In the current study, imiquimod-induced psoriasiform dermatitis was used for monitoring the changes in skin thickness, transepidermal water loss, body weight, blood perfusion and drug permeability for a topical cream formulation of caffeine, both in wild type and in knock out mice. Morphological characterization of control and diseased tissues was performed by scanning electron microscopy and two-photon microscopy. The chemically induced psoriatic group showed increased skin permeability for the model drug during disease progression. In wild type and TRPA1 KO mice, however, enhanced skin thickness and hyperkeratosis blocked further increase of drug penetration at the late phase (96 h). These results indicate that topical drug therapy can be more effective in early phases of plaque development, when skin thickness is lower. Although paracellular connections (tight junctions) are looser in the advanced phase, hyperkeratosis blocks drug delivery through the transappendageal routes. Novel drug formulations may have the potency for effective drug delivery across the epidermal barrier even in the advanced phase. For development of more effective topical drugs, further research is proposed to explore drug penetration both in healthy and diseased conditions.
Assuntos
Dispositivos Lab-On-A-Chip , Psoríase , Animais , Modelos Animais de Doenças , Epiderme , Camundongos , Imagem Óptica , Permeabilidade , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , PeleRESUMO
Scots pine (SO) and clove (CO) essential oils (EOs) are commonly used by inhalation, and their main components are shown to reduce inflammatory mediator production. The aim of our research was to investigate the chemical composition of commercially available SO and CO by gas chromatography-mass spectrometry and study their effects on airway functions and inflammation in an acute pneumonitis mouse model. Inflammation was evoked by intratracheal endotoxin and EOs were inhaled three times during the 24 h experimental period. Respiratory function was analyzed by unrestrained whole-body plethysmography, lung inflammation by semiquantitative histopathological scoring, myeloperoxidase (MPO) activity and cytokine measurements. α-Pinene (39.4%) was the main component in SO, and eugenol (88.6%) in CO. Both SO and CO significantly reduced airway hyperresponsiveness, and prevented peak expiratory flow, tidal volume increases and perivascular edema formation. Meanwhile, inflammatory cell infiltration was not remarkably affected. In contrast, MPO activity and several inflammatory cytokines (IL-1ß, KC, MCP-1, MIP-2, TNF-α) were aggravated by both EOs. This is the first evidence that SO and CO inhalation improve airway function, but enhance certain inflammatory parameters. These results suggest that these EOs should be used with caution in cases of inflammation-associated respiratory diseases.
Assuntos
Asma , Óleos Voláteis , Pinus sylvestris , Pneumonia , Syzygium , Animais , Endotoxinas/toxicidade , Inflamação/tratamento farmacológico , Camundongos , Óleos Voláteis/química , Pneumonia/induzido quimicamente , Syzygium/químicaRESUMO
There is a current imperative to reveal more precisely the molecular pathways of early onset of systemic autoimmune diseases (SADs). The investigation of newly diagnosed drug-naive SAD patients might contribute to identify novel disease-specific and prognostic markers. The multiplex analysis of 30 plasma proteins in 60 newly diagnosed drug-naive SADs, such as RA (rheumatoid arthritis, n = 31), SLE (systemic lupus erythematosus, n = 19), and SSc (systemic scleroderma, n = 10) patients, versus healthy controls (HCs, n = 40) was addressed. Thirty plasma cytokines were quantified using the Procarta Plex™ panel. The higher expression of IL-12p40, IL-10, IL-13, IFN-γ, M-CSF, IL-4, NTproBNP, IL-17A, BMP-9, PYY (3-36), GITRL, MMP-12, and TNFRSF6 was associated with RA; IL-12p40, M-CSF, IL-4, GITRL, and NTproBNP were higher in SLE; or NTproBNP, PYY (3-36), and MMP-12 were increased in SSc over HCs, respectively. The cleaved peptide tyrosine tyrosine (PYY 3-36) was elevated in RA (361.6 ± 47.7 pg/ml) vs. HCs (163.96 ± 14.5 pg/ml, mean ± SEM, ∗∗∗ p = 4 × 10-5). The CI (95%) was 268.05-455.16 pg/ml for RA vs. 135.55-192.37 pg/ml for HCs. The elevated PYY (3-36) level correlated significantly with the increased IL-4 or GITRL concentration but not with the clinical scores (DAS28, CRP, ESR, RF, aMCV). We are the first to report cleaved PYY (3-36) as a specific plasma marker of therapy-naive RA. Additionally, the multiplex plasma protein analysis supported a disease-specific cytokine pattern in RA, SLE, and SSc, respectively.
Assuntos
Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/fisiopatologia , Biomarcadores/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/imunologia , Tirosina/química , Idoso , Citocinas/metabolismo , Feminino , Humanos , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Reconhecimento Automatizado de Padrão , PrognósticoRESUMO
Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.
Assuntos
Luteína/análogos & derivados , Macrófagos/efeitos dos fármacos , Melilotus/química , Células Receptoras Sensoriais/efeitos dos fármacos , Animais , Luteína/análise , Luteína/isolamento & purificação , Luteína/farmacologia , Macrófagos/citologia , Camundongos , Ratos , Células Receptoras Sensoriais/citologiaRESUMO
The Transient Receptor Potential Ankyrin 1 (TRPA1) cation channel expressed on capsaicin-sensitive afferents, immune and endothelial cells is activated by inflammatory mediators and exogenous irritants, e.g., endotoxins, nicotine, crotonaldehyde and acrolein. We investigated its involvement in acute and chronic pulmonary inflammation using Trpa1 gene-deleted (Trpa1-/-) mice. Acute pneumonitis was evoked by intranasal Escherichia coli endotoxin (lipopolysaccharide: LPS) administration, chronic bronchitis by daily cigarette smoke exposure (CSE) for 4 months. Frequency, peak inspiratory/expiratory flows, minute ventilation determined by unrestrained whole-body plethysmography were significantly greater, while tidal volume, inspiratory/expiratory/relaxation times were smaller in Trpa1-/- mice. LPS-induced bronchial hyperreactivity, myeloperoxidase activity, frequency-decrease were significantly greater in Trpa1-/- mice. CSE significantly decreased tidal volume, minute ventilation, peak inspiratory/expiratory flows in wildtypes, but not in Trpa1-/- mice. CSE remarkably increased the mean linear intercept (histopathology), as an emphysema indicator after 2 months in wildtypes, but only after 4 months in Trpa1-/- mice. Semiquantitative histopathological scores were not different between strains in either models. TRPA1 has a complex role in basal airway function regulation and inflammatory mechanisms. It protects against LPS-induced acute pneumonitis and hyperresponsiveness, but is required for CSE-evoked emphysema and respiratory deterioration. Further research is needed to determine TRPA1 as a potential pharmacological target in the lung.
Assuntos
Bronquite Crônica/fisiopatologia , Fumar Cigarros/efeitos adversos , Pneumonia/fisiopatologia , Canal de Cátion TRPA1/metabolismo , Animais , Bronquite Crônica/induzido quimicamente , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Feminino , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Peroxidase/metabolismo , Pletismografia Total , Pneumonia/induzido quimicamente , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Testes de Função Respiratória , Canal de Cátion TRPA1/genéticaRESUMO
Thyme (TO), cinnamon (CO), and Ceylon type lemongrass (LO) essential oils (EOs) are commonly used for inhalation. However, their effects and mechanisms on inflammatory processes are not well-documented, and the number of in vivo data that would be important to determine their potential benefits or risks is low. Therefore, we analyzed the chemical composition and investigated the activity of TO, CO, and LO on airway functions and inflammatory parameters in an acute pneumonitis mouse model. The components of commercially available EOs were measured by gas chromatography-mass spectrometry. Airway inflammation was induced by intratracheal endotoxin administration in mice. EOs were inhaled during the experiments. Airway function and hyperresponsiveness were determined by unrestrained whole-body plethysmography on conscious animals. Myeloperoxidase (MPO) activity was measured by spectrophotometry from lung tissue homogenates, from which semiquantitative histopathological scores were assessed. The main components of TO, CO, and LO were thymol, cinnamaldehyde, and citronellal, respectively. We provide here the first evidence that TO and CO reduce inflammatory airway hyperresponsiveness and certain cellular inflammatory parameters, so they can potentially be considered as adjuvant treatments in respiratory inflammatory conditions. In contrast, Ceylon type LO inhalation might have an irritant effect (e.g., increased airway hyperresponsiveness and MPO activity) on the inflamed airways, and therefore should be avoided.
Assuntos
Anti-Inflamatórios/farmacologia , Endotoxinas/efeitos adversos , Óleos Voláteis/farmacologia , Óleos de Plantas/farmacologia , Thymus (Planta)/química , Animais , Anti-Inflamatórios/química , Biomarcadores , Modelos Animais de Doenças , Feminino , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Óleos Voláteis/química , Óleos de Plantas/química , Doenças Respiratórias/tratamento farmacológico , Doenças Respiratórias/etiologia , Doenças Respiratórias/patologiaRESUMO
Multiple sclerosis is a chronic inflammatory, demyelinating degenerative disease of the central nervous system. Current treatments target pathological immune responses to counteract the inflammatory processes. However, these drugs do not restrain the long-term progression of clinical disability. For this reason, new therapeutic approaches and identification of novel target molecules are needed to prevent demyelination or promote repair mechanisms. Transient Receptor Potential Ankyrin 1 (TRPA1) is a nonselective cation channel with relatively high Ca2+ permeability. Its pathophysiological role in central nervous system disorders has not been elucidated yet. In the present study, we aimed to assess the distribution of TRPA1 in the mouse brain and reveal its regulatory role in the cuprizone-induced demyelination. This toxin-induced model, characterized by oligodendrocyte apoptosis and subsequent primary demyelination, allows us to investigate the nonimmune aspects of multiple sclerosis. We found that TRPA1 is expressed on astrocytes in the mouse central nervous system. Interestingly, TRPA1 deficiency significantly attenuated cuprizone-induced demyelination by reducing the apoptosis of mature oligodendrocytes. Our data suggest that TRPA1 regulates mitogen-activated protein kinase pathways, as well as transcription factor c-Jun and a proapoptotic Bcl-2 family member (Bak) expression resulting in enhanced oligodendrocyte apoptosis. In conclusion, we propose that TRPA1 receptors enhancing the intracellular Ca2+ concentration modulate astrocyte functions, and influence the pro or anti-apoptotic pathways in oligodendrocytes. Inhibition of TRPA1 receptors might successfully diminish the degenerative pathology in multiple sclerosis and could be a promising therapeutic target to limit central nervous system damage in demyelinating diseases. GLIA 2016;64:2166-2180.
Assuntos
Apoptose/efeitos dos fármacos , Encéfalo , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Inibidores da Monoaminoxidase/toxicidade , Oligodendroglia/efeitos dos fármacos , Canal de Cátion TRPA1/deficiência , Polipose Adenomatosa do Colo/metabolismo , Animais , Apoptose/genética , Peso Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Doenças Desmielinizantes/genética , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Gliose/induzido quimicamente , Gliose/genética , Camundongos , Camundongos Knockout , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Allergic contact dermatitis (ACD) is an inflammatory skin disease induced by allergen exposure and characterized by erythema, oedema and immune cell infiltration. The sensory peptide galanin (GAL) and its three receptors (GAL1-3 ) are involved in regulating inflammation. As GAL and its receptors are expressed in human and murine skin and GAL expression is increased in oxazolone-induced contact allergy, it could play a role in dermatitis. As GAL reduces neurogenic plasma extravasation in the mouse skin via GAL3 activation, the role of GAL3 in the oxazolone-induced dermatitis model was explored. Following topical challenge with oxazolone, GAL3 gene-deficient mice showed a trend towards reduced ear thickness. Plasma extravasation and neutrophil infiltration increased considerably upon oxazolone challenge in both GAL3 knockout animals and wild-type controls without any observable effect of the gene deletion. We conclude that a lack of GAL3 does not influence oxazolone-induced ACD.
Assuntos
Dermatite Alérgica de Contato/metabolismo , Modelos Animais de Doenças , Receptor Tipo 3 de Galanina/metabolismo , Animais , Dermatite Alérgica de Contato/etiologia , Camundongos , OxazolonaRESUMO
OBJECTIVE, DESIGN: Mast cell tryptase (MCT) is elevated in arthritic joints, but its direct effects are not known. Here, we investigated MCT-evoked acute inflammatory and nociceptive mechanisms with behavioural, in vivo imaging and immunological techniques. MATERIAL AND SUBJECTS: Neurogenic inflammation involving capsaicin-sensitive afferents, transient receptor potential vanilloid 1 receptor (TRPV1), substance P (SP), neurokinin A (NKA) and their NK1 tachykinin receptor were studied using gene-deleted mice compared to C57Bl/6 wildtypes (n = 5-8/group). TREATMENT: MCT was administered intraarticularly or topically (20 µl, 12 µg/ml). Capsaicin-sensitive afferents were defunctionalized with the TRPV1 agonist resiniferatoxin (RTX; 30-70-100 µg/kg s.c. pretreatment). METHODS: Knee diameter was measured with a caliper, synovial perfusion with laser Doppler imaging, mechanonociception with aesthesiometry and weight distribution with incapacitance tester over 6 h. Cytokines and neuropeptides were determined with immunoassays. RESULTS: MCT induced synovial vasodilatation, oedema, impaired weight distribution and mechanical hyperalgesia, but cytokine or neuropeptide levels were not altered at the 6-h timepoint. Hyperaemia was reduced in RTX-treated and TRPV1-deleted animals, and oedema was absent in NK1-deficient mice. Hyperalgesia was decreased in SP/NKA- and NK1-deficient mice, weight bearing impairment in RTX-pretreated, TRPV1- and NK1-deficient animals. CONCLUSIONS: MCT evokes synovial hyperaemia, oedema, hyperalgesia and spontaneous pain. Capsaicin-sensitive afferents and TRPV1 receptors are essential for vasodilatation, while tachykinins mediate oedema and pain.
Assuntos
Artrite/induzido quimicamente , Edema/induzido quimicamente , Hiperalgesia/induzido quimicamente , Inflamação Neurogênica/induzido quimicamente , Dor/induzido quimicamente , Triptases , Animais , Artrite/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina , Citocinas/metabolismo , Edema/metabolismo , Edema/patologia , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inflamação Neurogênica/metabolismo , Inflamação Neurogênica/patologia , Dor/metabolismo , Dor/patologia , Precursores de Proteínas/genética , Receptor PAR-2/metabolismo , Receptores da Neurocinina-1/genética , Substância P/metabolismo , Canais de Cátion TRPV/genética , Taquicininas/genética , TatoRESUMO
OBJECTIVE AND DESIGN: The function of the neurokinin 1 (NK1) receptor was investigated in the DSS-induced mouse colitis model using NK1 receptor-deficient mice and the selective antagonist netupitant. SUBJECTS: Colitis was induced by oral administration of 20 mg/ml DSS solution for 7 days in C57BL/6 and Tacr1 KO animals (n = 5-7). TREATMENT: During the induction, one-half of the C57BL/6 and Tacr1 KO group received one daily dose of 6 mg/kg netupitant, administered intraperitoneally, the other half of the group received saline, respectively. METHODS: Disease activity index (DAI), on the basis of stool consistency, blood and weight loss, was determined over 7 days. Histological evaluation, myeloperoxidase (MPO) measurement, cytokine concentrations and receptor expression analysis were performed on the colon samples. RESULTS: NK1 receptors are up-regulated in the colon in response to DSS treatment. DSS increased DAI, histopathological scores, BLC, sICAM-1, IFN-γ, IL-16 and JE in wildtype mice, which were significantly reduced in NK1 receptor-deficient ones. NK1 receptor antagonism with netupitant significantly diminished DAI, inflammatory histopathological alterations, BLC, IFN-γ, IL-13 and IL-16 in wildtype mice, but not in the NK1-deficient ones. MPO was similarly elevated and netupitant significantly decreased its activity in both groups. CONCLUSIONS: NK1 receptor antagonism could be beneficial for colitis via inhibiting different inflammatory mechanisms.
Assuntos
Colite/tratamento farmacológico , Antagonistas dos Receptores de Neurocinina-1/uso terapêutico , Piridinas/uso terapêutico , Receptores da Neurocinina-1/fisiologia , Animais , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Citocinas/análise , Sulfato de Dextrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/metabolismo , Piridinas/farmacologia , Receptores da Neurocinina-1/genética , Índice de Gravidade de DoençaRESUMO
In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Hepatócitos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Cultivadas , Galinhas , Técnicas de Cocultura , Citocinas/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/metabolismo , Agentes de Imunomodulação/farmacologia , Agentes de Imunomodulação/química , Lipopolissacarídeos/farmacologia , Poli I-C/farmacologia , Ácidos Teicoicos/farmacologiaRESUMO
Introduction: Host defense peptides (HDPs) are increasingly referred to as promising candidates for the reduction of the use of conventional antibiotics, thereby combating antibiotic resistance. As HDPs have been described to exert various immunomodulatory effects, cecropin A (CecA) appears to be a potent agent to influence the host inflammatory response. Methods: In the present study, a chicken primary hepatocyte-non-parenchymal cell co-culture was used to investigate the putative immunomodulatory effects of CecA alone and in inflammatory conditions evoked by polyinosinic-polycytidylic acid (Poly I:C). To examine the viability of the cells, the extracellular lactate dehydrogenase (LDH) activity was determined by colorimetric assay. Inflammatory markers interleukin (IL)-8 and transforming growth factor-ß1 (TGF-ß1) were investigated using the ELISA method, whereas concentrations of IL-6, IL-10, and interferon-γ (IFN-γ) were assayed by Luminex xMAP technology. Extracellular H2O2 and malondialdehyde levels were measured by fluorometric and colorimetric methods, respectively. Results: Results of the lower concentrations suggested the safe application of CecA; however, it might contribute to hepatic cell membrane damage at its higher concentrations. We also found that the peptide alleviated the inflammatory response, reflected by the decreased production of the pro-inflammatory IL-6, IL-8, and IFN-γ. In addition, CecA diminished the levels of anti-inflammatory IL-10 and TGF-ß1. The oxidative markers measured remained unchanged in most cases of CecA exposure. Discussion: CecA displayed a multifaceted immunomodulatory but not purely anti-inflammatory activity on the hepatic cells, and might be suggested to maintain the hepatic inflammatory homeostasis in Poly I:C-triggered immune response. To conclude, our study suggests that CecA might be a promising molecule for the development of new immunomodulatory antibiotic-substitutive agents in poultry medicine; however, there is still a lot to clarify regarding its cellular effects.
RESUMO
As the threat posed by antimicrobial resistance grows more crucial, the development of compounds that can replace antibiotics becomes increasingly vital. Chicken cathelicidin-2 (Cath-2) belongs to the group of Host Defense Peptides (HDPs), which could provide a feasible solution for the treatment of gastrointestinal infections in poultry. It is a small peptide produced by the heterophil granulocytes of chickens as part of the innate immune response, and its immunomodulatory activity has already been demonstrated in several cell types. In this study, the effects of Cath-2 on the intestinal immune response were examined using ileal explant cultures isolated from chicken. Regarding our results, Cath-2 displayed a potent anti-inflammatory effect as it alleviated the LTA-caused elevation of interleukin (IL)-6 and IL-2 concentrations, and that of the IFN-γ/IL-10 ratio, furthermore, it increased the concentration of IL-10, alleviating the LTA-evoked decreased level of the anti-inflammatory cytokine. Moreover, when applied alone, it elevated the concentrations of IL-6, CXCLi2, and IL-2, providing evidence of its complex immunomodulatory mechanisms. In summary, Cath-2 was able to modulate the immune response of the intestinal wall not only by reducing pro-inflammatory cytokine release, but also through immune stimulation, demonstrating that it has the ability to improve innate immunity via a complex mechanism that may make it a suitable candidate for the control of intestinal infections.
Assuntos
Galinhas , Íleo , Animais , Galinhas/imunologia , Íleo/efeitos dos fármacos , Íleo/imunologia , Citocinas/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologiaRESUMO
BACKGROUND AND PURPOSE: To date, there are limited options for severe Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 virus. As ADP-ribosylation events are involved in regulating the life cycle of coronaviruses and the inflammatory reactions of the host; we have, here, assessed the repurposing of registered PARP inhibitors for the treatment of COVID-19. EXPERIMENTAL APPROACH: The effects of PARP inhibitors on virus uptake were assessed in cell-based experiments using multiple variants of SARS-CoV-2. The binding of rucaparib to spike protein was tested by molecular modelling and microcalorimetry. The anti-inflammatory properties of rucaparib were demonstrated in cell-based models upon challenging with recombinant spike protein or SARS-CoV-2 RNA vaccine. KEY RESULTS: We detected high levels of oxidative stress and strong PARylation in all cell types in the lungs of COVID-19 patients, both of which negatively correlated with lymphocytopaenia. Interestingly, rucaparib, unlike other tested PARP inhibitors, reduced the SARS-CoV-2 infection rate through binding to the conserved 493-498 amino acid region located in the spike-ACE2 interface in the spike protein and prevented viruses from binding to ACE2. In addition, the spike protein and viral RNA-induced overexpression of cytokines was down-regulated by the inhibition of PARP1 by rucaparib at pharmacologically relevant concentrations. CONCLUSION AND IMPLICATIONS: These results point towards repurposing rucaparib for treating inflammatory responses in COVID-19.
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Many bench-top flow cytometers (b-FCs) are compatible with microsphere-based multiplexed assays. Disciplines implementing b-FCs-based assays are expanding; they include monitoring and validating food quality. A multiplexed platform protocol was evaluated for poly-mycotoxin assays, which is compatible with a variety of b-FC models. The seven instruments included: BD FACSCalibur(™) , BD FACSArray(™) Bioanalyzer, Accuri C6, Partec CyFlow(®) Space, Beckman Coulter FC 500, Guava EasyCyte Mini, and Luminex 100 (™) . Current reports related to the food industry describe fungal co-infections leading to poly-mycotoxin contamination in grain (Sulyok M, Berthiller F, Krska R, Schuhmacher R, Rapid Commun Mass Spectrom 2006;20:2649-2659). It is imperative to determine whether b-FC-based assays can replace traditional single-mycotoxin enzyme-linked immunosorbent assay (ELISA). A six-plexed poly-mycotoxin kit was tested on seven different b-FCs. The modified kit was initially developed for the BD FACSArray(™) Bioanalyzer (BD Biosciences) (Czeh A, Mandy F, Feher-Toth S, Torok L, Mike Z, Koszegi B, Lustyik G, J Immunol Methods 2012;384:71-80). With the multiplexed platform, it is possible to identify up to six mycotoxin contaminants simultaneously at regional grain collection/transfer/inspection facilities. In the future, elimination of contaminated food threat may be better achieved with the inclusion of b-FCs in the food protection arsenal. A universal protocol, matched with postacquisition software, offers an effective alternative platform compared to using a series of ELISA kits. To support side-by-side evaluation of seven flow cytometers, an instrument-independent fluorescence emission calibration was added to the protocol. All instrument performances were evaluated for strength of agreement based on paired sets of evaluation to predicate method. The results suggest that all b-FCs were acceptable of performing with the multiplexed kit for five of six mycotoxins. For OTA, the detection sensitivity was consistent only for five of the seven instruments.
Assuntos
Citometria de Fluxo/instrumentação , Micotoxinas/análise , Calibragem , Citometria de Fluxo/normas , Microbiologia de Alimentos , Humanos , Kit de Reagentes para Diagnóstico/normas , Padrões de Referência , Reprodutibilidade dos Testes , SoftwareRESUMO
INTRODUCTION: The small intestine is one of the most sensitive organs to ischemia-reperfusion injury during transplantation. Cytoprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) is well known. The aim of our study was to measure changes of PACAP-38-like immunoreactivities and cytokine levels in intestinal grafts stored PACAP-38 containing preservation solution. MATERIAL AND METHODS: Small-bowel autotransplantation was performed on male Wistar rats (n = 56). Grafts were stored in University of Wisconsin (UW) solution at 4 °C for 1 (GI), 3 (GII), and 6 hours (GIII); and in PACAP-38 containing UW solution for 1 (GIV), 3 (GV), and 6 hours (GVI). Reperfusion lasted 3 hours in each group. Intestinal PACAP-38 immunoreactivities were measured by radioimmunoassay. To measure cytokine from tissue homogenates we used rat cytokine array and Luminex Multiplex Immunoassay. RESULTS: Levels of PACAP-38-like and PACAP-27-like immunoreactivities decreased by preservation time compared to control. This decrease was significant following 6 hours cold storage (p < 0.05). Values remained significantly higher in grafts stored in PACAP-38 containing UW. Expressions of sICAM-1, L-selectin, tissue inhibitor of metalloproteinase-1 were increased in GIII and were decreased in GVI. CONCLUSION: PACAP-38 increased tissue levels of PACAP-38 and PACAP-27, and decreased cytokine expression. This indicates that PACAP-38 has anti-inflammatory and cytoprotective effects in intestinal autotransplantation model.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Citocinas/metabolismo , Citoproteção/efeitos dos fármacos , Sobrevivência de Enxerto/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Intestino Delgado/transplante , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Adenosina , Alopurinol , Animais , Regulação para Baixo , Glutationa , Insulina , Molécula 1 de Adesão Intercelular/metabolismo , Intestino Delgado/metabolismo , Selectina L/metabolismo , Masculino , Preservação de Órgãos/métodos , Soluções para Preservação de Órgãos , Radioimunoensaio , Rafinose , Ratos , Ratos Wistar , Traumatismo por Reperfusão/etiologia , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Transplante AutólogoRESUMO
The use of natural feed supplements is an alternative tool to diminish the damage caused by certain bacteria, improving animal health and productivity. The present research aimed to investigate the proinflammatory effect of flagellin released from the bacterial flagellum of Salmonella enterica serovar Typhimurium and to attenuate the induced inflammation with luteolin as a plant-derived flavonoid on a chicken primary hepatocyte-non-parenchymal cell co-culture. Cells were cultured in a medium supplemented with 250 ng/mL flagellin and 4 or 16 µg/mL luteolin for 24 h. Cellular metabolic activity, lactate dehydrogenase (LDH) activity, interleukin-6, 8, 10 (IL-6, IL-8, IL-10), interferon-α, γ (IFN-α, IFN-γ), hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were determined. Flagellin significantly increased the concentration of the proinflammatory cytokine IL-8 and the ratio of IFN-γ/IL-10, while it decreased the level of IL-10, indicating that the model served adequate to study inflammation in vitro. Luteolin treatment at 4 µg/mL did not prove to be cytotoxic, as reflected by metabolic activity and extracellular LDH activity, and significantly reduced the flagellin-triggered IL-8 release of the cultured cells. Further, it had a diminishing effect on the concentration of IFN-α, H2O2 and MDA and restored the level of IL-10 and the ratio of IFN-γ/IL-10 when applied in combination with flagellin. These results suggest that luteolin at lower concentrations may protect hepatic cells from an excessive inflammatory response and act as an antioxidant to attenuate oxidative damage.
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IDR-1002, a synthetic host defense peptide (HDP), appears to be a potential candidate for the treatment of bacterial infections and the consequent inflammatory response due to its potent immunomodulatory activity. This is of relevance to the emerging issue of antimicrobial resistance in the farming sector. In this study, the effects of IDR-1002 were investigated on a chicken hepatocyteânon-parenchymal cell co-culture, and the results revealed that IDR-1002 had complex effects on the regulation of the hepatic innate immunity. IDR-1002 increased the levels of both RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and Macrophage colony stimulating factor (M-CSF), suggesting the peptide plays a role in the modulation of macrophage differentiation, also reflected by the reduced concentrations of interleukin (IL)-6 and IL-10. The pro-inflammatory cytokine release triggered by the bacterial cell wall component lipoteichoic acid (LTA) was ameliorated by the concomitantly applied IDR-1002 based on the levels of IL-6, chicken chemotactic and angiogenic factor (CXCLi2) and interferon (IFN)-γ. Moreover, the production of nuclear factor erythroid 2-related factor 2 (Nrf2), an essential transcription factor in the antioxidant defense pathway, was increased after IDR-1002 exposure, while protein carbonyl (PC) levels were also elevated. These findings suggest that IDR-1002 affects the interplay of the cellular immune response and redox homeostasis, thus the peptide represents a promising tool in the treatment of bacterially induced inflammation in chickens.