RESUMO
The rise of multidrug resistant fungal infections highlights the need to identify and develop novel antifungal agents. Occidiofungin is a nonribosomally synthesized glycolipopeptide that has a unique mechanism of action, disrupting actin-mediated functions and inducing cellular apoptosis. Antifungal activity has been observed in vitro against various fungal species, including multidrug resistant Candida auris, and in vivo efficacy has been demonstrated in a murine vulvovaginal candidiasis model. Occidiofungin, a cyclic glycolipopeptide, is composed of eight amino acids and in previous studies, an asparagine residue was assigned at position 7 (ASN7). In this study, new structural variants of occidiofungin have been characterized which have aspartic acid (ASP7), glutamine (GLN7), or glutamic acid (GLU7) at position 7. The side chain of the ASP7 variant contains the only terminal carboxylic acid in the peptide and provides a useful site for selective chemical modifications. Analogues were synthesized at the ASP7 position and tested for antifungal activity. These analogues were shown to be more active as compared to the ASP7 variant against a panel of Candida species. The naturally occurring variants of occidiofungin with a side chain containing a carboxylic acid at the seventh amino acid position can be used to develop semisynthetic analogues with enhanced therapeutic properties.
Assuntos
Antifúngicos , Burkholderia , Glicopeptídeos , Peptídeos Cíclicos , Camundongos , Animais , Antifúngicos/química , Burkholderia/química , Ácidos Carboxílicos , Testes de Sensibilidade MicrobianaRESUMO
Part of the mechanism by which 2,3,7.8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses immune function involves induction of regulatory T cells and suppression of effector T cells. The goal of this project was to examine whether TCDD's suppression of effector T cells was due in part to inducing B regulatory cells (Bregs). TCDD's potential to increase the percentage and/or function of CD24+CD38+ B cells was assessed in response to lipopolysaccharide (LPS) + interleukin (IL)-4 in vitro and in a mild model of experimental autoimmune encephalomyelitis (EAE) in vivo. In vitro, TCDD did not consistently increase the percentage of CD19+CD24+CD38+ cells using splenocytes, purified B cells or bone marrow (BM) cells. However, TCDD increased IL-10 in all three culture preparations, and TCDD increased the percentage of CD5+CD24+CD38+ cells producing IL-10. In EAE, TCDD did not affect the percentage of the CD24+CD38+ cell population in CD19, B220 or CD5 B cells in splenocytes (SPLC), lymph nodes (LN) nor BM cells at end-stage disease. On the other hand, TCDD increased the CD19+CD24+CD38+ percentage in the spinal cord (SC) in EAE. Moreover, TCDD-treated B cells isolated from spleens or TCDD-treated BM cells in EAE mice modestly reduced the ability of naïve effector T cells to express interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Together these data show that TCDD can induce regulatory functions in B cells, although it was not obvious simply by examining the expression of regulatory markers but by assessing function by cytokine production or mixed lymphocyte responses.
Assuntos
Encefalomielite Autoimune Experimental , Dibenzodioxinas Policloradas , Animais , Linfócitos B , Interferons , Interleucina-10 , Lipopolissacarídeos , Camundongos , Dibenzodioxinas Policloradas/toxicidade , Linfócitos T , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: The active experimental autoimmune encephalomyelitis (EAE) model is often initiated using myelin oligodendrocyte glycoprotein (MOG) immunization followed by pertussis toxin (PTX) to study multiple sclerosis. However, PTX inactivates G protein-coupled receptors, and with increasing knowledge of the role that various G protein-coupled receptors play in immune homeostasis, it is valuable to establish neuroimmune endpoints for active EAE without PTX. METHODS: Female C57BL/6 mice were immunized with MOG35-55 peptide in Complete Freund's Adjuvant and neuroinflammation, including central nervous system B-cell infiltration, was compared to saline-injected mice. Since it was anticipated that disease onset would be slower and less robust than EAE in the presence of PTX, both cervical and lumbosacral sections of the spinal cord were evaluated. RESULTS: Immunohistochemical analysis showed that EAE without PTX induced immune infiltration, CCL2 and VCAM-1 upregulation. Demyelination in the cervical region correlated with the infiltration of CD19+ B cells in the cervical region. There was upregulation of IgG, CD38, and PDL1 on B cells in cervical and lumbosacral regions of the spinal cord in EAE without PTX. Interestingly, IgG was expressed predominantly by CD19- cells. CONCLUSIONS: These data demonstrate that many neuroimmune endpoints are induced in EAE without PTX and although clinical disease is mild, this can be used as an autoimmune model when PTX inactivation of G protein-coupled receptors is not desired.
Assuntos
Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Inflamação/imunologia , Toxina Pertussis/farmacologia , Medula Espinal/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Feminino , Região Lombossacral , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/farmacologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Toxina Pertussis/imunologia , Fenótipo , Medula Espinal/efeitos dos fármacosRESUMO
The endocannabinoids (eCBs) are endogenous arachidonoyl-containing lipid mediators with important roles in host defense. Macrophages are first-line defenders of the innate immune system and biosynthesize large amounts of eCBs when activated. The cellular levels of eCBs are controlled by the activities of their biosynthetic enzymes and catabolic enzymes, which include members of the serine hydrolase (SH) superfamily. The physiologic activity of SHs can be assessed in a class-specific way using chemoproteomic activity-based protein profiling (ABPP) methods. Here, we have examined avian (chicken) HD11 macrophages, a widely used cell line in host-pathogen research, using gel-based ABPP and ABPP-multidimensional protein identification technology (MudPIT) to profile the changes in SH activities under baseline, chemical-inhibitor-treated, and pathogen-challenged conditions. We identified α/ß-hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH) as the principal SHs responsible for 2-arachidonoylglycerol (2AG) hydrolysis, thereby regulating the concentration of this lipid in HD11 cells. We further discovered that infection of HD11 macrophages by Salmonella Typhimurium caused the activities of these 2AG hydrolases to be downregulated in the host cells. ABHD6 and FAAH were potently inhibited by a variety of small-molecule inhibitors in intact live cells, and thus these compounds might be useful host-directed adjuvants to combat antimicrobial resistance in agriculture. 2AG was further shown to augment the phagocytic function of HD11 macrophages, which suggests that pathogen-induced downregulation of enzymes controlling 2AG hydrolytic activity might be a physiological mechanism to increase 2AG levels, thus enhancing phagocytosis. Together these results define ABHD6 and FAAH as 2AG hydrolases in avian macrophages that can be inactivated pharmacologically and decreased in activity during Salmonella Typhimurium infection.
Assuntos
Amidoidrolases/antagonistas & inibidores , Proteínas Aviárias/antagonistas & inibidores , Galinhas/metabolismo , Inibidores Enzimáticos/farmacologia , Macrófagos/enzimologia , Monoacilglicerol Lipases/antagonistas & inibidores , Infecções por Salmonella/enzimologia , Salmonella typhimurium/metabolismo , Amidoidrolases/metabolismo , Animais , Proteínas Aviárias/metabolismo , Galinhas/microbiologia , Endocanabinoides/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Monoacilglicerol Lipases/metabolismo , Infecções por Salmonella/patologiaRESUMO
The transcription factor Nurr1 is a member of the steroid hormone nuclear receptor superfamily. Ablation of Nurr1 expression arrests mesencephalic dopamine neuron differentiation while attenuation of Nurr1 in the subiculum and hippocampus impairs learning and memory. Additionally, reduced Nurr1 expression has been reported in patients with Parkinson's disease and Alzheimer's disease. In order to better understand the overall function of Nurr1 in the brain, quantitative immunohistochemistry was used to measure cellular Nurr1 protein expression, across Nurr1 immunoreactive neuronal populations. Additionally, neuronal Nurr1 expression levels were compared between different brain regions in wild-type mice (+/+) and Nurr1 heterozygous mice (+/-). Regional Nurr1 protein was also investigated at various time points after a seizure induced by pentylenetetrazol (PTZ). Nurr1 protein is expressed in various regions throughout the brain, however, a wide range of Nurr1 expression levels were observed among various neuronal populations. Neurons in the parietal and temporal cortex (secondary somatosensory, insular, auditory, and temporal association cortex) had the highest relative Nurr1 expression (100%) followed closely by the claustrum/dorsal endopiriform cortex (85%) and then subiculum (76%). Lower Nurr1 protein levels were found in neurons in the substantia nigra pars compacta and ventral tegmental area (39%) followed by CA1 (25%) and CA3 (19%) of the hippocampus. Additionally, in the parietal and temporal cortex, two distinct populations of high and medium Nurr1 expressing neurons were observed. Comparisons between +/- and +/+ mice revealed Nurr1 protein was reduced in +/- mice by 27% in the parietal/temporal cortex, 49% in the claustrum/dorsal endopiriform cortex, 25% in the subiculum, 33% in substantia nigra pars compacta, 22% in ventral tegmental area, and 21% in CA1 region of the hippocampus. Based on these data, regional mechanisms appear to exist which can compensate for a loss of a Nurr1 allele. Following a single PTZ-induced seizure, Nurr1 protein in the dentate gyrus peaked around 2 h and returned to baseline by 8 h. Since altered Nurr1 expression has been implicated in neurologic disorders and Nurr1 agonists have showed protective effects, understanding regional protein expression of Nurr1, therefore, is necessary to understand how changes in Nurr1 expression can alter brain function.
RESUMO
Previously we demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed experimental autoimmune encephalomyelitis (EAE), a model to study multiple sclerosis (MS), through induction of regulatory T cells (Tregs) and suppression of effector T cell function in the spleen. Since B cells and specifically regulatory B cells (Bregs) have been shown to be so critical in the pathology associated with EAE and MS, we wanted to determine whether TCDD could also induce Bregs. We specifically hypothesized that a Fas ligand (FasL)+ Breg population would be induced by TCDD in EAE thereby triggering apoptosis in Fas-expressing effector T cells as one mechanism to account for inhibition of T cell function by TCDD. TCDD (0.1-2.5 µg/kg/day administered orally for 12 days) modestly increased the percentage of FasL + B cells in the spleen and spinal cord in TCDD-treated EAE mice. However, we did not detect significant increases in percentages of FasL + B cells using TCDD in vitro in mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). Part of the modest effect by TCDD was likely related to the localized expression of FasL; for instance, in the spleen, FasL was more highly expressed by IgMhiIgDlo marginal zone (MZ) B cells, but IgMloIgDhi follicular (FO) B cells were more responsive to TCDD. Consistent with our observation of modest upregulation of FasL, we also observed modest changes in mitochondrial membrane potential in T cells co-cultured with isolated total B cells or IgM-depleted (i.e., FO-enriched) B cells from TCDD-treated EAE mice. These data suggest that while small microenvironments of apoptosis might be occurring in T cells in response to TCDD-treated B cells, it is not a major mechanism by which T cell function is compromised by TCDD in EAE. TCDD did robustly suppress IgG production systemically and in spleen and spinal cord B cells at end stage disease. Thus, these studies show that TCDD's primary effect on B cells in EAE is compromised IgG production but not FasL + Breg induction.
Assuntos
Linfócitos B/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/prevenção & controle , Proteína Ligante Fas/biossíntese , Imunoglobulina G/metabolismo , Dibenzodioxinas Policloradas/uso terapêutico , Animais , Linfócitos B/efeitos dos fármacos , Células Cultivadas , Poluentes Ambientais/farmacologia , Poluentes Ambientais/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dibenzodioxinas Policloradas/farmacologiaRESUMO
In this study cannabidiol (CBD) was administered orally to determine its effects and mechanisms in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). We hypothesized that 75 mg/kg of oral CBD given for 5 days after initiation of disease would reduce EAE severity through suppression of either the early peripheral immune or late neuroimmune response. EAE was induced in C57BL/6 mice at two different magnitudes, and peripheral inflammatory and neuroinflammatory responses were measured at days 3, 10, and 18. Th1, Th17, Tc1, Tc17, Tregs, and myeloid derived suppressor cells (MDSC) were identified from the lymph nodes and spleens of each mouse to determine if CBD altered the suppressor cell or inflammatory cell populations in secondary lymphoid tissues. Additionally, neuroinflammation was identified in brain and spinal cord tissues using various immunohistochemical techniques and flow cytometry. Early treatment of EAE with oral CBD reduced clinical disease at the day 18 timepoint which correlated with a significant decrease in the percentage of MOG35-55 specific IFN-γ producing CD8+ T cells in the spleen at day 10. Analysis of both T cell infiltration and lesion size within the spinal cord also showed a moderate reduction in neuroinflammation within the central nervous system (CNS). These results provide evidence that oral CBD suppressed the peripheral immune response that precedes neuroinflammation; however, analysis of the neuroinflammatory endpoints also suggest that the modest reduction in neuroinflammation was only partially responsible for CBD's neuroprotective capability. Graphical Abstract CBD was administered orally for the first 5 days following initiation of EAE. CBD attenuated clinical disease, and we found that CBD suppressed IFN-γ producing CD8+ T cells in the spleen at day 10. There was also modest suppression of neuroinflammation. Together these data demonstrate that early, oral administration of CBD protected mice from disease, but the modest effects on neuroinflammation suggest other mechanisms participate in CBD's neuroprotective effect in EAE.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Canabidiol/farmacologia , Encefalomielite Autoimune Experimental/imunologia , Baço/efeitos dos fármacos , Baço/imunologia , Animais , Encéfalo/imunologia , Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Inflamação/imunologia , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
BACKGROUND: Pentoxifylline (PTX) is a methylxanthine phosphodiesterase inhibitor that is used as a hemorrheologic and anti-inflammatory agent in veterinary and human medicine. In human studies, PTX has been shown to decrease T-cell production of cytokines such as IL-2 and IFN-γ. A RT-qPCR assay to measure activated T-cell gene expression of IL-2 and IFN-γ has been validated in dogs. OBJECTIVES: The goal of this study was to utilize this assay to investigate the effects of PTX on in vitro cytokine gene expression in canine whole blood. METHODS: Whole blood from seven healthy dogs was collected and incubated with various concentrations of PTX for 1 hr before activation. PTX concentrations spanned and exceeded blood concentrations achieved when administered at clinically relevant dosages (1, 2, 10, 50 and 200 µg/ml). Cyclosporine was used at a concentration of 500 ng/ml as a positive control. All blood samples, including untreated activated baseline samples, were then activated with phorbol myristate acetate and ionomycin for 5 hrs. RESULTS: Analysis of activated whole blood by RT-qPCR revealed that there was not a significant suppression of IL-2 or IFN-γ gene expression at any concentration of PTX when evaluating ΔCt values. All samples exposed to cyclosporine showed significant changes from untreated activated baseline samples, demonstrating marked suppression as the positive control. Cytokine expression, presented as a percentage of untreated activated baseline samples, was also evaluated. After exposure to the highest concentration of PTX (200 µg/ml), median percentage cytokine expression was suppressed to just below 50% of baseline values. This concentration, however, is much higher than blood concentrations reported to be achieved at standardly used pentoxifylline doses. CONCLUSIONS: PTX does not appear to significantly suppress T-cell cytokine production in samples from most dogs at clinically relevant drug concentrations. Further testing is needed to establish the full effects of PTX on the immune system in dogs.
Assuntos
Anti-Inflamatórios/farmacocinética , Cães/genética , Expressão Gênica/imunologia , Interferon gama/genética , Interleucina-2/genética , Pentoxifilina/farmacologia , Inibidores de Fosfodiesterase/farmacocinética , Animais , Cães/sangue , Cães/metabolismo , Relação Dose-Resposta a Droga , Interferon gama/metabolismo , Interleucina-2/metabolismoRESUMO
Eicosanoids are cellular metabolites, which shape the immune response, including inflammatory processes in macrophages. The effects of these lipid mediators on inflammation and bacterial pathogenesis are not clearly understood. Certain eicosanoids are suspected to act as molecular sensors for the recruitment of neutrophils, while others regulate bacterial uptake. In this study, gene expression analyses indicated that genes involved in eicosanoid biosynthesis including COX-1, COX-2, DAGL, and PLA-2 are differentially regulated in THP-1 human macrophages infected with Salmonella enterica Typhimurium or Yersinia enterocolitica. By using targeted metabolomics approach, we found that the eicosanoid precursor, arachidonic acid (AA) as well as its derivatives, including prostaglandins (PGs) PGF2α or PGE2/PGD2, and thromboxane TxB2, are rapidly secreted from macrophages infected with these Gram-negative pathogenic bacteria. The magnitude of eicosanoid biosynthesis in infected host cells depends on the presence of virulence factors of Y. enterocolitica and S. Typhimurium strains, albeit in an opposite way in Y. enterocolitica compared to S. Typhimurium infection. Trials with combinations of EP2/EP4 PGE2 receptor agonists and antagonists suggest that PGE2 signaling in these infection models works primarily through the EP4 receptor. Downstream of EP4 activation, PGE2 enhances inflammasome activation and represses M2 macrophage polarization while inducing key M1-type markers. PGE2 also led to a decreased numbers of Y. enterocolitica within macrophages. To summarize, PGE2 is a potent autocrine/paracrine activator of inflammation during infection in Gram-negative bacteria, and it affects macrophage polarization, likely controlling bacterial clearance by macrophages.
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The transcription factor Nurr1 is essential for dopamine neuron differentiation and is important in maintaining dopamine synthesis and neurotransmission in the adult. Reduced Nurr1 function, due to the Nurr1-null heterozygous genotype (+/-), impacts dopamine neuron function in a region specific manner resulting in a decrease in dopamine synthesis in the dorsal and ventral striatum and a decrease in tissue dopamine levels in the ventral striatum. Additionally, maintenance of tissue dopamine levels in the dorsal striatum and survival of nigrostriatal dopamine neurons with aging (>15 months) or after various toxicant treatments are impaired. To further investigate the effects of aging and the Nurr1-null heterozygous genotype, we measured regional tissue dopamine levels, dopamine neuron numbers, body weight, open field activity and rota-rod performance in young (3-5 months) and aged (15-17 months) wild-type +/+ and +/- mice. Behavioral tests revealed no significant differences in rota-rod performance or basal open field activity as a result of aging or genotype. The +/- mice did show a significant increase in open field activity after 3 min of restraint stress. No differences in tissue dopamine levels were found in the dorsal striatum. However, there were significant reductions in tissue dopamine levels in the ventral striatum, which was separated into the nucleus accumbens core and shell, in the aged +/- mice. These data indicate that the mesoaccumbens system is more susceptible to the combination of aging and the +/- genotype than the nigrostriatal system. Additionally, the effects of aging and the +/- genotype may be dependent on genetic background or housing conditions. As Nurr1 mutations have been implicated in a number of diseases associated with dopamine neurotransmission, further data is needed to understand why and how Nurr1 can have differential functions across different dopamine neuron populations in aging.
RESUMO
Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder, characterized by demyelination of neurons in the central nervous system. To investigate the pathogenicity of various T cell types in MS, especially IFN-γ- or IL-17-producing CD4(+ )cells (TH1 or TH17 cells, respectively), the mouse model, experimental autoimmune encephalomyelitis (EAE), is commonly used. One method by which EAE is induced is immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (MOG35-55) followed by subsequent injections of pertussis toxin (PTX) as an adjuvant. We have an interest in the mechanisms by which EAE occurs in the absence of PTX because it induces a milder disease state more consistent with autoimmune disease onset and PTX inactivates Gi/o protein-coupled receptors, many of which contribute to immune homeostasis. Another receptor that plays a role in immune homeostasis is the aryl hydrocarbon receptor (AHR). In fact, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to attenuate EAE pathogenesis by affecting CD4(+ )T and regulatory T (Treg) cells in an AHR-dependent manner. However, many of these studies have been conducted with an acute high dose TCDD. Thus, the goal of this work was to investigate the modulation of MOG-specific immune responses with subchronic low dose TCDD (0.1-1.0 µg/kg/d for 12 days) in EAE without PTX. The results demonstrate that subchronic, low dose exposure of TCDD attenuates the immune responses in EAE development in the absence of PTX, which is due in part to suppression of MOG-specific IL-17A and IFN-γ responses.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Imunossupressores/farmacologia , Interferon gama/metabolismo , Interleucina-17/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Interferon gama/imunologia , Interleucina-17/imunologia , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Fatores de TempoRESUMO
Tick-borne borreliae include Lyme disease and relapsing fever agents, and they are transmitted primarily by ixodid (hard) and argasid (soft) tick vectors, respectively. Tick-host interactions during feeding are complex, with host immune responses influenced by biological differences in tick feeding and individual differences within and between host species. One of the first encounters for spirochetes entering vertebrate host skin is with local antigen-presenting cells, regardless of whether the tick-associated Borrelia sp. is pathogenic. In this study, we performed a basic comparison of cytokine responses in THP-1-derived macrophages after exposure to selected borreliae, including a nonpathogen. By using THP-1 cells, differentiated to macrophages, we eliminated variations in host response and reduced the system to an in vitro model to evaluate the extent to which the Borrelia spp. influence cytokine production. Differentiated THP-1 cells were exposed to four Borrelia spp., Borrelia hermsii (DAH), Borrelia burgdorferi (B31), B. burgdorferi (NC-2), or Borrelia lonestari (LS-1), or lipopolysaccharides (LPS) (activated) or media (no treatment) controls. Intracellular and secreted interferon (IFN)-γ, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α were measured using flow cytometric and Luminex-based assays, respectively, at 6, 24, and 48 h postexposure time points. Using a general linear model ANOVA for each cytokine, treatment (all Borrelia spp. and LPS compared to no treatment) had a significant effect on secreted TNF-α only. Time point had a significant effect on intracellular IFN-γ, TNF-α and IL-6. However, we did not see significant differences in selected cytokines among Borrelia spp. TREATMENTS: Thus, in this model, we were unable to distinguish pathogenic from nonpathogenic borreliae using the limited array of selected cytokines. While unique immune profiles may be detectable in an in vitro model and may reveal predictors for pathogenicity in borreliae of unknown pathogenicity, a larger panel of cytokines would be desirable to test.
Assuntos
Borrelia/patogenicidade , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Monócitos/metabolismo , Borrelia/classificação , Linhagem Celular , Citocinas/genética , Humanos , Lipopolissacarídeos/toxicidade , Monócitos/efeitos dos fármacos , Monócitos/microbiologiaRESUMO
Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.
Assuntos
Neurônios Dopaminérgicos/citologia , Microdissecção e Captura a Laser/métodos , Área Tegmentar Ventral/citologia , Animais , Imuno-Histoquímica , CamundongosRESUMO
Although protein ubiquitination has been shown to regulate multiple processes during host response to Salmonella enterica serovar Typhimurium infection, specific functions of host deubiquitinating enzymes remain unknown in this bacterial infection. By using chemical proteomics approach, in which deubiquitinating enzymes were labeled by an active-site probe and analyzed by quantitative proteomics, we identified novel deubiquitinases in chicken macrophages based on their reactivity with the probe. Also, we detected down-regulation of UCH-L3, and USP4 as well as up-regulation of USP5 and UCH-L5 deubiquitinating enzymes in macrophages infected with Salmonella Typhimurium. We showed that decrease in either UCH-L5 activity, or in UCH-L5 protein amount in chicken and human macrophages infected or stimulated with LPS/nigericin, led to decreased IL-1ß release. These data point towards a putative role of UCH-L5 in inflammasome regulation during Salmonella infection. Because inflammasome activation is important in innate resistance to these bacteria, one would expect that naturally occurring or therapeutically induced alteration in UCH-L5 activation would influence disease outcome and could represent a target for new therapeutic approaches.
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Inflamassomos/metabolismo , Macrófagos/metabolismo , Proteômica/métodos , Animais , Linhagem Celular , Galinhas , Humanos , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , UbiquitinaçãoRESUMO
Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice.