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The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in humans through clinical signs that include fever, unthriftiness, and in some cases the development of an eschar. The guinea pig is the smallest animal model for SFR that allows the collection of multiple blood and skin samples antemortem for longitudinal studies. This unit provides the basic protocols necessary to establish, maintain, and utilize a guinea pig-tick-Rickettsia model for monitoring the course of infection and immune response to an infection by spotted fever group Rickettsia (SFGR) that can be studied at biosafety level 2 (BSL-2) and arthropod containment level 2 (ACL-2); adaptations must be made for BSL-3 agents. The protocols cover methods for tick feeding and colony development, laboratory infection of ticks, tick transmission of Rickettsia to guinea pigs, and monitoring of the course of infection through clinical signs, rickettsial burden, and immune response. It should be feasible to adapt these methods to study other tick-borne pathogens. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tick transmission of SFGR to guinea pigs Support Protocol 1: Laboratory infection of ticks by injection Alternate Protocol 1: Needle inoculation of SFGR to guinea pigs Basic Protocol 2: Monitoring the course of guinea pig rickettsial infection: clinical signs Basic Protocol 3: Monitoring the course of guinea pig rickettsial infection: collection of biological specimens Support Protocol 2: Guinea pig anesthesia Basic Protocol 4: Monitoring rickettsial burden in guinea pigs by multiplex qPCR Basic Protocol 5: Monitoring guinea pig immune response to infection: blood leukocytes by flow cytometry Basic Protocol 6: Monitoring immune response to guinea pig rickettsial infection: leukocyte infiltration of skin at the tick bite site by flow cytometry Basic Protocol 7: Monitoring the immune response to guinea pig rickettsial infection: antibody titer by ELISA Support Protocol 4: Coating ELISA Plates Alternate Protocol 2: Monitoring immune response to guinea pig rickettsial infection: antibody titer by immunofluorescence assay.
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Rickettsiose do Grupo da Febre Maculosa , Carrapatos , Animais , Cobaias , Humanos , Modelos Animais de Doenças , Imunidade , Infecção Laboratorial , Rickettsia/fisiologia , Rickettsiose do Grupo da Febre Maculosa/diagnóstico , Rickettsiose do Grupo da Febre Maculosa/imunologia , Carrapatos/microbiologiaRESUMO
Macrophages are scavenger cells and a fundamental part of innate and adaptive immune responses, and they are important in wound repair and tissue remodeling. The functions of macrophages include engulfing and killing invading pathogens, processing and presenting antigens, initiation of inflammation, secreting cytokines and other inflammatory mediators, and participating in the maintenance and repair of tissues. Based on functional differences and surface and intracellular marker expression, macrophages can be generally divided into either M1 (inflammatory) or M2 (wound healing); the M2 type can be further divided into M2a, M2b, M2c, and M2d. However, due to the time, effort, and cost of establishing a panel of markers that could thoroughly assess polarization, the characterization of types and subtypes is usually done using three markers or fewer. This can lead to problems, because the expression of some of the most widely used polarization markers can be altered by commonly used inflammatory or immunological stimuli. We have developed and optimized an eleven-color polychromatic flow cytometric assay for macrophage subtype identification that prevents mischaracterization due to stimulus-induced changes in individual markers by using partially redundant markers for which at least one is not substantially affected by a commonly used inflammatory stimulus (LPS). We polarized 3 × 105 RAW 264.7 cells, a mouse macrophage cell line, with IFN-γ (± LPS), IL-4 or IL-10 to derive M1, M2a, or M2c macrophage subtypes, respectively. The TNF-α concentration in cell supernatants was tested by ELISA to verify polarization. Then polarized cells were labeled with the following antibodies and assessed by flow cytometry to identify marker expression: F4/80, Arginase 1, TLR4, CD86, VEGF, CD14, CD206, MHC Class II, and TNF-α (surface and internal). Here we have identified clear distinctions between macrophage subtypes using these markers, and we anticipate that this panel will help disclose more details of the macrophage's role in the immune response and will save investigators the time and cost usually required to identify appropriate antibodies that do not interfere with each other or lead to difficult color compensation issues.
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Macrófagos , Fator de Necrose Tumoral alfa , Animais , Camundongos , Citometria de FluxoRESUMO
Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-Amblyomma-Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan® qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork.
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Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host-pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans.
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Assessing cells, proteins, and total RNA in the spinal cord is vital for advancing our understanding of neuroinflammation and neurodegenerative diseases. For instance, immune cells infiltrate the spinal cord in the experimental autoimmune encephalomyelitis (EAE) model, commonly used to study multiple sclerosis. Thus, it is valuable to assess total RNA to determine the neuronal and inflammatory profiles in the spinal cord. Further, RNA profiles are useful for deciphering the effects of drugs or chemicals on neuroinflammation and neurodegenerative diseases such as EAE. The purpose of this protocol and the online video illustrating it is to describe and demonstrate the expulsion of the spinal cord from the mouse spinal column and homogenization of the spinal cord using liquid nitrogen for optimal RNA isolation. Although we present this method with spinal cords from EAE mice, the technique is broadly applicable, including RNA isolation from the spinal cords of healthy mice. Proper performance of these steps is critical to achieving a sufficient yield of transcriptomic-quality spinal cord RNA when combined with final isolation using commercially available kits. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of the spinal cord from the spinal column Support Protocol: Preparation of blunt-end needle for spinal cord isolation Basic Protocol 2: Spinal cord homogenization using liquid nitrogen Basic Protocol 3: Assessment of RNA purity, quantification, and integrity.
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Encefalomielite Autoimune Experimental , Transcriptoma , Animais , Encefalomielite Autoimune Experimental/genética , Camundongos , Doenças Neuroinflamatórias , RNA/genética , Medula EspinalRESUMO
Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever group Rickettsia (SFGR) species. The immunofluorescence antibody assay (IFA) has been the gold standard for the serodiagnosis of rickettsial infections such as spotted fever rickettsiosis (SFR). However, the IFA is semi-quantitative and subjective, requiring a high level of expertise to interpret it correctly. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Rickettsia parkeri infection in the guinea pig. Our ELISA is an objective, quantitative, and high-throughput assay that shows greater sensitivity and resolution in observed titers than the IFA. We methodically optimized relevant parameters in sequence for optimal signal-to-noise ratio and low coefficient of variation% values. We used a guinea pig model as it is a part of our overall research efforts to understand the immunological and clinical response to SFGR species after tick transmission. Guinea pigs are a useful model to study SFR and show clinical signs of SFR, such as fever and eschars. We anticipate that this assay will be easily adapted to other hosts, including humans and other SFGR species.
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The guinea pig (Cavia porcellus) has an established track record as an animal model, with its utility in rickettsial research documented as early as the turn of the 20th century. From identifying Rickettsia rickettsii as the agent of Rocky Mountain spotted fever and ticks as the natural transmission route to evaluating protective immunity and treatment for tick-borne rickettsiae, guinea pigs have been essential for advances in our understanding of spotted fever rickettsioses (SFR). Tick feeding on guinea pigs is feasible and results in transmission of tick-borne rickettsiae. The resulting infection leads to the recapitulation of SFR as defined by clinical signs that include fever, unthrift, and in the case of transmission by a Rickettsia parkeri-infected Amblyomma maculatum tick, a characteristic eschar at the site of the bite. No other small animal model recapitulates SFR, is large enough to collect multiple blood and skin samples for longitudinal studies, and has an immune system as similar to the human immune system. In the 1980s, the use of the guinea pig was significantly reduced due to advances made to the more reproductively prolific and inexpensive murine model. These advances included the development of genetically modified murine strains, which resulted in the expansion of murine-specific reagents and assays. Still, the advantages of the guinea pig as a model for SFR persist, novel assays are being developed to better monitor guinea pig immune responses, and tools, like CRISPR/Cas9, are now available. These technical advances allow guinea pigs to again contribute to our understanding of SFR. Importantly, returning to the guinea pig model with enhanced tools will enable rickettsial researchers to corroborate and potentially refine results acquired using mice. This minireview summarizes Cavia porcellus as an animal model for human tick-borne rickettsial diseases.
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Modelos Animais de Doenças , Cobaias , Rickettsiose do Grupo da Febre Maculosa/microbiologia , Animais , Rickettsiose do Grupo da Febre Maculosa/imunologiaRESUMO
American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.
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Amblyomma/fisiologia , Amblyomma/parasitologia , Vetores Aracnídeos/fisiologia , Vetores Aracnídeos/parasitologia , Eucoccidiida/isolamento & purificação , Animais , Coccidiose/parasitologia , Coccidiose/veterinária , Doenças do Cão/parasitologia , Cães , Mississippi , Densidade DemográficaRESUMO
Guinea pigs are an ideal animal model for the study of several infectious diseases, including tuberculosis, legionellosis, brucellosis, and spotted fever rickettsiosis. In comparison to the murine model, clinical signs in guinea pigs are more representative of disease in humans, the guinea pig immune system is more similar to that of the human, and their large size offers logistic advantages for sample collection while following disease progression. Unfortunately, the advantage of using guinea pigs in biomedical research, particularly in understanding the immune response to infectious agents, is limited in large part by the paucity of available reagents and lack of genetically manipulated strains. Here, we expand the utility of guinea pigs in biomedical research by establishing an optimized five-color/seven-parameter polychromatic flow cytometric assay for immunophenotyping lymphocytes. This assay fills a need for immunophenotyping peripheral blood lymphocytes and is an improvement over current published flow cytometry assays for guinea pigs. We anticipate that our approach will be an important starting point for developing new assays to evaluate the cellular immune response to infectious diseases in the guinea pig model. Importantly, we are currently using this assay for evaluating immunity to spotted fever rickettsiosis in a guinea pig-tick-Rickettsia system, where CD8+ T cells are a critical contributor to the immune response. Developing resources to utilize the guinea pig more effectively will enhance our ability to understand infectious diseases where the guinea pig would otherwise be the ideal model.
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Citometria de Fluxo/veterinária , Imunofenotipagem/veterinária , Linfócitos/imunologia , Animais , Modelos Animais de Doenças , Citometria de Fluxo/instrumentação , Corantes Fluorescentes , Cobaias , Imunofenotipagem/instrumentação , Masculino , Infecções por Rickettsia/imunologia , Infecções por Rickettsia/veterináriaRESUMO
The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.
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Rickettsia parkeri, a causative agent of spotted fever rickettsiosis, is transmitted by Amblyomma maculatum (Gulf Coast tick), a tick that may also carry a non-pathogenic spotted fever group Rickettsia, "Candidatus Rickettsia andeanae". Here, we evaluated R. parkeri and "Candidatus R. andeanae" in tissues from A. maculatum prior to, during, and after blood feeding on rabbits. Using colony-reared A. maculatum that were capillary-fed uninfected cells, R. parkeri, "Candidatus R. andeanae", or both rickettsiae, we detected higher levels of Rickettsia spp. in the respective treatment groups. Rickettsial levels increased during blood feeding for both R. parkeri and "Candidatus R. andeanae", with a greater increase in R. parkeri in co-infected ticks compared to singly-infected ticks. We detected transovarial transmission of "Candidatus R. andeanae" in egg and larval cohorts and confirmed vertical transmission of R. parkeri in one group of larvae. Rabbits from all Rickettsia-exposed groups seroconverted on immunofluorescent antibody testing using R. parkeri antigen. Visualization of "Candidatus R. andeanae" in tick salivary glands suggested potential transmission via tick feeding. Here, rickettsial levels in artificially infected ticks demonstrate changes during feeding and transovarial transmission that may be relevant for interpreting rickettsial levels detected in wild A. maculatum.
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Transmissão Vertical de Doenças Infecciosas/veterinária , Ixodidae/microbiologia , Ixodidae/fisiologia , Coelhos/parasitologia , Infecções por Rickettsia/veterinária , Rickettsia/fisiologia , Animais , Feminino , Ixodidae/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/microbiologia , Larva/fisiologia , Masculino , Óvulo/crescimento & desenvolvimento , Óvulo/microbiologia , Infecções por Rickettsia/microbiologia , Infecções por Rickettsia/transmissãoRESUMO
Amblyomma maculatum is the primary vector for the spotted fever group rickettsiae, Rickettsia parkeri, a known pathogen, and "Candidatus Rickettsia andeanae," currently considered nonpathogenic. Spotted fever group rickettsiae are typically endothelial cell associated and rarely circulate in the blood. Horizontal transmission to naïve ticks through blood feeding from an infected host is likely rare. Cofeeding provides an opportunity for rickettsial transmission to naïve ticks in the absence of circulating rickettsiae. We evaluated R. parkeri transmission through cofeeding between A. maculatum adults and nymphs on beef calves. Six calves in each of two trials were infested with A. maculatum that had been capillary fed R. parkeri. Four days later, calves each received recipient A. maculatum that were either capillary fed "Ca. R. andeanae" or not capillary fed before infestation. Trials differed by whether we included a barrier to minimize adjacent feeding between recipient and donor ticks. After cofeeding, we detected R. parkeri in 27% of "Ca. R. andeanae"-free recipient ticks, whereas R. parkeri was not detected in any recipient ticks that were capillary fed "Ca. R. andeanae." Rickettsia parkeri transmission efficiency to naïve ticks was greater when ticks freely cofed in proximity. No rickettsial DNA was detected in calf blood. Results confirm cofeeding as a method of horizontal transmission of R. parkeri in the absence of host rickettsemia and suggest no evidence of transmission by cofeeding when recipient ticks are first exposed to "Ca. R. andeanae" through capillary feeding. While cofeeding may provide an opportunity for maintaining the pathogen, R. parkeri, the mechanisms driving any potential effect of "Ca. R. andeanae" on R. parkeri transmission are unclear.
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Doenças dos Bovinos/parasitologia , Ixodidae/microbiologia , Rickettsia/fisiologia , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Comportamento Alimentar , Ixodidae/fisiologia , Ninfa , Infecções por Rickettsia/microbiologia , Infecções por Rickettsia/transmissão , Infecções por Rickettsia/veterináriaRESUMO
Hepatic steatosis is recognized as an independent risk factor for the development of cardiovascular disease. While obesity and type 2 diabetes are well-established risk factors in the development of hepatic steatosis, recent studies have revealed exposure to mixtures of persistent organic pollutants (POPs), which are environmental contaminants in various fatty foods, can promote steatosis. Thus, the present study was designed to determine if exposure to a defined mixture of prevalent polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides or their metabolites promote hepatic steatosis in a genetically induced model of type 2 diabetes, the leptin-deficient ob/ob mouse. Male C57BL/6J wild type (WT) or ob/ob mice were administered an environmentally relevant mixture of PCBs and OCs for 7 weeks via oral gavage. Exposure to POPs did not significantly alter fasting serum glucose or insulin levels. However, POPs exposure significantly increased hepatic triglyceride content in ob/ob animals, while decreasing serum triglyceride levels. This POPs-mediated increase in hepatic triglyceride content did not appear to be associated with significantly increased inflammation in either the liver or adipose. Exposure to POPs significantly induced the expression of cytochrome P450 3a11 in WT animals, yet the expression of this cytochrome was significantly downregulated in ob/ob animals regardless of POPs exposure. Taken together, the present data indicate exposure to an environmentally relevant mixture of both PCBs and OC pesticides in ob/ob mice promotes hepatic steatosis while decreasing hypertriglyceridemia, which demonstrates exposure to a defined mixture of POPs alters systemic lipid metabolism in a genetically induced model of obesity and type 2 diabetes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1399-1411, 2017.
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Poluentes Ambientais/toxicidade , Fígado Gorduroso/induzido quimicamente , Praguicidas/toxicidade , Bifenilos Policlorados/toxicidade , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos , Masculino , Síndrome Metabólica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo , Obesidade/patologia , Triglicerídeos/metabolismoRESUMO
Many effects of the non-psychoactive cannabinoid, cannabidiol (CBD), have been described in immune responses induced by strong immunological stimuli. It has also been shown that CBD enhances IL-2 production in response to low-level T cell stimulation. Since IL-2, in combination with TGF-ß1, are critical for Treg induction, we hypothesized that CBD would induce CD4+CD25+FOXP3+ Tregs in response to low-level stimulation. Low-level T cell stimulation conditions were established based on minimal CD25 expression in CD4+ cells using suboptimal PMA/Io (4nM/0.05µM, S/o), ultrasuboptimal PMA/Io (1nM/0.0125µM, Us/o) or soluble anti-CD3/28 (400-800ng each, s3/28). CBD increased CD25+FOXP3+ cells from CD4+, CD4+CD25+, and CD4+CD25- T cells, as well as in CD4+ T cells derived from FOXP3-GFP mice. Most importantly, the Us/o+CBD-induced CD4+CD25+ Tregs robustly suppressed responder T cell proliferation, demonstrating that the mechanism by which CBD is immunosuppressive under low-level T cell stimulation involves induction of functional Tregs.
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Canabidiol/farmacologia , Imunossupressores/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Terapia de Imunossupressão , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Reguladores/imunologia , Acetato de Tetradecanoilforbol/imunologiaRESUMO
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.
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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
Exposure to p,p'-DDE (DDE), the main bioaccumulative metabolite of the organochlorine insecticide p,p'-DDT, is associated with a higher prevalence of obesity, dyslipidemia, insulin resistance, metabolic syndrome, and immunomodulation. The present study was carried out to determine whether DDE perturbs adipose tissue homeostasis through modulation of macrophage function. Treatment with DDE or a cyclooxygenase-2 inhibitor prior to lipopolysaccharide exposure significantly decreased production of prostaglandins (PG) from J774a.1 macrophages in vitro. Similarly, J774A.1 cell lysates incubated with DDE or a specific cyclooxygenase-2 inhibitor (NS-398) produced significantly less PGE2 and PGF2α. Macrophage polarization studies revealed a pattern of DDE effects that were not fully consistent with a purely pro- or purely anti- M1 or M2 effect. However, DDE suppressed expression of two M1 markers (induced by an M1 stimulus) and enhanced expression of an M2 marker (induced by an M2 stimulus). Further studies including assessment of macrophage function are needed to fully characterize the effects of DDE on macrophage polarization. Obesity is characterized by an increase in the number of resident adipose tissue macrophages. To assess monocyte/macrophage recruitment to the adipose tissue in vivo, male C57Bl/6H mice were treated with 2 mg/kg DDE or corn oil vehicle for 5 days by gavage. Epididymal fat pads were digested and macrophage populations were analyzed by flow cytometry. In DDE-treated animals, there was a significant increase (37%) in F4/80(+)CD11b(+) macrophages/g of epididymal adipose over vehicle (P < .05). Together, these results suggest a role for DDE in the enhancement of adipose tissue macrophage recruitment and/or proliferation, as well as modulation of immune cell function that may contribute to the etiology of metabolic diseases associated with organochlorine exposure.
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Tecido Adiposo/efeitos dos fármacos , Diclorodifenil Dicloroetileno/toxicidade , Dinoprostona/biossíntese , Poluentes Ambientais/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Antígenos de Diferenciação/imunologia , Arginase/genética , Antígeno CD11b/imunologia , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Epididimo/efeitos dos fármacos , Epididimo/imunologia , Epididimo/metabolismo , Citometria de Fluxo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Fosfolipases A2/metabolismoRESUMO
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
BACKGROUND: Relaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars. METHODS: Spermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA. RESULTS: Both receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa. CONCLUSIONS: Immunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Relaxina/metabolismo , Espermatozoides/metabolismo , Suínos/metabolismo , Animais , Epididimo/metabolismo , Citometria de Fluxo , Imuno-Histoquímica , Masculino , Receptores Acoplados a Proteínas G/análise , Receptores de Peptídeos/análise , Testículo/metabolismoRESUMO
Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 µM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox) levels and enhanced p47(phox) membrane localization compared to that in vehicle-treated cells. p47(phox) is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47(phox). In addition, ATK also blocked OC-induced p47(phox) serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation.