RESUMO
Many viruses have evolved structured RNA elements that can influence transcript abundance and translational efficiency, and help evade host immune factors by hijacking cellular machinery during replication. Here, we evaluated the functional impact of sub-genomic flaviviral RNAs (sfRNAs) known to stall exoribonuclease activity, by incorporating these elements into recombinant adeno-associated viral (AAV) genome cassettes. Specifically, sfRNAs from Dengue, Zika, Japanese Encephalitis, Yellow Fever, Murray Valley Encephalitis, and West Nile viruses increased transcript stability and transgene expression compared to a conventional woodchuck hepatitis virus element (WPRE). Further dissection of engineered transcripts revealed that sfRNA elements (i) require incorporation in cis within the 3' untranslated region (UTR) of AAV genomes, (ii) require minimal dumbbell structures to exert the observed effects, and (iii) can stabilize AAV transcripts independent of 5'-3' exoribonuclease 1 (XRN1)-mediated decay. Additionally, preliminary in vivo assessment of AAV vectors bearing sfRNA elements in mice achieved increased transcript abundance and expression in cardiac tissue. Leveraging the functional versatility of engineered viral RNA elements may help improve the potency of AAV vector-based gene therapies. IMPORTANCE: Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.
Assuntos
Dependovirus , Vetores Genéticos , RNA Viral , Dependovirus/genética , Animais , RNA Viral/genética , RNA Viral/metabolismo , Vetores Genéticos/genética , Camundongos , Humanos , Estabilidade de RNA , Flaviviridae/genética , Transgenes , Células HEK293 , Genoma Viral , Regiões 3' não Traduzidas/genética , Exorribonucleases/metabolismo , Exorribonucleases/genéticaRESUMO
This article provides an overview of the history of the Japanese Society of Biofeedback Research (JSBR) and presents some of its recent advances. Most of the research papers published in the JSBR journal (Biofeedback Kenkyu) have been written in Japanese, and therefore have had very few opportunities to reach global readers. We would like to present some of important findings previously published there. First, we present the history of the JSBR. Secondly, we will focus on paced breathing, which is instrumental in achieving relaxation in heart rate variability biofeedback (HRV-BF). We will look back on the origin of slow-paced breathing in Japan, that could be attributed to the concept of Tanden breathing (abdominal paced breathing) practiced in Zen meditation. Thirdly, we will introduce some of the current research progresses of JSBR, especially focusing on the development of a non-contact sensing technology and relaxation device. Finally, we will explain about a very recent trial, the "Suu-Haa" Relaxation Technique, which we hope may be useful for helping people cope with the SARS-CoV-2 (COVID-19) crisis.
Assuntos
COVID-19 , Biorretroalimentação Psicológica , Frequência Cardíaca , Humanos , Japão , Taxa Respiratória , SARS-CoV-2RESUMO
This study aimed to evaluate the perception of Brazilian dairy processors (n = 31) concerning food defense. The results showed that respondents consider the implementation of control procedures related to facilities, products, materials, and individuals as important measures in food defense. The higher agreement rates (strongly agreed + slightly agreed) of the companies in relation to the perception of food defense were 84% for external security, followed by personnel security (82%), generalities (81%), and internal security (74%). Thus, protecting facilities and controlling the traffic flow were considered to be the most important actions under the participants' perspectives. Employee satisfaction and identification of end products and raw materials are also considered relevant in the food defense program. Although food defense is not a formal requirement in Brazilian law, the results show that there is adequate awareness of this topic by the Brazilian dairy companies.
Assuntos
Indústria de Laticínios/economia , Contaminação de Alimentos/prevenção & controle , Inocuidade dos Alimentos , Animais , Brasil , HumanosRESUMO
We developed a simple method for identifying resonance frequency by focusing on the spectral peak of the low-frequency (LF) component of heart rate variability (HRV) and examined the hypothesis that paced breathing at an accurate resonance frequency increases HRV and baroreflex sensitivity (BRS). We assessed a peak frequency of the LF component of the resting HRV by using power spectral analysis under respiratory control at 0.25 Hz, and a resonance frequency, which was evaluated by using the standard breathing maneuver (Lehrer 2007). We examined the effects of paced breathing at the peak frequency of the LF component (Spectral condition) and paced breathing at the resonance frequency as determined by the standard breathing maneuver (Standard condition) on HRV and BRS in 28 healthy college students and young adults. Electrocardiogram, respiration, and noninvasive continuous blood pressure was recorded during a 5-min baseline, followed by a 5-min paced breathing session. Results indicated that the BRS increased during the breathing session under both conditions, but the increase in BRS under the Spectral condition was higher than the Standard condition (p < .05). The LF amplitude increased during the breathing session under both conditions (p < .001), although the difference between the conditions was not significant. These results suggest that paced breathing at the peak frequency of the LF component enhanced the autonomic baroreflex function. Moreover, assessment of the LF-peak may provide more accurate information on resonance frequency for paced breathing during HRV biofeedback.
Assuntos
Sistema Nervoso Autônomo/fisiologia , Barorreflexo/fisiologia , Exercícios Respiratórios , Frequência Cardíaca/fisiologia , Taxa Respiratória/fisiologia , Adulto , Biorretroalimentação Psicológica/fisiologia , Eletrocardiografia , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Dysphagia and malnutrition seem to be associated, but little research in detail has been reported. We aimed to clarify the association between dysphagia and malnutrition by adopting accurate diagnosis and mathematical evaluation of dysphagia using videofluorography and nutritional assessment calculated by a well-established nutritional risk index. We conducted a retrospective analysis of 165 enrolled patients who were admitted to our hospital for acute diseases and underwent videofluorography on suspicion of dysphagia in the year 2016. We diagnosed high-risk dysphagia in patients with 8-point penetration-aspiration scale (PAS) score over 4. We used the geriatric nutritional risk index (GNRI) as a nutritional assessment tool. A GNRI score less than 91.2 corresponds to malnutrition. The median age of 165 enrolled patients was 76.0, and the number of female patients was 53. The mean GNRI was 81.2, and 134 patients (81.2%) had malnutrition. The number of the patients with a diagnosis of high-risk dysphagia was 54 (32.7%). The GNRI of patients with high-risk dysphagia was significantly less than that of patients without (mean value 77.7 ± 10.5 vs. 83.0 ± 10.5, P = 0.003). GNRI < 91.2 was independently and significantly associated with high-risk dysphagia (OR 3.094; CI 1.057-9.058; P = 0.039). Based on the current study, the authors propose evaluating nutritional status to predict dysphagia risk of patients in the acute phase.
Assuntos
Transtornos de Deglutição/complicações , Desnutrição/etiologia , Avaliação Nutricional , Estado Nutricional , Idoso , Idoso de 80 Anos ou mais , Feminino , Avaliação Geriátrica , Humanos , Masculino , Desnutrição/epidemiologia , Estudos Retrospectivos , Fatores de RiscoRESUMO
BACKGROUND: Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors. METHODS: Twenty-one tamoxifen-selected cell lines were developed through cloning by limiting dilution of an MCF-7 cell culture treated with 1 µM tamoxifen for 6 months. The parent (MCF-7) and tamoxifen-selected cell lines were characterized for protein expression of ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) using immunohistochemistry (IHC). The mRNA levels of ER, DOK7, and ELF5 were assessed using quantitative RT-PCR. Promoter methylation levels of DOK7 and ELF5 were determined by pyrosequencing of bisulfite-modified DNA. The relationship between hormone receptor status and promoter methylation of DOK7 and ELF5 was further examined using available methylation array data (Illumina HM450) from a set of paired primary and second breast tumors from 24 women. RESULTS: All 21 of the novel tamoxifen-selected cell lines are ER-positive, and HER2-negative, and 18 of the cell lines are PR-negative while the MCF-7 cells were scored as ER-positive, modestly PR-positive and HER2 negative. Expression of DOK7 and ELF5 is significantly up-regulated in half of the tamoxifen-selected cell lines as compared to the parental MCF-7. In contrast, the previously established ER-negative TMX2-28 cell line has decreased expression of both DOK7 and ELF5 and increased DNA methylation in the transcriptional start site region of these genes. ELF5 methylation was lower in second versus primary tumors in women who received anti-estrogen treatment, in PR-negative versus PR-positive tumors, and in the subset of PR-positive first tumors from the group of women who had second PR-negative tumors as compared to those who had second PR-positive tumors. CONCLUSIONS: The distinct ELF5 methylation of PR-positive primary tumors from women who had a PR-negative recurrence indicates the possibility of stratification of women for tailored treatment in the early stages of disease.
RESUMO
Calcineurin-inhibitor refractory bronchiolitis obliterans (BO) represents the leading cause of late graft failure after lung transplantation. T helper (Th)2 and Th17 lymphocytes have been associated with BO development. Taking advantage of a fully allogeneic trachea transplantation model in mice, we addressed the pathogenicity of Th cells in obliterative airway disease (OAD) occurring in cyclosporine A (CsA)-treated recipients. We found that CsA prevented CD8(+) T cell infiltration into the graft and downregulated the Th1 response but affected neither Th2 nor Th17 responses in vivo. In secondary mixed lymphocyte cultures, CsA dramatically decreased donor-specific IFN-γ production, enhanced IL-17 production and did not affect IL-13. As CD4(+) depletion efficiently prevented OAD in CsA-treated recipients, we further explored the role of Th2 and Th17 immunity in vivo. Although IL-4 and IL-17 deficient untreated mice developed an OAD comparable to wild-type recipients, a single cytokine deficiency afforded significant protection in CsA-treated recipients. In conclusion, CsA treatment unbalances T helper alloreactivity and favors Th2 and Th17 as coexisting pathways mediating chronic rejection of heterotopic tracheal allografts.
Assuntos
Bronquiolite Obliterante/induzido quimicamente , Ciclosporina/toxicidade , Rejeição de Enxerto/induzido quimicamente , Interleucina-17/fisiologia , Transplante de Pulmão/efeitos adversos , Células Th2/imunologia , Traqueia/transplante , Animais , Western Blotting , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Citocinas/metabolismo , Citometria de Fluxo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Técnicas Imunoenzimáticas , Imunossupressores/toxicidade , Interferon gama/fisiologia , Interleucina-4/fisiologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traqueia/efeitos dos fármacos , Traqueia/imunologia , Transplante Heterotópico , Transplante HomólogoRESUMO
The present study was designed to examine the effect of heart rate variability (HRV) biofeedback on the cardiorespiratory resting function during sleep in daily life. Forty-five healthy young adults were randomly assigned to one of three groups: HRV biofeedback, Autogenic Training(AT), and no-treatment control. Participants in the HRV biofeedback were instructed to use a handheld HRV biofeedback device before their habitual bedtime, those in the AT were asked to listen to an audiotaped instruction before bedtime,and those in the control were asked to engage in their habitual activity before bedtime. Pulse wave signal during sleep at their own residences was measured continuously with a wrist watch-type transdermal photoelectric sensor for three time points. Baseline data were collected on the first night of measurements, followed by two successive nights for HRV biofeedback, AT, or control. Cardiorespiratory resting function was assessed quantitatively as the amplitude of high frequency(HF) component of pulse rate variability, a surrogate measure of respiratory sinus arrhythmia. HF component increased during sleep in the HRV biofeedback group,although it remained unchanged in the AT and control groups. These results suggest that HRV biofeedback before sleep may improve cardiorespiratory resting function during sleep.
Assuntos
Biorretroalimentação Psicológica/métodos , Frequência Cardíaca/fisiologia , Respiração , Descanso/fisiologia , Sono/fisiologia , Adolescente , Adulto , Feminino , Humanos , MasculinoRESUMO
Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.
Assuntos
Capsídeo , Dependovirus , Animais , Humanos , Capsídeo/química , Dependovirus/genética , Terapia Genética/métodos , Técnicas de Transferência de Genes , Proteínas do Capsídeo/genética , Vetores Genéticos , Transdução GenéticaRESUMO
Allograft acceptance and tolerance can be achieved by different approaches including inhibition of effector T cell responses through CD28-dependent costimulatory blockade and induction of peripheral regulatory T cells (Tregs). The observation that Tregs rely upon CD28-dependent signals for development and peripheral expansion, raises the intriguing possibility of a counterproductive consequence of CTLA4-Ig administration on tolerance induction. We have investigated the possible negative effect of CTLA4-Ig on Treg-mediated tolerance induction using a mouse model of single MHC class II-mismatched skin grafts in which long-term acceptance was achieved by short-term administration of IL-2/anti-IL-2 complex. CTLA4-Ig treatment was found to abolish Treg-dependent acceptance in this model, restoring skin allograft rejection and Th1 alloreactivity. CTLA4-Ig inhibited IL-2-driven Treg expansion, and prevented in particular the occurrence of ICOS(+) Tregs endowed with potent suppressive capacities. Restoring CD28 signaling was sufficient to counteract the deleterious effect of CTLA4-Ig on Treg expansion and functionality, in keeping with the hypothesis that costimulatory blockade inhibits Treg expansion and function by limiting the delivery of essential CD28-dependent signals. Inhibition of regulatory T cell function should therefore be taken into account when designing tolerance protocols based on costimulatory blockade.
Assuntos
Rejeição de Enxerto/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoconjugados/administração & dosagem , Interleucina-2/administração & dosagem , Linfócitos T Reguladores/imunologia , Abatacepte , Animais , Antígenos CD28/metabolismo , Teste de Histocompatibilidade , Interleucina-2/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Immune responses in newborn mice are known to be biased toward the helper type 2 phenotype. This may account for their propensity to develop tolerance. Herein, we evaluated the effects of IL-4 deprivation on CD4(+) T-cell activities elicited by neonatal exposure to allogeneic spleen cells. We showed that chimerism, Th2-type polarization and pathology, as well as skin allograft acceptance were inhibited in BALB/c mice immunized at birth with (A/J x BALB/c) F(1) spleen cells upon in vivo IL-4 neutralization. While IL-4 neutralization inhibited the development of Th2 cells in this model, it led to the accumulation of IL-17A, IL-17F, IL-22, IL-6 and RORγt mRNA in the spleen or graft tissues. Moreover, IL-4 deprivation led to the differentiation of donor-specific Th17 cells with a concomitant Th1 response characterized by IFN-γ production. The Th17-type response emerging in IL-4-deprived mice was found to mediate both intragraft neutrophil infiltration and the abrogation of B-cell chimerism. Neutralization of this Th17 response failed however to restore functional skin graft acceptance. Collectively, our observations indicate that the neonatal Th2 response opposes the development of Th17 cells, and that Th17 cells are responsible for controlling lymphoid chimerism in mice neonatally injected with semiallogeneic cells.
Assuntos
Animais Recém-Nascidos , Quimerismo , Interleucina-17/imunologia , Interleucina-4/genética , Animais , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques. We discover a highly potent, cross-species compatible variant (AAV.cc47) that shows improved attributes benchmarked against AAV serotype 9 as evidenced by robust reporter and therapeutic gene expression, Cre recombination and CRISPR genome editing in normal and diseased mouse models. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. We envision that ccAAV vectors may not only improve predictive modeling in preclinical studies, but also clinical translatability by broadening the therapeutic window of AAV based gene therapies.
Assuntos
Dependovirus , Edição de Genes , Animais , Dependovirus/metabolismo , Terapia Genética , Vetores Genéticos/genética , Humanos , Macaca/genética , Camundongos , Suínos , Transdução GenéticaRESUMO
Azodicarbonamide was recently identified as a new anti-HIV agent that targets the zinc finger domains of the HIV-1 NCp7 nucleocapsid protein. Here, we demonstrate that azodicarbonamide inhibits in a dose-dependent manner the responses of purified human CD4+ T lymphocytes stimulated either by monoclonal antibodies against CD3 and CD28 or by allogeneic dendritic cells. These suppressive effects involve a direct action on the calcium mobilization machinery, as azodicarbonamide strongly inhibited the calcium influx induced either by antibodies against CD3 and CD28 or the chemokine RANTES, as well as by thapsigargin, an activator of depletion-activated calcium channels. In vivo, administration of azodicarbonamide into mice blunted their response to polyclonal T-cell activation induced by the injection of monoclonal antibody against CD3 and resulted in delayed rejection of skin allografts. In addition to its anti-HIV properties, azodicarbonamide is a new immunosuppressive agent that might have therapeutic applications in T cell-mediated inflammatory disorders.
Assuntos
Fármacos Anti-HIV/farmacologia , Compostos Azo/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Tolerância Imunológica , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Feminino , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Pele/imunologiaRESUMO
The primary structure of A/J anti-p-azophenylarsonate (anti-Ars) antibodies expressing the major A-strain cross-reactive idiotype (CRIA) has provided important insights into issues of antibody diversity and the molecular basis of idiotypy in this important model system. Until recently, this idiotype was thought to be rarely, if ever, expressed in BALB/c mice. Indeed, it has been reported that BALB/c mice lack the heavy chain variable segment (VH) gene that is utilized by the entire family of anti-Ars antibodies expressing the A/J CRI. Recently, however, it has been possible to elicit CRIA+, Ars binding antibodies in the BALB/c strain by immunizing first with anti-CRI and then with antigen. Such BALB/c, CRIA+ anti-Ars antibodies can be induced occasionally with antigen alone. VH region amino acid sequences are described for two CRIA+ hybridoma products derived from BALB/c mice. While remarkably similar to each other, their VH segments (1-98) differ from the VH segments of A/J CRIA+, anti-Ars antibodies in over 40 positions. Rather than the usual JH2 gene segment used by most A/J CRIA+ anti-Ars antibodies, one BALB/c CRIA+ hybridoma utilizes a JH1 gene segment, while the other uses a JH4. However, the D segments of both of the BALB/c antibodies are remarkably homologous to the D segments of several A/J CRIA+ antibodies sequenced previously, as are the amino terminal amino acid sequences of their light chains. These data imply that BALB/c mice express the A/J CRIA by producing antibodies with very similar, if not identical, light chain and heavy chain D segments, but in the context of different VH and JH gene segments than their A/J counterparts. The results document that molecules that share serologic specificities can have vastly different primary structures.
Assuntos
Anticorpos Monoclonais/análise , Compostos Azo/imunologia , Cadeias Pesadas de Imunoglobulinas/análise , Idiótipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/análise , p-Azobenzenoarsonato/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/análise , Anticorpos Anti-Idiotípicos/genética , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/genética , Diversidade de Anticorpos , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Reações Cruzadas , Epitopos/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/classificação , Idiótipos de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/análise , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , CoelhosRESUMO
We have shown that, by suitable idiotypic manipulation, BALB/c mice can express the major cross-reactive idiotype (CRI) of A/J mice in response to azophenylarsonate (Ars). In order to know if the CRIA idiotype is present in the potential repertoire of BALB/c before any intentional selection, we used polyclonal activation in vitro and limiting dilution analysis. The readout was done with two monoclonal anti-CRIA antibodies that recognize distinct idiotopes on a CRIA+ A/J germline-encoded monoclonal antibody. We studied the frequency of CRIA+ lipopolysaccharide (LPS)-reactive cells in the spleens of nonimmune and immune A/J mice and in the spleens of naive and manipulated (i.e., producing CRIA+ antibodies) BALB/c mice. A/J and BALB/c naive individuals presented very high frequencies of Ars-specific B cells while the frequency of CRIA+ B cells was only a minor subset (0.5%) of the total Ars-specific subset in the two strains. When A/J mice were immunized with Ars-keyhole limpet hemocyanin, a clear preferential expansion of the CRIA+ minor subset of A/J mice was observed (100x). No such enhancement was observed in BALB/c mice similarly treated. Manipulated BALB/c mice presented a higher frequency of CRIA+ anti-Ars B cells than naive or antigen-immunized BALB/c individuals.
Assuntos
Compostos Azo/imunologia , Idiótipos de Imunoglobulinas/imunologia , p-Azobenzenoarsonato/imunologia , Animais , Anticorpos Anti-Idiotípicos/fisiologia , Anticorpos Monoclonais/fisiologia , Células Produtoras de Anticorpos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Contagem de Células , Reações Cruzadas , Hemocianinas/imunologia , Idiótipos de Imunoglobulinas/biossíntese , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , CoelhosRESUMO
Glucocorticoids (GCs) affect peripheral immune responses by inhibiting T cell immunity at several stages of the activation cascade, causing impaired cytokine production and effector function. The recent demonstration that the thymic epithelium and possibly thymocytes themselves produce steroids suggests that endogenous GCs also play a role in the control of T cell development. As both peripheral responsiveness and thymic differentiation appear to be regulated by the quantity and quality of intracellular signals issued by antigen-major histocompatibility complex-engaged T cell receptor (TCR) complexes, we investigated the effects of GCs on the signaling properties of T cells stimulated by anti-CD3 monoclonal antibodies or agonist peptides. We demonstrate in this work that dexamethasone, a synthetic GC, inhibits the early signaling events initiated upon TCR ligation, such as tyrosine phosphorylation of several TCR-associated substrates including the zeta chain, the ZAP70 kinase, and the transmembrane adapter molecule linker for activation of T cells. Hypophosphorylation was not a consequence of reduced kinase activity of src protein tyrosine kinases, but was correlated with an altered- membrane compartmentalization of these molecules. These observations indicate that in addition to their well-described ability to interfere with the transcription of molecules involved in peripheral responses, GCs inhibit T cell activation by affecting the early phosphorylating events induced after TCR ligation.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Imunossupressores/farmacologia , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Regulação para Baixo/efeitos dos fármacos , Hibridomas , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Timo/citologia , Tirosina/metabolismoRESUMO
Dendritic cells (DC) are described as "nature's adjuvant," since they have the capacity to sensitize T cells in vivo upon first encounter with the antigen. The potent accessory properties of DC appear to develop sequentially. In particular, the ability to process antigens and to sensitize native T cells develops in sequence, a process termed "maturation" that is well described in vitro. Here, we obtain evidence for maturation in vivo in response to the bacterial product lipopolysaccharide (LPS). Before LPS treatment, many DC are found at the margin between the red and white pulp. These cells lack the M342 and DEC-205 markers, but process soluble proteins effectively. 6 h after LPS, DC with the M342 and DEC-205 markers are found in increased numbers in the T cell areas. These cells have a reduced capacity to process proteins, but show increases in the B7 costimulator and T cell stimulatory capacity. 48 h after LPS, the number of DC in the spleen is reduced markedly. We interpret these findings to mean that LPS can cause DC in the marginal zone to mature and to migrate into and then out of the T cell areas.
Assuntos
Células Dendríticas/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Baço/imunologia , Animais , Antígenos de Diferenciação , Antígeno B7-1/biossíntese , Diferenciação Celular , Movimento Celular , Separação Celular , Células Dendríticas/efeitos dos fármacos , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/biossíntese , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Baço/anatomia & histologia , Baço/efeitos dos fármacos , Regulação para CimaRESUMO
The aim of this study was to develop an immunization procedure avoiding external adjuvant. Data are presented showing that syngeneic dendritic cells (DC), which have been pulsed in vitro with antigen, induce a strong antibody response in mice. By contrast, antigen (Ag)-pulsed low-density B cells, although equally able to induce interleukin 2 secretion by an Ag-specific T cell hybridoma in vitro, only weakly prime the mice in vivo. Moreover, we show that the injection of Ag-pulsed DC induces the synthesis of isotypes similar to the immunoglobulin classes detected after immunization with the same Ag in complete Freund's adjuvant. Importantly, high amounts of IgG2a antibodies are produced, suggesting that T helper type 1 cells are activated. Collectively, these data indicate that DC can initiate a primary humoral response and that they may be used as physiological adjuvant in vivo.
Assuntos
Formação de Anticorpos , Células Apresentadoras de Antígenos/imunologia , Células Dendríticas/imunologia , Mioglobina/imunologia , gama-Globulinas/imunologia , Animais , Células Cultivadas , Feminino , Humanos , Hibridomas/imunologia , Interleucina-2/biossíntese , Cinética , Camundongos , Camundongos Endogâmicos DBA , Baço/imunologia , Linfócitos T/imunologia , BaleiasRESUMO
Injection of adult mice with high doses of monomeric human gamma globulins (dHGG) has been previously shown to produce a state of peripheral tolerance in both B and T cells. To gain insight into the mechanism of induction and maintenance of adult tolerance in this model, we have analyzed the pattern of lymphokines produced by control and tolerant animals in response to the tolerogen. The data presented indicate that HGG-specific, interleukin 2 (IL-2)- and interferon gamma (IFN-gamma)-producing T cells (thus referred to as T helper type 1 [Th1] cells) are rendered unresponsive after in vivo administration of soluble HGG. In contrast, antigenic stimulation of T cells isolated from tolerant adult mice leads to increased production of IL-4 in vitro. In vivo challenge of dHGG-treated adult animals with hapten-coupled HGG (p-azophenylarsonate [ARS]-HGG) induced a significant ARS-specific antibody response, suggesting that tolerance induction in this model does not completely abrogate tolerogen-specific Th activity in vivo. In agreement with the in vitro data, hapten-specific antibody response of tolerant animals is characterized by a selective deficiency in the IFN-gamma-dependent IgG2a subclass. Injection of immunogenic forms of HGG into tolerant animals also produced an IL-4-dependent increase in total serum IgE levels, indicative of an increased activity of HGG-specific Th2 cells in these animals. The finding that tolerance induction differentially affects Th subpopulations suggests that crossregulation among lymphocyte subsets may play a role in the induction and/or maintenance of acquired tolerance in adults.
Assuntos
Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T/imunologia , gama-Globulinas/imunologia , Animais , Anticorpos/análise , Ensaio de Imunoadsorção Enzimática , Imunoglobulina E/análise , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos A , Radioimunoensaio , Valores de Referência , gama-Globulinas/administração & dosagemRESUMO
Anti-idiotypic antibodies (Ab2) were raised in allotype-matched rabbits against anti-carbohydrate or anti-tobacco mosaic virus antibodies (Ab1). Several Ab2 were purified and injected into a third series of rabbits III which synthesized antiantiidiotypic antibodies (Ab3). Antigen was then given for the first time in those rabbits who had synthesized Ab3. The specific antibody synthesized in rabbits III was called Ab1'. Anti-idiotypic antibodies were raised against purified Ab3 antibodies (Ab4). In most cases, Ab1' antibodies are sharing idiotypic specificities with Ab1. Ab3 did not react with antigen but shared idiotopes with Ab1 and Ab1' because Ab4 antibodies, which are anti-idiotypes to Ab3 do recognize specifically Ab1 and Ab1' antibodies belonging to the same chain of immunization. It seems therefore that Ab3 looks idiotypically like Ab1 and Ab4 displays the same behaviour as Ab2. A general view of the functioning of the immune system is presented.