RESUMO
Somatic mutations are major genetic contributors to cancers and many other age-related diseases. Many disease-causing somatic mutations can initiate clonal growth prior to the appearance of any disease symptoms, yet experimental models that can be used to examine clonal abnormalities are limited. We describe a mosaic analysis system with Cre or Tomato (MASCOT) for tracking mutant cells and demonstrate its utility for modeling clonal hematopoiesis. MASCOT can be induced to constitutively express either Cre-GFP or Tomato for lineage tracing of a mutant and a reference group of cells simultaneously. We conducted mosaic analysis to assess functions of the Id3 and/or Tet2 gene in hematopoietic cell development and clonal hematopoiesis. Using Tomato-positive cells as a reference population, we demonstrated the high sensitivity of this system for detecting cell-intrinsic phenotypes during short-term or long-term tracking of hematopoietic cells. Long-term tracking of Tet2 mutant or Tet2/Id3 double-mutant cells in our MASCOT model revealed a dynamic shift from myeloid expansion to lymphoid expansion and subsequent development of lymphoma. This work demonstrates the utility of the MASCOT method in mosaic analysis of single or combined mutations, making the system suitable for modeling somatic mutations identified in humans.
Assuntos
Integrases/genética , Modelos Genéticos , Mutação/genética , Solanum lycopersicum/genética , Animais , Hematopoiese Clonal/genética , Técnicas Genéticas , Linfoma/genética , Camundongos , Camundongos Transgênicos , Mosaicismo , Análise de Sequência de DNARESUMO
BACKGROUND: Obesity is associated with impaired primary and secondary immune responses to influenza infection, with T cells playing a critical role. T-cell function is highly influenced by the cellular metabolic state; however, it remains unknown how altered systemic metabolism in obesity alters T-cell metabolism and function to influence immune response. Our objective was to identify the altered cellular metabolic state of T cells from obese mice so that we may target T-cell metabolism to improve immune response to infection. METHODS: Mice were fed normal chow or high-fat diet for 18-19 weeks. Changes in T-cell populations were analyzed in both adipose tissue and spleens using flow cytometry. Splenic T cells were further analyzed for nutrient uptake and extracellular metabolic flux. As changes in T-cell mitochondrial oxidation were observed in obesity, obese mice were treated with metformin for 6 weeks and compared to lean control mice or obese mice undergoing weight loss through diet switch; immunity was measured by survival to influenza infection. RESULTS: We found changes in T-cell populations in adipose tissue of high-fat diet-induced obese mice, characterized by decreased proportions of Treg cells and increased proportions of CD8+ T cells. Activated CD4+ T cells from obese mice had increased glucose uptake and oxygen consumption rate (OCR), compared to T cells from lean controls, indicating increased mitochondrial oxidation of glucose. Treatment of isolated CD4+ T cells with metformin was found to inhibit OCR in vitro and alter the expression of several activation markers. Last, treatment of obese mice with metformin, but not weight loss, was able to improve survival to influenza in obesity. CONCLUSIONS: T cells from obese mice have an altered metabolic profile characterized by increased glucose oxidation, which can be targeted to improve survival against influenza infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Obesidade/imunologia , Infecções por Orthomyxoviridae/imunologia , Estresse Oxidativo , Tecido Adiposo/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Dieta Hiperlipídica , Vírus da Influenza A Subtipo H1N1 , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Baço/imunologiaRESUMO
The critical importance of the thymus for generating new naive T cells that protect against novel infections and are tolerant to self-antigens has led to a recent revival of interest in monitoring thymic function in species other than humans and mice. Nonhuman primates such as rhesus macaques (Macaca mulatta) provide particularly useful animal models for translational research in immunology. In this study, we tested the performance of a 15-marker multicolor Ab panel for flow cytometric phenotyping of lymphocyte subsets directly from rhesus whole blood, with validation by thymectomy and T cell depletion. Immunohistochemical and multiplex RNA expression analysis of thymus tissue biopsies and molecular assays on PBMCs were used to further validate thymus function. Results identify Ab panels that can accurately classify rhesus naive T cells (CD3+CD45RA+CD197+ or CD3+CD28+CD95-) and recent thymic emigrants (CD8+CD28+CD95-CD103+CD197+) using just 100 µl of whole blood and commercially available fluorescent Abs. An immunohistochemical panel reactive with pan-cytokeratin (CK), CK14, CD3, Ki-67, CCL21, and TdT provides histologic evidence of thymopoiesis from formalin-fixed, paraffin-embedded thymus tissues. Identification of mRNAs characteristic of both functioning thymic epithelial cells and developing thymocytes and/or molecular detection of products of TCR gene rearrangement provide additional complementary methods to evaluate thymopoiesis, without requiring specific Abs. Combinations of multiparameter flow cytometry, immunohistochemistry, multiplex gene expression, and TCR excision circle assays can comprehensively evaluate thymus function in rhesus macaques while requiring only minimal amounts of peripheral blood or biopsied thymus tissue.
Assuntos
Citometria de Fluxo , Macaca mulatta , Timo , Animais , Timo/imunologia , Timo/metabolismo , Timo/citologia , Imuno-Histoquímica , Imunofenotipagem , Masculino , Feminino , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , TimectomiaRESUMO
OBJECTIVE: Cardiac surgery, especially when employing cardiopulmonary bypass and deep hypothermic circulatory arrest, is associated with systemic inflammatory responses that significantly affect morbidity and mortality. Intestinal perfusion abnormalities have been implicated in such responses, but the mechanisms linking local injury and systemic inflammation remain unclear. Intestinal mast cells are specialized immune cells that secrete various preformed effectors in response to cellular stress. We hypothesized that mast cells are activated in a microenvironment shaped by intestinal ischemia/reperfusion, and investigated local and systemic consequences. DESIGN: Rat model of deep hypothermic circulatory arrest. SETTING: University research laboratory. SUBJECTS: Twelve- to 14-week-old male Sprague-Dawley rats. INTERVENTIONS: Rats were anesthetized and cooled to 16°C to 18°C on cardiopulmonary bypass before instituting deep hypothermic circulatory arrest for 45 minutes. Specimens were harvested following rewarming and 2 hours of recovery. MEASUREMENTS AND MAIN RESULTS: Significant intestinal barrier disruption was found, together with macro- and microscopic evidence of ischemia/reperfusion injury in ileum and colon, but not in the lungs or kidneys. Immunofluorescence and toluidine blue staining revealed increased numbers of mast cells and their activation in the gut. In animals pretreated with the mast cell stabilizer, cromolyn sodium, mast cell degranulation was blocked, and intestinal morphology and barrier function were preserved following deep hypothermic circulatory arrest. Furthermore, cromolyn sodium treatment was associated with reduced intestinal neutrophil influx and blunted systemic release of proinflammatory cytokines. CONCLUSION: Our data provide primary evidence that intestinal ischemia/reperfusion is a leading pathophysiologic process in a rat model of deep hypothermic circulatory arrest, and that intestinal injury, and local and systemic inflammatory responses are critically dependent on mast cell activation. This identifies intestinal mast cells as central players in deep hypothermic circulatory arrest-associated responses, and opens novel therapeutic possibilities for patients undergoing this procedure.
Assuntos
Parada Circulatória Induzida por Hipotermia Profunda , Intestinos/irrigação sanguínea , Mastócitos/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Animais , Ponte Cardiopulmonar , Parada Circulatória Induzida por Hipotermia Profunda/efeitos adversos , Modelos Animais de Doenças , Hipotermia Induzida , Masculino , Mastócitos/imunologia , Ratos , Ratos Sprague-Dawley , Síndrome de Resposta Inflamatória Sistêmica/etiologiaRESUMO
Central tolerance ensures autoreactive T cells are eliminated or diverted to the regulatory T cell lineage, thus preventing autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their T-cell receptors (TCRs) for autoreactivity against the diverse self-antigens displayed by antigen-presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, synchronized positive selection assays, two-photon time-lapse microscopy, and quantification of TCR-signaled apoptotic thymocytes, demonstrate that CCR4 and CCR7 promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets in mice. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and is required for medullary localization and negative selection of mature thymocytes. In addition, CCR4 and CCR7 differentially enforce self-tolerance, with CCR4 enforcing tolerance to self-antigens presented by activated APCs, which express CCR4 ligands. Our findings show that CCR7 expression is not synonymous with medullary localization and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of negative selection, respectively, via interactions with distinct APC subsets.
Autoimmune diseases occur when immune cells mistakenly identify the body's own tissues as 'foreign' and attack them. To reduce the risk of this happening, the body has multiple ways of removing self-reactive immune cells, including T cells. One such way, known as central tolerance, occurs in the thymus the organ where T cells develop. In the center of the thymus the medulla specialized cells display fragments of the majority of proteins expressed by healthy cells throughout the body. Developing T cells enter the medulla, where they scan these specialized cells to determine if they recognize the presented protein fragments. If an immature T cell recognizes and binds to these 'self-antigens' too strongly, it is either destroyed, or it develops into a regulatory cell, capable of actively suppressing T cell responses to that self-antigen. This ensures that T cells won't attack healthy cells in the body that make those self-antigens, and therefore, it is important that T cells enter the medulla and carry out this scanning process efficiently. T cells are recruited to the medulla from the outer region of the thymus by chemical signals called chemokines. These signals are recognized by chemokine receptors on T cells, which are expressed at different times during T cell development. Previous work has shown that one of these receptors, called CCR7, guides T cells to the medulla. Although it was thought that CCR7 was solely responsible for this migration, prior work suggests another receptor, CCR4, may also contribute to T cell migration into the medulla and central tolerance. To determine whether CCR7 and CCR4 play the same or different roles in central tolerance, Li, Tipan et al. used a combination of experimental methods, including live imaging of the thymus, to study T cell development in mice. The experiments revealed that CCR4 is expressed first, and this receptor alone guides immature T cells into the medulla and ensures that they are the first to be checked for self-reactivity. In contrast, CCR7 is expressed by more mature developing T cells two to three days later, ensuring they also accumulate within the medulla and become tolerant to self-antigens. Both receptors are required for protection from autoimmunity, with results suggesting that CCR4 and CCR7 promote tolerance against different tissues. Taken together, the findings provide new information about the distinct requirement for CCR4 and CCR7 in guiding immature T cells into the medulla and ensuring central tolerance to diverse tissues. One outstanding question is whether defects in T cells entering the medulla earlier or later alter tolerance to distinct self-antigens and lead to different autoimmune diseases. Future work will also investigate whether these observations hold true in humans, potentially leading to therapies for autoimmune diseases.
Assuntos
Timócitos , Timo , Animais , Camundongos , Autoantígenos/metabolismo , Diferenciação Celular , Tolerância Central , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CCR7/metabolismo , Timócitos/metabolismo , Timo/metabolismoRESUMO
Inflammatory bowel disease lacks a long-lasting and broadly effective therapy. Here, by taking advantage of the anti-infection and anti-inflammatory properties of natural antibodies against the small-molecule epitope phosphorylcholine (PC), we show in multiple mouse models of colitis that immunization of the animals with self-assembling supramolecular peptide nanofibres bearing PC epitopes induced sustained levels of anti-PC antibodies that were both protective and therapeutic. The strength and type of immune responses elicited by the nanofibres could be controlled through the relative valency of PC epitopes and exogenous T-cell epitopes on the nanofibres and via the addition of the adjuvant CpG. The nanomaterial-assisted induction of the production of therapeutic antibodies may represent a durable therapy for inflammatory bowel disease.
RESUMO
Kaposi's sarcoma (KS) is the major AIDS-associated malignancy. It is characterized by the proliferation of spindle cells, inflammatory infiltrate, and aberrant angiogenesis caused by Kaposi's sarcoma herpesvirus (KSHV) infection. Small GTPase Rac1, an inflammatory signaling mediator triggering reactive oxygen species (ROS) production by NADPH-oxidases, is implicated in carcinogenesis and tumor angiogenesis. Here, we show that expression of a constitutively active Rac1 (RacCA) driven by the alpha-smooth muscle actin promoter in transgenic mice is sufficient to cause KS-like tumors through mechanisms involving ROS-driven proliferation, up-regulation of AKT signaling, and hypoxia-inducible factor 1-alpha-related angiogenesis. RacCA-induced tumors expressed KS phenotypic markers; displayed remarkable transcriptome overlap with KS lesions; and were, like KS, associated with male gender. The ROS scavenging agent N-acetyl-cysteine inhibited angiogenesis and completely abrogated transgenic RacCA tumor formation, indicating a causal role of ROS in tumorigenesis. Consistent with a pathogenic role in KS, immunohistochemical analysis revealed that Rac1 is overexpressed in KSHV(+) spindle cells of AIDS-KS biopsies. Our results demonstrate the direct oncogenicity of Rac1 and ROS and their contribution to a KS-like malignant phenotype, further underscoring the carcinogenic potential of oxidative stress in the context of chronic infection and inflammation. They define the RacCA transgenic mouse as a model suitable for studying the role of oxidative stress in the pathogenesis and therapy of KS, with relevance to other inflammation-related malignancies. Our findings suggest host and viral genes triggering Rac1 or ROS production as key determinants of KS onset and potential KS chemopreventive or therapeutic targets.
Assuntos
Antioxidantes/metabolismo , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/enzimologia , Síndrome da Imunodeficiência Adquirida/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Ativação Enzimática , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sarcoma de Kaposi/irrigação sanguínea , Sarcoma de Kaposi/etiologia , Transcrição Gênica/genética , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Immunity Related GTPases (IRG) are a family of proteins produced during infection that regulate membrane remodeling events in cells, particularly autophagy and mitophagy. The human IRGM gene has been strongly associated with Crohn's disease and other inflammatory diseases through Genome-Wide Association studies. Absence of Irgm1 in mice prompts intestinal inflammation, autoimmunity, and impaired immune control of pathogenic bacteria and protozoa. Although prior work has focused on a prominent role for IRGM/Irgm1 in regulating macrophage function, the work described here addresses a potential role of Irgm1 in regulating the function of mature T cells. Irgm1 was found to be highly expressed in T cells in a manner that varied with the particular T cell subset and increased with activation. Mice with a complete lack of Irgm1, or a conditional lack of Irgm1 specifically in T cells, displayed numerous changes in T cell numbers and function in all subsets examined, including CD4+ (Th1 and Treg) and CD8+ T cells. Related to changes in T cell number, apoptosis was found to be increased in Irgm1-deficient CD4+ and CD8+ T cells. Altered T cell metabolism appeared to be a key driver of the phenotypes: Glucose metabolism and glycolysis were increased in Irgm1-deficient CD4+ and CD8+ T cells, and muting these effects with glycolytic inhibitors partially restored T cell function and viability.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/fisiologia , Animais , Apoptose/genética , Autofagia/genética , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/genética , Glucose/metabolismo , Glicólise , Ativação Linfocitária/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subpopulações de Linfócitos T/imunologiaRESUMO
Thymic epithelial cells (TECs) and hematopoietic antigen presenting cells (HAPCs) in the thymus microenvironment provide essential signals to self-reactive thymocytes that induce either negative selection or generation of regulatory T cells (Treg), both of which are required to establish and maintain central tolerance throughout life. HAPCs and TECs are comprised of multiple subsets that play distinct and overlapping roles in central tolerance. Changes that occur in the composition and function of TEC and HAPC subsets across the lifespan have potential consequences for central tolerance. In keeping with this possibility, there are age-associated changes in the cellular composition and function of T cells and Treg. This review summarizes changes in T cell and Treg function during the perinatal to adult transition and in the course of normal aging, and relates these changes to age-associated alterations in thymic HAPC and TEC subsets.
Assuntos
Envelhecimento/imunologia , Tolerância Central , Timo/imunologia , Fatores Etários , Células Apresentadoras de Antígenos/imunologia , Células Epiteliais/imunologia , Humanos , Linfócitos T Reguladores/imunologiaRESUMO
Human age-related thymus involution is characterized by loss of developing thymocytes and the thymic epithelial network that supports them, with replacement by adipose tissue. The mechanisms that drive these changes are difficult to study in vivo due to constant trafficking to and from the thymus. We hypothesized that the loss of thymocytes that occurs during human thymic organ cultures could model some aspects of thymus involution and begin to identify mechanisms that drive age-related changes in the thymic microenvironment. Potential mechanistically important candidate molecules were initially identified by screening conditioned media from human thymus organ cultures using antibody microarrays. These candidates were further validated using cultured tissue extracts and conditioned media. Results were compared with gene expression studies from a panel of well-characterized (non-cultured) human thymus tissues from human donors aged 5 days to 78 years. L-selectin released into conditioned media was identified as a biomarker for the content of viable thymocytes within the cultured thymus. Levels of the chemokines CCL21 and CXCL12, likely produced by surviving thymic epithelial cells, increased markedly in conditioned media as thymocytes were lost during culture. Native non-cultured thymus from adults older than 18 years also showed a strong trend toward increased CCL21 expression, in conjunction with significant decreases in thymocyte-related mRNAs compared with thymus from subjects younger than 18 years. Together, these findings demonstrate that use of postnatal human thymus organ cultures can model some aspects of human age-related thymic involution.
Assuntos
Tecido Adiposo , Timócitos , Biomarcadores , Expressão Gênica , Humanos , TimoRESUMO
Hair loss (alopecia) can result from a variety of metabolic, endocrine, immunologic, and environmental causes. This investigation was undertaken to determine the mechanisms underlying the sporadic development of alopecia in litters from C57BL/6 interleukin-10-deficient (Il10(-/-)) mice. All pups in affected litters demonstrated alopecia by postnatal days 17-19, with hair loss from their trunks but not from their head, base of tail, or feet. Histopathology revealed distorted hair follicles containing broken hair shafts and prominent dermal infiltrates containing increased numbers of activated mast cells. Hair re-growth began soon after weaning, suggesting that the alopecia was triggered by factors transmitted during lactation. Milk from Il10(-/-) dams induced macrophage secretion of pro-inflammatory cytokines in vitro regardless of whether or not their pups developed alopecia. Feeding dams a diet containing 3-6 ppm iron increased the percentage of litters with alopecia to 100% for pups with mast cells, with 0% alopecia in mast cell-deficient pups. When dams were fed a diet containing 131 ppm iron, significantly lower haemoglobin and hematocrit values were observed in pups from litters with alopecia (71%; 5 of 7 litters) compared to litters without alopecia. Genetic or pharmacologic inhibition of c-kit that resulted in depletion of mast cells in pups prevented hair loss in at-risk litters. These studies demonstrate that maternal iron-restricted diets enhance the incidence of alopecia in IL-10-deficient mouse pups and suggest mast cells as potential effector cells. Further studies are indicated to further explore the mechanisms involved and to determine how mast cells may contribute to alopecia in humans.
Assuntos
Alopecia/etiologia , Interleucina-10/deficiência , Deficiências de Ferro , Proteínas Proto-Oncogênicas c-kit/genética , Alopecia/genética , Alopecia/patologia , Anemia/complicações , Anemia/patologia , Animais , Animais Lactentes , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Degranulação Celular , Linhagem Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Dieta , Feminino , Folículo Piloso/patologia , Interleucina-10/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Leite/imunologia , Gravidez , Proteínas Proto-Oncogênicas c-kit/imunologia , Pele/patologiaRESUMO
Patients with inflammatory bowel disease (IBD) have an increased risk of colorectal cancer, particularly in ulcerative colitis (UC) when the majority of colon epithelial cells may be exposed to inflammation-associated mutagenesis. In addition to mutagenesis generated by oxidative stress, inflammation can induce activation-induced cytidine deaminase (Aicda), a mutator enzyme in the APOBEC family, within colon epithelial cells. This study tested the hypothesis that deletion of the Aicda gene could protect against the development of inflammation-associated colorectal cancers, using a model of UC-like colitis in "T/I" mice deficient in TNF and IL10. Results showed that T/I mice that were additionally Aicda-deficient ("TIA" mice) spontaneously developed moderate to severe UC-like colitis soon after weaning, with histologic features and colon inflammation severity scores similar those in T/I mice. Although the mean survival of TIA mice was decreased compared to T/I mice, multivariable analysis that adjusted for age when neoplasia was ascertained showed a decreased numbers of neoplastic colorectal lesions in TIA mice, with a trend toward decreased incidence of neoplasia. Aicda deficiency increased serum IL1α and slightly decreased IL12p40 and M-CSF, as compared with T/I mice, and led to undetectable levels of IgA, IgG1, IgG2a, IgG2b, and IgG3. Taken together, these studies show that Aicda deficiency can decrease the number of neoplastic lesions but is not sufficient to prevent the risk of inflammation-associated colorectal neoplasia in the setting of severe UC-like inflammation. The TIA model may also be useful for assessing the roles of antibody class-switch recombination deficiency and somatic hypermutation on regulation of microbiota and inflammation in the small intestine and colon, as well as the pathogenesis of colitis associated with hyper-IgM syndrome in humans. Further studies will be required to determine the mechanisms that drive early mortality in TIA mice.
Assuntos
Colite Ulcerativa/genética , Neoplasias Colorretais/genética , Citidina Desaminase/genética , Animais , Colite Ulcerativa/complicações , Colite Ulcerativa/patologia , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/etiologia , Citidina Desaminase/deficiência , Feminino , Deleção de Genes , Imunoglobulinas/sangue , Interleucina-1/sangue , Interleucina-10/genética , Interleucina-12/sangue , Fator Estimulador de Colônias de Macrófagos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/genéticaRESUMO
The maintenance and propagation of complex mixtures of cells in vitro in the form of native organs or engineered organoids has contributed to understanding mechanisms of cell and organ development and function which can be translated into therapeutic benefits. For example, allogeneic cultured postnatal human thymus tissue has been shown to support production of naïve recipient T cells when transplanted into patients with complete DiGeorge anomaly and other genetic defects that result in congenital lack of a thymus. Patients receiving such transplants typically exhibit reversal of their immunodeficiency and normalization of their peripheral blood T cell receptor V-beta repertoire, with long-term survival. This study was designed to assess the histopathologic changes that occur in postnatal human thymus slices when cultured according to protocols used for transplanted tissues. Results showed that as thymic organ cultures progressed from days 0 through 21, slices developed increasing amounts of necrosis, increasing condensation of thymic epithelium, and decreasing numbers of residual T cells. The architecture of the thymic epithelial network remained generally well-preserved throughout the 21 days of culture, with focal expression of cytokeratin 14, a putative biomarker of thymic epithelial cells with long-term organ-repopulating potential. All organ slices derived from the same donor thymus closely resembled one another, with minor differences in size, shape, and relative content of cortex versus medulla. Similarly, slices derived from different donors showed similar histopathologic characteristics when examined at the same culture time point. Taken together, these results demonstrate that diagnostic criteria based on structural features of the tissue identifiable via hematoxylin and eosin staining and cytokeratin immunohistochemistry can be used to evaluate the quality of slices transplanted into patients with congenital athymia.
Assuntos
Timo/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Hospedeiro Imunocomprometido , Lactente , Queratina-14/metabolismo , Técnicas de Cultura de Órgãos , Linfócitos T/citologia , Linfócitos T/patologia , Timo/metabolismo , Fatores de TempoRESUMO
Autoimmune thyroid disease occurs in some complete DiGeorge anomaly patients after thymus transplantation. This study was designed to assess the effect of culture of thymus tissue on the expression of genes involved in the development of autoimmunity. The expression of autoimmune regulator (AIRE), thyroglobulin (TG), thyroid peroxidase (TPO), and cytokeratin RNAs was examined in thymocytes and thymus tissue on the day of thymus harvest and after 14 and 21 days of culture. Immunohistochemistry was used to evaluate the cytokeratin expression in the thymus tissue. AIRE, TG, TPO, and cytokeratin mRNAs were found in harvest-day, 14-day and 21-day cultured tissues. Levels of AIRE, TG, and cytokeratin mRNAs were mostly higher after culture compared to expression on the harvest day, likely secondary to thymocyte depletion.
Assuntos
Expressão Gênica , Linfócitos T/metabolismo , Timo/metabolismo , Timo/transplante , Animais , Meios de Cultura/farmacologia , Humanos , Iodeto Peroxidase/genética , Queratinas/genética , Técnicas de Cultura de Órgãos/normas , Linfócitos T/efeitos dos fármacos , Timo/efeitos dos fármacos , Tireoglobulina/genética , Fatores de Transcrição/genética , Proteína AIRERESUMO
Infection of mouse colonies with Helicobacter spp. has become an increasing concern for the research community. Although Helicobacter infection may cause clinical disease, investigators may be unaware that their laboratory mice are infected because the pathology of Helicobacter species is host-dependent and may not be recognized clinically. The effects of Helicobacter infections are not limited to the gastrointestinal system and can affect reproduction, the development of cancers in gastrointestinal organs and remote organs such as the breast, responses to vaccines, and other areas of research. The data we present in this review show clearly that unintentional Helicobacter infection has the potential to significantly interfere with the reliability of research studies based on murine models. Therefore, frequent screening of rodent research colonies for Helicobacter spp. and the eradication of these pathogens should be key goals of the research community.
Assuntos
Criação de Animais Domésticos/normas , Animais de Laboratório/microbiologia , Infecções por Helicobacter/veterinária , Helicobacter/fisiologia , Doenças dos Roedores/microbiologia , Roedores/microbiologia , Criação de Animais Domésticos/métodos , Animais , Antibacterianos/uso terapêutico , Cricetinae , Modelos Animais de Doenças , Helicobacter/efeitos dos fármacos , Helicobacter/isolamento & purificação , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/prevenção & controle , Humanos , Programas de Rastreamento/veterinária , Camundongos , Murinae , Ratos , Reprodutibilidade dos Testes , Doenças dos Roedores/diagnóstico , Doenças dos Roedores/prevenção & controleRESUMO
While exposure to radiation can be lifesaving in certain settings, it can also potentially result in long-lasting adverse effects, particularly to hematopoietic and immune cells. This study investigated hematopoietic recovery and immune function in rhesus macaques Cross-sectionally (at a single time point) 2 to 5 years after exposure to a single large dose (6.5 to 8.4 Gray) of total body radiation (TBI) derived from linear accelerator-derived photons (2 MeV, 80 cGy/minute) or Cobalt 60-derived gamma irradiation (60 cGy/min). Hematopoietic recovery was assessed through measurement of complete blood counts, lymphocyte subpopulation analysis, and thymus function assessment. Capacity to mount specific antibody responses against rabies, Streptococcus pneumoniae, and tetanus antigens was determined 2 years after TBI. Irradiated macaques showed increased white blood cells, decreased platelets, and decreased frequencies of peripheral blood T cells. Effects of prior radiation on production and export of new T cells by the thymus was dependent on age at the time of analysis, with evidence of interaction with radiation dose for CD8+ T cells. Irradiated and control animals mounted similar mean antibody responses to proteins from tetanus and rabies and to 10 of 11 serotype-specific pneumococcal polysaccharides. However, irradiated animals uniformly failed to make antibodies against polysaccharides from serotype 5 pneumococci, in contrast to the robust responses of non-irradiated controls. Trends toward decreased serum levels of anti-tetanus IgM and slower peak antibody responses to rabies were also observed. Taken together, these data show that dose-related changes in peripheral blood cells and immune responses to both novel and recall antigens can be detected 2 to 5 years after exposure to whole body radiation. Longer term follow-up data on this cohort and independent validation will be helpful to determine whether these changes persist or whether additional changes become evident with increasing time since radiation, particularly as animals begin to develop aging-related changes in immune function.
Assuntos
Raios gama/efeitos adversos , Sistema Hematopoético/efeitos da radiação , Imunidade/efeitos da radiação , Irradiação Corporal Total/efeitos adversos , Adulto , Animais , Formação de Anticorpos/efeitos da radiação , Contagem de Células Sanguíneas , Relação Dose-Resposta à Radiação , Hematopoese/efeitos da radiação , Humanos , Subpopulações de Linfócitos/efeitos da radiação , Macaca mulatta , Masculino , Lesões Experimentais por Radiação/etiologia , Linfócitos T/efeitos da radiação , Timo/efeitos da radiaçãoRESUMO
Bromelain, a mixture of proteases derived from pineapple stem, has been reported to have therapeutic benefits in a variety of inflammatory diseases, including murine inflammatory bowel disease. The purpose of this work was to understand potential mechanisms for this anti-inflammatory activity. Exposure to bromelain in vitro has been shown to remove a number of cell surface molecules that are vital to leukocyte trafficking, including CD128a/CXCR1 and CD128b/CXCR2 that serve as receptors for the neutrophil chemoattractant IL-8 and its murine homologues. We hypothesized that specific proteolytic removal of CD128 molecules by bromelain would inhibit neutrophil migration to IL-8 and thus decrease acute responses to inflammatory stimuli. Using an in vitro chemotaxis assay, we demonstrated a 40% reduction in migration of bromelain- vs. sham-treated human neutrophils in response to rhIL-8. Migration to the bacterial peptide analog fMLP was unaffected, indicating that bromelain does not induce a global defect in leukocyte migration. In vivo bromelain treatment generated a 50-85% reduction in neutrophil migration in 3 different murine models of leukocyte migration into the inflamed peritoneal cavity. Intravital microscopy demonstrated that although in vivo bromelain treatment transiently decreased leukocyte rolling, its primary long-term effect was abrogation of firm adhesion of leukocytes to blood vessels at the site of inflammation. These changes in adhesion were correlated with rapid re-expression of the bromelain-sensitive CD62L/L-selectin molecules that mediate rolling following in vivo bromelain treatment and minimal re-expression of CD128 over the time period studied. Taken together, these studies demonstrate that bromelain can effectively decrease neutrophil migration to sites of acute inflammation and support the specific removal of the CD128 chemokine receptor as a potential mechanism of action.
Assuntos
Bromelaínas/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Inflamação/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
Infection with Helicobacter species is endemic in many animal facilities and may alter the penetrance of inflammatory bowel disease (IBD) phenotypes. However, little is known about the relative pathogenicity of H. typhlonius, H. rodentium, and combined infection in IBD models. We infected adult and neonatal IL10-/- mice with H. typhlonius, H. rodentium, or both bacteria. The severity of IBD and incidence of inflammation-associated colonic neoplasia were assessed in the presence and absence of antiHelicobacter therapy. Infected IL10-/- mice developed IBD with severity of noninfected (minimal to no inflammation) < H. rodentium < H. typhlonius Assuntos
Neoplasias do Colo/etiologia
, Infecções por Helicobacter/complicações
, Helicobacter/patogenicidade
, Doenças Inflamatórias Intestinais/etiologia
, Interleucina-10/deficiência
, Animais
, Animais Recém-Nascidos
, Antibacterianos/uso terapêutico
, Neoplasias do Colo/imunologia
, Modelos Animais de Doenças
, Feminino
, Helicobacter/classificação
, Infecções por Helicobacter/tratamento farmacológico
, Infecções por Helicobacter/imunologia
, Infecções por Helicobacter/patologia
, Doenças Inflamatórias Intestinais/tratamento farmacológico
, Doenças Inflamatórias Intestinais/imunologia
, Doenças Inflamatórias Intestinais/patologia
, Interleucina-10/genética
, Masculino
, Camundongos
, Camundongos Endogâmicos C57BL
, Camundongos Knockout
, Especificidade da Espécie
RESUMO
Infections with a variety of Helicobacter species have been documented in rodent research facilities, with variable effects on rodent health. Helicobacter typhlonius has been reported to cause enteric disease in immunodeficient and IL10(-/-) mice, whereas H. rodentium has only been reported to cause disease in immunodeficient mice coinfected with other Helicobacter species. The effect of Helicobacter infections on murine reproduction has not been well studied. The reproductive performance of C57BL/6 IL10(-/-) female mice intentionally infected with H. typhlonius, H. rodentium, or both was compared with that of age-matched uninfected controls or similarly infected mice that received antihelicobacter therapy. The presence of Helicobacter organisms in stool and relevant tissues was detected by PCR assays. Helicobacter infection of IL10(-/-) female mice markedly decreased pregnancy rates and pup survival. The number of pups surviving to weaning was greatest in noninfected mice and decreased for H. rodentium > H. typhlonius >> H. rodentium and H. typhlonius coinfected mice. Helicobacter organisms were detected by semiquantitative real-time PCR in the reproductive organs of a subset of infected mice. Treatment of infected mice with a 4-drug regimen consisting of amoxicillin, clarithromycin, metronidazole, and omeprazole increased pregnancy rates, and pup survival and dam fecundity improved. We conclude that infection with H. typhlonius, H. rodentium, or both decreased the reproductive performance of IL10(-/-) mice. In addition, antihelicobacter therapy improved fecundity and enhanced pup survival.
Assuntos
Infecções por Helicobacter/fisiopatologia , Helicobacter/isolamento & purificação , Interleucina-10/metabolismo , Reprodução , Doenças dos Roedores/fisiopatologia , Animais , Antibacterianos/uso terapêutico , DNA Bacteriano/isolamento & purificação , Feminino , Fertilidade , Helicobacter/genética , Helicobacter/patogenicidade , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/transmissão , Infecções por Helicobacter/veterinária , Transmissão Vertical de Doenças Infecciosas , Interleucina-10/deficiência , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovário/microbiologia , Ovário/fisiopatologia , Gravidez , Reprodução/efeitos dos fármacos , Doenças dos Roedores/tratamento farmacológico , Doenças dos Roedores/microbiologia , Útero/microbiologia , Útero/fisiopatologiaRESUMO
Resource limitation is a fundamental factor governing the composition and function of ecological communities. However, the role of resource supply in structuring the intestinal microbiome has not been established and represents a challenge for mammals that rely on microbial symbionts for digestion: too little supply might starve the microbiome while too much might starve the host. We present evidence that microbiota occupy a habitat that is limited in total nitrogen supply within the large intestines of 30 mammal species. Lowering dietary protein levels in mice reduced their faecal concentrations of bacteria. A gradient of stoichiometry along the length of the gut was consistent with the hypothesis that intestinal nitrogen limitation results from host absorption of dietary nutrients. Nitrogen availability is also likely to be shaped by host-microbe interactions: levels of host-secreted nitrogen were altered in germ-free mice and when bacterial loads were reduced via experimental antibiotic treatment. Single-cell spectrometry revealed that members of the phylum Bacteroidetes consumed nitrogen in the large intestine more readily than other commensal taxa did. Our findings support a model where nitrogen limitation arises from preferential host use of dietary nutrients. We speculate that this resource limitation could enable hosts to regulate microbial communities in the large intestine. Commensal microbiota may have adapted to nitrogen-limited settings, suggesting one reason why excess dietary protein has been associated with degraded gut-microbial ecosystems.