RESUMEN
We investigated whether decellularized pig tracheas could regenerate in vivo, without being recellularized before transplantation, using the own body as bioreactor. Decellularized pig tracheal scaffolds were intraoperative conditioned with mononuclear cells and growth and differentiation factors. During the postoperative period, the in situ regeneration was boosted by administering bioactive molecules to promote peripheral mobilization and differentiation of stem/progenitor cells and ultimately the regenerative process. Results revealed, after 2 weeks, a nearly normal trachea, with respiratory epithelium and a double-banded cartilage but without any mechanical differences compared to the native tissue. The growth factor administration resulted in a mobilization of progenitor and stem cells into the peripheral circulation and in an up-regulation of anti-apoptotic genes. Isolated stem/progenitor cells could be differentiated in vitro into several cell types, proving their multipotency. We provide evidence that the own body can be used as bioreactor to promote in vivo tissue engineering replacement. Moreover, we demonstrated the beneficial effect of additional pharmaceutical intervention for an improved engraftment of the transplant.
Asunto(s)
Ingeniería de Tejidos/métodos , Tráquea/fisiología , Animales , Fenómenos Biomecánicos , Separación Celular , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Inflamación/patología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sus scrofa , Tráquea/patologíaRESUMEN
The adipocyte-derived hormone leptin is a critical regulator of many physiological functions, ranging from satiety to immunity. Surprisingly, very little is known about the transcriptional pathways that regulate adipocyte-specific expression of leptin. Here, we report studies in which we pursued a strategy integrating BAC transgenic reporter mice, reporter assays, and chromatin state mapping to locate an adipocyte-specific cis-element upstream of the leptin (LEP) gene in human fat cells. Quantitative proteomics with affinity enrichment of protein-DNA complexes identified the transcription factor FOS-like antigen 2 (FOSL2) as binding specifically to the identified region, a result that was confirmed by ChIP. Knockdown of FOSL2 in human adipocytes decreased LEP expression, and overexpression of Fosl2 increased Lep expression in mouse adipocytes. Moreover, the elevated LEP expression observed in obesity correlated well with increased FOSL2 levels in mice and humans, and adipocyte-specific genetic deletion of Fosl2 in mice reduced Lep expression. Taken together, these data identify FOSL2 as a critical regulator of leptin expression in adipocytes.
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Adipocitos/citología , Antígeno 2 Relacionado con Fos/metabolismo , Leptina/biosíntesis , Animales , Diferenciación Celular , Cromatina/química , Mapeo Cromosómico , Femenino , Perfilación de la Expresión Génica , Genes Reporteros , Genómica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Ratones Transgénicos , Modelos Genéticos , Proteómica/métodosRESUMEN
Epidemiological studies have indicated that obesity is associated with a higher risk for certain cancers caused by elevated levels of adipocyte-derived hormones. Leptin, one such hormone produced by adipocytes, is a major regulator of metabolism and has also been shown to modulate immunity. However, its role in regulating human natural killer (NK) cell functions is largely unknown. Here, we show that the leptin receptor (Ob-R) is expressed on 5% of NK cells isolated from blood donors, as measured with flow cytometry, and expression of the signal-transducing long form of the leptin receptor Ob-Rb was confirmed with quantitative PCR. The Ob-R+ subpopulation displayed a lower expression of CD16, a cell surface receptor mediating antibody-dependent activation. Short-term stimulation with leptin increased IFNγ secretion, CD69 activation marker expression, and cytotoxic lysis of tumor cells; this was mediated by an improved conjugate forming between NK cells and tumor cells as well as higher expression of tumor necrosis factor-related apoptosis-inducing ligand. On the contrary, long-term incubation with leptin significantly impaired these NK cell immune functions and decreased cell proliferation. In addition, phosphorylation of Jak-2 after leptin stimulation was reduced in peripheral mononuclear blood cells from obese humans compared with normal-weight controls. NK cells represent an immune cell population that is crucial for an effective antitumor response. Here, we show that long-term exposure to leptin, similarly to the situation in obese individuals with elevated serum leptin levels, significantly impairs integral parts of NK cell immune functions, possibly linking leptin to increased cancer susceptibility in obesity.
Asunto(s)
Citofagocitosis , Células Asesinas Naturales/inmunología , Leptina/metabolismo , Obesidad/inmunología , Receptores de Leptina/metabolismo , Células 3T3-L1 , Adipocitos/inmunología , Adipocitos/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Interferón gamma/sangre , Interferón gamma/metabolismo , Células Asesinas Naturales/metabolismo , Leptina/sangre , Leptina/genética , Ratones , Neoplasias/complicaciones , Neoplasias/inmunología , Obesidad/sangre , Obesidad/complicaciones , Obesidad/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de IgG/metabolismo , Receptores de Leptina/química , Receptores de Leptina/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
BACKGROUND: Thermal sensitivity in uraemia is decreased. Non-selective synthetic nitric oxide synthase (NOS) inhibitors significantly attenuate thermal hyperalgesia in preclinical models. The aim of our study was to evaluate the effect of experimental uraemia, which is associated with an increase of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), on thermal sensitivity in rats. Furthermore, we intended to study the effect of chronic ADMA infusion alone on thermal sensitivity. METHODS: Male Sprague-Dawley rats (n = 54), 10 weeks old, weight 370-430 g, were randomly assigned to three groups receiving either (i) isotonic saline or (ii) ADMA via osmotic mini pumps or (iii) underwent 5/6 nephrectomy (Nx). After 14 days, 50% of all animals from all groups underwent thermal sensitivity testing and terminal blood draw. After 28 days, the remaining animals underwent the same procedures. Thermal sensitivity examination was performed by the hot-plate test, measuring time from heat exposition to first paw licking or jumping of the animal. RESULTS: While the median [interquartile range] latency time between heat exposition to first paw licking or jumping of the animal in the NaCl infusion group remained unchanged between Day 14 (8.4 [6.75-11.50] s) and Day 28 (7.35 [6.10-7.90] s) both, ADMA infusion and 5/6 nephrectomy tended to increase the thermal pain threshold at Day 14 (9.25 [6.55-12.18] s) and (9.50 [5.8 ± 11.0] s), respectively, compared to NaCl on Day 14 (8.4 [6.75-11.50] s). This difference became statistical significant at Day 28 where the median latency time in the ADMA group (13.10 [11.85-15.95] s) and in the 5/6 Nx group (13.50 [10.85-17.55] s) were significantly higher than in the NaCl group (7.35 [6.10-7.90] s). CONCLUSIONS: Induction of progressive renal failure in rats by 5/6 nephrectomy, which is accompanied by a marked increase of the serum levels of the endogenous NOS inhibitor ADMA, leads to a significantly increased heat pain threshold at 28 days. The sole infusion of ADMA into healthy rats leads to the same increase in heat pain threshold.
Asunto(s)
Arginina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/fisiopatología , Umbral del Dolor/efectos de los fármacos , Dolor/tratamiento farmacológico , Dolor/etiología , Animales , Arginina/farmacología , Calor , Masculino , Nefrectomía , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
Even now, sepsis remains a major problem in modern clinical medicine, leading to systemic inflammatory response including altered leukocyte subset distribution and increased cytokine release. As immune cells are known to express NPY receptors, we investigated the effects of a specific NPY Y(2) receptor agonist (NPY(13-36)) and/or the corresponding Y(2) receptor antagonist BIIE0246 treatment on blood (by FACS analyses) and tissue (by immunohistochemistry) leukocyte subsets as well as on levels of IL-4, IL-6, IL-10, TNF-α, INF-γ (by Cytometric Bead Array) in healthy and acutely endotoxemic rats. Results show a significant decrease in blood monocytes after LPS challenge in endotoxemic control animals (by 93%), in endotoxemic NPY(13-36) treated animals (by 83%) and in endotoxemic BIIE0246 treated animals (by 88%) as compared to the corresponding healthy controls. Endotoxemic control animals showed a significant increase of TNF-α (by 98%) as compared to the healthy control group. A treatment with NPY(13-36) significantly stabilized TNF-α level in endotoxemic animals. This study indicates distinct subset- and cytokine-specific in vivo effects induced by an NPY Y(2) receptor specific treatment after a short-term LPS challenge.
Asunto(s)
Arginina/análogos & derivados , Benzazepinas/farmacología , Citocinas/inmunología , Endotoxemia/inmunología , Leucocitos/inmunología , Neuropéptido Y/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Neuropéptido Y/antagonistas & inhibidores , Animales , Arginina/farmacología , Células Cultivadas , Leucocitos/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas LewRESUMEN
To analyse the hypothesis as to whether there is a functional relationship between human cationic amino acid transporters (hCATs, y(+) transporter, the main transporter of L-arginine and L-lysine) and human ß-defensin (important components of immune function) production on the ocular surface, arginase and nitrate monoxide synthase (NOS), enzymes that compete for L-arginine, were inhibited by norNOHA (N(omega)-hydroxy-nor-L-arginine) and/or L-NAME (NG-nitro-L-arginine methyl ester) in cultured human corneal epithelial cells. In addition, the transport activity of hCAT proteins was inhibited or activated through α-tocopherol or PMA (phorbol myristate acetate), respectively. Concentrations of the human inducible ß-defensins (hBD) 2 and 3 were determined by ELISA experiments. The basic expression of hBD3 in non-stimulated HCE cells significantly exceeded that of hBD2. Both ß-defensins also differed as to how readily their excretion could be stimulated. HBD2 excretion rate was 3.5 time more by L-NAME, whereas norNOHA had no effect. In contrast, hBD3 excretion was increased by norNOHA by a factor of 1.5 but L-NAME alone had no effect. The excretion of both ß-defensins was increased 3- and 6-fold by combined administration of L-NAME, norNOHA and interleukin (IL)-1ß. Administration of α-tocopherol increased hBD2 excretion twofold. No effect was observed for hBD3. With PMA, on the other hand, a reduction in secretion for both ß-defensins was observed. These in vitro findings provide evidence of a functional association between CAT proteins and ß-defensins 2 and 3 opening up a new field of research with pharmacological perspectives for treatment of inflammatory diseases such as keratitis or dry eye disease.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Células Epiteliales/metabolismo , Epitelio Corneal/metabolismo , beta-Defensinas/metabolismo , Línea Celular , HumanosRESUMEN
OBJECTIVE: In the industrialized world, obesity is an increasing socioeconomic health problem. Obese subjects have a higher risk of developing several types of cancer. NK cells are an integral component of the innate immune system, able to destruct tumor cells. The adipokine leptin plays a crucial role in the development of obesity and its related diseases. Peripheral leptin signaling is modulated by the liver. METHODS: The aim of this study was to evaluate the number of hepatic NK cells (CD56+) and the number of leptin-receptor positive (Ob-R+) cells in the livers of five normal-weight and five obese humans. Livers were removed during autopsy and accurately defined sections were stained immunohistochemically and CD56+, Ob-R+, and double-positive cells were quantified. RESULTS: Results revealed a dramatic reduction of NK cells and Ob-R-expressing NK cells in the livers of obese individuals. CONCLUSIONS: The present study demonstrates, for the first time, body-weight-dependent numbers of hepatic NK cells. This supports the hypothesis of obesity-associated alterations of immune cell numbers in different human organs.
Asunto(s)
Células Asesinas Naturales/metabolismo , Obesidad/metabolismo , Obesidad/patología , Receptores de Leptina/biosíntesis , Adolescente , Adulto , Anciano , Recuento de Células , Humanos , Inmunohistoquímica , Células Asesinas Naturales/patología , Hígado/metabolismo , Hígado/patología , Persona de Mediana Edad , Adulto JovenRESUMEN
The LEW/Ztm-ci2 rat is an animal model for syndromal deafness that arose from a spontaneous mutation. Homozygous animals show locomotor abnormalities like lateralized circling behavior. Additionally, an impaired vision can be observed in some animals through behavioral studies. Syndromal deafness as well as retinal degeneration are features of the Usher syndrome in humans. In the present study, the mutation was identified as a base substitution (T->C) in exon 56 of Myo15, leading to an amino acid exchange from leucine (Leu) to proline (Pro) within the carboxy-terminal MyTH4 domain in the proteins' tail region. Myo15 mRNA was expressed in the retina as demonstrated for the first time with the help of in-situ hybridization and PCR. To characterize the visual phenotype, rats were examined by scotopic and photopic electroretinography and, additionally, histological analyses of the retinas were conducted. The complete loss of sight was detected along with a severe degeneration of photoreceptor cells. Interestingly, the manifestation of the disease does not solely depend on the mutation, but also on environmental factors. Since the LEW/Ztm-ci2 rat features the entire range of symptoms of the human Usher syndrome we think that this strain is an appropriate model for this disease. Our findings display that mutations in binding domains of myosin XV do not only cause non-syndromic hearing loss but can also lead to syndromic disorders including retinal dysfunction.
Asunto(s)
Mutación/genética , Miosinas/genética , Síndromes de Usher/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Electrorretinografía , Ambiente , Exones/genética , Femenino , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Hibridación in Situ , Luz , Masculino , Datos de Secuencia Molecular , Miosinas/química , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Mutantes , Degeneración Retiniana/complicaciones , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiología , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndromes de Usher/complicaciones , Síndromes de Usher/fisiopatología , Visión Ocular/efectos de la radiaciónRESUMEN
The underlying mechanisms controlling food intake and satiety are thoroughly controlled, but seem to be insufficient under conditions of almost unlimited food supply. Hence, overweight and obesity are serious problems especially in industrialized countries. To assess the possible influence of CD26, exerting a dipeptidyl peptidase activity (DPP4) cleaving several energy homeostasis-relevant peptides, we investigated wild type and DPP4-deficient dark agouti rats in a model of diet-induced obesity and found a reduced weight gain in DPP4-deficient rats. When investigating the specific increase of whole body fat volume by MRI to assess the distribution pattern (subcutaneous vs. intraabdominal), there was an altered ratio under dietary conditions only in DPP4-deficient rats, which was due to lower intraabdominal fat amounts. Furthermore, we investigated the number of cells immunopositive for the leptin receptor (OB-R), the orexigenic leptin antagonist neuropeptide Y (NPY), as well as of the NPY receptors Y1, Y2, and Y5 within hypothalamic nuclei. Independent from the body weight, higher levels of NPY and all receptors were expressed in DPP4-deficent rats. Under obese conditions, hypothalamic Y2-levels were reduced in both strains. Concerning NPY and Y1, there were partly oppositional effects, with reduced hypothalamic Y1 levels only in wild types, and increased NPY levels only in DPP4-deficient rats. These effects might be responsible for unaltered food intake in DPP4-deficent rats compared to wild types, despite reduced weight gain. However, since the food intake remained unaffected, these effects suggest that DPP4 exerts its effects on intraabdominal fat also via peripheral actions.
Asunto(s)
Dipeptidil Peptidasa 4/deficiencia , Grasa Intraabdominal/fisiología , Neuropéptido Y/metabolismo , Obesidad/enzimología , Aumento de Peso/genética , Animales , Regulación del Apetito/genética , Regulación del Apetito/fisiología , Grasas de la Dieta/metabolismo , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/fisiología , Modelos Animales de Enfermedad , Ingestión de Energía/genética , Técnicas de Inactivación de Genes , Masculino , Obesidad/genética , Obesidad/metabolismo , Ratas , Ratas Endogámicas , Receptores de Neuropéptido Y/metabolismo , Aumento de Peso/fisiologíaAsunto(s)
Síndromes de Inmunodeficiencia/epidemiología , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/epidemiología , Gripe Humana/inmunología , Obesidad/complicaciones , Obesidad/inmunología , Adipocitos/fisiología , Adipoquinas/metabolismo , Animales , Enfermedad Crítica , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Ratas , Transducción de SeñalRESUMEN
The adipocyte-derived catabolic protein leptin alters cell-mediated immunity and cytokine crosstalk. This may provide new insights into the altered immune response, seen in obese individuals. Therefore, we determined the tissue distribution of immune cells in diet-induced obese (dio) and normal weight F344 rats challenged with MADB106 tumor cells or leptin. Immune cell distribution in blood (by FACS analysis) and tissues (NK cells in spleen and liver, immunohistologically) as well as pro-inflammatory cytokines (IL-6, TNF-α; by flow cytometry) were investigated in 28 normal weight and 28 dio rats (n = 4-6/group). Pro-inflammatory cytokines were increased 3-fold for IL-6 and 7-fold for TNF-α in obese animals. Higher numbers of blood monocytes and NK cells were found in obese as compared to normal weight animals. In dio rats challenged with leptin and MADB106 tumor cells, monocyte numbers were decreased as compared to the obese control animals. Immunohistochemistry revealed an altered NK cell distribution in a compartment-, treatment-, and bodyweight-specific manner. In conclusion, our data reveal a distinct distribution pattern of monocytes and NK cells in dio rats as compared to normal weight littermates and an additional modulatory effect of a leptin- and MADB106 tumor cell challenge.
Asunto(s)
Interleucina-6/inmunología , Células Asesinas Naturales/inmunología , Leptina/inmunología , Monocitos/inmunología , Obesidad Abdominal/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adenocarcinoma/inmunología , Animales , Peso Corporal/inmunología , Femenino , Citometría de Flujo , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-6/sangre , Leptina/farmacología , Masculino , Neoplasias Mamarias Animales/inmunología , Trasplante de Neoplasias , Obesidad Abdominal/metabolismo , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/farmacología , Microambiente Tumoral/inmunología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
In obesity, the regulatory effects of leptin, a primarily adipocyte-derived hormone, are severely disturbed affecting the control of energy homeostasis and immune functions. In addition, recent studies indicate that specific immune cells can affect glucose and lipid metabolism of liver. However, the contribution of body weight and immune cells, such as Natural Killer (NK) cells, to the regulation of the leptin-receptor expression remains elusive. Therefore, we investigated the expression of the signal-transducing long form of the leptin receptor (Ob-Rb) in diet-induced obesity and after adoptive cross-over NK cell transfer between normal weight and obese male F344 rats. Expression of Ob-Rb was significantly increased in liver in diet-induced obese rats as compared to normal weight littermates. Similarly, the expression of Ob-Rb was higher in liver of obese animals that received NK cells from either obese or normal weight donors as compared to normal weight animals that received NK cells from normal weight donors. Interestingly, normal weight animals that were transferred with NK cells from obese donors also showed a tendency towards a higher Ob-Rb expression. In contrast to the findings in liver, the expression of Ob-Rb in spleen or lung remained unaffected by changes in body weight or cross-over NK cell transfer. Our results suggest that the expression of Ob-Rb mRNA in liver, but not in spleen or lung, is dependent on the body weight but can also be influenced by NK cells, thereby indicating a bidirectional cross-talk between the metabolic and the immune system.
Asunto(s)
Células Asesinas Naturales/fisiología , Hígado/fisiología , Obesidad/fisiopatología , Receptores de Leptina/fisiología , Traslado Adoptivo , Animales , Peso Corporal/fisiología , Citometría de Flujo , Regulación de la Expresión Génica/fisiología , Pulmón/fisiología , Masculino , ARN Mensajero/genética , Ratas , Ratas Endogámicas F344 , Receptores de Leptina/biosíntesis , Receptores de Leptina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Bazo/fisiologíaRESUMEN
Breast cancer continues to be a major cause of cancer deaths in women. Estrogen, which is also produced by the adipose tissue, is held responsible for the elevated risk of breast cancer in obese women. However, the adipose tissue secrets hormones and adipokines such as leptin and IGF-I and these substances could also contribute to an increased breast cancer risk for obese women. In this study, the impact of obesity on cell proliferation was investigated. The carcinogen 7, 12, dimethylbenz[a]anthracene (DMBA) was administered to normal weight and diet-induced obese female Sprague-Dawley rats. Cell proliferation was evaluated by immunohistological staining of BrdU-incorporation. In the mammary glands and inguinal lymphatic nodes of the obese rats, cell proliferation was significantly increased, indicating a significant influence of obesity on breast cancer. Effects of leptin, estrogen, and IGF-I on the proliferation of MCF-7 cells in vitro were assessed using an MTT assay. Cell culture experiments demonstrated a mitogenic role of these three mediators on cell proliferation. Our data demonstrate a stimulative effect of substances produced by the adipose tissue on breast cancer. Body weight specific cell proliferation suggests that obesity-related adipokines and mediators enhance cell proliferation and increase the risk for breast cancer.
Asunto(s)
Proliferación Celular , Estradiol/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Leptina/metabolismo , Neoplasias Mamarias Experimentales/patología , Obesidad/patología , 9,10-Dimetil-1,2-benzantraceno , Adenocarcinoma/patología , Análisis de Varianza , Animales , Peso Corporal , Neoplasias de la Mama/patología , Carcinógenos , Línea Celular Tumoral , Grasas de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Estradiol/administración & dosificación , Femenino , Ingle , Humanos , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Leptina/administración & dosificación , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática , Neoplasias Mamarias Experimentales/inducido químicamente , Obesidad/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
In diet-induced obese rats, leptin-mediated natural killer (NK) cell activation has been demonstrated to be impaired by abrogated intracellular JAK2-STAT3 signaling. The contribution of the obese microenvironment to this NK cell dysfunction and its reversibility remains elusive. In this study, the functions of NK cells from diet-induced obese rats after adoptive transfer into lean littermates were investigated using in vivo and in vitro approaches. Endogenous NK cells of normal-weight and diet-induced obese F344 rats were depleted in vivo. Then, NK cells from either normal-weight or obese donors were transferred. The numbers of peripheral blood NK cells were analyzed by fluorescence-activated cell sorting (FACS) and the distribution pattern of NK cells in lung and spleen by immunohistochemistry. Ob-R expression was evaluated by immunohistology and activation of intracellular target proteins of Ob-R by immunoblotting. The numbers of NK cells in blood and lung were significantly higher in obese animals compared to lean ones after transfer of NK cells from obese F344 rats. This was correlated with increased postreceptor signaling (JAK-2p, PKBpT308, ERK-2p) without altered Ob-R expression in those NK cells transferred to lean (ob-->nw) vs. obese (ob-->ob) animals. These results show for the first time that the altered phenotype of NK cells from obese rats can be normalized by generation of a physiological (metabolic) environment of lean rats.
Asunto(s)
Células Asesinas Naturales/inmunología , Obesidad/inmunología , Traslado Adoptivo , Animales , Citometría de Flujo , Immunoblotting , Inmunohistoquímica , Janus Quinasa 2/sangre , Células Asesinas Naturales/patología , Hígado/inmunología , Hígado/patología , Pulmón/inmunología , Pulmón/patología , Masculino , Obesidad/sangre , Obesidad/patología , Fenotipo , Distribución Aleatoria , Ratas , Ratas Endogámicas F344 , Receptores de Leptina/inmunología , Factor de Transcripción STAT3/sangre , Transducción de Señal , Organismos Libres de Patógenos Específicos , Bazo/inmunología , Bazo/patologíaRESUMEN
BACKGROUND: Treatment of diabetes type 2 using chronic pharmacological inhibition of dipeptidyl peptidase 4 (DP4) still requires an in-depth analysis of models for chronic DP4 deficiency, because adverse reactions induced by some DP4 inhibitors have been described. METHODS: In the present study, a novel congenic rat model of DP4 deficiency on a "DP4-high" DA rat genetic background was generated (DA.F344-Dpp4(m)/ SvH rats) and comprehensively phenotyped. RESULTS: Similar to chronic pharmacological inhibition of DP4, DP4 deficient rats exhibited a phenotype involving reduced diet-induced body weight gain and improved glucose tolerance associated with increased levels of glucagon-like peptide-1 (GLP-1) and bound leptin as well as decreased aminotransferases and triglycerides. Additionally, DA.F344-Dpp4(m)/SvH rats showed anxiolytic-like and reduced stress-like responses, a phenomenon presently not targeted by DP4 inhibitors. However, several immune alterations, such as differential leukocyte subset composition at baseline, blunted natural killer cell and T-cell functions, and altered cytokine levels were observed. CONCLUSIONS: While this animal model confirms a critical role of DP4 in GLP-1-dependent glucose regulation, genetically induced chronic DP4 deficiency apparently also affects stress-regulatory and immuneregulatory systems, indicating that the use of chronic DP4 inhibitors might have the potential to interfere with central nervous system and immune functions in vivo.
Asunto(s)
Dipeptidil Peptidasa 4/inmunología , Dipeptidil Peptidasa 4/metabolismo , Animales , Animales Congénicos , Peso Corporal , Citocinas/inmunología , Dipeptidil Peptidasa 4/deficiencia , Dipeptidil Peptidasa 4/genética , Modelos Animales de Enfermedad , Femenino , Péptido 1 Similar al Glucagón/inmunología , Células Asesinas Naturales/inmunología , Leptina/inmunología , Fenotipo , Ratas , Ratas Endogámicas F344 , Linfocitos T/inmunología , Transaminasas/metabolismo , Triglicéridos/metabolismoRESUMEN
Leptin, a hormone mainly generated by adipocytes, acts centrally in the hypothalamus to regulate body weight and energy expenditure. However, there is strong evidence that leptin is also involved in cell-mediated immunity and cytokine crosstalk. In the present study the effects of diet-induced obesity and central and peripheral leptin treatment on leukocyte subsets and cytokine production was investigated. Leptin was injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) in male endotoxaemic or vehicle-treated healthy LEW-rats. Numbers of blood leukocyte subsets were analysed by FACS and cytokines (TNF-alpha and IL-6) by ELISA. Results showed that peripheral rather than central leptin treatment was able to significantly increase numbers of granulocytes, NK cells and monocytes. Three-colour staining revealed that the increase of ED9(+) monocytes was most likely due to the mobilization of two distinct monocyte subsets, predominantly ED9(+)CD4(-)NKR-P1A(+) and ED9(+)CD4(+)NKR-P1A(+). ELISA analysis revealed significantly elevated TNF-alpha levels in obese animals compared to their lean littermates, while IL-6 failed to show notable changes. In conclusion, the data of the present study revealed that leptin application induces a nutrition- and application-site dependent increase of circulating NK cells, granulocytes and specific monocyte subsets.
Asunto(s)
Granulocitos/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Leptina/administración & dosificación , Monocitos/efectos de los fármacos , Obesidad/sangre , Delgadez/sangre , Animales , Modelos Animales de Enfermedad , Endotoxemia/sangre , Endotoxemia/inmunología , Ingestión de Energía , Granulocitos/inmunología , Inyecciones Intravenosas , Inyecciones Intraventriculares , Células Asesinas Naturales/inmunología , Leptina/fisiología , Recuento de Leucocitos , Masculino , Monocitos/clasificación , Monocitos/inmunología , Obesidad/etiología , Obesidad/inmunología , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/administración & dosificación , Delgadez/inmunología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Leptin acts not only as an anorexigenic hormone but also regulates cell-mediated immunity via leptin receptors (Ob-R) expressed on T and B lymphocytes. However, the impact of leptin on natural killer (NK) cells is currently elusive. We evaluated leptin effects on NK cells in relation to the body weight in rats using in vivo and in vitro approaches. Leptin was injected iv in male lean and diet-induced obese Lewis and F344 rats. NK cell numbers were analyzed in blood and spleen by fluorescence activated cell sorting and immunohistochemistry, and the activity of NK cells was measured by chromium release assay. Ob-R expression was investigated by confocal laser scanning and quantitative RT-PCR. To compare leptin-dependent intracellular signaling under basal and leptin- and tumor cell (MADB106)-stimulated conditions, intracellular target proteins of NK cells were evaluated by Western blotting. Number and distribution pattern of splenic NK cells were significantly different in lean and obese animals. Leptin administration resulted in a 4-fold higher stimulation of the NK activity in lean than obese animals. This was not due to a decreased expression of Ob-R because quantitative RT-PCR revealed significantly higher Ob-Rb mRNA levels in NK cells from obese rats. In contrast, postreceptor signaling is differentially abrogated in obese animals with significantly lower activation of postreceptor signaling components (Janus kinase-2p, protein kinase B pT308, AMPalphapT172) after an in vivo leptin challenge. In conclusion, the results for the first time assign leptin a central role as a modulator of NK cell number and activity only in lean but not obese subjects. The differential role of leptin has important implications for the influence of body weight in the response to systemic inflammations and in the immunological defense of cancer.
Asunto(s)
Janus Quinasa 2/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Leptina/farmacología , Obesidad/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Recuento de Células , Línea Celular Tumoral , Carbohidratos de la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Citometría de Flujo , Immunoblotting , Inyecciones Intravenosas , Células Asesinas Naturales/metabolismo , Leptina/administración & dosificación , Leptina/metabolismo , Masculino , Microscopía Confocal , Obesidad/etiología , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Bazo/citologíaRESUMEN
Obese patients have an increased incidence of systemic infections and higher morbidity and mortality rates than normal weight subjects. Ghrelin is a potent orexigenic signal from the stomach and seems to play a role in the generation and control of immune interactions. To examine a possible benefit of a single ghrelin application on acute endotoxemia, chronic intravenous (i.v.) cannulated lean and diet-induced obese male LEW rats were treated with a bolus injection of either ghrelin (10 nmol/kg) or vehicle, 10 min prior to a challenge with a sublethal bolus of endotoxin (100 microg/kg) or vehicle. Multiple blood samples were taken within a period from 24 h before the experiment up to 24 h after the endotoxin challenge to measure ghrelin and cytokine levels. Additionally, food consumption was recorded and ghrelin expression in fore- and glandular stomach was evaluated immunohistochemically. Despite higher serum ghrelin levels, the food consumption was significantly decreased in obese endotoxemic rats compared to lean littermates after ghrelin treatment. Furthermore we could show an increase of anti-inflammatory IL-10 serum levels after ghrelin treatment of normal weight endotoxemic and an opposite effect in obese animals. As the therapy of disease-associated cachexia and various immunological problems in endotoxemia is still insufficient, peptides such as ghrelin with their modulating abilities for the endocrine and the immune system are of special interest. However, the present study shows that the beneficial effects of ghrelin were attenuated in obese endotoxemic animals. These data further document the necessity to differentiate between normal weight and obese subjects in the attempt to establish ghrelin as a therapeutic target in endotoxemia.
Asunto(s)
Citocinas/inmunología , Ingestión de Alimentos , Endotoxemia , Mucosa Gástrica , Obesidad , Hormonas Peptídicas , Estómago , Animales , Peso Corporal/efectos de los fármacos , Citocinas/sangre , Endotoxemia/complicaciones , Endotoxemia/tratamiento farmacológico , Endotoxemia/inmunología , Mucosa Gástrica/metabolismo , Ghrelina , Inmunohistoquímica , Masculino , Obesidad/complicaciones , Obesidad/inmunología , Hormonas Peptídicas/biosíntesis , Hormonas Peptídicas/sangre , Hormonas Peptídicas/inmunología , Hormonas Peptídicas/uso terapéutico , Ratas , Ratas Endogámicas Lew , Estómago/inmunologíaRESUMEN
PURPOSE: Laparoscopy has been associated with lower inflammatory responses. However, it has been postulated that minilaparotomy, in contrast to full laparotomy, is equally minimally invasive. OBJECTIVE: The aim of this study was to investigate local, systemic, and distant organ immune responses after different surgical approaches to the abdominal cavity, such as minilaparotomy, full laparotomy, and laparoscopy, in a small animal model. METHODS: Male Lewis rats received a permanent central venous catheter and were randomized to 4 groups (n = 6 per group). The animals were subjected to anesthesia alone (control), minilaparotomy (1 cm), full laparotomy (7 cm), or laparoscopy for 60 minutes. Blood was collected via the central venous catheter before as well as 1 hour and 6 hours after the start of intervention. Peritoneal and bronchoalveolar lavages, as well as heart puncture, were performed after 24 hours. RESULTS: All surgical interventions led to a significant migration of polymorphonucleocytes into the abdominal cavity. Full laparotomy resulted in a significant increase in nitric oxide production by peritoneal macrophages as compared with control. Macrophage nitric oxide production after laparoscopy and minilaparotomy was not significantly different. A shift in the expression of OX-6 and CD54 was only detected after full laparotomy. Systemically, O(2)(-) release by circulating mononuclear cells was significantly increased after minilaparotomy and full laparotomy, but not after laparoscopy. The systemic levels of IL6 were significantly accelerated only after full laparotomy, with a maximum after 6 hours. In the lungs, function of alveolar macrophages was not altered in any group. CONCLUSIONS: Any approach to the peritoneal cavity causes local inflammatory responses. Full laparotomy alters peritoneal macrophage functions more pronouncedly than does minilaparotomy or laparoscopy. Systemic inflammatory responses, such as free oxygen radical release, are significantly increased by both minilaparotomy and full laparotomy, whereas laparoscopy preserves systemic immune function. Our results may lead to further preference for the laparoscopic approach over minilaparotomy and full laparotomy.
Asunto(s)
Abdomen/cirugía , Sistema Inmunológico/fisiopatología , Laparoscopía , Laparotomía/métodos , Pulmón/inmunología , Cavidad Peritoneal , Animales , Formación de Anticuerpos , Antígenos de Superficie/metabolismo , Molécula 1 de Adhesión Intercelular , Interleucina-6/sangre , Interleucina-6/metabolismo , Pulmón/metabolismo , Macrófagos Peritoneales/metabolismo , Masculino , Monocitos/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico/sangre , Periodo Posoperatorio , Ratas , Ratas Endogámicas Lew , Superóxidos/metabolismo , Factores de TiempoRESUMEN
Central venous devices are frequently used in children to monitor haemodynamic status, to administer fluids, medication, parenteral nutrition and for blood sampling. Life-threatening complications that may occur on insertion if the central venous catheter (CVC) is misplaced, are cardiac tamponade or a hydro-/haemopericardium. There is still controversy over the optimum catheter tip position in paediatric patients, whether to place the CVC tip in the superior vena cava, outside the pericardial boundaries or in the right atrium. However, the exact location of the pericardium cannot be seen on a normal chest x-ray. The carina is a radiographic marker for CVC placement, suggested on the basis of studies with conserved and fresh adult cadavers. In order to confirm this landmark for children, the present study was performed with 31 fresh cadavers of small children (mean age 12.5+/-3.4 months) that had been selected for autopsy in the Institute of Legal Medicine. Results clearly demonstrate that the carina was 0.5+/-0.04 cm above the pericardial duplication as it transversed the SVC. In no infant cadaver was the carina inferior to the pericardium. Thus, the results are analogous to those in adults and confirm that the carina is a simple anatomical-radiological landmark, superior to the pericardial reflection, that can be used to identify the placement of CVC even in newborn and small children.