RESUMEN
Increased secretion of antimicrobial peptides and cytokines is present in atopic skin. The purpose of this study was to compare the production of ß-defensin (cBD)3-like, cathelicidin (cCath) and cytokines in atopic and healthy canine keratinocytes. Seven atopic house dust mites (HDMs) sensitive and five healthy age-matched beagles were used. Keratinocytes were stimulated for 24 h, and the supernatant was collected. A significantly higher production of cBD3-like was present at baseline in atopic compared with healthy keratinocytes, but cBD3-like did not increase after stimulation. IL-17 and lipopolysaccharide increased cBD3-like in healthy compared with atopic keratinocytes. cCath increased in both groups after stimulation. Atopic keratinocytes exposed to HDM produced more IL-8 and keratinocyte-derived chemokine-like than healthy keratinocytes. Exposure to HDM induced an increased IL-8 in atopic keratinocytes and a decreased IFN-γ in healthy keratinocytes. These results may suggest an over sensitization of atopic keratinocytes and a possible impairment of the cutaneous defense against micro-organisms.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/biosíntesis , Citocinas/biosíntesis , Dermatitis Atópica/veterinaria , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/metabolismo , Queratinocitos/inmunología , Queratinocitos/metabolismo , Animales , Células Cultivadas , Dermatitis Atópica/inmunología , Dermatitis Atópica/metabolismo , Perros , Pyroglyphidae/inmunologíaRESUMEN
BACKGROUND: Antimicrobial peptides (AMPs) are small immunomodulatory peptides produced by epithelial and immune cells. ß-Defensins (BDs) and cathelicidins (Caths) are the most studied AMPs. Recently, increased cutaneous expression of AMPs was reported in atopic humans and in beagles with experimentally induced atopy. HYPOTHESIS/OBJECTIVES: Our goal was to analyse mRNA expression and protein levels of canine (c)BD1-like, cBD2-like/122, cBD3-like, cBD103 and cCath in healthy and naturally affected atopic dogs, with and without active skin infection, along with their distribution in the epidermis using indirect immunofluorescence. ANIMALS: Skin biopsies were taken from 14 healthy and 11 atopic privately owned dogs. METHODS: The mRNA levels of cBD1-like, cBD2-like/122, cBD3-like, cBD103 and cCath were quantified using quantitative real-time PCR. The protein levels of cBD3-like and cCath were analysed by relative competitive inhibition enzyme-linked immunosorbent assay, while the distributions of cBD2-like/122, cBD3-like and cCath were detected by indirect immunofluorescence. RESULTS: Dogs with atopic dermatitis had significantly greater mRNA expression of cBD103 (P = 0.04) than control dogs. Furthermore, atopic skin with active infection had a higher cBD103 mRNA expression (P = 0.01) and a lower cBD1-like mRNA expression (P = 0.04) than atopic skin without infection. No significant differences in protein levels (cBD3-like and cCath) or epidermal distribution of AMPs (cBD2-like/122, cBD3-like and cCath) were seen between healthy and atopic dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Expression of cBD103 mRNA was greater, while expression of cBD1-like mRNA was lower in dogs with atopic dermatitis that had active infections. Work is needed to clarify the biological mechanisms and possible therapeutic options to maintain a healthy canine skin.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Infecciones Bacterianas/veterinaria , Dermatitis Atópica/veterinaria , Enfermedades de los Perros/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/genética , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/patología , Enfermedad Crónica , Dermatitis Atópica/metabolismo , Enfermedades de los Perros/etiología , Perros , Femenino , Regulación de la Expresión Génica , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.
Asunto(s)
Suplementos Dietéticos , Ácidos Docosahexaenoicos/farmacología , Fertilidad/efectos de los fármacos , Linoleoil-CoA Desaturasa/deficiencia , Linoleoil-CoA Desaturasa/genética , Espermatogénesis/efectos de los fármacos , Animales , Ácido Araquidónico/administración & dosificación , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Colestadienos/metabolismo , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/metabolismo , Grasas de la Dieta/farmacología , Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Docosahexaenoicos/metabolismo , Femenino , Flagelos/efectos de los fármacos , Flagelos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Recuento de Espermatozoides , Cabeza del Espermatozoide/efectos de los fármacos , Cabeza del Espermatozoide/metabolismo , Motilidad Espermática/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (-/-), which enabled us to study HUFA deficiency without depleting their precursors. In -/-, no in vivo AA synthesis was detected after administration of [U-(13)C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the -/- developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The -/- also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male -/- lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in -/- declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in -/-. DPAn-6 was absent in -/- brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (-/-) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.
Asunto(s)
Intestinos/patología , Linoleoil-CoA Desaturasa/deficiencia , Linoleoil-CoA Desaturasa/genética , Reproducción/genética , Úlcera Cutánea/genética , Úlcera/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dermatitis/genética , Suplementos Dietéticos , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Hepatomegalia/genética , Infertilidad Masculina/genética , Linoleoil-CoA Desaturasa/metabolismo , Masculino , Ratones , Especificidad de Órganos , Fenotipo , Úlcera Cutánea/etiología , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Esplenomegalia/genética , Úlcera/etiología , Úlcera/metabolismo , Úlcera/patologíaRESUMEN
This report reviews decade two of the lifetime diet restriction study of the dog. Labrador retrievers (n 48) were paired at age 6 weeks by sex and weight within each of seven litters, and assigned randomly within the pair to control-feeding (CF) or 25 % diet restriction (DR). Feeding began at age 8 weeks. The same diet was fed to all dogs; only the quantity differed. Major lifetime observations included 1.8 years longer median lifespan among diet-restricted dogs, with delayed onset of late life diseases, especially osteoarthritis. Long-term DR did not negatively affect skeletal maturation, structure or metabolism. Among all dogs, high static fat mass and declining lean body mass predicted death, most strongly at 1 year prior. Fat mass above 25 % was associated with increasing insulin resistance, which independently predicted lifespan and chronic diseases. Metabolizable energy requirement/lean body mass most accurately explained energy metabolism due to diet restriction; diet-restricted dogs required 17 % less energy to maintain each lean kilogram. Metabonomics-based urine metabolite trajectories reflected DR-related differences, suggesting that signals from gut microbiota may be involved in the DR longevity and health responses. Independent of feeding group, increased hazard of earlier death was associated with lower lymphoproliferative responses to phytohaemagglutinin, concanavalin A, and pokeweed mitogen; lower total lymphocytes, T-cells, CD4 and CD8 cells; lower CD8 percentages and higher B-cell percentages. When diet group was taken into account, PWM responses and cell counts and percentages remained predictive of earlier death.
Asunto(s)
Envejecimiento/fisiología , Restricción Calórica/veterinaria , Perros/fisiología , Longevidad , Tejido Adiposo/metabolismo , Envejecimiento/inmunología , Animales , Antioxidantes/análisis , Linfocitos B/inmunología , Glucemia/metabolismo , Composición Corporal , Relación CD4-CD8 , Metabolismo Energético , Femenino , Insulina/metabolismo , Intestinos/microbiología , Lípidos/sangre , Masculino , Mitógenos/farmacología , Neoplasias/veterinaria , Proteínas/análisis , Reproducción , Linfocitos T/inmunología , TiempoRESUMEN
Caloric restriction (CR) has been shown to retard immunosenescence and to extend median and maximum life span in rodent species. Longitudinal effects of CR on the canine immune system are presented in this report. A group of 48 Labrador Retrievers, divided at weaning into weight- and sex-matched pairs, were maintained on a diet restriction protocol from age 8 weeks until death. Each restricted dog received 75% of the total food consumed by its control-fed pair mate. Immune parameters were monitored from 4 to 13 years. CR retarded age-related declines in both lymphoproliferative responses and absolute numbers of lymphocytes and the T, CD4, and CD8-cell subsets. In females, CR attenuated the age-related increase in T-cell percentages and marginally retarded the age-related increase in memory cell percentages. Age-related changes in B-cell percentages and numbers were augmented by CR. No direct effect of CR on phagocytic activity of PMN, antibody production or NK cell activity, was observed. Lower lymphoproliferative responses, lower numbers of lymphocytes, T, CD4 and CD8 cells, lower CD8 percentages and higher B-cell percentages were all found to be significantly associated with a decreased likelihood of survival in these dogs.
Asunto(s)
Envejecimiento/inmunología , Restricción Calórica/veterinaria , Perros/inmunología , Leucocitos Mononucleares/inmunología , Animales , Anticuerpos/sangre , Femenino , Citometría de Flujo/veterinaria , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/citología , Estudios Longitudinales , Activación de Linfocitos , Recuento de Linfocitos/veterinaria , Subgrupos Linfocitarios/inmunología , Masculino , Mitógenos/inmunología , Fagocitosis/inmunología , Modelos de Riesgos ProporcionalesRESUMEN
Conventional drug development for treatment of cocaine addiction is greatly hindered by the extreme difficulty in designing a selective cocaine antagonist. We employed anti-idiotypic (anti-Id) antibodies to generate cocaine antagonists. The purpose of this study was to investigate the feasibility of this alternative approach. Herein, we describe the molecular cloning, bacterial expression, and functional properties of an anti-Id monoclonal antibody (mAb), designated K2-3f, which possesses an internal image of cocaine within its variable regions. The heavy and light chain variable domains of K2-3f were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and a single chain antibody variable fragment (scFv) was assembled for expression in Escherichia coli. The scFv bound to the human dopamine transporter (hDAT) with moderate affinity (K(a)=5.3 x 10(6) M(-1)) and excellent mimicry of the cocaine molecule completely inhibited cocaine binding at a molar concentration closely resembling in vivo conditions while allowing approximately 90% of equimolar dopamine uptake. Our data suggest that the use of anti-Id antibody as a template for generation of a cocaine antagonist is a promising approach well worth pursuing. If this strategy is successful, it could be applied to potential ligand-receptor interactions in the treatment of other diseases.
Asunto(s)
Anticuerpos Antiidiotipos/genética , Anticuerpos Monoclonales/genética , Cocaína/metabolismo , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso , Secuencia de Aminoácidos , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Afinidad de Anticuerpos , Sitios de Unión de Anticuerpos , Cromatografía de Afinidad , Clonación Molecular , Cocaína/inmunología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Espectrometría de Masas , Proteínas de Transporte de Membrana/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/inmunología , Anticuerpos de Cadena ÚnicaRESUMEN
OBJECTIVE: To evaluate the effects of 25% diet restriction on life span of dogs and on markers of aging. DESIGN: Paired feeding study. ANIMALS: 48 Labrador Retrievers. PROCEDURES: Dogs were paired, and 1 dog in each pair was fed 25% less food than its pair-mate from 8 weeks of age until death. Serum biochemical analyses were performed, body condition was scored, and body composition was measured annually until 12 years of age. Age at onset of chronic disease and median (age when 50% of the dogs were deceased) and maximum (age when 90% of the dogs were deceased) life spans were evaluated. RESULTS: Compared with control dogs, food-restricted dogs weighed less and had lower body fat content and lower serum triglycerides, triiodothyronine, insulin, and glucose concentrations. Median life span was significantly longer for dogs in which food was restricted. The onset of clinical signs of chronic disease generally was delayed for food-restricted dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 25% restriction in food intake increased median life span and delayed the onset of signs of chronic disease in these dogs.
Asunto(s)
Envejecimiento/fisiología , Enfermedades de los Perros/prevención & control , Perros/fisiología , Privación de Alimentos/fisiología , Longevidad , Animales , Composición Corporal/fisiología , Enfermedad Crónica , Femenino , Esperanza de Vida , Masculino , Distribución Aleatoria , Factores de TiempoRESUMEN
Polychlorinated biphenyls (PCBs) are environmental contaminants known to cause adverse health effects to biological systems. Limited data are available on their effects on the immune system of wildlife species. Previously, we found that 4 and 6-week-old white-footed mice (Peromyscus leucopus) born from dams injected with a single dose (300 mg/kg) of Aroclor 1254, had altered immunological, hematological, and biochemical responses. Here, we examined the effect of transplacental, lactational and postnatal exposure to Aroclor 1254, at a concentration similar to that found at contaminated sites, on various physiological parameters of 22-week-old white-footed mice. Liver weight and liver somatic index of PCB treated animals were significantly higher, the combined weights of the adrenal glands were significantly lower and EROD and BROD enzyme activity was significantly higher compared to control values. The number of thymocytes of the treated mice was significantly lower than that of the controls; however, thymocytes of treated mice had a higher proliferative response to the mitogen Con A. These alterations were correlated with the PCBs body burdens. Some toxic effects of chronic exposure to PCBs, at levels comparable to exposure found in contaminated sites in the USA, are still evident in adult P. leucopus.
Asunto(s)
/toxicidad , Inmunidad/efectos de los fármacos , Animales , Carga Corporal (Radioterapia) , Recuento de Células , División Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Dieta , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Celular/efectos de los fármacos , Recuento de Leucocitos , Mitógenos/toxicidad , Tamaño de los Órganos/efectos de los fármacos , Peromyscus , Embarazo , Efectos Tardíos de la Exposición PrenatalRESUMEN
Use of soy-based infant formulas and soy/isoflavone supplements has aroused concern because of potential estrogenic effects of the soy isoflavones genistein and daidzein. Here we show that s.c. genistein injections in ovariectomized adult mice produced dose-responsive decreases in thymic weight of up to 80%. Genistein's thymic effects occurred through both estrogen receptor (ER) and non-ER-mediated mechanisms, as the genistein effects on thymus were only partially blocked by the ER antagonist ICI 182,780. Genistein decreased thymocyte numbers up to 86% and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis. Genistein injection caused decreases in relative percentages of thymic CD4(+)CD8(-) and double-positive CD4(+)CD8(+) thymocytes, providing evidence that genistein may affect early thymocyte maturation and the maturation of the CD4(+)CD8(-) helper T cell lineage. Decreases in the relative percentages of CD4(+)CD8(-) thymocytes were accompanied by decreases in relative percentages of splenic CD4(+)CD8(-) cells and a systemic lymphocytopenia. In addition, genistein produced suppression of humoral immunity. Genistein injected at 8 mg/kg per day produced serum genistein levels comparable to those reported in soy-fed human infants, and this dose caused significant thymic and immune changes in mice. Critically, dietary genistein at concentrations that produced serum genistein levels substantially less than those in soy-fed infants produced marked thymic atrophy. These results raise the possibility that serum genistein concentrations found in soy-fed infants may be capable of producing thymic and immune abnormalities, as suggested by previous reports of immune impairments in soy-fed human infants.