RESUMEN
Health monitoring of laboratory rodents not only improves animal health but also enhances the validity of animal experiments. In particular, infections of laboratory animals with murine parvoviruses influence biomedical research data. Despite strict barrier housing, prevalence remains high in animal facilities, leading to increased risk of parvovirus introduction after the import of contaminated mice. Unfortunately, hygienic rederivation can be challenging, since gametes often contain residual virus material. Consequently, the process has to be closely monitored with highly sensitive diagnostic methods to verify parvovirus decontamination of the rederived progeny. However, diagnostic sensitivity of traditional methods is often low and requires testing of large animal cohorts. Therefore, we aimed to develop a powerful quantitative real-time polymerase chain reaction (qPCR) assay for the fast and reliable detection of murine parvoviruses in different sample materials. We validated the assay within an infection experiment and systematically analysed various animal-derived and environmental sample materials. We further developed a strategic risk assessment procedure for parvovirus monitoring after embryo transfer. Our novel qPCR assay reliably detected parvovirus DNA in a broad variety of sample materials, with environmental samples dominating in the acute phase of infection, whereas animal-derived samples were more suitable to detect low virus loads in the chronic phase. Here, the assay served as a highly sensitive screening method for parvovirus contamination in mouse colonies, requiring significantly lower sample sizes than traditional methods like conventional PCR and serology. Thus, the use of our novel qPCR assay substantially improves parvovirus diagnostics, enhancing research validity according to the 6Rs.
Asunto(s)
Infecciones por Parvoviridae , Parvovirus , Enfermedades de los Roedores , Animales , Ratones , Infecciones por Parvoviridae/diagnóstico , Parvovirus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Medición de Riesgo , Enfermedades de los Roedores/diagnósticoRESUMEN
Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.
Asunto(s)
Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Microbiota/inmunología , Inmunidad Adaptativa/inmunología , Inmunidad Adaptativa/fisiología , Animales , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/microbiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microbiota/fisiología , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal/inmunologíaRESUMEN
For a wide range of biomedical approaches, an accurate estimate of the age of embryos or pups is important. Overnight mating is the method that is mostly used to establish timed pregnancies. The oestrus cycle in mice repeats every four to five days. So, not all females will get pregnant because they are not in oestrus. Therefore, the aim of this study was to analyse whether polygamous mating could increase the rate of timed pregnancies per breeding cage and female. We compared overnight timed mating regimes with up to four females per male, using C57BL/6 and BALB/c mice as well as F1 hybrids of these two strains. The number of vaginal plugs, number of females that gave birth and weaned litter (including size and weaning weight) were recorded. Our results showed that the plug and pregnancy rate decreased, but the productivity per breeding cage increased for polygamous mating regimes. The proportion of females with vaginal plugs and females that gave birth was significantly higher in monogamous mating. The proportion of plugged females that gave birth, as well as litter size and weaning weight, were not influenced by the mating regime. After analysing 513 breeding cages with a total of 1090 females, we found that polygamous mating with up to three females per male can increase the number of timed pregnancies. However, in the mating regime with more than three females, the rate of timed pregnancy as well as number of pups per female declined.
RESUMEN
Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced (p < 0.001) in KO skin explants compared to WT control skin but did not differ (p = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group (p = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.
Asunto(s)
Colágeno/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Piel/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Dipéptidos/farmacología , Masculino , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones , Proteolisis , Piel/efectos de los fármacosRESUMEN
Embryo transfer of pre-implantation embryos to surrogate dams is a key technique for the hygienic sanitation of strains, cryopreservation, in vitro fertilization, genetic modification and engineering. However, the effects of several parameters, such as the number of transferred embryos, on the success of embryo transfer are not well studied. In this retrospective study, we reanalysed 1320 embryo transfers of two-cell embryos originating from genetically altered donors, which were performed under routine conditions in our facility over a period of 5 years. Of them, 453 embryo transfers were done with freshly collected embryos and 867 transfers were performed with cryopreserved embryos. Despite the fact that the genetic background of the embryo donors was quite heterogeneous, we found that the transfer of ≥ 21 embryos reduced the success of embryo transfers for freshly collected embryos in correlation with the number of pregnancies and born pups, whereas this was not the case for transfer in the cryopreservation group. Most pregnancies were achieved after embryo transfer of 10-20 freshly collected embryos (90.4%), which dropped to 37.5% if more embryos were transferred. The highest pregnancy rates in the cryopreservation group were achieved if 15-17 embryos were transferred (62.9%). Despite the fact that the precise substrains were only rarely defined, we confirmed that beside the number of transferred embryos, the genetic background of the donors had an influence on the success of embryo transfer. Significantly more embryos in a C57BL/6 background developed to term than embryos on a BALB/c background.
Asunto(s)
Criopreservación/métodos , Transferencia de Embrión/métodos , Embrión de Mamíferos/fisiología , Animales , Desarrollo Embrionario , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Embarazo , Estudios RetrospectivosRESUMEN
The laboratory mouse is the most used animal model in biomedical research. Several artificial reproductive techniques, such as revitalization of cryopreserved strains, rederivation after hygienic contaminations and the production of transgenic mouse models, require the transfer of preimplantation embryos to surrogate mothers. Pseudopregnancy is essential in recipient females and is induced by mating with sterile males. Commonly, surgically vasectomized males are used for this purpose. As an alternative, genetically modified mouse strains have been identified, in which homozygous infertile males are sexually active. Here, we investigated the suitability of genetically infertile Gapdhstm1Dao males under routine laboratory conditions with respect to plug rates, pregnancy rates and frequency of born offspring after embryo transfer. Our results showed no significant differences for these aspects between Gapdhstm1Dao and vasectomized CD2F1 males. In addition, we evaluated the efforts to obtain a defined number of sterile males either by breeding of sterile mutants or surgical vasectomy, and addressed the impact of both options on animal welfare. In conclusion, infertile males of the Gapdhstm1Dao line are a reliable alternative to vasectomized males for the induction of pseudopregnancy, and can contribute to the refinement of the procedure by avoiding surgical interventions.
Asunto(s)
Transferencia de Embrión/veterinaria , Infertilidad Masculina/veterinaria , Ratones Noqueados , Modelos Animales , Seudoembarazo/veterinaria , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Embarazo , Índice de Embarazo , VasectomíaRESUMEN
Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-ß and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.
RESUMEN
The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1ß gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60-80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.
Asunto(s)
Antiinflamatorios/farmacología , Factores Inmunológicos/farmacología , Elastasa de Leucocito/metabolismo , Enfisema Pulmonar/tratamiento farmacológico , alfa 1-Antitripsina/farmacología , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/inmunología , Adulto , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/deficiencia , Lipopolisacáridos/toxicidad , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Enfisema Pulmonar/genética , Enfisema Pulmonar/inmunología , Proteínas Recombinantes/farmacología , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/inmunologíaRESUMEN
Helicobacter pylori maintains colonization in its human host using a limited set of taxis sensors. TlpD is a proposed energy taxis sensor of H. pylori and dominant under environmental conditions of low bacterial energy yield. We studied the impact of H. pylori TlpD on colonization in vivo using a gerbil infection model which closely mimics the gastric physiology of humans. A gerbil-adapted H. pylori strain, HP87 P7, showed energy-dependent behavior, while its isogenic tlpD mutant lost it. A TlpD-complemented strain regained the wild-type phenotype. Infection of gerbils with the complemented strain demonstrated that TlpD is important for persistent infection in the antrum and corpus and suggested a role of TlpD in horizontal navigation and persistent corpus colonization. As a part of the full characterization of the model and to gain insight into the genetic basis of H. pylori adaptation to the gerbil, we determined the complete genome sequences of the gerbil-adapted strain HP87 P7, two HP87 P7 tlpD mutants before and after gerbil passage, and the original human isolate, HP87. The integrity of the genome, including that of a functional cag pathogenicity island, was maintained after gerbil adaptation. Genetic and phenotypic differences between the strains were observed. Major differences between the gerbil-adapted strain and the human isolate emerged, including evidence of recent recombination. Passage of the tlpD mutant through the gerbil selected for gain-of-function variation in a fucosyltransferase gene, futC (HP0093). In conclusion, a gerbil-adapted H. pylori strain with a stable genome has helped to establish that TlpD has important functions for persistent colonization in the stomach.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Helicobacter pylori/metabolismo , Adaptación Fisiológica , Animales , Proteínas Bacterianas/genética , Portador Sano , Genoma Bacteriano , Gerbillinae , Helicobacter pylori/genética , Movimiento , MutaciónRESUMEN
The immunoglobulin-like glycoprotein CD226 represents a receptor activating cytotoxic T and NK cells taking part in tumour surveillance. In addition, CD226 is involved in the differentiation of naïve CD4(+) T cells into effector cells. CD155 that is widely over-expressed on tumour cells, was identified as a counter-receptor of CD226 rendering many cancer cells sensitive to NK driven elimination. However, CD155 was also assigned a role in the establishment of follicular helper T cells in the small intestine and the final maturation of CD8 positive thymocytes. Here we show that mice lacking CD226 are distinguished by virtually identical phenotypes as already reported for CD155 deficient mice: a paucity of follicular helper T cells in Peyer's Patches and of terminally matured CD8 T cells in thymus. Moreover, like CD155, CD226 is involved in negative selection of CD8 thymocytes. These observations establish a firm link between the functions of CD155 and CD226 in several T cell differentiation steps.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD8-positivos/inmunología , Ganglios Linfáticos Agregados/inmunología , Receptores Virales/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Diferenciación Celular , Movimiento Celular/inmunología , Células Cultivadas , Selección Clonal Mediada por Antígenos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Unión Proteica/inmunología , Timocitos/inmunología , Timo/inmunologíaRESUMEN
BACKGROUND: Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain. RESULTS: After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαß+ as well as CD8+/CD3+/TCRαß+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat. CONCLUSION: The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.
Asunto(s)
Endonucleasas/metabolismo , Marcación de Gen , Proteínas de Homeodominio/genética , Dedos de Zinc , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Embrión de Mamíferos/metabolismo , Citometría de Flujo , Mutación del Sistema de Lectura/genética , Genotipo , Células Germinativas , Proteínas de Homeodominio/química , Humanos , Depleción Linfocítica , Tejido Linfoide/patología , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Ratas MutantesRESUMEN
BACKGROUND: Cell lines represent a key tool in cancer research allowing the generation of neoplasias which resemble initial tumours in in-vivo animal models. The characterisation of early tumour development is of major interest in order to evaluate the efficacy of therapeutic agents. Magnetic resonance imaging (MRI) based in-vivo characterisation allows visualisation and characterisation of tumour development in early stages prior to manual palpation. Contrast agents for MRI such as superparamagnetic iron oxide nanoparticles (SPIOs) and manganese chloride (MnCl2) represent powerful tools for the in-vivo characterisation of early stage tumours. In this experimental study, we labelled prostate cancer cells with MnCl2 or SPIOs in vitro and used 1 T MRI for tracing labelled cells in-vitro and 7 T MRI for tracking in an in-vivo animal model. METHODS: Labelling of prostate cancer cells CT1258 was established in-vitro with MnCl2 and SPIOs. In-vitro detection of labelled cells in an agar phantom was carried out through 1 T MRI while in-vivo detection was performed using 7 T MRI after subcutaneous (s.c.) injection of labelled cells into NOD-Scid mice (n = 20). The animals were scanned in regular intervals until euthanization. The respective tumour volumes were analysed and corresponding tumour masses were subjected to histologic examination. RESULTS: MnCl2in-vitro labelling resulted in no significant metabolic effects on proliferation and cell vitality. In-vitro detection-limit accounted 105 cells for MnCl2 as well as for SPIOs labelling. In-vivo 7 T MRI scans allowed detection of 103 and 104 cells. In-vivo MnCl2 labelled cells were detectable from days 4-16 while SPIO labelling allowed detection until 4 days after s.c. injection. MnCl2 labelled cells were highly tumourigenic in NOD-Scid mice and the tumour volume development was characterised in a time dependent manner. The amount of injected cells correlated with tumour size development and disease progression. Histological analysis of the induced tumour masses demonstrated characteristic morphologies of prostate adenocarcinoma. CONCLUSIONS: To the best of our knowledge, this is the first study reporting direct in-vitro MnCl2 labelling and 7 T based in-vivo MRI tracing of cancer cells in a model of prostate cancer. MnCl2 labelling was found to be suitable for in-vivo tracing allowing long detection periods. The labelled cells kept their highly tumourigenic potential in-vivo. Tumour volume development was visualised prior to manual palpation allowing tumour characterisation in early stages of the disease.
Asunto(s)
Cloruros , Medios de Contraste , Compuestos Férricos , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita , Compuestos de Manganeso , Neoplasias de la Próstata/diagnóstico , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de la Próstata/patologíaRESUMEN
A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LODâ=â3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1(ter)/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders.
Asunto(s)
Infertilidad/genética , Neoplasias Ováricas/genética , Mutación Puntual , Proteínas de Unión al ARN/genética , Teratoma/genética , Neoplasias Testiculares/genética , Animales , Femenino , Genotipo , Células Germinativas/metabolismo , Células Germinativas/patología , Gónadas/metabolismo , Gónadas/patología , Infertilidad/patología , Masculino , Neoplasias Ováricas/patología , Ratas , Teratoma/patología , Neoplasias Testiculares/patologíaRESUMEN
Pluripotent cells referred to as embryonic germ cells (EGCs) can be derived from the embryonic precursors of the mature gametes: the primordial germ cells (PGCs). A homozygous mutation (ter) of the dead-end homolog 1 gene (Dnd1) in the rat causes gonadal teratocarcinogenesis and sterility due to neoplastic transformation and loss of germ cells. We mated heterozygous ter/+ WKY-Dnd1(ter)/Ztm rats and were able to cultivate the first genital ridge-derived EGCs of the rat embryo at day 14.5 post coitum (pc). Genotyping revealed that ten EGC lines were Dnd1 deficient, while only one wild type cell line had survived in culture. This suggests that the inactivation of the putative tumor suppressor gene Dnd1 facilitates the immortalization of late EGCs in vitro. Injection of the wild type EGCs into blastocysts resulted in the first germ-line competent chimeras. These new pluripotent rat EGCs offer an innovative approach for studies on germ cell tumor development as well as a new tool for genetic manipulations in rats.
Asunto(s)
Células Germinativas/citología , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Animales , Células Cultivadas , Femenino , Genes Supresores de Tumor , Células Germinativas/metabolismo , Masculino , Proteínas de Unión al ARN/genética , Ratas , Ratas Endogámicas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
OBJECTIVE: The pleiotropic cytokine interleukin (IL)-6 seems to play a pivotal role in sepsis, but contradictory findings in animal models impede a rationale for therapies directed against IL-6. IL-6 signals by two mechanisms via the ubiquitous transmembrane glycoprotein 130 (gp130): "classic" signaling using membrane-bound IL-6 receptor (IL-6R) and trans-signaling using soluble IL-6R (sIL-6R). Trans-signaling is selectively inhibited by soluble gp130 (sgp130). The aim of this study was to systematically compare complete blockade of IL-6 signaling (using a neutralizing anti-IL-6 antibody) and selective blockade of IL-6 trans-signaling (using a fusion protein of sgp130 and the crystallizable fragment of immunoglobulin G1, sgp130Fc) in a standardized cecal ligation and puncture (CLP) sepsis model. DESIGN: Animal study. SETTING: Animal laboratory. SUBJECTS: C57BL/6J mice. INTERVENTIONS: We performed a 96-hr dose-response study and a 24-hr study to investigate short-term mechanisms. In the 96-hr study, CLP was performed in 120 randomized mice (20 mice received vehicle, 10 mice per dose group). Mice were treated with equimolar doses of sgp130Fc (0.01/0.1/1/10 mg/kg) or anti-IL-6 (0.008/0.08/0.8/8 mg/kg) 24 hrs before CLP. Two additional groups received 0.5 mg/kg sgp130Fc 24 hrs before or 1 mg/kg sgp130Fc 24 hrs after CLP. Survival and activity scores were obtained daily until 96 hrs after CLP. In the 24-hr study, mice were randomized into four groups with 10 animals each (sham/vehicle, CLP/vehicle, CLP/anti-IL-6 [0.8 mg/kg], and CLP/sgp130Fc [1 mg/kg]) and killed after 24 hrs. MEASUREMENTS AND MAIN RESULTS: In contrast to anti-IL-6, pretreatment with sgp130Fc significantly and dose-dependently increased survival from 45% to 100%. In addition, 1 mg/kg sgp130Fc administered 24 hrs after CLP increased survival from 45% to 80%. Mechanistically, sgp130Fc efficacy was reflected by complete prevention of epithelial cell apoptosis in the jejunum after CLP, which was not achieved with anti-IL-6. CONCLUSION: Selective inhibition of IL-6 trans-signaling by sgp130Fc has considerable potential for the treatment of sepsis and related disorders.
Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Interleucina-6/antagonistas & inhibidores , Interleucina-6/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Sepsis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Sepsis/etiología , Sepsis/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The number of rat strains increased considerably in the last decade and will increase continuously during the next years. This requires enough space for maintaining vital strains and techniques for cryobanking, which can be applied not only in specialised rat resource centres but also in regular animal houses. Here we describe an easy and fast method for the cryopreservation and transplantation of frozen-thawed ovaries of the rat. With dimethyl sulfoxide as cryoprotectant rat ovaries can be stored at -196 degrees C for unlimited time. For revitalisation thawed ovaries have to be orthotopically transplanted into appropriate ovarectomised recipients. Reestablishment of the reproductive cycle in the recipients can be confirmed by vaginal cytology shortly after transplantation. The recipients are able to produce 2-3 litters after mating with males of an appropriate strain. Cyropreservation of ovaries thus can be considered a reliable method to preserve scientifically and economically important stocks and strains of rats that are currently not required.
Asunto(s)
Criopreservación/métodos , Criopreservación/veterinaria , Ovario/trasplante , Animales , Criopreservación/economía , Crioprotectores , Dimetilsulfóxido , Femenino , Masculino , Ovario/citología , Ratas , Vagina/citologíaRESUMEN
It is still a major problem to achieve successful therapy in polymicrobial sepsis. Stimulation of the innate immune system via Toll-like receptors (TLRs) 2 and 6 had beneficial effects on chronic inflammatory disorders and a severe peritonitis model when administered 4 days before induction. In the present study, the hypothesis whether the TLR-2 and TLR-6 pathway can also be used as a therapeutic agent parallel to sepsis induction and several hours after the induction was tested. Therefore, the TLR-2 and TLR-6 agonist macrophage-activating lipopeptide 2 (MALP-2) was applied simultaneous to cecal ligation and puncture-sepsis induction and 6 h thereafter. Vehicle-treated animals served as controls. Survival, activity, cytokine levels at different time points, and pulmonary neutrophil infiltration were determined. Improved survival was found after both MALP-2 treatments in comparison with untreated controls. The treatment resulted in reduced monocyte chemotactic protein 1 levels in the plasma; furthermore, pulmonary infiltration by neutrophils was decreased. These results demonstrate a beneficial effect of MALP-2 as a therapeutic agent in polymicrobial sepsis in the cecal ligation and puncture mouse model.
Asunto(s)
Lipopéptidos/uso terapéutico , Sepsis/prevención & control , Animales , Ciego , Quimiocinas/inmunología , Citocinas/sangre , Ligadura , Pulmón/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Peritonitis/inmunología , Sepsis/inmunología , Sepsis/mortalidad , Receptor Toll-Like 2RESUMEN
Solitary intestinal lymphoid tissue (SILT) comprises a spectrum of phenotypically diverse lymphoid aggregates interspersed throughout the small intestinal mucosa. Manifestations of SILT range from tiny lymphoid aggregates almost void of mature lymphocytes to large structures dominated by B cells. Large SILT phenotypically resemble a single Peyer's patch follicle, suggesting that SILT might contribute to intestinal humoral immune responses. In this study, we track the fate of individual SILT in vivo over time and analyze SILT formation and function in chemokine receptor CXCR5-deficient mice. We show that, in analogy to Peyer's patches, formation of SILT is invariantly determined during ontogeny and depends on CXCR5. Young CXCR5-deficient mice completely lack SILT, suggesting that CXCR5 is essential for SILT formation during regular postnatal development. However, microbiota and other external stimuli can induce the formation of aberrant SILT distinguished by impaired development of B cell follicles in CXCR5-deficient mice. Small intestinal transplantation and bone marrow transplantation reveal that defect follicle formation is due to impaired B cell homing. Moreover, oral immunization with cholera toxin or infection with noninvasive Salmonella fail to induce efficient humoral immune responses in CXCR5-deficient mice. Bone marrow transplantation of CXCR5-deficient recipients with wild-type bone marrow rescued B cell follicle formation in SILT but failed to restore full humoral immune responses. These results reveal an essential role of CXCR5 in Peyer's patch and SILT development and function and indicate that SILT do not fully compensate for the lack of Peyer's patches in T cell-dependent humoral immune responses.
Asunto(s)
Linfocitos B/inmunología , Quimiotaxis de Leucocito/inmunología , Inmunoglobulina A/biosíntesis , Mucosa Intestinal/inmunología , Tejido Linfoide/inmunología , Receptores CXCR5/fisiología , Administración Oral , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Trasplante de Médula Ósea/inmunología , Toxina del Cólera/uso terapéutico , Mucosa Intestinal/metabolismo , Tejido Linfoide/crecimiento & desarrollo , Tejido Linfoide/metabolismo , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores CXCR5/deficiencia , Receptores CXCR5/genética , Salmonelosis Animal/inmunología , Salmonelosis Animal/patología , Salmonella typhimurium/inmunología , Linfocitos T/inmunologíaRESUMEN
The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl(-) and lactate outward currents with an EC(50) of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.
Asunto(s)
Eritrocitos/metabolismo , Eritrocitos/parasitología , Activación del Canal Iónico , Malaria/metabolismo , Plasmodium falciparum/metabolismo , Albúmina Sérica/metabolismo , Animales , Caseínas/farmacología , Canales de Cloruro/metabolismo , Eritrocitos/efectos de los fármacos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Ácido Láctico/metabolismo , Ovalbúmina/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Albúmina Sérica Bovina/farmacologíaRESUMEN
BACKGROUND: Prostate cancer is a frequent finding in man. In dogs, malignant disease of the prostate is also of clinical relevance, although it is a less common diagnosis. Even though there are numerous differences in origin and development of the disease, man and dog share many similarities in the pathological presentation. For this reason, the dog might be a useful animal model for prostate malignancies in man.Although prostate cancer is of great importance in veterinary medicine as well as in comparative medicine, there are only few cell lines available. Thus, it was the aim of the present study to determine whether the formerly established prostate carcinoma cell line CT1258 is a suitable tool for in vivo testing, and to distinguish the growth pattern of the induced tumours. METHODS: For characterisation of the in vivo behaviour of the in vitro established canine prostate carcinoma cell line CT1258, cells were inoculated in 19 NOD.CB17-PrkdcScid/J (in the following: NOD-Scid) mice, either subcutaneously or intraperitoneally. After sacrifice, the obtained specimens were examined histologically and compared to the pattern of the original tumour in the donor. Cytogenetic investigation was performed. RESULTS: The cell line CT 1258 not only showed to be highly tumourigenic after subcutaneous as well as intraperitoneal inoculation, but also mimicked the behaviour of the original tumour. CONCLUSION: Tumours induced by inoculation of the cell line CT1258 resemble the situation in naturally occurring prostate carcinoma in the dog, and thus could be used as in vivo model for future studies.