RESUMEN
Aberrant loss of oocytes following cancer treatments or genetic mutations leads to premature ovarian insufficiency (POI) associated with endocrine-related disorders in 1% of women. Therefore, understanding the mechanisms governing oocyte death is crucial for the preservation of female fertility. Here, we report the striking reproductive features of a novel mouse model of POI obtained through oocyte-specific inactivation (ocKO) of Omcg1/Zfp830 encoding a nuclear zinc finger protein involved in pre-mRNA processing. Genetic ablation of OMCG1 in early growing oocytes leads to reduced transcription, accumulation of DNA double-strand breaks and subsequent c-Abl/TAp63-dependent oocyte death, thus uncovering the key role of OMCG1 for oocyte genomic integrity. All adult Omcg1(ocKO) females displayed complete elimination of early growing oocytes and sterility. Unexpectedly, mutant females exhibited a normal onset of puberty and sexual receptivity. Detailed studies of Omcg1(ocKO) ovaries revealed that the ovarian somatic compartment underwent a dramatic structural and functional remodeling. This allowed the cooperation between oocyte-depleted follicles and interstitial tissue to produce estradiol. Moreover, despite early folliculogenesis arrest, mutant mice exhibited sexual cyclicity as shown by cyclical changes in estrogen secretion, vaginal epithelium cytology and genital tract weight. Collectively, our findings demonstrate the key role of Omcg1 for oocyte survival and highlight the contribution of p63 pathway in damaged oocyte elimination in adulthood. Moreover, our findings challenge the prevailing view that sexual cyclicity is tightly dependent upon the pace of folliculogenesis and luteal differentiation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Inestabilidad Genómica/fisiología , Proteínas Nucleares/metabolismo , Oocitos/metabolismo , Ovario/metabolismo , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Muerte Celular , Supervivencia Celular , Estrógenos/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Oocitos/citología , Ovario/citología , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Transactivadores/genéticaRESUMEN
Helicobacter pylori infection is the major cause of gastroduodenal pathologies including gastric cancer. The long persistence of bacteria and the type of immune and inflammatory response determine the clinical issue. In this study, the global gene expression profile after 6 and 12 months of H. pylori infection was investigated in the mouse stomach, using the Affymetrix GeneChip Mouse Expression Array A430. Genes related to the inflammatory and immune responses were focused. Levels of selected transcripts were confirmed by reverse transcription polymerase chain reaction. Twenty- five and nineteen percent of the differentially expressed genes observed at 6 and 12 months post-infection respectively, were related to immune response. They are characterized by an interferon (IFN)gamma-dependent expression associated to a T helper 1 (Th1) polarised response. In-depth analysis revealed that an up-regulation of IL-23p19, took place in the stomach of H. pylori infected-mice. Strong IL-23p19 levels were also confirmed in gastric biopsies from H. pylori-infected patients with chronic gastritis, as compared to healthy subjects. Our microarray analysis revealed also, a high decrease of H+K+-ATPase transcripts in the presence of the H. pylori infection. Association of gastric Th1 immune response with hypochlorhydria through the down-regulation of H+K+-ATPase contributes to the genesis of lesions upon the H. pylori infection. Our data highlight that the up-regulation of IL-23 and of many IFNgamma signature transcripts occur early on during the host response to H. pylori, and suggest that this type of immune response may promote the severity of the induced gastric lesions.
Asunto(s)
Perfilación de la Expresión Génica , Infecciones por Helicobacter/inmunología , Helicobacter pylori , Interferón gamma/fisiología , Interleucina-23/genética , Animales , Mucosa Gástrica/metabolismo , Regulación de la Expresión Génica , ATPasa Intercambiadora de Hidrógeno-Potásio/fisiología , Infecciones por Helicobacter/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Regulación hacia ArribaRESUMEN
We aimed to set up and validate a new in vitro model of placental histocultures, for the evaluation of cytokine and chemokine profiles of the placental environment, over a long culture period. Micro-explant cultures from 6 early and 6 term placentae were set up on collagen sponge gel supports at a liquid/air interface. At various times during culture, we analyzed tissue morphology and cell death by microscopy and quantified beta-hCG production and mRNA levels for beta-hCG and insulin-like 4 (INSL4). Levels of IL-6, LIF, TNF alpha, IL-10, IFN-gamma, IL-16 and RANTES in the medium were measured by ELISA on days 1, 4 and 7 of culture. SDF-1 mRNA expression was determined by real-time PCR at the same time points. Histocultures from early and term placentae remained viable until day 10. High levels of IL-6 and LIF production, low levels of TNF alpha, IL-10 and IFN-gamma production and significant SDF-1 expression were observed. These data indicate that placental histoculture is a suitable and reliable in vitro model for studying the placental environment.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Quimiocinas/metabolismo , Vellosidades Coriónicas/metabolismo , Primer Trimestre del Embarazo , Nacimiento a Término , Adulto , Apoptosis , Supervivencia Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas/análisis , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Vellosidades Coriónicas/anatomía & histología , Vellosidades Coriónicas/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Etiquetado Corte-Fin in Situ , Embarazo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Shigella infections lead to severe inflammation associated with destruction of colonic mucosa. We assessed the effect of in vivo blockade of CD14 on the outcome of experimental Shigella infection in rabbits. A total of 17 rabbits were divided into two groups: 8 received a single i.v. dose of anti-rabbit CD14 monoclonal antibody prior to infection with an invasive Shigella flexneri strain; the remainder served as controls. The anti-CD14-treated rabbits exhibited more severe tissue destruction and a 50-fold increase in bacterial invasion of the intestinal mucosa when compared to controls. Similar numbers of polymorphonuclear leukocytes were recruited to the intestinal mucosa in both groups despite the massive bacterial invasion seen in the CD14-blocked group. No statistically significant differences were seen in levels of IL-1beta nor in the ratio of IL-1RA/IL-1beta for either group. In contrast, higher quantities of TNF-alpha were observed in the CD14-blocked group. To conclude, anti-CD14 treatment had a detrimental effect on the capacity of Shigella-infected animals to clear the infection.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Disentería Bacilar/inmunología , Receptores de Lipopolisacáridos/fisiología , Shigella flexneri/patogenicidad , Animales , Degranulación de la Célula , Colon/patología , Citocinas/análisis , Disentería Bacilar/microbiología , Disentería Bacilar/patología , Humanos , Inmunohistoquímica , Interleucina-1/análisis , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Receptores de Lipopolisacáridos/inmunología , Macrófagos/inmunología , Mutación , Conejos , Shigella flexneri/genética , Shigella flexneri/inmunología , Shigella flexneri/aislamiento & purificación , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in young cattle. Here we demonstrate BRSV persistence at low levels in tracheobronchial and mediastinal lymph nodes up to 71 days after the experimental infection of calves. Positive results were obtained on viral genomic RNA and messenger RNA coding for the nucleoprotein, glycoprotein (G), and fusion protein (F). G and F proteins were also detected in the pulmonary lymph nodes by immunohistochemistry. Double-staining experiments revealed that viral antigen was present in B-lymphocytes. Coculture experiments with the lymph node cells showed that the virus was still able to infect permissive target cells, even though no cytopathic effect was recorded. In vitro studies indicate that BRSV was still able to replicate in bovine B-lymphocyte cell lines 6 months after infection. These results may also be relevant to the understanding not only of the epidemiology and the peculiarities of the immune response of BRSV infections but also of human respiratory syncytial virus infections.
Asunto(s)
Linfocitos B/virología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Bovino/fisiología , Latencia del Virus/fisiología , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Bronquios/citología , Bovinos , Línea Celular , Técnicas de Cocultivo , Inmunohistoquímica , Lipopolisacáridos/farmacología , Ganglios Linfáticos/citología , Activación de Linfocitos , Mediastino , Mitógenos/farmacología , Fitohemaglutininas/farmacología , ARN Viral/análisis , Virus Sincitial Respiratorio Bovino/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tráquea/citologíaRESUMEN
Cold agglutinins (CA) are autoantibodies that bind to erythrocyte carbohydrates at low temperatures and induce complement-mediated cell lysis, thus causing hemolytic anemia. Tolerance mechanisms towards CA-expressing B cells and the factors inducing pathogenic CA production are unknown. In order to develop an animal model for CA disease, we have produced transgenic mice expressing the heavy or the light chain of a human CA, previously shown to be pathogenic to the mouse. Expression of the human H chain alone resulted in a B cell maturation block at the pro-B stage, and did not induce allelic exclusion. In double-transgenic mice, co-expression of the human H and L chains restored B cell development but the majority of bone marrow cells expressing the human IgM were eliminated by deletion. In the periphery, B cells were depleted, and a large proportion of the remaining cells co-expressed a human and a murine H chain, secreting "mixed" IgM. A few autoreactive cells, predominating in the peritoneal cavity, escaped tolerance mechanisms and secreted transgenic IgM. The autoreactive B cells are amenable to polyclonal stimulation, making these transgenic mice a suitable model for a human autoimmune disease.
Asunto(s)
Aglutininas/fisiología , Linfocitos B/fisiología , Tolerancia Inmunológica , Aglutininas/genética , Animales , Enfermedades Autoinmunes/etiología , Células de la Médula Ósea/fisiología , Crioglobulinas , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Bazo/citologíaRESUMEN
In contrast to pathogenic human immunodeficiency virus and simian immunodeficiency virus (SIV) infections, chronic SIVagm infections in African green monkeys (AGMs) are characterized by persistently low peripheral and tissue viral loads that correlate with the lack of disease observed in these animals. We report here data on the dynamics of acute SIVagm infection in AGMs that exhibit remarkable similarities with viral replication patterns observed in peripheral blood during the first 2 weeks of pathogenic SIVmac infections. Plasma viremia was evident at day 3 postinfection (p.i.) in AGMs, and rapid viral replication led by days 7 to 10 to peak viremias characterized by high levels of antigenemia (1.2 to 5 ng of p27/ml of plasma), peripheral DNA viral load (10(4) to 10(5) DNA copies/10(6) peripheral blood mononuclear cells [PBMC]), and plasma RNA viral load (2 x 10(6) to 2 x 10(8) RNA copies/ml). The lymph node (LN) RNA and DNA viral load patterns were similar to those in blood, with peaks observed between day 7 and day 14. These values in LNs (ranging from 3 x 10(5) to 3 x 10(6) RNA copies/10(6) LN cell [LNC] and 10(3) to 10(4) DNA copies/10(6) LNC) were at no time point higher than those observed in the blood. Both in LNs and in blood, rapid and significant decreases were observed in all infected animals after this peak of viral replication. Within 3 to 4 weeks p. i., antigenemia was no longer detectable and peripheral viral loads decreased to values similar to those characteristic of the chronic phase of infection (10(2) to 10(3) DNA copies/10(6) PBMC and 2 x 10(3) to 2 x 10(5) RNA copies/ml of plasma). In LNs, viral loads declined to 5 x 10(1) to 10(3) DNA copies and 10(4) to 3 x 10(5) RNA copies per 10(6) LNC at day 28 p.i. and continued to decrease until day 84 p.i. (<10 to 3 x 10(4) RNA copies/10(6) LNC). Despite extensive viremia during primary infection, neither follicular hyperplasia nor CD8(+) cell infiltration into LN germinal centers was detected. Altogether, these results indicate that the nonpathogenic outcome of SIVagm infection in its natural host is associated with a rapidly induced control of viral replication in response to SIVagm infection, rather than with a poorly replicating virus or a constitutive host genetic resistance to virus replication.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Replicación Viral , Animales , Chlorocebus aethiops , ADN Viral/sangre , Humanos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , ARN Viral/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Carga Viral , Viremia/virologíaRESUMEN
Experimental infection of mice with Helicobacter felis reproduces many aspects of the gastritis observed in Helicobacter pylori-infected humans. The development of gastric inflammatory lesions in chronically infected inbred mice is host-dependent; in BALB/c mice, gastric B-cell MALT lymphomas were observed, whilst other murine hosts (e.g. C57BL/6) developed severe glandular hyperplasia. The aims of this investigation were to characterize and immunophenotype Helicobacter-induced inflammatory lesions in mice with an outbred genetic background. Swiss mice (n=10 per group) were either inoculated with a suspension of H. felis or left untreated. H. felis-inoculated mice and age-matched control animals were killed 13 months later. The severity of gastric inflammatory lesions in the animals was graded and the number and distribution of B (CD45R(+)) and T (CD3(+)) lymphocytes in lymphoid tissues was determined by immunohistochemistry. Compared with control mice, animals with long-term H. felis infection developed severe hyperplastic gastritis (0.80+/-0.63 vs. 2.7+/-0.68), with epithelial dedifferentiation (0. 40+/-0.52 vs. 2.3+/-0.82) and lengthening of the pits and glands (0. 46+/-0.05 vs. 0.8+/-0.19). Gastric CD45R(+) and CD3(+) lymphocyte scores were significantly elevated (r=0.803) in infected animals, while lymphoepithelial lesions and polymorphonuclear leucocyte infiltrates were absent. Although prominent lymphoid follicles were present in the tissues of all infected animals, and in one control animal, only a proportion (55%) of the mucosal follicles had a dominant B-cell phenotype (defined as > or =75% CD45R(+) labelling), and all were poorly labelled with anti-mouse immunoglobulin antibodies. It was concluded that the lesions in outbred Swiss mice differed from B-cell MALT lymphomas. In contrast to inbred mice, outbred animals developed both glandular and lymphoid tissue lesions to chronic H. felis infection. It is suggested that the default T-helper phenotype of the host influences glandular lesion formation or B-cell lymphomagenesis in this model of infection.
Asunto(s)
Gastritis/microbiología , Infecciones por Helicobacter/patología , Tejido Linfoide/patología , Estómago/patología , Animales , Subgrupos de Linfocitos B/inmunología , Complejo CD3/análisis , Enfermedad Crónica , Gastritis/inmunología , Gastritis/patología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Hiperplasia/inmunología , Hiperplasia/microbiología , Hiperplasia/patología , Técnicas para Inmunoenzimas , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Antígenos Comunes de Leucocito/análisis , Tejido Linfoide/inmunología , Tejido Linfoide/microbiología , Linfoma de Células B de la Zona Marginal/microbiología , Ratones , Especificidad de la Especie , Organismos Libres de Patógenos Específicos , Estómago/inmunología , Estómago/microbiología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Shigella is a diarrheal pathogen that causes disease through invasion of the large intestinal mucosa. The endotoxin of the invading bacterium may play a key role in the disease process by causing inflammation and tissue injury during infection. Earlier studies have shown that various animal species lacking functional CD14 were protected against endotoxin-mediated shock. Rabbits experimentally infected with Shigella were used to test the hypothesis that blockade of endotoxin-induced cell activation with anti-CD14 mAb would diminish inflammation and thus disease severity. Unexpectedly, we observed that the intestinal mucosa of anti-CD14-treated animals exhibited a 50-fold increase in bacterial invasion and more severe tissue injury compared with controls. Despite higher bacterial loads in treated animals, the numbers of polymorphonuclear leukocytes that were recruited to the infection site were similar to those in controls. Furthermore, the phagocytic cells of CD14-blocked animals produced IL-1 and TNF-alpha. Moreover, in vitro blockade of CD14 did not impede bactericidal activity. Thus, anti-CD14 treatment interfered with host defense mechanisms involved with removal/eradication of Shigella.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Disentería Bacilar/inmunología , Disentería Bacilar/patología , Receptores de Lipopolisacáridos/inmunología , Shigella flexneri/patogenicidad , Animales , Degranulación de la Célula/inmunología , Movimiento Celular/inmunología , Citocinas/biosíntesis , Disentería Bacilar/microbiología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Conejos , Shigella flexneri/crecimiento & desarrollo , Shigella flexneri/inmunologíaRESUMEN
As a means of avoiding the host immune response, the tick-borne relapsing fever spirochete Borrelia turicatae undergoes antigenic variation in its abundant surface lipoproteins. In this study, B. turicatae strain Oz1, serotype B, was subcultured in vitro and cloned by limited dilutions after 50 passages. Four different serotypes (serotypes A, B, E, and F) differing by their expressed Vsp lipoproteins were isolated. Using pulsed-field gel electrophoresis, we showed that the variability in surface-exposed proteins is correlated with rearrangement between different linear plasmids, defining serotype-specific plasmid profiles. Moreover, we determined the nucleotide sequence of genes encoding the VspE and VspF lipoproteins, corresponding to the two novel serotypes E and F, respectively. Our results showed that antigenic variation in B. turicatae occurs spontaneously in vitro, in the absence of immune selection.
Asunto(s)
Variación Antigénica , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Borrelia/genética , Lipoproteínas/genética , Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Southern Blotting , Borrelia/química , Borrelia/clasificación , Electroforesis en Gel de Campo Pulsado , Lipoproteínas/química , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Análisis de Secuencia de ADN , SerotipificaciónRESUMEN
The purpose of the current study was to characterize parasite-containing cells located in spleens of BALB/c mice infected with Leishmania donovani. In particular, expression of MHC class II molecules by these cells was examined to determine whether they could potentially act as cells capable of immunostimulating Leishmania-reactive CD4+ T lymphocytes. To this end, an immunohistological analysis of spleens taken at various time points after infection was undertaken. Using this approach, we observed, in the red pulp, the formation of small cellular infliltrates containing heavily infected macrophages that could be stained with the monoclonal antibodies MOMA-2 and FA/11. All of them expressed high levels of MHC class II molecules. Parasites were also detected in the white pulp, especially in MOMA-2+, FA/11+ and MHC class II+ macrophages of the periarteriolar lymphocyte sheath and in MOMA-2+ marginal zone macrophages. Infected cells were further characterized by fluorescence microscopy after their enrichment by adherence. All infected mononuclear cells recovered by this procedure could be stained with MOMA-2 and FA/11 and thus very probably belonged to the mononuclear phagocyte lineage. Furthermore, all of them strongly expressed both MHC class II as well as H-2M molecules, regardless of the time points after infection. Analysis of the parasitophorous vacuoles (PV) by confocal microscopy showed that these compartments were surrounded by a membrane enriched in lysosomal glycoproteins lamp-1 and lamp-2, in macrosialin (a membrane protein of prelysosomes recognized by FA/11) and in MOMA-2 antigen. About 80% of the PV also had MHC class II and H-2M molecules on their membrane. Altogether, these data indicate that in the spleens of L. donovani-infected mice, a high percentage of amastigotes are located in macrophages expressing MHC class II molecules and that they live in PV exhibiting properties similar to those of PV detected in mouse bone marrow-derived macrophages exposed to a low dose of interferon gamma (IFN-gamma) and infected in vitro.
Asunto(s)
Leishmania donovani , Leishmaniasis Visceral/inmunología , Macrófagos/parasitología , Bazo/parasitología , Animales , Antígenos CD/análisis , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/análisis , Antígenos de Diferenciación Mielomonocítica/metabolismo , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Clase II/análisis , Inmunohistoquímica , Leishmania donovani/aislamiento & purificación , Proteínas de Membrana de los Lisosomas , Macrófagos/metabolismo , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Factores de Tiempo , Vacuolas/metabolismo , Vacuolas/parasitologíaAsunto(s)
Anemia Hemolítica Autoinmune/genética , Modelos Animales de Enfermedad , Sistema del Grupo Sanguíneo ABO/inmunología , Animales , Apoptosis , Autoanticuerpos/genética , Autoanticuerpos/inmunología , Autoinmunidad , Ceramidas/inmunología , Anergia Clonal , Supresión Clonal , Frío , Membrana Eritrocítica/inmunología , Genes de Inmunoglobulinas , Antígenos H-2/genética , Antígenos H-2/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Inmunológicos , Imitación Molecular , Mycoplasma pneumoniae/inmunología , Ácido N-Acetilneuramínico/inmunología , TransgenesRESUMEN
Transferrin (Tf), the iron transport protein, is essential for the growth and differentiation of cells. Therefore, it provides an excellent model to analyze the regulatory mechanisms controlling the expression of a eukaryotic gene in different cell types and during fetal and adult life. In this study, the tissue-specific and developmental regulation of the Tf gene in vivo were analyzed. Human Tf mRNA was detected mainly in fetal and adult liver. A weaker expression was observed in adult and fetal brain and in fetal spleen. By in situ hybridization the presence of mouse Tf mRNA was detected in the hepatic primordia. This is the first observation pointing out Tf as an early marker of hepatic differentiation, prior to the formation of the liver. Thus, TF may be an important tool to follow the hepatic specification of the gut endoderm. Mouse Tf mRNA was also detected in the liver bud and subsequently in the liver throughout fetal life, and in newborn and adult animals. No expression of the Tf gene was observed in the mouse fetal central nervous system (CNS). In contrast, Tf mRNA was detected from the 5th day after birth in the derivatives of the caudal part of the neural tube and subsequently in the derivatives of the rhomboencephalon and that of the prosencephalon. These results indicate that Tf gene expression correlates with the postnatal development of oligodendrocytes in the mouse CNS. To test whether the control elements of the human gene previously found in ex vivo experiments were also active in vivo during fetal and adult life, we fused the -4000/+395' flanking region of the human gene to the coding region of the lacZ gene and generated transgenic mice. The expression of the reporter gene during development was analyzed.
Asunto(s)
Encéfalo/fisiología , Regulación del Desarrollo de la Expresión Génica , Hígado/fisiología , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Oligodendroglía/fisiología , Transferrina/biosíntesis , Envejecimiento , Animales , Animales Recién Nacidos , Biomarcadores , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Diferenciación Celular , Desarrollo Embrionario y Fetal , Endodermo/fisiología , Humanos , Hígado/embriología , Hígado/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Sistema Nervioso/citología , Oligodendroglía/citología , Proteínas Recombinantes/biosíntesis , Bazo/embriología , Bazo/crecimiento & desarrollo , Bazo/metabolismo , Transferrina/análisis , beta-Galactosidasa/biosíntesisRESUMEN
The Escherichia coli beta-galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level. Here we report a procedure for monitoring beta-galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 degrees C. After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl-beta-D galactopyranoside (X-Gal). This procedure allows both the retention of a high level of beta-galactosidase activity and the preservation of good tissue morphology. Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection.
Asunto(s)
Colorantes , Adhesión en Parafina/métodos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , Animales , Arterias/anatomía & histología , Arterias/metabolismo , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/metabolismo , Endotelio/anatomía & histología , Endotelio/metabolismo , Escherichia coli/genética , Genes Reporteros , Corazón/anatomía & histología , Inmunohistoquímica , Riñón/anatomía & histología , Riñón/metabolismo , Pulmón/anatomía & histología , Pulmón/metabolismo , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Rombencéfalo/anatomía & histología , Rombencéfalo/metabolismo , Fijación del Tejido/métodos , Ganglio del Trigémino/anatomía & histología , Ganglio del Trigémino/metabolismoRESUMEN
In this study we further characterized the phenotype at the homeostasis of mice genetically deficient in Tumor Necrosis Factor-alpha and Lymphotoxin-alpha (LT-alpha TNF-alpha -/-). As initially observed in LT-alpha -/- mice, these mice are devoid of lymph nodes and Peyer's patches, while in their spleen the white and red pulp domains are no more detectable. In the blood the leukocytosis dominated by lymphocytosis is not solely due to the absence of lymph nodes. Indeed, this abnormality was shown to be correctable by the transfer of wild type bone marrow in the absence of lymph node. We now report that the metallophilic macrophages of the marginal zone are no more detectable with an antibody reactive to sialoadhesin, a macrophage restricted transmembrane molecule known to bind myeloid and lymphoid cells. The absence of sialoadhesin within the marginal zone, a critical domain for lymphocyte trafficking towards the white pulp suggests a possible cellular basis for the observed blood leukocytosis. In addition, in the peritoneal cavity of LT-alpha TNF-alpha-/- mice, the size of the resident leukocyte population is increased. By their amplitudes these leukocytosis are similar within the blood and the peritoneal compartments.
Asunto(s)
Leucocitosis/patología , Linfotoxina-alfa/genética , Bazo/patología , Factor de Necrosis Tumoral alfa/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Cavidad Peritoneal/patologíaRESUMEN
The gene encoding the secreted alkaline protease, a suspected virulence factor of Aspergillus fumigatus, was inactivated by gene disruption. The disruption was performed by transformation of a pathogenic strain of the fungus with a linear DNA fragment carrying the gene from which the central part was replaced by the selectable Escherichia coli hygromycin B dominant resistance marker. Two transformants were shown to produce no alkaline protease. Restriction fragment analysis of the DNA of these two transformants was consistent for chromosomal integration of the disrupted gene by homologous recombination. Both isogenic alkaline protease-producing and non-producing A. fumigatus strains invaded lung tissues, causing comparable mortality in immunosuppressed mice. A significant residual proteolytic activity observed in alkaline protease non-producing strain cultures could play a role in the invasion of the tissues by the fungus.
Asunto(s)
Aspergillus fumigatus/patogenicidad , Serina Endopeptidasas/deficiencia , Animales , Aspergillus fumigatus/enzimología , Mapeo Cromosómico , Clonación Molecular , Colágeno/metabolismo , Farmacorresistencia Microbiana , Elastina/metabolismo , Electroforesis en Gel de Poliacrilamida , Eliminación de Gen , Genes Fúngicos/fisiología , Higromicina B , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos , Polimorfismo de Longitud del Fragmento de Restricción , Biosíntesis de Proteínas , Serina Endopeptidasas/genética , Transformación Genética , Virulencia/fisiologíaRESUMEN
Protothecosis are uncommon infections caused by Prototheca, considered to be achlorophylous algae. Nearly 80 human cases are reported in the literature since the first case described by Davies and Wakelin in 1964 in Sierra Leone (11). The disease have been identified in Europe, Asia (Japan, Thailand, China), Oceania and in the United States with 40 cases, particularly in the Southeast. Clinically, three clinical manifestations can be observed: 1) cutaneous lesions: papules, plaques or eczematoid, papulo-nodular areas of the extremities, 2) olecranon bursitis which occurred in 25% of cases, 3) systemic protothecosis. An immunosuppressive factor local or general can be found in half of the cases and the first description of algal meningitis was reported in a patient with AIDS in association with Cryptococcus neoformans. Because the disease is clinically not suspected, the diagnosis is often provided by histology showing a dermic granuloma with endospores. The characteristic feature of protothecosis in tissues is the presence of specific mature sporangia of Prototheca wickerhamii with the pattern of morula. The organism was PAS, Grocott and mucicarmin positive. The ecology was studied by Clark (7), Pidoux (23), Pore (25) and Sudman (27). Prototheca are ubiquitous inhabitants of sewages and are found in slime flux and animal wastes contaminating different aquatic systems. The transmission generally occurred by traumatic inoculation. Prototheca are also found in the digestive system of man and animals without never invasion of the epithelium and mucosae in experimental models. The pathogenicity and virulence are moderate and they are considered as rare opportunistic agents.
Asunto(s)
Prototheca , Animales , Bursitis/etiología , Humanos , Infecciones/diagnóstico , Infecciones/epidemiología , Infecciones/transmisión , Prototheca/clasificación , Prototheca/aislamiento & purificación , Prototheca/patogenicidad , Enfermedades de la Piel/etiología , AguaRESUMEN
The authors present five new cases of entomophthoromycosis observed during three years of histopathology in Cameroon. Three classic cases of rhinoentomophthoromycosis. In one case, Conidiobolus coronatus was isolated. Two cases of subcutaneous entomophthoromycosis, without isolation of the germ. In one observation, the clinical and histological aspect suggested mucormycosis post entomophthoromycosis. An immunofluorescence technique showed the Basidiobolus haptosporus to be the real cause.
Asunto(s)
Entomophthora/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Micosis/diagnóstico , Adulto , Anciano , Niño , Eosinófilos/patología , Femenino , Humanos , Masculino , Micosis/microbiología , Micosis/patologíaRESUMEN
Infective and invasive processes of entomopathogenic fungi expected to be used in biological control shall be analysed in detail. Histological and ultrastructural studies are therefore essential. This paper is devoted to the field of light microscopy; its purpose is to show that coating, in a 4% agar solution, the insects infected by a fungus, prior to fixation and embedding, allows the preservation of the fungal structures present on the cuticle, especially the germinating conidia. This is useful when observing the initial stages of infection. The relevance of this coating technique is illustrated with the example of Pea aphid, Acyrthosiphon pisum, infected by Conidiobolus osmodes. This contribution is the first histological study of infective processes of this entomophthoralean fungus in an insect.
Asunto(s)
Áfidos/microbiología , Entomophthora/fisiología , Animales , Áfidos/anatomía & histología , Entomophthora/ultraestructuraRESUMEN
The delta virus (DV) was shown to be the predominant but not exclusive aetiology in 124 cases of fulminating hepatitis (FH) in the Central African Republic. The condition occurs in an endemo-epidemic form in young adults with no apparent risk factors. The mortality is high (88 p. 100) and the clinical features well established. After a short prodromic period, the patients develop severe jaundice, hepatic encephalopathy, and, in over 1/3 of cases, severe haemorrhage. Biologically, the main changes are related to hepatic cytolysis and insufficiency. Histology shows appearances of spongiocytic (steatosic) hepatitis in 81 p. 100 of cases. HBS antigen is found in 90 p. 100 of cases and DV markers are present in 53 p. 100 of these. The HBS Ag also occurs in 60 to 90 p. 100 of the close relatives of the patients and anti-DV antibodies occur in 40 to 90 p. 100 of these subjects. To the authors' knowledge, this is the first published series to demonstrate FH associated with DV in the African continent.