RESUMEN
BACKGROUND: The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY. RESULTS: During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations. CONCLUSIONS: This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.
Asunto(s)
Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Enterotoxinas/genética , Mutagénesis Insercional/genética , Factores de Transcripción/genética , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Supervivencia Celular/efectos de los fármacos , Pared Celular/genética , Chlorocebus aethiops , Clostridioides difficile/metabolismo , Clostridioides difficile/fisiología , Enterotoxinas/metabolismo , Enterotoxinas/toxicidad , Regulación Bacteriana de la Expresión Génica , Células HT29 , Humanos , Mutagénesis Insercional/inmunología , Transcripción Genética , Regulación hacia Arriba , Células VeroRESUMEN
Unless stimulated by a chronic inflammatory agent, such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions: What do inflammatory tissues provide to promote mutagenesis? And what is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements--ecotropic and polytropic murine leukemia virus and intracisternal A particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to ten de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice.
Asunto(s)
Emolientes/efectos adversos , Aceite Mineral/efectos adversos , Mutagénesis Insercional , Neoplasias Experimentales , Plasmacitoma , Retroelementos , Animales , Línea Celular , Emolientes/farmacología , Ratones , Ratones Endogámicos BALB C , Aceite Mineral/farmacología , Mutagénesis Insercional/efectos de los fármacos , Mutagénesis Insercional/inmunología , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Plasmacitoma/inducido químicamente , Plasmacitoma/genética , Plasmacitoma/inmunología , Plasmacitoma/patologíaRESUMEN
Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.
Asunto(s)
Regulación Viral de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Antígenos del Núcleo de la Hepatitis B/genética , Virus de la Hepatitis B/química , Mutagénesis Insercional , Fragmentos de Péptidos/genética , Multimerización de Proteína , Proteínas del Núcleo Viral/química , Cristalización , Cristalografía por Rayos X , Antígenos del Núcleo de la Hepatitis B/química , Antígenos del Núcleo de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Mutagénesis Insercional/genética , Mutagénesis Insercional/inmunología , Fragmentos de Péptidos/química , Multimerización de Proteína/fisiología , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/aislamiento & purificaciónRESUMEN
Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1-binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1-binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding-like ß3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum-exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1-specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration.
Asunto(s)
Anticuerpos Bloqueadores/metabolismo , Antígenos de Protozoos/metabolismo , Membrana Eritrocítica/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Mutagénesis Insercional/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/metabolismo , Animales , Anticuerpos Bloqueadores/genética , Antígenos de Protozoos/clasificación , Antígenos de Protozoos/genética , Sitios de Unión de Anticuerpos/genética , Adhesión Celular/genética , Adhesión Celular/inmunología , Secuencia Conservada/genética , Secuencia Conservada/inmunología , Reacciones Cruzadas/inmunología , Membrana Eritrocítica/genética , Membrana Eritrocítica/inmunología , Genómica/métodos , Células HEK293 , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Mutagénesis Insercional/genética , Plasmodium falciparum/genética , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , RatasRESUMEN
The genetic modification of lymphocytes is an important topic in the emerging field of gene therapy. Many clinical trials targeting immunodeficiency syndromes or cancer have shown therapeutic benefit; further applications address inflammatory and infectious disorders. Retroviral vector development requires a detailed understanding of the interactions with the host. Most researchers have used simple gammaretroviral vectors to modify lymphocytes, either directly or via hematopoietic stem and progenitor cells. Lentiviral, spumaviral (foamyviral) and alpharetroviral vectors were designed to reduce the necessity for cell stimulation and to utilize potentially safer integration properties. Novel surface modifications (pseudotyping) and transgenes, built using synthetic components, expand the retroviral toolbox, altogether promising increased specificity and potency. Product consistency will be an important criterion for routine clinical use.
Asunto(s)
Vectores Genéticos/genética , Vectores Genéticos/inmunología , Retroviridae/genética , Retroviridae/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virología , Animales , Ensayos Clínicos como Asunto , Vectores Genéticos/uso terapéutico , Humanos , Mutagénesis Insercional/genética , Mutagénesis Insercional/inmunología , Mutagénesis Insercional/métodos , Subgrupos de Linfocitos T/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm1a(KOMP)Wtsi) allele (Spns2(tm1a)). The Spns2(tm1a/tm1a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm1a/tm1a) mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm1a/tm1a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.
Asunto(s)
Proteínas de Transporte de Anión/fisiología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/patología , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Proteínas de Transporte de Anión/deficiencia , Proteínas de Transporte de Anión/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Cruzamientos Genéticos , Marcación de Gen , Inmunofenotipificación , Subgrupos Linfocitarios/metabolismo , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Lisofosfolípidos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Insercional/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Esfingosina/genética , Esfingosina/metabolismoRESUMEN
Mutations in the Sonic hedgehog limb enhancer, the zone of polarizing activity regulatory sequence (ZRS, located within the gene LMBR1), commonly called the ZRS), cause limb malformations. In humans, three classes of mutations have been proposed based on the limb phenotype; single base changes throughout the region cause preaxial polydactyly (PPD), single base changes at one specific site cause Werner mesomelic syndrome, and large duplications cause polysyndactyly. This study presents a novel mutation-a small insertion. In a Swedish family with autosomal-dominant PPD, we found a 13 base pair insertion within the ZRS, NG_009240.1:g.106934_106935insTAAGGAAGTGATT (traditional nomenclature: ZRS603ins13). Computational transcription factor-binding site predictions suggest that this insertion creates new binding sites and a mouse enhancer assay shows that this insertion causes ectopic gene expression. This study is the first to discover a small insertion in an enhancer that causes a human limb malformation and suggests a potential mechanism that could explain the ectopic expression caused by this mutation.
Asunto(s)
Deformidades Congénitas de la Mano/genética , Proteínas de la Membrana/genética , Mutagénesis Insercional/inmunología , Polidactilia/genética , Pulgar/patología , HumanosRESUMEN
Mast cell activation is one of the most dramatic immune-mediated responses the body can encounter. In the worst scenario (i.e., anaphylaxis), this response is fatal. However, the importance of mast cells as initiators and effectors of both innate and adaptive immunity in healthy individuals has recently been appreciated. It was reported that mast cell activation can be used as an adjuvant to promote Ag-specific humoral immune responses upon vaccination. In this study, we have used a clinically relevant mucosal adjuvant, cholera toxin A1 subunit (CTA1)-DD, which is a fusion protein composed of CTA1, the ADP-ribosylating part of cholera toxin, and DD, two Ig-binding domains derived from Staphylococcus aureus protein A. CTA1-DD in combination with polyclonal IgG induced degranulation and production of TNF-alpha from mouse mast cells. Furthermore, CTA1-DD and polyclonal IgG complex induced mast cell degranulation in mouse skin tissue and nasal mucosa. We also found that intranasal immunization with hapten (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled to chicken gammaglobulin admixed with CTA1-DD complexed with polyclonal IgG greatly enhanced serum IgG anti-NP Ab responses and stimulated higher numbers of NP-specific plasma cells in the bone marrow as compared with that observed in mice immunized with NP-chicken gammaglobulin with CTA1-DD alone. This CTA1-DD/IgG complex-mediated enhancement was mast cell dependent because it was absent in mast cell-deficient Kit(W-sh/W-sh) mice. In conclusion, our data suggest that a clinically relevant adjuvant, CTA1-DD, exerts additional augmenting effects through activation of mucosal mast cells, clearly demonstrating that mast cells could be further exploited for improving the efficacy of mucosal vaccines.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Complejo Antígeno-Anticuerpo/metabolismo , Toxina del Cólera/administración & dosificación , Inmunidad Mucosa , Inmunoglobulina G/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Adyuvantes Inmunológicos/fisiología , Animales , Complejo Antígeno-Anticuerpo/biosíntesis , Células Cultivadas , Toxina del Cólera/fisiología , Inmunidad Mucosa/genética , Inmunoglobulina G/biosíntesis , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Mastocitos/trasplante , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Mutagénesis Insercional/inmunología , Proteínas Proto-Oncogénicas c-kit/deficiencia , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.
Asunto(s)
Flavivirus/inmunología , Vacunas contra la Encefalitis Japonesa/inmunología , Mutagénesis Insercional/inmunología , Vacunas Sintéticas/inmunología , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/inmunología , Chlorocebus aethiops , Virus de la Encefalitis Japonesa (Especie)/inmunología , Epítopos/inmunología , Ratones , Células Vero , Vacunas contra el Virus del Nilo Occidental/inmunologíaRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/fas(lpr/lpr) mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas(lpr/lpr) mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas(lpr/lpr) B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas(lpr/lpr) greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas(lpr/lpr) mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) theta, pol eta and pol zeta, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/fas(lpr/lpr) mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.
Asunto(s)
Citidina Desaminasa/biosíntesis , Roturas del ADN , Cambio de Clase de Inmunoglobulina/inmunología , Lupus Eritematoso Sistémico/enzimología , Lupus Eritematoso Sistémico/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Animales , Secuencia de Bases , Lupus Eritematoso Sistémico/inmunología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Datos de Secuencia Molecular , Mutagénesis Insercional/inmunología , Ganglios Linfáticos Agregados/inmunología , Mutación Puntual/inmunología , Recombinación Genética/inmunología , Eliminación de Secuencia/inmunología , Bazo/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
In humans, two types of chitinases have been identified: chitotriosidase I (CHIT1) and acid mammalian chitinase (AMCase). They are enzymes that cleave chitin, a polysaccharide contained in many different human parasites. So far, only little is known about their function in human and especially in human diseases. Recently we have described association of polymorphisms of AMCase with bronchial asthma in a pediatric population. In this study we were interested in whether CHIT1 is also involved in the genetics of asthma. The amino acid variants Gly102Ser and Ala442Gly, as well as a 24 bp duplication within CHIT1, were typed by means of restriction fragment length polymorphisms on 322 children with asthma and 270 randomly chosen adult controls. Statistical analyses made use of the Armitage's trend test; haplotypes were calculated by FAMHAP and FASTEHPLUS. The amino acid variants showed no association with bronchial asthma. The 24 bp duplication, previously shown to completely demolish CHIT1 activity, was also evenly distributed between asthmatics and controls. Finally, the haplotype showed no association with the disease. We conclude from our results that CHIT1 does not play a major role in the development of bronchial asthma in Caucasian children. The results might also imply that the two human chitinases that have been identified so far have quite distinct functions in human diseases even though they have the same substrate.
Asunto(s)
Sustitución de Aminoácidos , Asma/genética , Hexosaminidasas/genética , Polimorfismo de Longitud del Fragmento de Restricción , Adolescente , Adulto , Sustitución de Aminoácidos/inmunología , Asma/enzimología , Asma/etnología , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Hexosaminidasas/inmunología , Humanos , Masculino , Mutagénesis Insercional/genética , Mutagénesis Insercional/inmunología , Población BlancaRESUMEN
X-linked lymphoproliferative disease (XLP) is an immunodeficiency resulting from mutations in SH2D1A, which encodes signalling lymphocytic activation molecule (SLAM)-associated protein (SAP). In addition to SLAM, SAP associates with several other cell-surface receptors including 2B4 (CD244), Ly9 (CD229), CD84 and NTB-A. SAP contains a single src-homology-2 domain and acts as an intracellular adaptor protein by recruiting the protein tyrosine kinase FynT to the cytoplasmic domains of some of these receptors, which results in the initiation of specific downstream signal transduction pathways. XLP is likely to result from perturbed signalling through one or more of these SAP-associating receptors. In this study, we identified missense (Y54C, I84T and F87S) and insertion (fs82 --> X103) mutations in four different kindreds affected by XLP. Each mutation dramatically reduced the half-life of SAP, thus diminishing its expression in primary lymphocytes as well as in transfected cell lines. Interestingly, although the Y54C and F87S mutations compromised the ability of SAP to associate with different receptors, the I84T mutation had no effect on the ability of SAP to bind SLAM, CD84 or 2B4. However, signalling downstream of SLAM was reduced in the presence of SAP bearing the I84T mutation. These findings indicate that, irrespective of the type of mutation, signalling through SAP-associating receptors in XLP can be impaired by reducing the expression of SAP, the ability of SAP to bind surface receptors and/or its ability to activate signal transduction downstream of the SLAM-SAP complex.
Asunto(s)
Regulación de la Expresión Génica/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación Missense , Agregación de Receptores/genética , Transducción de Señal/genética , Antígenos CD/inmunología , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Semivida , Humanos , Péptidos y Proteínas de Señalización Intracelular/inmunología , Trastornos Linfoproliferativos , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Mutagénesis Insercional/genética , Mutagénesis Insercional/inmunología , Mutación Missense/inmunología , Agregación de Receptores/inmunología , Transducción de Señal/inmunología , Proteína Asociada a la Molécula de Señalización de la Activación LinfocitariaRESUMEN
Natural killer (NK) cell development in the bone marrow is not fully understood. Following lineage commitment, these cells appear to advance through a series of developmental stages that are beginning to be characterized. We previously reported a selective deficiency of NK cells in a C57BL/6 mouse with a transgenic construct consisting of the cDNA for the Ly49A major histocompatibility complex (MHC) class 1-specific inhibitory receptor driven by the granzyme A gene. This mouse has few NK cells in peripheral tissues with relative preservation of other immune cells, including T and B cells. Herein we demonstrate that these mice have an accumulation of NK cells with an immature phenotype in the bone marrow, consistent with a block at a previously proposed stage in normal NK-cell development. The phenotype is associated with transgenic insertion into Atf2, the gene for the basic leucine zipper (bZIP) transcription factor family member ATF-2. Although analysis of Atf2-null NK cells shows no defect, the transgenic mice express abnormal truncated Atf2 transcripts that may mediate a repressor effect because ATF2 can heterodimerize with other bZIP molecules. The defect is cell intrinsic, suggesting that certain bZIP molecules play significant roles in NK-cell development.
Asunto(s)
Factor de Transcripción Activador 2/inmunología , Diferenciación Celular/inmunología , Células Asesinas Naturales/inmunología , Mutagénesis Insercional/inmunología , Sitios de Carácter Cuantitativo/inmunología , Transgenes/inmunología , Factor de Transcripción Activador 2/genética , Animales , Antígenos Ly/genética , Antígenos Ly/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Trasplante de Médula Ósea , Diferenciación Celular/genética , Células Asesinas Naturales/citología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ratones , Ratones Transgénicos , Mutagénesis Insercional/genética , Subfamilia A de Receptores Similares a Lectina de Células NK , Sitios de Carácter Cuantitativo/genética , Receptores Similares a Lectina de Células NK , Transgenes/genética , Quimera por Trasplante/genética , Quimera por Trasplante/inmunologíaRESUMEN
In spite of the availability of drugs and a vaccine, tuberculosis--one of man's medical nemeses--remains a formidable public health problem, particularly in the developing world. The persistent nature of the tubercle bacillus, with one third of the world's population is estimated to be infected, combined with the emergence of multi drug-resistant strains and the exquisite susceptibility of HIV-positive individuals, has underscored the urgent need for in-depth study of the biology of Mycobacterium tuberculosis address the resurgence of TB. In aiming to understand the mechanisms by which mycobacteria react to their immediate environments, molecular genetic tools have been developed from naturally occurring genetic elements. These include protein expressing genes, and episomal and integrating elements, which have been derived mainly from prokaryotic but also from eukaryotic organisms. Molecular genetic tools that had been established as routine procedures in other prokaryotic genera were thus mimicked. Knowledge of the underlying mechanisms greatly expedited the harnessing of these elements for mycobacteriological research and has brought us to a point where these molecular genetic tools are now employed routinely in laboratories worldwide.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Vectores Genéticos/genética , Infecciones por VIH/complicaciones , VIH , Mycobacterium tuberculosis/genética , Plásmidos/genética , Tuberculosis Resistente a Múltiples Medicamentos/etiología , Replicación del ADN/genética , Elementos Transponibles de ADN/genética , Elementos Transponibles de ADN/inmunología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/genética , Genes Reporteros/genética , Marcadores Genéticos/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Mutagénesis Insercional/genética , Mutagénesis Insercional/inmunología , Mycobacterium tuberculosis/inmunología , Transgenes/genética , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
The TNFR family member BAFF-R facilitates peripheral B cell development, although it is unclear whether it promotes survival of B cells, or also initiates a differentiation program. We show that disruption of the BAFF-R encoding gene Tnfrsf13c in strain A/WySnJ mice causes a progressive decline in peripheral B cell numbers, beginning at the transitional 1 developmental stage and continuing through the mature peripheral B cell stage. Bcl-x(L) overexpression in A/WySnJ B cells decreased the turnover of transitional B cells, as determined by 5-bromo-2'-deoxyuridine labeling, and restored follicular B cell development. We conclude that the mutant A/WySnJ allele of Tnfrsf13c can be complemented through the survival signal provided by Bcl-x(L).
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Regulación de la Expresión Génica/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores del Factor de Necrosis Tumoral/genética , Bazo/inmunología , Bazo/patología , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Apoptosis/inmunología , Factor Activador de Células B , Receptor del Factor Activador de Células B , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Femenino , Prueba de Complementación Genética , Vectores Genéticos/síntesis química , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos A , Ratones Mutantes , Datos de Secuencia Molecular , Mutagénesis Insercional/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Retroelementos/inmunología , Retroviridae/genética , Bazo/citología , Bazo/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteína bcl-XRESUMEN
Autoantibodies are often detected in hepatocellular carcinoma (HCC), and these responses may represent recognition of tumor Ags that are associated with transformation events. The identities of these Ags, however, are less well known. Using serological analysis of recombinant cDNA expression libraries (SEREX) from four HCC patients, we identified 55 independent cDNA sequences potentially encoding HCC tumor Ags. Of these genes, 15 are novel. Two such proteins, HCA587 and HCA661, were predominantly detected in testis, but not in other normal tissues, except for a weak expression in normal pancreas. In addition to HCC, these two Ags can be found in cancers of other histological types. Therefore, they can be categorized as cancer-testis (CT) Ags. Two other Ags (HCA519 and HCA90) were highly overexpressed in HCC and also expressed in cancer cell lines of lung, prostate, and pancreas, but not in the respective normal tissues. Four other Ags were identified to be expressed in particular types of cancer cell lines (HCA520 in an ovarian cancer cell line, HCA59 and HCA67 in a colon cancer cell line, HCA58 in colon and ovarian cancer cell lines), but not in the normal tissue counterpart(s). In addition, abundant expression of complement inactivation factors was found in HCC. These results indicate a broad range expression of autoantigens in HCC patients. Our findings open an avenue for the study of autoantigens in the transformation, metastasis, and immune evasion in HCC.
Asunto(s)
Anticuerpos Antineoplásicos/análisis , Antígenos de Carbohidratos Asociados a Tumores/análisis , Autoanticuerpos/análisis , Carcinoma Hepatocelular/inmunología , Proteínas de Ciclo Celular , Neoplasias Hepáticas/inmunología , Anciano , Reacciones Antígeno-Anticuerpo/genética , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Northern Blotting , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/genética , ADN Complementario/biosíntesis , Inhibidor de la Unión a Diazepam/análisis , Inhibidor de la Unión a Diazepam/inmunología , Biblioteca de Genes , Humanos , Isoantígenos/análisis , Isoantígenos/genética , Isoantígenos/inmunología , Neoplasias Hepáticas/química , Neoplasias Hepáticas/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutagénesis Insercional/inmunología , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Eliminación de Secuencia/inmunología , Factor de Transcripción DP1 , Factores de Transcripción/análisis , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Células Tumorales CultivadasRESUMEN
Recently, pathogenicity models that involve the killing of the genetically tractable nematode Caenorhabditis elegans by human pathogens have been developed. From the perspective of the pathogen, the advantage of these models is that thousands of mutagenized bacterial clones can be individually screened for avirulent mutants on separate petri plates seeded with C. elegans. The advantages of using C. elegans to study host responses to pathogen attack are the extensive genetic and genomic resources available and the relative ease of identifying C. elegans mutants that exhibit altered susceptibility to pathogen attack. The use of Caenorhabditis elegans as the host for a variety of human pathogens is discussed.
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Actinomycetales/fisiología , Infecciones Bacterianas/fisiopatología , Proteínas Bacterianas/inmunología , Caenorhabditis elegans/fisiología , Expresión Génica , Interacciones Huésped-Parásitos/inmunología , Interacciones Huésped-Parásitos/fisiología , Mutagénesis Insercional/inmunología , Pseudomonas aeruginosa/metabolismo , Transducción de Señal , Actinomycetales/inmunología , Actinomycetales/patogenicidad , Animales , Proteínas Bacterianas/farmacología , Proteínas Bacterianas/toxicidad , Caenorhabditis elegans/parasitología , Infecciones por Bacterias Grampositivas/inmunología , Humanos , Pseudomonas aeruginosa/patogenicidad , VirulenciaRESUMEN
Murine studies implicate Ikaros proteins as regulators of hemopoiesis, particularly in the lymphoid lineages. High homology between murine and human Ikaros suggests that Ikaros expression in the two might be similar. However, initial human studies that focused on leukemia detected novel Ikaros transcripts in patient samples. Thus, novel Ikaros splice forms and DNA nonbinding isoforms were linked with malignancy. We undertook an extensive analysis of normal human Ikaros expression to determine whether novel mRNAs are expressed as proteins and the extent to which these splice variants are unique to leukemia. Here we show that both mRNA and protein for DNA nonbinding Ikaros isoforms and splice variants previously linked to leukemia are expressed in normal human cells. However, our studies identify a new Ikaros isoform not previously described in mouse or human. This isoform is the predominant Ikaros protein in normal human cells, but not in leukemia cell lines.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Factores de Transcripción/biosíntesis , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Sangre Fetal/citología , Sangre Fetal/inmunología , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/inmunología , Humanos , Factor de Transcripción Ikaros , Células Jurkat , Leucemia/inmunología , Leucemia/metabolismo , Mutagénesis Insercional/inmunología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Eliminación de Secuencia/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/inmunología , Células Tumorales CultivadasRESUMEN
Ag receptor gene loci contain many V gene segments, each of which is recombined and expressed at a different frequency and is subject to allelic exclusion. To probe the parameters that mediate the different levels of regulation of V gene rearrangement, a Vbeta gene segment together with 3.6-kb 5' and 0.7-kb 3' flanking sequences was inserted 6.8 kb upstream of the Dbeta1 gene segment in the murine TCRbeta locus. Despite its proximity to the Dbeta gene segments and the Ebeta enhancer, the inserted Vbeta segment underwent VDJ recombination at the same frequency as the natural copy located 470 kb upstream. However, the inserted Vbeta segment was no longer under allelic exclusion control as it recombined at a similar frequency in the presence of a TCRbeta transgene. These results suggest that while the inserted fragment contains the necessary cis-regulatory elements for determining the frequency of Vbeta rearrangement, additional cis-regulatory elements are required for mediating Vbeta allelic exclusion. Interestingly, most of the inserted Vbeta rearrangements were not transcribed and expressed in the presence of a TCRbeta transgene, suggesting that TCRbeta allelic exclusion can also be achieved by blocking the transcription of the rearranged gene segments. These findings provide strong evidence for distinct control of the frequency and allelic exclusion of Vbeta gene rearrangement.
Asunto(s)
Alelos , Frecuencia de los Genes/inmunología , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Animales , Marcación de Gen , Ganglios Linfáticos/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Insercional/inmunología , Regiones Promotoras Genéticas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Recombinación Genética/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transgenes/inmunologíaRESUMEN
The identification of the "blank" allele HLA-B*5111N, which was detected in German and Czech individuals, is described. In the pedigree analysis this new allele segregates with the serological haplotype HLA-A2; B-; DR4 which is frequent in Czech population. The non-expression of B*5111N is caused by the insertion of an additional cytosine molecule at the cytosine island between the nucleotides 621-626 (codons 183-185, first three codons of exon 4) leading to a frame shift that creates a stop codon at codon 196. This insertion may be explained either by conversion with the pseudogene HLA-J or by slipped-strand mispairing. In order not to overlook the presence of alleles with altered expression in case of hematopoietic stem cell transplantation, both serological and DNA-based typing should be performed (Note).