RESUMEN
CD8+ T cells are perceived to play a major role in the pathogenesis of type 1 diabetes (T1D). In this study, we characterized the function and phenotype of circulating CD8+ memory T cells in samples from individuals at different stages of T1D progression using flow cytometry and single-cell multiomics. We observed two distinct CD8+ T-cell signatures during progression of T1D within the highly differentiated CD27-CD8+ memory T cell subset. A proinflammatory signature, with an increased frequency of IFN-γ+TNF-α+ CD27-CD8+ memory T cells, was observed in children with newly diagnosed T1D (stage 3) and correlated with the level of dysglycemia at diagnosis. In contrast, a co-inhibitory signature, with an increased frequency of KLRG1+TIGIT+ CD27-CD8+ memory T cells, was observed in islet autoantibody-positive children who later progressed to T1D (stage 1). No alterations within CD27-CD8+ memory T cells were observed in adults with established T1D or in children during the initial seroconversion to islet autoantibody positivity. Single-cell multiomics analyses suggested that CD27-CD8+ T cells expressing the IFNG+TNF+ proinflammatory signature may be distinct from those expressing the KLRG1+TIGIT+ co-inhibitory signature at the single-cell level. Collectively, our findings suggest that distinct blood CD8+ T-cell signatures could be employed as potential biomarkers of T1D progression.
RESUMEN
BACKGROUND: Allergen-specific type 2 CD4+ TH2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the TH2 response has only recently been appreciated. OBJECTIVE: We sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4+ T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy. METHODS: Dog allergen-specific memory CD4+ T cells were detected ex vivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing. RESULTS: Dog allergen-specific T-cell responses in allergic subjects were dominantly of TH2 type. TH2 cells could be phenotypically further divided into 3 subsets, which consisted of TH2-like (CCR6-CXCR3-CRTH2-), TH2 (CCR6-CXCR3-CRTH2+CD161-), and TH2A (CCR6-CXCR3-CRTH2+CD161+CD27-) cells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the TH2-biased signature in allergen-specific T cells from allergic subjects and revealed a TH1/TH17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors. Finally, TCR and transcriptomic analyses revealed a close relationship between TH2-like, TH2, and TH2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype. CONCLUSIONS: Our study demonstrates heterogeneity within allergen-specific TH2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches.
Asunto(s)
Alérgenos , Perros , Células Th2 , Animales , Desensibilización Inmunológica , Humanos , Receptores de Antígenos de Linfocitos T/metabolismo , Células TH1 , Células Th2/metabolismoRESUMEN
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which the insulin-producing ß cells within the pancreas are destroyed. Identification of target Ags and epitopes of the ß cell-reactive T cells is important both for understanding T1D pathogenesis and for the rational development of Ag-specific immunotherapies for the disease. Several studies suggest that proinsulin is an early and integral target autoantigen in T1D. However, proinsulin epitopes recognized by human CD4+ T cells have not been comprehensively characterized. Using a dye dilution-based T cell cloning method, we generated and characterized 24 unique proinsulin-specific CD4+ T cell clones from the peripheral blood of 17 individuals who carry the high-risk DR3-DQ2 and/or DR4-DQ8 HLA class II haplotypes. Some of the clones recognized previously reported DR4-restricted epitopes within the C-peptide (C25-35) or A-chain (A1-15) of proinsulin. However, we also characterized DR3-restricted epitopes within both the B-chain (B16-27 and B22-C3) and C-peptide (C25-35). Moreover, we identified DQ2-restricted epitopes within the B-chain and several DQ2- or DQ8-restricted epitopes within the C-terminal region of C-peptide that partially overlap with previously reported DQ-restricted epitopes. Two of the DQ2-restricted epitopes, B18-26 and C22-33, were shown to be naturally processed from whole human proinsulin. Finally, we observed a higher frequency of CDR3 sequences matching the TCR sequences of the proinsulin-specific T cell clones in pancreatic lymph node samples compared with spleen samples. In conclusion, we confirmed several previously reported epitopes but also identified novel (to our knowledge) epitopes within proinsulin, which are presented by HLA class II molecules associated with T1D risk.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DQ/inmunología , Proinsulina/inmunología , Adolescente , Secuencia de Aminoácidos , Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Niño , Preescolar , Humanos , Lactante , Insulina/inmunología , Células Secretoras de Insulina/inmunología , Bazo/inmunologíaAsunto(s)
Asma , Hipersensibilidad , Alérgenos , Animales , Epítopos , Ratones , Linfocitos T/inmunologíaRESUMEN
Novel hot electron-emitting working electrodes and conventional counter electrodes were created by screen printing. Thus, low-cost disposable electrode chips for bioaffinity assays were produced to replace our older expensive electrode chips manufactured by manufacturing techniques of electronics from silicon or on glass chips. The present chips were created by printing as follows: (i) silver lines provided the electronic contacts, counter electrode and the bottom of the working electrode and counter electrode, (ii) the composite layer was printed on appropriate parts of the silver layer, and (iii) finally a hydrophobic ring was added to produce the electrochemical cell boundaries. The applicability of these electrode chips in bioaffinity assays was demonstrated by an immunoassay of human C-reactive protein (i) using Tb(III) chelate label displaying long-lived hot electron-induced electrochemiluminescence (HECL) and (ii) now for the first time fluorescein isothiocyanate (FITC) was utilized as an a low-cost organic label displaying a short-lived HECL in a real-world bioaffinity assay.
Asunto(s)
Electroquímica/métodos , Electrones , Inmunoensayo/métodos , Mediciones Luminiscentes/métodos , Proteína C-Reactiva/metabolismo , Calibración , Electrodos , Humanos , Propiedades de SuperficieRESUMEN
Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the ß-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution.
Asunto(s)
Alérgenos/química , Lipocalinas/química , Multimerización de Proteína , Alérgenos/inmunología , Lipocalinas/inmunología , Espectrometría de Masas , Modelos Moleculares , Conformación ProteicaRESUMEN
BACKGROUND: The recently identified dog lipocalin allergen Can f 4 is an important respiratory allergen. OBJECTIVE: We sought to comprehensively characterize the memory CD4(+) T-cell responses of allergic and nonallergic subjects to Can f 4. METHODS: Can f 4-specific CD4(+)CD45RO(+) T-cell lines (TCLs) from allergic and healthy subjects were established and characterized by their functional and phenotypic properties. The epitope specificity of the TCLs was tested with 48 overlapping 16-mer peptides spanning the sequence of Can f 4. HLA restriction of the specific TCLs and the binding capacity of the epitope-containing peptides to common HLA class II molecules were studied. RESULTS: Can f 4-specific memory CD4(+) TCLs were obtained at an 8-fold higher frequency from allergic than from nonallergic subjects. Functionally, the TCLs of allergic subjects exhibited a higher T-cell receptor avidity and expression of CD25 and predominantly produced IL-4 and IL-5. The TCLs of nonallergic subjects mostly secreted IFN-γ and IL-10, with high CXCR3 expression. Several distinct T-cell epitope regions along the allergen were identified. Importantly, the peptides from the region between amino acids 43 and 67 showed promiscuous HLA-binding capacity and induced memory CD4(+) T-cell responses in 90% of the allergic donors. CONCLUSION: Productive TH2-deviated memory T-cell responses to Can f 4 are observed in allergic but not nonallergic subjects. A 19-mer peptide sequence covering the core of the immunodominant region of the allergen is a potential target for the development of peptide-based allergen immunotherapy.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Memoria Inmunológica , Lipocalinas/inmunología , Células Th2/inmunología , Alérgenos/farmacología , Animales , Línea Celular , Citocinas/inmunología , Perros , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Hipersensibilidad/patología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Lipocalinas/farmacología , Masculino , Receptores CXCR3/inmunologíaRESUMEN
PURPOSE: Divergent results on the IgE reactivity of dog-allergic subjects to Can f 4 have been reported. The aim of this study was to evaluate the significance of Can f 4 in dog allergy and to develop an immunochemical method for measuring Can f 4 content in environmental samples. METHODS: We purified the natural dog allergen Can f 4 from a dog dander extract by monoclonal antibody-based affinity chromatography and generated its variant in a recombinant form. Sixty-three dog-allergic patients and 12 nonallergic control subjects were recruited in the study. The IgE-binding capacity of natural Can f 4 and its recombinant variant was assessed by ELISA, immunoblotting, and skin prick tests (SPT). RESULTS: Eighty-one percent of the dog-allergic patients showed a positive result to the immunoaffinity-purified natural Can f 4 in IgE ELISA, but only 46% in IgE immunoblotting. Respective results with the recombinant Can f 4 variant were 54% and 49%. SPT results reflected those obtained in ELISA and immunoblotting. The overall IgE reactivity of the immunoaffinity-purified natural Can f 4 was found to depend strongly on the integrity of the allergen's conformation. A sandwich ELISA based on monoclonal antibodies was found to be functional for measuring Can f 4 in environmental samples. CONCLUSIONS: Can f 4 is a major allergen of dog together with Can f 1 and Can f 5. In combination with other dog allergens, it improves the reliability of allergy tests in dog allergy.
RESUMEN
Four out of six officially recognized dog allergens are members of the lipocalin protein family. So far, a three-dimensional structure has been determined for only one dog allergen, Can f 2, which is a lipocalin protein. We present here the crystal structure of a second lipocalin allergen from dog, a variant of Can f 4. Moreover, we have compared and analyzed the structures of these two weakly homologous (amino acid identity 21%) dog allergens. The size and the amino acid composition of the ligand-binding pocket indicate that Can f 4 is capable of binding only relatively small hydrophobic molecules which are different from those that Can f 2 is able to bind. The crystal structure of Can f 4 contained both monomeric and dimeric forms of the allergen, suggesting that Can f 4 is able to form transient (weak) dimers. The existence of transient dimers in solution was confirmed by use of native mass spectrometry. The dimeric structure of Can f 4 is formed when the ends of four ß-strands are packed against the same strands from the second monomer. The residues in the interface are mainly hydrophobic and the formation of the dimer is similar to the major horse allergen Equ c 1. Interestingly, the crystal structure of dog Can f 2 has been reported to show a different type of dimer formation. The capability of these allergens to form dimers may be important for the development of immediate allergic reaction (mast cell activation) because oligomeric allergens can effectively present multivalent epitopes.
Asunto(s)
Alérgenos/química , Lipocalinas/química , Multimerización de Proteína , Estructura Terciaria de Proteína , Alérgenos/genética , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Alérgenos Animales/química , Alérgenos Animales/inmunología , Perros , Immunoblotting , Ligandos , Lipocalinas/genética , Lipocalinas/inmunología , Masculino , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Lipocalin allergens form a notable group of proteins, as they contain most of the significant respiratory allergens from mammals. The basis for the allergenic capacity of allergens in the lipocalin family, that is, the development of T-helper type 2 immunity against them, is still unresolved. As immunogenicity has been proposed to be a decisive feature of allergens, the purpose of this work was to examine human CD4+ T cell responses to the major dog allergen Can f 1 and to compare them with those to its human homologue, tear lipocalin (TL). For this, specific T cell lines were induced in vitro from the peripheral blood mononuclear cells of Can f 1-allergic and healthy dog dust-exposed subjects with peptides containing the immunodominant T cell epitopes of Can f 1 and the corresponding TL peptides. We found that the frequency of Can f 1 and TL-specific T cells in both subject groups was low and close to each other, the difference being about two-fold. Importantly, we found that the proliferative responses of both Can f 1 and TL-specific T cell lines from allergic subjects were stronger than those from healthy subjects, but that the strength of the responses within the subject groups did not differ between these two antigens. Moreover, the phenotype of the Can f 1 and TL-specific T cell lines, determined by cytokine production and expression of cell surface markers, resembled each other. The HLA system appeared to have a minimal role in explaining the allergenicity of Can f 1, as the allergic and healthy subjects' HLA background did not differ, and HLA binding was very similar between Can f 1 and TL peptides. Along with existing data on lipocalin allergens, we conclude that strong antigenicity is not decisive for the allergenicity of Can f 1.
Asunto(s)
Alérgenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Lipocalina 1/inmunología , Alérgenos/química , Animales , Estudios de Casos y Controles , Línea Celular , Citocinas/biosíntesis , Perros , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Hipersensibilidad/inmunología , Lipocalina 1/química , Activación de Linfocitos/inmunología , Péptidos/química , Péptidos/inmunología , Fenotipo , Unión Proteica , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
The responses of allergen-specific CD4(+) T cells of allergic and healthy individuals are still incompletely understood. Our objective was to investigate the functional and phenotypic properties of CD4(+) T cells of horse-allergic and healthy subjects specific to the immunodominant epitope region of the major horse allergen Equ c 1. Specific T-cell lines (TCLs) and clones were generated from peripheral blood mononuclear cells with Equ c 1(143-160), the peptide containing the immunodominant epitope region of Equ c 1. The frequency, proliferative response, cytokine production and HLA restriction of the cells were examined. The frequency of Equ c 1-specific CD4(+) T cells was low (approximately 1 per 10(6) CD4(+) T cells) in both allergic and non-allergic subjects. The cells of allergic subjects had a stronger proliferative capacity than those of non-allergic subjects, and they predominantly emerged from the memory T-cell pool and expressed the T helper type 2 cytokine profile, whereas the cells of non-allergic subjects emerged from the naive T-cell pool and produced low levels of interferon-γ and interleukin-10. T-cell response to Equ c 1(143-160) was restricted by several common HLA class II molecules from both DQ and DR loci. As the phenotypic and functional properties of Equ c 1-specific CD4(+) T cells differ between allergic and non-allergic subjects, allergen-specific T cells appear to be tightly implicated in the development of diseased or healthy outcome. Restriction of the specific CD4(+) T-cell response by multiple HLA alleles suggests that Equ c 1(143-160) is a promising candidate for peptide-based immunotherapy.
Asunto(s)
Alérgenos/inmunología , Epítopos de Linfocito T/inmunología , Glicoproteínas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Hipersensibilidad/inmunología , Células Th2/inmunología , Animales , Línea Celular , Proliferación Celular , Femenino , Caballos , Humanos , Hipersensibilidad/patología , Interferón gamma/inmunología , Interleucina-10/inmunología , Lipocalinas , Masculino , Células Th2/patologíaRESUMEN
Allergic sensitization results from a complex interaction between genetic and environmental factors. Earlier studies have shown that highly polymorphic HLA genes are associated with a variety of allergies. Several important respiratory allergens belong to the family of lipocalin proteins. These include occupational sensitizers, such as cow Bos d 2 or rat Rat n 1, and prevalent indoor sensitizers, such as dog Can f 1 or cockroach Bla g 4. HLA associations with sensitization to lipocalin allergens are incompletely known. In the present study we have investigated an association between HLA alleles and sensitization to the major cow allergen Bos d 2. The HLA-DR/DQ genotypes of 40 Bos d 2-sensitized subjects having occupational asthma were determined by polymerase chain reaction (PCR) and the results were compared with the genotypes of 151 unrelated Finnish subjects. The frequencies of HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501 were significantly higher among Bos d 2-sensitized than among control subjects. In addition, the allergic subjects expressed significantly lower frequencies of HLA-DRB1*0301 and DQB1*0201 alleles than did the control subjects. These data suggest that the HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501, and the haplotypes that include them, are associated with sensitization to the major cow allergen Bos d 2, whereas HLA-DRB1*0301 and DQB1*0201 are dissociated with it. Amino acid analysis provides a biologically plausible explanation for the HLA associations.
Asunto(s)
Antígenos de Plantas/inmunología , Asma Ocupacional/inmunología , Frecuencia de los Genes/inmunología , Cadenas beta de HLA-DQ/inmunología , Cadenas beta de HLA-DR/inmunología , Hipersensibilidad/inmunología , Adulto , Alelos , Alérgenos , Animales , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Asma Ocupacional/genética , Asma Ocupacional/metabolismo , Sitios de Unión , Estudios de Casos y Controles , Bovinos , Femenino , Genotipo , Cadenas beta de HLA-DQ/genética , Cadenas beta de HLA-DQ/metabolismo , Cadenas beta de HLA-DR/genética , Cadenas beta de HLA-DR/metabolismo , Haplotipos , Humanos , Hipersensibilidad/genética , Hipersensibilidad/metabolismo , Lipocalinas/genética , Lipocalinas/inmunología , Lipocalinas/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Unión ProteicaRESUMEN
Carbonic anhydrase VI (CAVI) is the only secreted isozyme of the α-carbonic anhydrase family, which catalyzes the reversible reaction [Formula in text]. It appears that CAVI protects teeth and gastrointestinal mucosa by neutralizing excess acidity. However, the evidence for this physiological function is limited, and CAVI may have additional functions that have yet to be discovered. To explore the functions of CAVI more fully, we generated Car6 (-/-) mice and analyzed Car6 (-/-) mutant phenotypes. We also examined transcriptomic responses to CAVI deficiency in the submandibular gland, stomach, and duodenum of Car6 (-/-) mice. Car6 (-/-) mice were viable and fertile and had a normal life span. Histological analyses indicated a greater number of lymphoid follicles in the small intestinal Peyer's patches. A total of 94, 56, and 127 genes were up- or down-regulated in the submandibular gland, stomach, and duodenum of Car6 (-/-) mice, respectively. The functional clustering of differentially expressed genes revealed a number of altered biological processes. In the duodenum, the significantly affected biological pathways included the immune system process and retinol metabolic processes. The response to oxidative stress and brown fat cell differentiation changed remarkably in the submandibular gland. Notably, the submandibular gland, stomach, and duodenum shared one important transcriptional susceptibility pathway: catabolic process. Real-time PCR confirmed an altered expression in 14 of the 16 selected genes. The generation and of Car6 (-/-) mice and examination of the effects of CAVI deficiency on gene transcription have revealed several affected clusters of biological processes, which implicate CAVI in catabolic processes and the immune system response.
Asunto(s)
Anhidrasas Carbónicas/deficiencia , Duodeno/metabolismo , Mucosa Gástrica/metabolismo , Perfilación de la Expresión Génica , Glándula Submandibular/metabolismo , Animales , Ratones , Análisis por Micromatrices , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although allergen-specific CD4(+) T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401-restricted responses of peripheral blood-derived memory (CD4(+)CD45RO(+)) and naïve (CD4(+)CD45RA(+)) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2(127-142)-specific memory T cells in the peripheral blood-derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2(127-142)-specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2(127-142)-specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long-term Bos d 2(127-142)-specific T-cell lines generated from both memory and naïve T-cell pools from individuals with allergy proliferated more strongly, produced more IL-4 and IL-10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T-cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen-specific T-cell repertoires differ between individuals with or without allergy.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Fragmentos de Péptidos/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Th2/metabolismo , Animales , Antígenos de Plantas , Antígenos CD4/biosíntesis , Bovinos/inmunología , Células Cultivadas , Citocinas/metabolismo , Antígenos HLA-DR/inmunología , Antígenos HLA-DR/metabolismo , Cadenas HLA-DRB1 , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/patología , Memoria Inmunológica , Inmunofenotipificación , Antígenos Comunes de Leucocito/biosíntesis , Unión Proteica , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR3/biosíntesis , Receptores CXCR3/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Células Th2/inmunología , Células Th2/patologíaAsunto(s)
Alérgenos/farmacología , Proliferación Celular/efectos de los fármacos , Hipersensibilidad/inmunología , Linfocitos T Reguladores/inmunología , Alérgenos/inmunología , Animales , Antígenos de Plantas , Perros , Femenino , Humanos , Hipersensibilidad/patología , Masculino , Linfocitos T Reguladores/patologíaRESUMEN
We examined the progression of the WSN influenza virus infection in isolated, multinucleated rat skeletal myofibers. Contrary to mononucleated cells, the adsorbed virions showed markedly delayed entry kinetics. Viral budding occurred on the sarcolemma, but the hemagglutinin envelope glycoprotein matured inefficiently and was poorly cleaved. Compatible with this, plaque assays indicated that infective viral particles were not formed. In situ hybridization studies showed that at low-dose infection, viral RNA production was restricted to one or a few nuclei within a myofiber. Dual in situ hybridization indicated that two different viral RNAs usually co-localized in the same nucleus or nuclei, suggesting that different viral genome segments replicated in the same nucleus. Newly synthesized viral ribonucleoprotein particles (vRNPs) did not re-enter virgin nuclei. Therefore, a single infected nucleus was able to support viral protein production, and notably, these proteins could reach hundreds of micrometers from the nucleus of origin. These results suggest that after viral disassembly in the endosome, the genome segments remained glued together and entered a myonucleus as a package. Spreading of the infection into virgin nuclei either by vRNPs or newly made virions did not occur, and thus the infection was abortive.
Asunto(s)
Fibras Musculares Esqueléticas/virología , Infecciones por Orthomyxoviridae/virología , Orthomyxoviridae/patogenicidad , Animales , Secuencia de Bases , Núcleo Celular/virología , Femenino , Hibridación in Situ , Técnicas In Vitro , Microscopía Electrónica de Transmisión , Modelos Biológicos , Fibras Musculares Esqueléticas/ultraestructura , Orthomyxoviridae/genética , Orthomyxoviridae/fisiología , Orthomyxoviridae/ultraestructura , Infecciones por Orthomyxoviridae/patología , ARN Viral/genética , ARN Viral/metabolismo , Ratas , Sarcolema/ultraestructura , Sarcolema/virología , Proteínas Virales/metabolismo , Internalización del Virus , Liberación del VirusRESUMEN
We have previously proposed that mammalian lipocalin allergens are recognized suboptimally by the human immune system due to their homology with endogenous lipocalins. Here, we have characterized in detail the human T cell recognition of one of the previously identified T cell epitopes of the major dog allergen Can f 1, contained in peptide p105-120. A panel of peptide analogues (altered peptide ligands, APLs) of p105-120 was tested on two specific T cell clones restricted by different human leukocyte antigen (HLA) alleles. Interestingly, we identified for both of the clones several heteroclitic APLs that were capable of stimulating them at 10-30-fold lower concentrations than the natural peptide. Moreover, one of the heteroclitic APLs identified with the T cell clones, L115F, was observed to induce a stronger polyclonal T cell response than the natural allergen peptide from the peripheral blood mononuclear cells (PBMCs) of six Can f 1-allergic subjects studied. The heteroclitic APLs bound with the same affinity as p105-120 to common HLA-DR- and HLA-DP-alleles, suggesting that their improved stimulatory capacity is attributable to a more efficient T cell receptor (TCR) recognition rather than increased HLA binding. Collectively, our data suggest that p105-120 is recognized suboptimally by human T cells. This may contribute to the allergenicity of Can f 1.
Asunto(s)
Alérgenos/inmunología , Epítopos de Linfocito T/inmunología , Hipersensibilidad/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Alelos , Alérgenos/genética , Animales , Antígenos de Plantas , Células Cultivadas , Perros , Relación Dosis-Respuesta a Droga , Epítopos de Linfocito T/genética , Antígenos HLA-DP/genética , Antígenos HLA-DP/inmunología , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Humanos , Hipersensibilidad/genética , Lipocalinas/genética , Lipocalinas/inmunología , Péptidos/genética , Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Three patients with neurofibromatosis 1 (NF1) were operated for congenital pseudarthrosis (PA) of the tibia. Three non-NF1 patients served as reference. Both NF1 mRNA and protein were detected in the PAs and in rows of osteoblasts and numerous osteoclasts next to the NF1-related PA arguing against inactivation of both NF1 alleles in the resident cells. Analyses on mesenchymal stem cells (MSCs) cultured from the red bone marrow of 1) next to PA of the affected NF1 tibiae, 2) the non-affected NF1 iliac crest of the same patients, and from 3) non-NF1 bone marrow demonstrated that the potential to form bone in vitro was the lowest in cells from the affected NF1-tibiae. The latter cells also displayed reduced levels of NF1 mRNA and protein, and upregulated phosphorylated p44/42 MAPK levels, consistent with an upregulated Ras-pathway. An exhaustive NF1 gene analysis detected constitutional mutation in each case, but no second hits or loss of heterozygosity were found. However, one patient displayed a mutation resulting in two potential active splice sites ultimately affecting exon 6. Interestingly, only one of the respective transcripts was detected in cells from the iliac crest, but two novel transcripts were detected in MSCs cultured from site next to PA. This finding may identify a novel mechanism how a single NF1 gene mutation may exert distinct effects on separate anatomical locations. The molecular pathogenesis of NF1-related PA apparently may not be entirely explained by second mutations or loss of heterozygosity of the NF1 gene.
Asunto(s)
Diferenciación Celular , Regulación de la Expresión Génica , Neurofibromatosis 1/complicaciones , Neurofibromina 1/genética , Osteoblastos/patología , Seudoartrosis/congénito , Seudoartrosis/complicaciones , Células de la Médula Ósea/patología , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Humanos , Inmunohistoquímica , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neurofibromatosis 1/enzimología , Neurofibromatosis 1/genética , Neurofibromatosis 1/cirugía , Neurofibromina 1/metabolismo , Osteoblastos/metabolismo , Seudoartrosis/enzimología , Seudoartrosis/cirugía , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radiografía , Tibia/diagnóstico por imagen , Tibia/patología , Tibia/cirugíaRESUMEN
The established protocols for in vitro studies of peripheral nerve myelination with rat embryonic dorsal root ganglia (DRG) and postnatal Schwann cell cocultures do not work with mouse cells. Consequently, the full potential of this model, which would allow to perform cell type-specific, mixed genotype cocultures without cross-breeding the animals, cannot be exploited. We determined the conditions required to promote full myelination in cocultures of pre-purified mouse embryonic DRG and neonatal Schwann cells, and present a method which consistently yields 50-200 mature myelin sheaths/culture. Causes for the failure of the existing protocols to yield satisfactory results with mouse cells fell into three categories: the lack of adherent support provided by the substratum, growth factor and hormone deficiencies, and the high serum content of the media. For optimal results, mouse cocultures require a 3-dimensional substratum, a myelination-promoting culture medium containing pituitary extract, N2 supplement and forskolin, and a low serum concentration.
Asunto(s)
Ganglios Espinales/citología , Proteína Básica de Mielina/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Neuronas/fisiología , Células de Schwann/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Técnicas de Cocultivo , Embrión de Mamíferos , Femenino , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Nervio Ciático/citología , Factores de TiempoRESUMEN
In northern Finland myotonia congenita is caused by three main mutations in the ClC-1 chloride channel. We studied the molecular basis of these mutations (1238T>G/F413C, 1592C>T/A531V, and 2680C>T/R894X). The mutated cDNAs were expressed either in L6 myotubes or in isolated rat myofibers using recombinant Semliki Forest virus. Experiments in L6 cells indicated that A531V and R894X proteins suffered from stability problems in these cells. Analysis in myofibers indicated that the A531V protein was totally retained in the endoplasmic reticulum (ER), whereas the export of the F413C protein was severely reduced. The C-terminal nonsense mutant (R894X), however, was normally transported to the Golgi elements in the myofibers. Defective export or reduced stability of the mutated proteins may thus be reasons for the myotonic symptoms.