RESUMEN
In the past, identification of HLA alleles was limited to sequencing the region of the gene coding for the peptide binding groove, resulting in a lack of sequence information in the HLA database, challenging HLA allele assignment software programs. We investigated full-length sequences of 19 HLA class I and 7 HLA class II alleles, and we extended another 47 HLA class I alleles with sequences of 5' and 3' UTR regions that were all not yet available in the IPD-IMGT/HLA database. We resolved 8638 unknown nucleotides in the coding sequence of HLA class I and 2139 of HLA class II. Furthermore, with full-length sequencing of the 26 alleles, more than 90 kb of sequence information was added to the non-coding sequences, whereas extension of the 47 alleles resulted in the addition of 5.5 kb unknown nucleotides to the 5' UTR and > 31.7 kb to the 3' UTR region. With this information, some interesting features were observed, like possible recombination events and lineage evolutionary origins. The continuing increase in the availability of full-length sequences in the HLA database will enable the identification of the evolutionary origin and will help the community to improve the alignment and assignment accuracy of HLA alleles.
Asunto(s)
Evolución Biológica , Nucleótidos , Alelos , Regiones no Traducidas 3'/genética , Membrana Celular , Nucleótidos/genéticaRESUMEN
The HLA-B15 typing by serological approaches defined the serological subgroups (or splits) B62, B63, B75, B76, B77 and B70 (B71 and B72). The scarcity of sera with specific anti-HLA antibodies makes the serological typing method difficult to discriminate a high variety of HLA antigens, especially between the B15 antigen subgroups. Advancements in DNA-based technologies have led to a switch from serological typing to high-resolution DNA typing methods. DNA sequencing techniques assign B15 specificity to all alleles in the HLA-B*15 allele group, without distinction of the serological split equivalents. However, the presence of antibodies in the patient defined as split B15 antigens urges the identification of HLA-B*15 allele subtypes of the donor, since the presence of donor-specific antibodies is an important contraindication for organ transplantation. Although the HLA dictionary comprises information regarding the serological subtypes of HLA alleles, there are currently 394 B15 antigens out of 516 in the IPD-IMGT/HLA database (3.38.0) without any assigned serological subtype. In this regard, we aimed to identify specific amino acid patterns for each B*15 serological split, in order to facilitate the assignment of B*15 alleles to serological equivalents after high-resolution molecular typing. As a result, serological specificities of 372/394 not yet assigned alleles could be predicted based on amino acid motifs. Furthermore, two new serological types were identified and added, B62-Bw4 and B71-Bw4.
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Dermatoglifia del ADN/métodos , Antígeno HLA-B15/genética , Antígeno HLA-B15/inmunología , Prueba de Histocompatibilidad/métodos , Linfocitos/inmunología , Donantes de Tejidos , Alelos , Secuencias de Aminoácidos , Antígeno HLA-B15/sangre , Antígeno HLA-B15/clasificación , HumanosRESUMEN
Whereas regular allocation avoids unacceptable mismatches on the donor organ, allocation to highly sensitized patients within the Eurotransplant Acceptable Mismatch (AM) program is based on the patient's HLA phenotype plus acceptable antigens. These are HLA antigens to which the patient never made antibodies, as determined by extensive laboratory testing. AM patients have superior long-term graft survival compared with highly sensitized patients in regular allocation. Here, we questioned whether the AM program also results in lower rejection rates. From the PROCARE cohort, consisting of all Dutch kidney transplants in 1995-2005, we selected deceased donor single transplants with a minimum of 1 HLA mismatch and determined the cumulative 6-month rejection incidence for patients in AM or regular allocation. Additionally, we determined the effect of minimal matching criteria of 1 HLA-B plus 1 HLA-DR, or 2 HLA-DR antigens on rejection incidence. AM patients showed significantly lower rejection rates than highly immunized patients in regular allocation, comparable to nonsensitized patients, independent of other risk factors for rejection. In contrast to highly sensitized patients in regular allocation, minimal matching criteria did not affect rejection rates in AM patients. Allocation based on acceptable antigens leads to relatively low-risk transplants for highly sensitized patients with rejection rates similar to those of nonimmunized individuals.
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Rechazo de Injerto/diagnóstico , Antígenos HLA/inmunología , Histocompatibilidad/inmunología , Inmunización/métodos , Fallo Renal Crónico/inmunología , Trasplante de Riñón/efectos adversos , Selección de Paciente , Donantes de Tejidos/provisión & distribución , Femenino , Estudios de Seguimiento , Rechazo de Injerto/etiología , Rechazo de Injerto/patología , Supervivencia de Injerto/inmunología , Antígenos HLA/química , Prueba de Histocompatibilidad , Humanos , Isoanticuerpos/efectos adversos , Fallo Renal Crónico/cirugía , Trasplante de Riñón/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Pronóstico , Factores de Riesgo , Obtención de Tejidos y Órganos/métodos , Inmunología del TrasplanteRESUMEN
The clinical significance of non-HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large-scale studies incorporating analysis of multiple non-HLA antibodies simultaneously. We developed a multiplex non-HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non-HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP-dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased-donor kidney (N = 3276) but not in recipients of a living-donor kidney (N = 1496). At 10 years after deceased-donor transplantation, recipients with anti-ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death-censored covariate-adjusted graft survival compared to the anti-ARHGDIB-negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32-2.53; P = .0003). These antibodies occur independently from donor-specific anti-HLA antibodies (DSA) or other non-HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non-HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti-ARHGDIB antibodies in all patients awaiting deceased-donor transplantation.
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Autoanticuerpos/inmunología , Rechazo de Injerto/mortalidad , Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Trasplante de Riñón/efectos adversos , Complicaciones Posoperatorias/mortalidad , Inhibidor beta de Disociación del Nucleótido Guanina rho/inmunología , Adulto , Femenino , Estudios de Seguimiento , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/etiología , Humanos , Isoanticuerpos/inmunología , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/mortalidad , Fallo Renal Crónico/cirugía , Donadores Vivos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/etiología , Pronóstico , Estudios Retrospectivos , Factores de RiesgoRESUMEN
BACKGROUND: Few studies have evaluated the effect of different immunosuppressive strategies on long-term kidney transplant outcomes. Moreover, as they were usually based on historical data, it was not possible to account for the presence of pretransplant donor-specific human-leukocyte antigen antibodies (DSA), a currently recognized risk marker for impaired graft survival. The aim of this study was to evaluate to what extent frequently used initial immunosuppressive therapies increase graft survival in immunological low-risk patients. METHODS: We performed an analysis on the PROCARE cohort, a Dutch multicentre study including all transplantations performed in the Netherlands between 1995 and 2005 with available pretransplant serum (n = 4724). All sera were assessed for the presence of DSA by a luminex single-antigen bead assay. Patients with a previous kidney transplantation, pretransplant DSA or receiving induction therapy were excluded from the analysis. RESULTS: Three regimes were used in over 200 patients: cyclosporine (CsA)/prednisolone (Pred) (n = 542), CsA/mycophenolate mofetil (MMF)/Pred (n = 857) and tacrolimus (TAC)/MMF/Pred (n = 811). Covariate-adjusted analysis revealed no significant differences in 10-year death-censored graft survival between patients on TAC/MMF/Pred therapy (79%) compared with patients on CsA/MMF/Pred (82%, P = 0.88) or CsA/Pred (79%, P = 0.21). However, 1-year rejection-free survival censored for death and failure unrelated to rejection was significantly higher for TAC/MMF/Pred (81%) when compared with CsA/MMF/Pred (67%, P < 0.0001) and CsA/Pred (64%, P < 0.0001). CONCLUSION: These results suggest that in immunological low-risk patients excellent long-term kidney graft survival can be achieved irrespective of the type of initial immunosuppressive therapy (CsA or TAC; with or without MMF), despite differences in 1-year rejection-free survival.
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Ciclosporina/uso terapéutico , Rechazo de Injerto , Terapia de Inmunosupresión/métodos , Trasplante de Riñón , Ácido Micofenólico/uso terapéutico , Tacrolimus/uso terapéutico , Adulto , Estudios de Cohortes , Supervivencia sin Enfermedad , Femenino , Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Humanos , Terapia de Inmunosupresión/efectos adversos , Inmunosupresores/uso terapéutico , Riñón/inmunología , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , PrednisolonaRESUMEN
BACKGROUND: Pre-transplant donor-specific anti-human leucocyte antigen (HLA) antibodies (DSAs) are associated with impaired kidney graft survival while the clinical relevance of non-donor-specific anti-HLA antibodies (nDSAs) is more controversial. The aim of the present paired kidney graft study was to compare the clinical relevance of DSAs and nDSAs. METHODS: To eliminate donor and era-dependent factors, a post hoc paired kidney graft analysis was performed as part of a Dutch multicentre study evaluating all transplantations between 1995 and 2005 with available pre-transplant serum samples. Anti-HLA antibodies were detected with a Luminex single-antigen bead assay. RESULTS: Among 3237 deceased donor transplantations, we identified 115 recipient pairs receiving a kidney from the same donor with one recipient being DSA positive and the other without anti-HLA antibodies. Patients with pre-transplant DSAs had a significantly lower 10-year death-censored graft survival (55% versus 82%, P=0.0001). We identified 192 pairs with one recipient as nDSA positive (against Class I and/or II) and the other without anti-HLA antibodies. For the patients with nDSAs against either Class I or II, graft survival did not significantly differ compared with patients without anti-HLA antibodies (74% versus 77%, P = 0.79). Only in patients with both nDSAs Class I and II was there a trend towards a lower graft survival (58%, P = 0.06). Lastly, in a small group of 42 recipient pairs, 10-year graft survival in recipients with DSAs was 49% compared with 68% in recipients with nDSAs (P=0.11). CONCLUSION: This paired kidney analysis confirms that the presence of pre-transplant DSAs in deceased donor transplantations is a risk marker for graft loss, whereas nDSAs in general are not associated with a lower graft survival. Subgroup analysis indicated that only in broadly sensitized patients with nDSAs against Class I and II, nDSAs may be a risk marker for graft loss in the long term.
Asunto(s)
Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Isoanticuerpos/sangre , Adulto , Femenino , Antígenos de Histocompatibilidad Clase I , Humanos , Trasplante de Riñón/mortalidad , Masculino , Persona de Mediana Edad , Países Bajos , Riesgo , Donantes de Tejidos , Adulto JovenRESUMEN
Background Complement-fixing antibodies against donor HLA are considered a contraindication for kidney transplant. A modification of the IgG single-antigen bead (SAB) assay allows detection of anti-HLA antibodies that bind C3d. Because early humoral graft rejection is considered to be complement mediated, this SAB-based technique may provide a valuable tool in the pretransplant risk stratification of kidney transplant recipients.Methods Previously, we established that pretransplant donor-specific anti-HLA antibodies (DSAs) are associated with increased risk for long-term graft failure in complement-dependent cytotoxicity crossmatch-negative transplants. In this study, we further characterized the DSA-positive serum samples using the C3d SAB assay.Results Among 567 pretransplant DSA-positive serum samples, 97 (17%) contained at least one C3d-fixing DSA, whereas 470 (83%) had non-C3d-fixing DSA. At 10 years after transplant, patients with C3d-fixing antibodies had a death-censored, covariate-adjusted graft survival of 60%, whereas patients with non-C3d-fixing DSA had a graft survival of 64% (hazard ratio, 1.02; 95% confidence interval, 0.70 to 1.48 for C3d-fixing DSA compared with non-C3d-fixing DSA; P=0.93). Patients without DSA had a 10-year graft survival of 78%.Conclusions The C3d-fixing ability of pretransplant DSA is not associated with increased risk for graft failure.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Complemento C3d/inmunología , Rechazo de Injerto/inmunología , Antígenos HLA/inmunología , Trasplante de Riñón/efectos adversos , Sistema de Registros , Adulto , Distribución por Edad , Suero Antilinfocítico/inmunología , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Rechazo de Injerto/epidemiología , Humanos , Incidencia , Trasplante de Riñón/métodos , Masculino , Persona de Mediana Edad , Cuidados Preoperatorios/métodos , Estudios Retrospectivos , Medición de Riesgo , Distribución por Sexo , Donantes de Tejidos , Receptores de Trasplantes/estadística & datos numéricos , Inmunología del TrasplanteRESUMEN
Natural killer (NK) cell-based immunotherapy is a promising novel approach to treat cancer. However, NK cell function has been shown to be potentially diminished by factors common in the tumor microenvironment (TME). In this study, we assessed the synergistic potential of antibody-dependent cell-mediated cytotoxicity (ADCC) and killer immunoglobin-like receptor (KIR)-ligand mismatched NK cells to potentiate NK cell antitumor reactivity in multiple myeloma (MM). Hypoxia, lactate, prostaglandin E2 (PGE2) or combinations were selected to mimic the TME. To investigate this, NK cells from healthy donors were isolated and NK cell ADCC capacity in response to MM cells was assessed in flow cytometry-based cytotoxicity and degranulation (CD107a) assays in the presence of TME factors. Hypoxia, lactate and PGE2 reduced cytotoxicity of NK cells against myeloma target cells. The addition of daratumumab (anti-CD38 antibody) augmented NK-cell cytotoxicity against target cells expressing high CD38, but not against CD38 low or negative target cells also in the presence of TME. Co-staining for inhibitory KIRs and NKG2A demonstrated that daratumumab enhanced degranulation of all NK cell subsets. Nevertheless, KIR-ligand mismatched NK cells were slightly better effector cells than KIR-ligand matched NK cells. In summary, our study shows that combination therapy using strategies to maximize activating NK cell signaling by triggering ADCC in combination with an approach to minimize inhibitory signaling through a selection of KIR-ligand mismatched donors, can help to overcome the NK-suppressive TME. This can serve as a platform to improve the clinical efficacy of NK cells.
Asunto(s)
ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Microambiente TumoralRESUMEN
Between 2001 and 2012, the number of unrelated donors registered worldwide increased from 7 to 21 million, and the number of public cord blood units increased to over 500,000. We addressed the question of whether this expansion resulted in higher percentages of patients reaching transplantation. Unrelated donor searches were evaluated for 3,124 eligible patients in the Netherlands in two cohorts (2001-2006, n=995; 2007-2012, n=2129), comparing results for patients of Northwestern European and non-Northwestern European origin. Endpoints were 'donor found' and 'transplantation reached'. The substantial growth of the donor inventory over the period studied did not increase the median number of potential unrelated donors (n=7) for non-Northwestern European patients, but almost doubled the number for Northwestern European patients from 42 to 71. Before and after 2007, an unrelated donor or cord blood was identified for 91% and 95%, respectively, of Northwestern European patients and for 65% and 82% of non-Northwestern European patients (P<0.0001). Non-Northwestern European patients more often needed a cord blood transplant. The degree of HLA matching was significantly lower for non-Northwestern European patients (P<0.0006). The time needed to identify a donor decreased for both populations. The percentage of Northwestern European patients reaching transplantation increased from 77% to 83% and for non-Northwestern European patients from 57% to 72% (P=0.0003). The increase of the global inventory resulted in more transplants for patients lacking a family donor, although the quality and quantity of (potential) haematopoietic cell grafts for patients of a non-Northwestern European descent remained inferior, indicating the need for adaptation of recruitment.
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Trasplante de Células Madre Hematopoyéticas , Sistema de Registros , Donantes de Tejidos , Adolescente , Adulto , Niño , Femenino , Trasplante de Células Madre Hematopoyéticas/estadística & datos numéricos , Prueba de Histocompatibilidad , Humanos , Masculino , Neoplasias/diagnóstico , Neoplasias/epidemiología , Neoplasias/terapia , Países Bajos , Grupos de Población , Adulto JovenRESUMEN
BACKGROUND AND AIM: Human leukocyte antigen (HLA) typing is an important step in the diagnostic algorithm for celiac disease (CD) and is also used for screening purposes. Collection of blood is invasive and accompanied with emotional impact especially in children. Genetic technological progress now enables HLA typing from buccal cell samples. This study evaluated the reliability and feasibility of HLA typing for CD-associated HLA polymorphisms using buccal swabs as routine test in high-risk individuals. METHODS: Blood and buccal swabs of 77 children and adolescents with high risk for CD were prospectively collected in this cohort study. Buccal swab collection was performed either by the investigator at the outpatient clinic or by the patient or its parents at home. To evaluate the possibility of self-administration, three families performed the test at home. DNA was extracted using an adapted QIAamp method. Quantity, quality, and purity of DNA were recorded. HLA-DRB1, HLA-DQA1, and HLA-DQB1 typing was examined on buccal cell-derived and blood-derived DNA at low and, if necessary, high resolution level, using sequence-specific oligonucleotide and sequence-based typing, respectively. RESULTS: DNA isolation using buccal swabs yielded a good quality and sufficient quantity of DNA to perform HLA-DQ typing in all individuals. HLA typing results on buccal cell-derived DNA were identical to typing on blood-derived DNA, also for the self-administered samples. CONCLUSION: Introduction of the buccal swab test for HLA typing of CD risk in routine diagnostics can omit the current venipuncture and enables self-administration at home. Therefore, the buccal swab test is beneficial for individuals with a clinical suspicion for CD, as well as for screening purposes in high-risk populations.
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Enfermedad Celíaca/diagnóstico , Antígenos de Histocompatibilidad Clase II/genética , Prueba de Histocompatibilidad/métodos , Mucosa Bucal/citología , Manejo de Especímenes/métodos , Adolescente , Enfermedad Celíaca/genética , Niño , Preescolar , Estudios de Factibilidad , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Servicios de Atención a Domicilio Provisto por Hospital , Humanos , Lactante , Masculino , Flebotomía , Polimorfismo Genético , Autocuidado/métodosRESUMEN
Immunotherapy with allogeneic natural killer (NK) cells offers therapeutic perspectives for multiple myeloma patients. Here, we aimed to refine NK cell therapy by evaluation of the relevance of HLA-class I and HLA-E for NK anti-myeloma reactivity. We show that HLA-class I was strongly expressed on the surface of patient-derived myeloma cells and on myeloma cell lines. HLA-E was highly expressed by primary myeloma cells but only marginally by cell lines. HLA-E(low) expression on U266 cells observed in vitro was strongly upregulated after in vivo (bone marrow) growth in RAG-2(-/-) γc(-/-) mice, suggesting that in vitro HLA-E levels poorly predict the in vivo situation. Concurrent analysis of inhibitory receptors (KIR2DL1, KIR2DL2/3, KIR3DL1 and NKG2A) and NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK cells degranulate more than matched subsets and that HLA-E abrogates degranulation of NKG2A+ subsets. Inhibition by HLA-class I and HLA-E was also observed with IL-2-activated NK cells and at low oxygen levels (0.6 %) mimicking hypoxic bone marrow niches where myeloma cells preferentially reside. Our study demonstrates that NKG2A-negative, KIR-ligand-mismatched NK cells are the most potent subset for clinical application. We envision that infusion of high numbers of this subclass will enhance clinical efficacy.
Asunto(s)
Separación Celular/métodos , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/trasplante , Mieloma Múltiple/terapia , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Animales , Degranulación de la Célula , Línea Celular Tumoral , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Proteínas de Unión al ADN/genética , Citometría de Flujo , Humanos , Interleucina-2/inmunología , Ratones , Ratones Noqueados , Mieloma Múltiple/inmunología , Trasplante de Neoplasias , Oxígeno/metabolismo , Antígenos HLA-ERESUMEN
BACKGROUND AIMS: Natural killer (NK) cell-based immunotherapy is a promising treatment for a variety of malignancies. However, generating sufficient cell numbers for therapy remains a challenge. To achieve this, optimization of protocols is required. METHODS: Mature NK cells were expanded from peripheral blood mononuclear cells PBMCs in the presence of anti-CD3 monoclonal antibody and interleukin-2. Additionally, NK-cell progenitors were generated from CD34(+) hematopoietic stem cells or different T/NK-cell progenitor populations. Generated NK cells were extensively phenotyped, and functionality was determined by means of cytotoxicity assay. RESULTS: Addition of ascorbic acid (AA) resulted in more proliferation of NK cells without influencing NK-cell functionality. In more detail, PBMC-derived NK cells expanded 2362-fold (median, range: 90-31,351) in the presence of AA and were capable of killing tumor cells under normoxia and hypoxia. Moreover, hematopoietic stem cell-derived progenitors appeared to mature faster in the presence of AA, which was also observed in the NK-cell differentiation from early T/NK-cell progenitors. CONCLUSIONS: Mature NK cells proliferate faster in the presence of phospho-L-AA, resulting in higher cell numbers with accurate functional capacity, which is required for adoptive immunotherapy.
Asunto(s)
Ácido Ascórbico/farmacología , Técnicas de Cultivo de Célula/métodos , Células Asesinas Naturales/citología , Células Asesinas Naturales/trasplante , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
Metastasis is the process by which cancers spread to distinct sites in the body. It is the principal cause of death in individuals suffering from cancer. For some types of cancer, early detection of metastasis at lymph nodes close to the site of the primary tumor is pivotal for appropriate treatment. Because it can be difficult to detect lymph node metastases reliably, many individuals currently receive inappropriate treatment. We show here that DNA microarray gene-expression profiling can detect lymph node metastases for primary head and neck squamous cell carcinomas that arise in the oral cavity and oropharynx. The predictor, established with an 82-tumor training set, outperforms current clinical diagnosis when independently validated. The 102 predictor genes offer unique insights into the processes underlying metastasis. The results show that the metastatic state can be deciphered from the primary tumor gene-expression pattern and that treatment can be substantially improved.
Asunto(s)
Carcinoma de Células Escamosas/diagnóstico , Perfilación de la Expresión Génica , Neoplasias de Cabeza y Cuello/diagnóstico , Metástasis Linfática/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Técnicas de Diagnóstico Molecular/métodos , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Orofaríngeas/diagnóstico , Neoplasias Orofaríngeas/genética , Neoplasias Orofaríngeas/patología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of those scores in relation to transplant outcome is unknown. In a large Dutch kidney transplant cohort, Ellipro scores of pretransplant donor antibodies that can be assigned to known eplets (donor epitope specific HLA antibodies [DESAs]) were compared between early graft failure and long surviving deceased donor transplants. We did not observe a significant Ellipro score difference between the two cohorts, nor significant differences in graft survival between transplants with DESAs having high versus low total Ellipro scores. We conclude that Ellipro scores cannot be used to identify DESAs associated with early versus late kidney graft loss in deceased donor transplants.
Asunto(s)
Enfermedades Renales , Trasplante de Riñón , Humanos , Supervivencia de Injerto , Alelos , Anticuerpos , Riñón , Epítopos , Rechazo de Injerto , Antígenos HLA , Donantes de TejidosRESUMEN
In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope-specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan-Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre-transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations (p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA-DR, and 1 of HLA-DQ, and most antibodies were directed to HLA-B (47%). Fifty-three patients (69.7%) had DESA against one donor epitope (range 1-5). Long-term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84-3.25) in deceased donation, and 2.22 (1.25-3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA-based risk analysis on antigen level.
Asunto(s)
Trasplante de Riñón , Humanos , Trasplante de Riñón/efectos adversos , Epítopos , Antígenos HLA/genética , Relevancia Clínica , Isoanticuerpos , Alelos , Donantes de Tejidos , Rechazo de InjertoRESUMEN
The primary goal of the HLA-DPA1 ~ promoter ~ HLA-DPB1 haplotype component of the 18th IHIWS was to characterise the extended haplotypes within the HLA-DP region and survey the extent of genetic diversity in this region across human populations. In this report, we analysed single-nucleotide polymorphisms (SNPs) in 255 subjects from 6 different cohorts. The results from the HLA-DP haplotype component have validated findings from the initial pilot study. SNPs in this region were inherited in strong linkage, particularly HLA-DPA1, SNP-linked promoter haplotypes and motifs in exon 2 of HLA-DPB1. We reported 17 SNP-linked haplotypes in the promoter region. Together with HLA-DPA1 and HLA-DPB1 alleles, they formed 74 distinct extended HLA-DP haplotypes in 438 sequences. We also observed the presence of region-specific alleles and promoter haplotypes. Our approach involved phasing extended SNPs including promoter SNPs, HLA-DPA1 and HLA-DPB1 alleles, in a 22 kb region, GRCh38/hg38 (chr6:33,064,111-33,086,679), followed by clustering of these SNPs as one extended haplotype. This hierarchical clustering revealed four major clades, suggesting that haplotypes within each clade may have diverged from a common ancestral haplotype and undergone similar evolutionary processes. The correlation between HLA-DPA1 and the promoter region raises questions about the role of HLA-DPA1 antigen in the heterodimer. This finding requires validation on a larger sample size specifically designed for anthropological analysis. Nevertheless, the results from this study highlight the clinical potential of selecting better-matched donors for patients awaiting haematopoietic stem cell transplants from genetically overlapping groups that share common ancestral haplotypes.
Asunto(s)
Inmunogenética , Humanos , Haplotipos , Frecuencia de los Genes , Proyectos Piloto , Alelos , Cadenas beta de HLA-DP/genética , Regiones Promotoras GenéticasRESUMEN
The HLA genes are amongst the most polymorphic in the human genome. Alternative splicing could add an extra layer of complexity, but has not been studied extensively. Here, we applied an RNA based approach to study the influence of allele polymorphism on alternative splicing of HLA-C in peripheral blood. RNA was isolated from these peripheral cells, converted into cDNA and amplified specifically for 12 common HLA-C allele groups. Through subsequent sequencing of HLA-C, we observed alternative splicing variants of HLA-C*04 and *16 that resulted in exon 5 skipping and were co-expressed with the mature transcript. Investigation of intron 4 sequences of HLA-C*04 and *16 compared with other HLA-C alleles demonstrated no effect on predicted splice sites and branch point. To further investigate if the unique polymorphic positions in exon 5 of HLA-C*04 or *16 may facilitate alternative splicing by acting on splicing regulatory elements (SRE), in-silico splicing analysis was performed. While the HLA-C*04 specific SNP in exon 5 had no effect on predicted exonic SRE, the HLA-C*16 specific exon 5 SNP did alter exonic SRE. Our findings provide experimental and theoretical support for the concept that polymorphisms within the HLA-C alleles influence the alternative splicing of HLA-C.
Asunto(s)
Empalme Alternativo , Antígenos HLA-C , Alelos , Exones/genética , Antígenos HLA-C/genética , Humanos , Intrones , Polimorfismo Genético , ARN/genéticaRESUMEN
Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD15 have been demonstrated to modulate the outcome of allogeneic hematopoietic stem cell transplantation (SCT). The effect of NOD2/CARD15 polymorphism is reported to be associated with type of donor (sibling or matched unrelated donor) as well as type of conditioning regimen. We reviewed NOD2/CARD15 SNPs in all donor/recipient pairs of 192 consecutive patients who received nonmyeloablative allogeneic SCT at our institution between 2002 and 2006. All patients were treated with fludarabine 30 mg/m(2)/day for 3 days followed by 200 cGy total-body irradiation (TBI) (n = 154) or TBI alone (n = 38) and received grafts from HLA-matched related (n = 132) or unrelated (n = 61) donors. NOD2/CARD15 polymorphisms were observed in 36 of 192 (19%) patients and in 35 of 192 (18%) donors. The incidences of acute and chronic graft-versus-host disease (aGVHD, cGVHD) were 39% and 49%, respectively, in patients with NOD2/CARD15 variants versus 51% and 61% in patients with wild type. The relapse rate at 3 years was 38% in patients with variants and 36% in patients with wild type. The incidence of transplant-related mortality was 22% for patients with variants and 21% for patients with wild type. Overall survival (OS) at 3 years was 56% in patients with variants and 64% in patients with wild-type NOD2/CARD15. There was no significant impact of NOD2/CARD15 mutations on clinical outcome (all P > .05, Kaplan-Meier and Fine and Gray's tests). These data indicate that mutations in the NOD2/CARD15 gene are not a risk factor for clinical outcome in nonmyeloablative allogeneic SCT. Therefore, screening for NOD2/CARD15 polymorphisms in patients or donors does not have additional value in patients undergoing nonmyeloablative SCT.
Asunto(s)
Neoplasias Hematológicas/genética , Trasplante de Células Madre Hematopoyéticas , Proteína Adaptadora de Señalización NOD2/genética , Adulto , Anciano , Femenino , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/cirugía , Humanos , Masculino , Persona de Mediana Edad , Proteína Adaptadora de Señalización NOD2/inmunología , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Acondicionamiento Pretrasplante , Resultado del Tratamiento , Vidarabina/administración & dosificación , Vidarabina/análogos & derivados , Irradiación Corporal Total , Adulto JovenRESUMEN
CD4+ T-helper cells play an important role in alloimmune reactions following transplantation by stimulating humoral as well as cellular responses, which might lead to failure of the allograft. CD4+ memory T-helper cells from a previous immunizing event can potentially be reactivated by exposure to HLA mismatches that share T-cell epitopes with the initial immunizing HLA. Consequently, reactivity of CD4+ memory T-helper cells toward T-cell epitopes that are shared between immunizing HLA and donor HLA could increase the risk of alloimmunity following transplantation, thus affecting transplant outcome. In this study, the amount of T-cell epitopes shared between immunizing and donor HLA was used as a surrogate marker to evaluate the effect of donor-reactive CD4+ memory T-helper cells on the 10-year risk of death-censored kidney graft failure in 190 donor/recipient combinations using the PIRCHE-II algorithm. The T-cell epitopes of the initial theoretical immunizing HLA and the donor HLA were estimated and the number of shared PIRCHE-II epitopes was calculated. We show that the natural logarithm-transformed PIRCHE-II overlap score, or Shared T-cell EPitopes (STEP) score, significantly associates with the 10-year risk of death-censored kidney graft failure, suggesting that the presence of pre-transplant donor-reactive CD4+ memory T-helper cells might be a strong indicator for the risk of graft failure following kidney transplantation.
Asunto(s)
Epítopos de Linfocito T/inmunología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Trasplante de Riñón , Linfocitos T/inmunología , Adulto , Anciano , Epítopos de Linfocito T/genética , Femenino , Rechazo de Injerto/genética , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Antígenos HLA/genética , Humanos , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/métodos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Linfocitos T/metabolismo , Donantes de Tejidos , Receptores de Trasplantes , Trasplante Homólogo , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. Ongoing developments on instrumentation and basecalling software have improved the per-base accuracy of 1D2 nanopore reads tremendously. In the current study, two validation panels of samples covering 70 of the 71 known HLA class I allele groups were used to compare third field sequences obtained by MinION, with Sanger sequence-based typing showing a 100% concordance between both data sets. In addition, the first validation panel was used to set the acceptance criteria for the use of MinION in a routine setting. The acceptance criteria were subsequently confirmed with the second validation panel. In summary, the present study describes validation and implementation of nanopore sequencing HLA class I typing method and illustrates that nanopore sequencing technology has advanced to a point where it can be used in routine diagnostics with high accuracy.