RESUMO
Background: Increasing resistance drives empirical use of less potent and previously reserved antibiotics, including for urinary tract infections (UTIs). Molecular profiling, without culture, might better guide early therapy. Objectives: To explore the potential of AusDiagnostics multiplex tandem (MT) PCR UTI assays. Methods: Two MT-PCR assays were developed successively, seeking 8 or 16 resistance genes. Amplification was tracked in real time, with melting temperatures used to confirm product identity. Assays were variously performed on: (i) extracted DNA; (ii) cultured bacteria; (iii) urine spiked with reference strains; and (iv) bacteria harvested from clinical urines. Results were compared with those from sequencing, real-time SybrGreen PCR or phenotypic susceptibility. Results: Performance was similar irrespective of whether DNA, cultures or urines were used, with >90% sensitivity and specificity with respect to common ß-lactamases, dfr genes and aminoglycoside resistance determinants except aadA1/A2/A3, for which carriage correlated poorly with streptomycin resistance. Fluoroquinolone-susceptible and -resistant Escherichia coli (but not other species) were distinguished by the melting temperatures of their gyrA PCR products. The time from urine to results was <3âh. Conclusions: The MT-PCR assays rapidly identified resistance genes from Gram-negative bacteria in urines as well as from cultivated bacteria. Used directly on urines, this assay has the potential to guide early therapy.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Enterobacteriaceae/urina , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Reação em Cadeia da Polimerase Multiplex/métodos , DNA Bacteriano/genética , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologiaRESUMO
OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (nâ=â10) and from healthy urines 'spiked' with multiresistant Escherichia coli (nâ=â5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Metagenômica/métodos , Testes de Sensibilidade Microbiana/métodos , Nanoporos , Infecções Urinárias/diagnóstico , Urina/microbiologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/microbiologia , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Fatores de Tempo , Infecções Urinárias/microbiologiaRESUMO
OBJECTIVES: Escherichia coli is the most common agent of bacteraemia, bacterial gastroenteritis and urinary tract infections (UTIs). Lineages causing UTIs and gastrointestinal disease are well defined, but less is known about those causing bacteraemia. We therefore investigated the population structure of E. coli from bacteraemia in the UK and Ireland between 2001 and 2010. METHODS: E. coli isolates (nâ=â2166) were submitted to the BSAC Bacteraemia Surveillance Programme from 18 UK and Irish centres from 2001 to 2010. Genotypes were analysed by MLST using the Achtman scheme; MICs, blaCTX-M group and patient demographics were previously determined in the BSAC surveillance. RESULTS: Four hundred and forty-eight STs were identified, but five of these, and their associated clonal complexes (CCs), accounted for 58.4% (1264 of 2166) of isolates: CC73 was the most common (20.7%), followed by CC131 (13.9%), CC95 (11.3%), CC69 (6.9%) and CC12 (5.5%). All these, except CC69 (group D), belong to phylogenetic group B2. CC131 isolates were much more often MDR than other STs were: they rose from 2.9% of isolates in 2001 to 20.5%-20.7% in 2007-08 and then declined to 14.3% in 2010. Resistance rates to cephalosporins, aminoglycosides and fluoroquinolones remained below 10% in other major CCs throughout. CONCLUSIONS: The five most prevalent bacteraemia STs have all been associated previously with UTIs. They dominated in all years, but their proportions fluctuated, most notably for ST131, a globally disseminated high-risk clone that is often MDR.
Assuntos
Bacteriemia/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Variação Genética , Genótipo , Adolescente , Bacteriemia/epidemiologia , Criança , Pré-Escolar , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Irlanda/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Prevalência , Reino Unido/epidemiologia , Adulto Jovem , beta-Lactamases/análiseRESUMO
Despite advances in surgical technique and clinical care, lung transplantation still remains a short-term solution for the treatment of end-stage lung disease. To date, there has been limited experience in experimental lung transplantation using nonhuman primate models. Therefore, we have endeavored to develop a long-term, nonhuman primate model of orthotopic lung transplantation for the ultimate purpose of designing protocols to induce tolerance of lung grafts. Here, we report our initial results in developing this model and our observation that the nonhuman primate lung is particularly prone to rejection. This propensity toward rejection may be a consequence of 1) upregulated nonspecific inflammation, and 2) a larger number of pre-existing alloreactive memory T cells, leading to augmented deleterious immune responses. Our data show that triple-drug immunosuppression mimicking clinical practice is not sufficient to prevent acute rejection in nonhuman primate lung transplantation. The addition of horse-derived anti-thymocyte globulin and a monoclonal antibody to the IL-6 receptor allowed six out of six lung recipients to be free of rejection for over 120 days.
Assuntos
Pneumopatias/cirurgia , Transplante de Pulmão , Animais , Soro Antilinfocitário/química , Teste de Histocompatibilidade , Cavalos , Tolerância Imunológica , Memória Imunológica/imunologia , Terapia de Imunossupressão , Inflamação/imunologia , Pulmão/patologia , Macaca fascicularis , Complexo Principal de Histocompatibilidade , Modelos Animais , Receptores de Interleucina-6/metabolismo , Linfócitos T/citologia , Transplante Autólogo , Transplante HomólogoRESUMO
Escherichia coli sequence types (STs) 69, 73, 95, and 131 are collectively responsible for a large proportion of E. coli urinary tract and bloodstream infections, and they differ markedly in their antibiotic susceptibilities. Here, we describe a novel PCR method to rapidly detect and distinguish these lineages. Three hundred eighteen published E. coli genomes were compared in order to identify signature sequences unique to each of the four major STs. The specificities of these sequences were assessed in silico by seeking them in an additional 98 genomes. A PCR assay was designed to amplify size-distinguishable fragments unique to the four lineages and was validated using 515 E. coli isolates of known STs. Genome comparisons identified 22 regions ranging in size from 335 bp to 26.5 kb that are unique to one or more of the four predominant E. coli STs, with two to 10 specific regions per ST. These regions predominantly harbor genes encoding hypothetical proteins and are within or adjacent to prophage sequences. Most (13/22) were highly conserved (>96.5% identity) in the genomes of their respective ST. The new assay correctly identified all 142 representatives of the four major STs in the validation set (n = 515), with only two ST12 isolates misidentified as ST95. Compared with MLST, the assay has 100% sensitivity and 99.5% specificity. The rapid identification of major extraintestinal E. coli STs will benefit future epidemiological studies and could be developed to tailor antibiotic therapy to the different susceptibilities of these dominant lineages.
Assuntos
Bacteriemia/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/genética , Tipagem de Sequências Multilocus/métodos , Infecções Urinárias/microbiologia , Antibacterianos/farmacologia , Bacteriemia/diagnóstico , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/diagnóstico , Genoma Bacteriano , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Infecções Urinárias/diagnósticoRESUMO
There are an estimated 17 million human diarrhoea cases annually in the United Kingdom. In 2008 and 2009, enteroaggregative E. coli (EAEC) were identified in 1.9% of stools. However, it remains unclear whether there is a causal link between presence of EAEC and disease. This study used bacterial load, the presence of co-infections and demographic data to assess if EAEC was independently associated with intestinal infectious disease. Quantitative real-time PCR data (Ct values) generated directly from stool specimens for several pathogen targets were analysed to identify multiple pathogens, including EAEC, in the stools of cases and healthy controls. Sensitivity and specificity using Ct value (60% and 60%) was not useful for identifying cases or controls, but an independent association between disease and EAEC presence was demonstrated: multivariate logistic regression for EAEC presence (odds ratio: 2.41; 95% confidence interval: 1.783.26; p<0.001). The population-attributable fraction was 3.3%. The group of bacteria known as EAEC are associated with gastrointestinal disease in at least half of the cases with EAEC positive stools. We conclude that the current definition of EAEC, by plasmid gene detection, includes true pathogens as well as non-pathogenic variants.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/isolamento & purificação , Enteropatias/microbiologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Coinfecção , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Fezes/microbiologia , Feminino , Humanos , Incidência , Enteropatias/epidemiologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Reação em Cadeia da Polimerase em Tempo Real , Reino Unido/epidemiologia , Adulto JovemRESUMO
We have previously shown that a short course of high-dose tacrolimus induces long-term tolerance to fully mismatched lung allografts procured from healthy MHC-inbred miniature swine. Here, we investigate whether donor brain death affects tolerance induction. Four recipient swine were transplanted with fully mismatched lung grafts from donors that were rendered brain dead and mechanically ventilated for 4 h before procurement (Group 1). These recipients were compared to two control groups (Group 2: 4 h of donor ventilation without brain death [n = 5]; and Group 3: no donor brain death with <1 h of ventilation [n = 6]). All recipients were treated with a 12-day course of tacrolimus. In contrast to both groups of control animals, the swine transplanted with lung allografts from brain dead donors all rejected their grafts by postoperative day 45 and showed persistent responsiveness to donor antigen by MLR. Several additional swine underwent brain death induction and/or mechanical ventilation alone to determine the effects of these procedures on the expression of proinflammatory molecules. Significant increases in serum concentrations of IL-1, TNF-α and IL-10 were seen after brain death. Upregulation of IL-1 and IL-6 gene expression was also observed.
Assuntos
Morte Encefálica/imunologia , Rejeição de Enxerto/imunologia , Tolerância Imunológica/imunologia , Transplante de Pulmão/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Doadores de Tecidos , Animais , Ensaio de Imunoadsorção Enzimática , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Modelos Animais , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Porco Miniatura , Transplante Homólogo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Using data from whole-genome projects, an updated multiplex PCR strategy was developed to assign Escherichia coli isolates rapidly to major phylogenetic groups. This assay accommodates sequence variations detected within target sequences, thereby increasing sensitivity and reliability. It was validated using 185 isolates of known sequence types and showed improved congruence with multilocus sequence typing data.
Assuntos
Técnicas Bacteriológicas/métodos , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Escherichia coli/genética , Humanos , Sensibilidade e EspecificidadeRESUMO
Difficulties in accurately identifying serovar 4,[5],12:i:- as monophasic variants of Salmonella enterica serovar Typhimurium mean there is confusion in the reporting of serovars Typhimurium and 4,[5],12:i:-. To gain insight into the prevalence and diversity of these monophasic variants in England and Wales, screening for fljB, hin and the serovar 4,[5],12:i:- DT193-associated genomic island was conducted on 609 S. enterica isolates designated as definitive phage type (DT) 193, and 142 isolates serologically-defined as monophasic variants of serovar Typhimurium but belonging to phage types other than DT193. All latter 142 isolates were subtyped by multilocus variable-number tandem repeat analysis (MLVA). MLVA was also applied to 70 DT193 serologically-defined monophasic variant isolates. Results indicate that serovar 4,[5],12:i:- accounted for 108 of 209 (52%) of DT193 isolates with available serological data and 99 of 142 (70%) monophasic variant isolates belonging to other phage types. Of 609 DT193 isolates, 463 (76%) lacked fljB and hin. Moreover, genetically-related isolates of DTs 120, 191, 191a, 195, phage types U311 and U323, and reacts but does not conform (RDNC) and untypable (UT) strains were also lacking either hin and/or fljB. Of note, the serovar 4,[5],12:i:- DT193-associated genomic island was identified in not only 458 of 463 (99%) monophasic DT193 isolates, but also 25 of 139 (18%) biphasic DT193 isolates and 56 of 76 (74%) monophasic variants of other phage types. Accurate monitoring of the emergence of serovar 4,[5],12:i:- isolates is important to ascertain the public health impact of these strains; since 2012 the Health Protection Agency's Salmonella Reference Unit has therefore begun determining full antigenic structures of all presumptive O:4 isolates in addition to routinely performing phage typing for identification of variants of serovar Typhimurium.
Assuntos
Tipagem de Bacteriófagos , Infecções por Salmonella/epidemiologia , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Salmonella typhimurium/genética , Sorotipagem/métodos , Inglaterra/epidemiologia , Ilhas Genômicas , Humanos , Tipagem de Sequências Multilocus , Prevalência , Infecções por Salmonella/diagnóstico , Infecções por Salmonella/microbiologia , Salmonella typhimurium/isolamento & purificação , Sequências de Repetição em Tandem , País de Gales/epidemiologiaRESUMO
BACKGROUND: Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming essential to optimal cancer care. METHODS: We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping. RESULTS: Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ≥1 mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%). Unanticipated mutations were observed at lower frequencies in IDH and ß-catenin. We observed several associations between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers. Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients with advanced disease toward a genotype-directed targeted therapy. CONCLUSIONS: Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are developed, such multiplexed molecular testing will become a standard part of practice.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genótipo , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase Multiplex , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Ensaios Clínicos como Assunto , Testes Diagnósticos de Rotina , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Terapia de Alvo Molecular , Mutação , Adulto JovemRESUMO
Salmonella enterica serovar (S.) Enteritidis is an important cause of food-borne infection in Europe and the United States. Further subtyping of isolates is necessary to support epidemiological data for the detection of outbreaks and identification of the vehicle of infection. Multilocus variable-number tandem-repeat analysis (MLVA) is reportedly more discriminatory and produces data that are easier to share via databases than other molecular subtyping methods. However, lack of standardisation of the methodology and interpretive criteria for data analysis has meant that comparison of data between laboratories can be problematic. On the basis of MLVA profiles of 298 S. Enteritidis isolates received at the Health Protection Agency's Salmonella Reference Unit and sequence analysis of selected isolates, we propose a MLVA scheme for S. Enteritidis based on five loci (SENTR4, SENTR5, SENTR6, SENTR7 and SE-3) that have been selected from previously published S. Enteritidis MLVA schemes. A panel of reference strains has been developed that can be used by laboratories to normalise their raw fragment data to actual fragment sizes. We also provide recommendations for analysing and interpreting MLVA data. We urge laboratories to consider implementing these guidelines, thereby allowing direct comparison of data between laboratories irrespective of the platform used for fragment analysis, to facilitate international surveillance and investigation of international outbreaks.
Assuntos
Impressões Digitais de DNA/métodos , DNA Bacteriano/genética , Repetições Minissatélites , Salmonella enteritidis/classificação , Salmonella enteritidis/genética , Sorotipagem/normas , Sequência de Bases , Surtos de Doenças , Eletroforese em Gel de Campo Pulsado , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/genética , Salmonella enteritidis/isolamento & purificação , Sorotipagem/métodosRESUMO
We conducted an unmatched retrospective casecontrol study to investigate an upsurge of non-travel-related sporadic cases of infection with Salmonella enterica subsp. enterica serotype Enteritidis phage type 14b with antimicrobial resistance to nalidixic acid and partial resistance to ciprofloxacin (S. Enteritidis PT 14b NxCp(L)) that was reported in England from 1 September to 31 December 2009. We analysed data from 63 cases and 108 controls to determine whether cases had the same sources of infection as those found through investigation of 16 concurrent local foodborne outbreaks in England and Wales. Multivariable logistic regression analysis adjusting for age and sex identified food consumption at restaurants serving Chinese or Thai cuisine (odds ratio (OR): 4.4; 95% CI: 1.314.8; p=0.02), egg consumed away from home (OR: 5.1; 95% CI: 1.321.2; p=0.02) and eating vegetarian foods away from home (OR: 14.6; 95% CI: 2.199; p=0.006) as significant risk factors for infection with S. Enteritidis PT 14b NxCp(L). These findings concurred with those from the investigation of the16 outbreaks, which identified the same Salmonella strain in eggs from a specified source outside the United Kingdom. The findings led to a prohibition of imports from this source, in order to control the outbreak.
Assuntos
Surtos de Doenças , Intoxicação Alimentar por Salmonella/epidemiologia , Fagos de Salmonella/isolamento & purificação , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Dieta Vegetariana/efeitos adversos , Ovos/intoxicação , Inglaterra/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Restaurantes/normas , Estudos Retrospectivos , Intoxicação Alimentar por Salmonella/diagnóstico , Intoxicação Alimentar por Salmonella/etiologia , Salmonella enteritidis/isolamento & purificação , Salmonella enteritidis/virologia , Adulto JovemRESUMO
p53 Arg72Pro, MDM2 T309G, and CCND1 G870A are functional single-nucleotide polymorphisms (SNPs) in key genes that regulate apoptosis and cell cycle. Variant genotypes of these SNPs have been associated with increased risk and earlier age of onset in some cancers. We investigated the association of these SNPs with susceptibility to esophageal adenocarcinoma in a large, North American case-control study. Three hundred and twelve cases and 454 cancer-free controls recruited in Boston, USA were genotyped for each of the three SNPs, and demographic and clinical data were collected. Genotype frequencies for each of the three SNPs did not deviate from the Hardy-Weinberg equilibrium, and did not differ between cases and controls. Odds ratios (OR), adjusted for clinical risk factors, for the homozygous variant genotypes were 0.99 (95% confidence interval [CI] 0.57-1.72) for p53 Pro/Pro, 0.81 (95% CI 0.52-1.28) for MDM2 G/G, and 0.97 (95% CI 0.64-1.49) for CCND1 A/A. The analysis was adequately powered (80%) to detect ORs of 1.37, 1.35, and 1.34 for each SNP, respectively. In contrast to the results of smaller published studies, no association between p53 Arg72Pro, MDM2 T309G, and CCND1 G870A SNPs and susceptibility to esophageal adenocarcinoma, age of onset, or stage of disease at diagnosis was detected.
Assuntos
Ciclina D1/genética , Neoplasias Esofágicas/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: We have previously reported the successful induction of renal allograft tolerance in non-human primates using a nonmyeloablative conditioning regimen to produce a mixed-chimeric state in the recipient. In the present study, we applied this same technique to lung allotransplantation in cynomolgus monkeys. METHODS: Nine pairs of fully major histocompatibility complex (MHC)-mismatched cynomolgus monkeys were used. The conditioning regimen consisted of total body irradiation, thymic irradiation, and antithymocyte globulin. The recipients underwent lung and bone marrow transplantation, followed by anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine. The regimen included anti-CD8 mAb in the last 5 recipients and alpha 1-antitripsin in the last 3 recipients. The results were compared with 8 recipients that received kidney allografts using the same regimen. RESULTS: Transient chimerism developed in all lung recipients, as was previously seen in the kidney recipients. Nonetheless, the lung recipients rejected their allografts significant earlier than the kidney recipients (P < .01). CONCLUSIONS: Despite the successful induction of mixed chimerism in recipients of fully MHC-mismatched lung allografts, we have not observed long-term graft survival, as has been seen in an analogous kidney model. Strategies to overcome this problem include organ-specific modifications of the transplant regimen.
Assuntos
Sobrevivência de Enxerto/imunologia , Tolerância Imunológica , Transplante de Rim/imunologia , Transplante de Pulmão/imunologia , Quimeras de Transplante , Sistema ABO de Grupos Sanguíneos , Animais , Soro Antilinfocitário/uso terapêutico , Transplante de Medula Óssea/imunologia , Teste de Histocompatibilidade , Macaca fascicularis , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Irradiação Corporal TotalRESUMO
AIMS: The aim of the study was to compare measures of the quality of life (QOL) after resection of a chordoma of the mobile spine with the national averages in the United States and to assess which factors influenced the QOL, symptoms of anxiety and depression, and coping with pain post-operatively in these patients. PATIENTS AND METHODS: A total of 48 consecutive patients who underwent resection of a primary or recurrent chordoma of the mobile spine between 2000 and 2015 were included. A total of 34 patients completed a survey at least 12 months post-operatively. The primary outcome was the EuroQol-5 Dimensions (EQ-5D-3L) questionnaire. Secondary outcomes were the Patient-Reported Outcome Measurement Information System (PROMIS) anxiety, depression and pain interference questionnaires. Data which were recorded included the indication for surgery, the region of the tumour, the number of levels resected, the status of the surgical margins, re-operations, complications, neurological deficit, length of stay in hospital and rate of re-admission. RESULTS: The median EQ-5D-3L score was 0.71 (interquartile range (IQR) 0.44 to 0.79) which is worse than the national average in the United States of 0.85 (p < 0.001). Anxiety (median: 55 (IQR 49 to 61), p = 0.031) and pain (median: 61 (IQR 56 to 68), p < 0.001) were also worse than the national average in the United States (50), while depression was not (median: 52 (IQR 38 to 57), p = 0.513). Patients who underwent a primary resection had better QOL and less anxiety, depression and pain compared with those who underwent resection for recurrent or residual disease. The one- and five-year probabilities were 0.96 and 0.74 for survival, 0.07 and 0.25 for tumour recurrence, and 0.02 and 0.16 for developing distant metastasis. A total of 25 local complications occurred in 20 patients (42%), and there were 50 systemic and other complications in 25 patients (52%) within 90 days. CONCLUSION: These patient reported outcomes and oncological and surgical outcomes can be used when counselling patients and to aid decision-making when planning surgery. Cite this article: Bone Joint J 2017;99-B:979-86.
Assuntos
Cordoma/psicologia , Cordoma/cirurgia , Qualidade de Vida , Neoplasias da Medula Espinal/psicologia , Neoplasias da Medula Espinal/cirurgia , Idoso , Ansiedade/psicologia , Cordoma/patologia , Depressão/psicologia , Avaliação da Deficiência , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Dor Pós-Operatória/psicologia , Medidas de Resultados Relatados pelo Paciente , Doses de Radiação , Neoplasias da Medula Espinal/patologia , Inquéritos e Questionários , Tomografia Computadorizada por Raios X , Estados UnidosRESUMO
Wild Escherichia coli are superbly adapted to survive in the intestines of their mammalian hosts and in the environment. E. coli K12 derivative (MG1655) encodes 4288 potential genes that provide the background genetic framework of this species. Particular E. coli clonal types encode additional chromosomal and extrachromosomal genes that facilitate the ability of E. coli to adapt to new environments. These additional genes are often clustered, have related functions (for example, virulence-associated genes in pathogenicity islands) and may be integrated at specific sites on the E. coli chromosome.
Assuntos
Escherichia coli/genética , Genoma Bacteriano , Cromossomos Bacterianos/genética , Genes BacterianosRESUMO
UNLABELLED: We have previously reported that tolerance to class I disparate lung allografts in miniature swine could be induced using an intensive 12-day course of tacrolimus and that pretransplant sensitization with immunogenic MHC class I allopeptides failed to block the induction of tolerance. We also have previously reported the importance of the presence of the thymus in the induction of tolerance to isolated heart, kidney, and combined heart-kidney transplants. In this study, we examined the impact of thymectomy on tolerance induction in lung transplantation. METHODS: Orthotopic left lung transplantation was performed using MHC class I-disparate donors. The recipients received a 12-day course of high-dose tacrolimus (n = 6). Total thymectomies were performed in three of the swine 21 days prior to transplantation. Lung grafts were monitored by chest radiography and serial open lung biopsy. RESULTS: All euthymic recipients maintained their grafts for over 1 year. None of the thymectomized recipients has experienced graft loss in the 6 to 10 months following transplantation. Although isolated lesions of obliterative bronchiolitis were occasionally seen in one thymectomized animal on biopsy, donor-specific unresponsiveness has been observed on assays of cell-mediated lymphocytotoxicity in all recipients. Moreover, co-culture assays have shown that recipient lymphocytes can strongly inhibit the normally robust response of naïve recipient-matched lymphocytes to donor antigen. This inhibition was not seen when using stimulators primed with third-party antigens against appropriate targets. CONCLUSIONS: These data suggest that thymus-independent peripheral regulatory mechanisms may be sufficient to induce and maintain long-term acceptance of the lung allografts.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Transplante de Pulmão/imunologia , Timectomia , Transplante Homólogo/imunologia , Animais , Genótipo , Rejeição de Enxerto/imunologia , Homozigoto , Imunossupressores/uso terapêutico , Transplante de Pulmão/patologia , Suínos , Porco Miniatura , Tacrolimo/uso terapêuticoRESUMO
UNLABELLED: Considerable evidence suggests that indirect recognition of MHC allopeptides plays an important role in solid-organ rejection. Here, we examine whether immunization with class I or class II allopeptides accelerates rejection in a fully MHC-mismatched lung transplant model in miniature swine. METHODS: Recipients were immunized with either donor-derived class I or class II peptides. Sensitization to the peptides was confirmed by DTH testing and in vitro proliferation assays. Nonimmunized control (n = 6), class I peptide-immunized (n = 3), and class II peptide-immunized (n = 3) swine were transplanted with fully mismatched lungs using only a 12-day course of tacrolimus. RESULTS: One control animal rejected its graft on postoperative day 103, while the others maintained their grafts for over 1 year. In the class I peptide-immunized group, two recipients rejected their grafts (days 14 and 52). The third animal has not rejected the graft (day 120, experiment is ongoing). In contrast, in the class II-peptide immunized group, only one animal rejected its graft on day 52, while the others maintained their grafts over 1 year. Both anti-donor IgM and IgG antibodies were detectable in all acute rejectors, although no alloantibody was detectable in long-term acceptors. Regardless of the fate of the graft, all animals have maintained their proliferative responses to the peptides. However, only acceptors maintained donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity and mixed lymphocyte reaction assays. CONCLUSIONS: Pretransplant sensitization of lung allograft recipients to donor allopeptides accelerates graft rejection. This appears particularly true for class I-derived allopeptides, suggesting that class II molecules may be less antigenic when presented indirectly.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Transplante de Pulmão/imunologia , Animais , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Antígenos HLA/imunologia , Teste de Histocompatibilidade , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Complexo Principal de Histocompatibilidade , Modelos Animais , Suínos , Porco MiniaturaRESUMO
OBJECTIVES: The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. We have induced robust tolerance to class I-disparate lung allografts in miniature swine using an intensive 12-day course of tacrolimus. Here, we tested whether a tolerant state can be induced in swine receiving fully mismatched lung allografts. METHODS: Orthotopic left lung allografts were performed using MHC class I-disparate (group 1: n = 3) or fully disparate (group 2: n = 6) donors. The recipients received a 12-day postoperative course of tacrolimus (continuous intravenous infusion; target level = 35-50 ng/mL) as their only immunosuppression. RESULTS: All swine in group 1 maintained their grafts long term without developing any lesions of chronic rejection (>497, >432, >451 days). These recipients exhibited donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity (CML) and mixed lymphocyte reaction (MLR) assays. In group 2, five of the six recipients maintained their grafts long term (sacrificed on postoperative days 515, 389, 429, 481, and 438 with viable grafts). Isolated lesions of obliterative bronchiolitis were occasionally seen on biopsy, and donor-specific hyporesponsiveness on assays was consistently observed. The remaining recipient rejected its graft on day 103 with histologic findings of obliterative bronchiolitis. CONCLUSIONS: We report long-term graft acceptance without chronic immunosuppression in five of six recipients across a full MHC disparity, albeit with some evidence of obliterative bronchiolitis. These data suggest that the class II disparity inherent in a fully mismatched transplant increases the requirement for tolerance induction.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica , Transplante de Pulmão/imunologia , Animais , Rejeição de Enxerto/patologia , Transplante de Pulmão/patologia , Complexo Principal de Histocompatibilidade , Modelos Animais , Suínos , Porco Miniatura , Transplante Homólogo/imunologiaRESUMO
BACKGROUND: Because there is no clear consensus as to the predictive value of K-ras gene mutation for survival in patients with lung cancer, we examined the occurrence of K-ras mutations in a large, prospective case series of non-small-cell lung cancer (NSCLC). Our goals were to define the patient characteristics associated with K-ras mutation and to determine whether mutation of this gene might be a biomarker of patient prognosis. METHODS: Consecutive, newly diagnosed patients with lung cancer treated with potentially curative resection over a 4-year period were recruited for study. The mutation status of K-ras codon 12 in each patient's tumor DNA was determined by means of polymerase chain reaction-restriction fragment length polymorphism analysis of archived pathology specimens. Analyses were restricted to adenocarcinoma. RESULTS: There was a statistically significant association between female sex and K-ras mutation after adjustment for carcinogen exposures (odds ratio = 3.3; 95% confidence interval [CI] = 1.3-7.9); mutations were found only in smokers. Comparison of Kaplan-Meier curves indicated a strong association between K-ras mutation and decreased patient survival (two-sided P =.009); analysis stratified by pathologic staging groups revealed that this association was statistically significant only for stage I tumors (two-sided P =.002). Cox proportional hazards modeling indicated that K-ras codon 12 mutation was a statistically significant predictor of patient survival, after adjustment for the effects of age, sex, and stage (risk ratio = 1.8; 95% CI = 1.1-3.1). CONCLUSIONS: After adjustment for environmental exposures, non-small-cell lung tumors in women appear to be more likely than those in men to harbor K-ras mutations, suggesting a possible role of estrogen exposure in either the initiation or the selection of K-ras mutant clones in adenocarcinoma. In addition, our data suggest that K-ras codon 12 mutation is a marker of aggressive NSCLC, as evidenced by its association with decreased patient survival, particularly for early-stage disease.