RESUMO
In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.
Assuntos
Transplante de Coração , Xenoenxertos , Transplante Heterólogo , Humanos , Animais , Suínos , Masculino , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/genética , Proteômica , Metabolômica , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/imunologia , Transcriptoma , Perfilação da Expressão Gênica , Linfócitos T/imunologia , Linfócitos T/metabolismo , Lipidômica , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , MultiômicaRESUMO
Tacrolimus metabolism is heavily influenced by the CYP3A5 genotype, which varies widely among African Americans (AA). We aimed to assess the performance of a published genotype-informed tacrolimus dosing model in an independent set of adult AA kidney transplant (KTx) recipients. CYP3A5 genotypes were obtained for all AA KTx recipients (n = 232) from 2010 to 2019 who met inclusion criteria at a single transplant center in Philadelphia, Pennsylvania, USA. Medical record data were used to calculate predicted tacrolimus clearance using the published AA KTx dosing equation and two modified iterations. Observed and model-predicted trough levels were compared at 3 days, 3 months, and 6 months post-transplant. The mean prediction error at day 3 post-transplant was 3.05 ng/mL, indicating that the model tended to overpredict the tacrolimus trough. This bias improved over time to 1.36 and 0.78 ng/mL at 3 and 6 months post-transplant, respectively. Mean absolute prediction error-a marker of model precision-improved with time to 2.33 ng/mL at 6 months. Limiting genotype data in the model decreased bias and improved precision. The bias and precision of the published model improved over time and were comparable to studies in previous cohorts. The overprediction observed by the published model may represent overfitting to the initial cohort, possibly limiting generalizability.
Assuntos
Negro ou Afro-Americano , Citocromo P-450 CYP3A , Genótipo , Imunossupressores , Transplante de Rim , Tacrolimo , Humanos , Tacrolimo/farmacocinética , Tacrolimo/administração & dosagem , Negro ou Afro-Americano/genética , Masculino , Feminino , Pessoa de Meia-Idade , Citocromo P-450 CYP3A/genética , Imunossupressores/farmacocinética , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Estudos Retrospectivos , Adulto , Transplantados , Idoso , Modelos BiológicosRESUMO
BACKGROUND: Oral microbiome sequencing has revealed key links between microbiome dysfunction and dental caries. However, these efforts have largely focused on Western populations, with few studies on the Middle Eastern communities. The current study aimed to identify the composition and abundance of the oral microbiota in saliva samples of children with different caries levels using machine learning approaches. METHODS: Oral microbiota composition and abundance were identified in 250 Saudi participants with high dental caries and 150 with low dental caries using 16 S rRNA sequencing on a NextSeq 2000 SP flow cell (Illumina, CA) using 250 bp paired-end reads, and attempted to build a classifier using random forest models to assist in the early detection of caries. RESULTS: The ADONIS test results indicate that there was no significant association between sex and Bray-Curtis dissimilarity (p ~ 0.93), but there was a significant association with dental caries status (p ~ 0.001). Using an alpha level of 0.05, five differentially abundant operational taxonomic units (OTUs) were identified between males and females as the main effect along with four differentially abundant OTUs between high and low dental caries. The mean metrics for the optimal hyperparameter combination using the model with only differentially abundant OTUs were: Accuracy (0.701); Matthew's correlation coefficient (0.0509); AUC (0.517) and F1 score (0.821) while the mean metrics for random forest model using all OTUs were:0.675; 0.054; 0.611 and 0.796 respectively. CONCLUSION: The assessment of oral microbiota samples in a representative Saudi Arabian population for high and low metrics of dental caries yields signatures of abundances and diversity.
Assuntos
Cárie Dentária , Microbiota , Masculino , Criança , Feminino , Humanos , Cárie Dentária/genética , Arábia Saudita , RNA Ribossômico 16S/genética , Microbiota/genética , SalivaRESUMO
BACKGROUND: Crohn's diseases and ulcerative colitis, both of which are chronic immune-mediated disorders of the gastrointestinal tract are major contributors to the overarching Inflammatory bowel diseases. It has become increasingly evident that the pathological processes of IBDs results from interactions between genetic and environmental factors, which can skew immune responses against normal intestinal flora. METHODS: The aim of this study is to assess and analyze the taxa diversity and relative abundances in CD and UC in the Saudi population. We utilized a sequencing strategy that targets all variable regions in the 16 S rRNA gene using the Swift Amplicon 16 S rRNA Panel on Illumina NovaSeq 6000. RESULTS: The composition of stool 16 S rRNA was analyzed from 219 patients with inflammatory bowel disease and from 124 healthy controls. We quantified the abundance of microbial communities to examine any significant differences between subpopulations of samples. At the genus level, two genera in particular, Veillonella and Lachnoclostridium showed significant association with CD versus controls. There were significant differences between subjects with CD versus UC, with the top differential genera spanning Akkermansia, Harryflintia, Maegamonas and Phascolarctobacterium. Furthermore, statistically significant taxa diversity in microbiome composition was observed within the UC and CD groups. CONCLUSIONS: In conclusion we have shown that there are significant differences in gut microbiota between UC, CD and controls in a Saudi Arabian inflammatory bowel disease cohort. This reinforces the need for further studies in large populations that are ethnically and geographically diverse. In addition, our results show the potential to develop classifiers that may have add additional richness of context to clinical diagnosis of UC and CD with larger inflammatory bowel disease cohorts.
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Colite Ulcerativa , Doença de Crohn , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Humanos , Microbioma Gastrointestinal/genética , Arábia Saudita , Doenças Inflamatórias Intestinais/microbiologia , Colite Ulcerativa/microbiologia , Doença de Crohn/microbiologiaRESUMO
Ischemic stroke represents a significant societal burden across the globe. Rare high penetrant monogenic variants and less pathogenic common single nucleotide polymorphisms (SNPs) have been described as being associated with risk of diseases. Genetic studies in Saudi Arabian patients offer a greater opportunity to detect rare high penetrant mutations enriched in these consanguineous populations. We performed whole exome sequencing on 387 ischemic stroke subjects from Saudi Arabian hospital networks with up to 20,230 controls from the Saudi Human Genome Project and performed gene burden analyses of variants in 177 a priori loci derived from knowledge-driven curation of monogenic and genome-wide association studies of stroke. Using gene-burden analyses, we observed significant associations in numerous loci under autosomal dominant and/or recessive modelling. Stroke subjects with modified Rankin Scale (mRSs) above 3 were found to carry greater cumulative polygenic risk score (PRS) from rare variants in stroke genes (standardized PRS mean > 0) compared to the population average (standardized PRS mean = 0). However, patients with mRS of 3 or lower had lower cumulative genetic risk from rare variants in stroke genes (OR (95%CI) = 1.79 (1.29-2.49), p = 0.0005), with the means of standardized PRS at or lower than 0. In conclusion, gene burden testing in Saudi stroke populations reveals a number of statistically significant signals under different disease inheritance models. However, interestingly, stroke subjects with mRS of 3 or lower had lower cumulative genetic risk from rare variants in stroke genes and therefore, determining the potential mRS cutoffs to use for clinical significance may allow risk stratification of this population.
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AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Sequenciamento do Exoma , Arábia Saudita , Estudo de Associação Genômica Ampla , Fatores de Risco , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia , Predisposição Genética para DoençaRESUMO
BACKGROUND: Epilepsy, a serious chronic neurological condition effecting up to 100 million people globally, has clear genetic underpinnings including common and rare variants. In Saudi Arabia, the prevalence of epilepsy is high and caused mainly by perinatal and genetic factors. No whole-exome sequencing (WES) studies have been performed to date in Saudi Arabian epilepsy cohorts. This offers a unique opportunity for the discovery of rare genetic variants impacting this disease as there is a high rate of consanguinity among large tribal pedigrees. RESULTS: We performed WES on 144 individuals diagnosed with epilepsy, to interrogate known epilepsy-related genes for known and functional novel variants. We also used an American College of Medical Genetics (ACMG) guideline-based variant prioritization approach in an attempt to discover putative causative variants. We identified 32 potentially causative pathogenic variants across 30 different genes in 44/144 (30%) of these Saudi epilepsy individuals. We also identified 232 variants of unknown significance (VUS) across 101 different genes in 133/144 (92%) subjects. Strong enrichment of variants of likely pathogenicity was observed in previously described epilepsy-associated loci, and a number of putative pathogenic variants in novel loci are also observed. CONCLUSION: Several putative pathogenic variants in known epilepsy-related loci were identified for the first time in our population, in addition to several potential new loci which may be prioritized for further investigation.
Assuntos
Epilepsia , Exoma , Humanos , Arábia Saudita/epidemiologia , Sequenciamento do Exoma , Exoma/genética , Epilepsia/epidemiologia , Epilepsia/genética , Epilepsia/diagnóstico , Linhagem , Predisposição Genética para DoençaRESUMO
BACKGROUND: Heart transplantation provides a significant improvement in survival and quality of life for patients with end-stage heart disease, however many recipients experience different levels of graft rejection that can be associated with significant morbidities and mortality. Current clinical standard-of-care for the evaluation of heart transplant acute rejection (AR) consists of routine endomyocardial biopsy (EMB) followed by visual assessment by histopathology for immune infiltration and cardiomyocyte damage. We assessed whether the sensitivity and/or specificity of this process could be improved upon by adding RNA sequencing (RNA-seq) of EMBs coupled with histopathological interpretation. METHODS: Up to 6 standard-of-care, or for-cause EMBs, were collected from 26 heart transplant recipients from the prospective observational Clinical Trials of Transplantation (CTOT)-03 study, during the first 12-months post-transplant and subjected to RNA-seq (n = 125 EMBs total). Differential expression and random-forest-based machine learning were applied to develop signatures for classification and prognostication. RESULTS: Leveraging the unique longitudinal nature of this study, we show that transcriptional hallmarks for significant rejection events occur months before the actual event and are not visible using traditional histopathology. Using this information, we identified a prognostic signature for 0R/1R biopsies that with 90% accuracy can predict whether the next biopsy will be 2R/3R. CONCLUSIONS: RNA-seq-based molecular characterization of EMBs shows significant promise for the early detection of cardiac allograft rejection.
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Transplante de Coração , Qualidade de Vida , Aloenxertos , Biópsia , Perfilação da Expressão Gênica , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/genética , Rejeição de Enxerto/patologia , Humanos , Miocárdio/patologia , Complicações Pós-Operatórias/patologia , Prognóstico , Estudos ProspectivosRESUMO
BACKGROUND AND OBJECTIVES: Next generation sequencing (NGS) has promising applications in transfusion medicine. Exome sequencing (ES) is increasingly used in the clinical setting, and blood group interpretation is an additional value that could be extracted from existing data sets. We provide the first release of an open-source software tailored for this purpose and describe its validation with three blood group systems. MATERIALS AND METHODS: The DTM-Tools algorithm was designed and used to analyse 1018 ES NGS files from the ClinSeq® cohort. Predictions were correlated with serology for 5 antigens in a subset of 108 blood samples. Discrepancies were investigated with alternative phenotyping and genotyping methods, including a long-read NGS platform. RESULTS: Of 116 genomic variants queried, those corresponding to 18 known KEL, FY and JK alleles were identified in this cohort. 596 additional exonic variants were identified KEL, ACKR1 and SLC14A1, including 58 predicted frameshifts. Software predictions were validated by serology in 108 participants; one case in the FY blood group and three cases in the JK blood group were discrepant. Investigation revealed that these discrepancies resulted from (1) clerical error, (2) serologic failure to detect weak antigenic expression and (3) a frameshift variant absent in blood group databases. CONCLUSION: DTM-Tools can be employed for rapid Kell, Duffy and Kidd blood group antigen prediction from existing ES data sets; for discrepancies detected in the validation data set, software predictions proved accurate. DTM-Tools is open-source and in continuous development.
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Alelos , Antígenos de Grupos Sanguíneos/análise , Antígenos de Grupos Sanguíneos/genética , Sequenciamento do Exoma/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Sistema do Grupo Sanguíneo Duffy/genética , Variação Genética , Técnicas de Genotipagem , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Metaloendopeptidases/genética , Receptores de Superfície Celular/genética , Transportadores de UreiaRESUMO
BACKGROUND: Ductal adenocarcinoma of the prostate is an aggressive subtype, with high rates of biochemical recurrence and overall poor prognosis. It is frequently found coincident with conventional acinar adenocarcinoma. The genomic features driving evolution to its ductal histology and the biology associated with its poor prognosis remain unknown. OBJECTIVE: To characterize genomic features distinguishing ductal adenocarcinoma from coincident acinar adenocarcinoma foci from the same patient. DESIGN, SETTING, AND PARTICIPANTS: Ten patients with coincident acinar and ductal prostate cancer underwent prostatectomy. Laser microdissection was used to separately isolate acinar and ductal foci. DNA and RNA were extracted, and used for integrative genomic and transcriptomic analyses. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Single nucleotide mutations, small indels, copy number estimates, and expression profiles were identified. Phylogenetic relationships between coincident foci were determined, and characteristics distinguishing ductal from acinar foci were identified. RESULTS AND LIMITATIONS: Exome sequencing, copy number estimates, and fusion genes demonstrated coincident ductal and acinar adenocarcinoma diverged from a common progenitor, yet they harbored distinct alterations unique to each focus. AR expression and activity were similar in both histologies. Nine of 10 cases had mutually exclusive CTNNB1 hotspot mutations or phosphatase and tensin homolog (PTEN) alterations in the ductal component, and these were absent in the acinar foci. These alterations were associated with changes in expression in WNT- and PI3K-pathway genes. CONCLUSIONS: Coincident ductal and acinar histologies typically are clonally related and thus arise from the same cell of origin. Ductal foci are enriched for cases with either a CTNNB1 hotspot mutation or a PTEN alteration, and are associated with WNT- or PI3K-pathway activation. These alterations are mutually exclusive and may represent distinct subtypes. PATIENT SUMMARY: The aggressive subtype ductal adenocarcinoma is closely related to conventional acinar prostate cancer. Ductal foci contain additional alterations, however, leading to frequent activation of two targetable pathways.
Assuntos
Carcinoma de Células Acinares/patologia , Carcinoma Ductal/patologia , Neoplasias Primárias Múltiplas/patologia , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/patologia , beta Catenina/genética , Idoso , Carcinoma de Células Acinares/genética , Carcinoma de Células Acinares/cirurgia , Carcinoma Ductal/genética , Carcinoma Ductal/cirurgia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/cirurgia , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Sequenciamento do ExomaRESUMO
INTRODUCTION: Securing an airway in maxillofacial injuries remains a challenge and is an important objective on the part of a maxillofacial surgeon to thoroughly understand its management. PURPOSE: The aim of this study was to evaluate the efficacy and complications of submental intubation in the management of midfacial/panfacial trauma patients where oro-endotracheal or naso-endotracheal intubation is contraindicated and tracheostomy can be avoided. PATIENTS AND METHODS: Twenty patients with maxillofacial injuries were selected for submental intubation who were admitted in Kamineni Hospital, Narketpally, Nalgonda during a 2 year period (2010-2012). The parameters used to assess the efficacy were; restoration of the occlusion, duration of the surgery, presence of scar, presence of infection, damage to vital structures or any post-operative salivary fistula. RESULTS: Submental intubation allowed reduction and fixation of all fractures without the interference of the tube during surgical procedure in all of the patients. There were no intra-operative complications and none of the patients required post-operative ventilation. There were no significant post-operative complications. However, in one of the cases (case 4) infection of submental wound was seen and in another case (case 11) salivary fistula was formed. Both the cases were appropriately managed without any difficulty. The submental scar was well accepted by all the patients. CONCLUSION: Submental intubation is a safe and extremely useful procedure in severe maxillofacial injuries. It presents a low incidence of operative and post operative complications. It allows both the surgeon and the anaesthetist to deliver a better quality of patient care.
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INTRODUCTION: Fibro-osseous lesions are a diverse group of bone disorders and include developmental, reactive or dysplastic diseases and neoplasms. They share overlapping clinical, radiographic and histopathologic features and demonstrate a wide range of biological behaviour. AIM: To evaluate the characteristics, treatment and outcome of benign fibro-osseous lesions of the jaws. PATIENTS AND METHOD: All patients with fibro-osseous lesions of the jaws treated at the department of Oral and Maxillofacial Surgery of the Kamineni Institute of Dental Sciences from 2007 to 2013 were included in this study. RESULTS: Six males and four females were treated. Juvenile ossifying fibroma was most often encountered (40 %), and the mandible was the most frequent location (70 %). Main clinical feature in most of the cases was a painless expansile swelling with facial asymmetry, and radiologically mixed (radiolucent and radiopaque lesions) were seen in majority of cases. All cases were surgically treated and histopathologically confirmed. Segmental ostectomy was performed in six cases; maxillectomy was done in one case and excision along with margin in three cases. Mean follow-up was of 3.3 years with no recurrence. CONCLUSIONS: Fibro-osseous lesions, although sharing similar microscopic features, exhibit a variety of clinical behavior rendering their treatment highly individualized. Radical treatment is the choice to achieve an outcome without recurrence.