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BACKGROUND: The coronal plane alignment of the knee (CPAK) classification system divides coronal knee anatomy into 9 phenotypes, suggesting different soft tissue balancing is needed for optimal outcomes. We investigated the interplay between CPAK phenotypes and gap stress curves throughout the knee's range of motion, aiming to understand their impact on total knee arthroplasty balancing. METHODS: There were 1,112 total knee arthroplasty cases from 2 imageless robotic assisted navigation systems using posterior stabilized implants that were classified into CPAK phenotypes. Medial and lateral initial gap values were measured throughout the knee flexion-extension arc, gap curve morphologies were generated, and mediolateral (ML) gap balance was calculated for each phenotype. The most common phenotypes were included in this study, CPAK I to VI. RESULTS: Each phenotype exhibited a distinct gap curve morphology. Type I maintained the largest ML gap difference (-3.6 to -2.1), with the medial compartment tightest in extension. Type II showed relative laxity in the lateral compartment compared to the medial gap (-1.0 to -1.9), with the medial compartment tightening through flexion. Type III had a looser medial and tighter lateral compartment in extension that inverts to a tighter medial compartment in deep flexion (2.1 to -0.8), while Type IV showed a decreasing compartment difference with increased flexion (-3.7 to 0.6). Type V had fluctuating tightness (-0.6 to 1.8), and Type VI had the medial compartment tightening more with flexion (0.6 to 1.8). CONCLUSIONS: The distinct stress curves and ML gap behavior provide a "fingerprint" for each corresponding CPAK phenotype. Investigating these morphologies can help determine the best phenotype-specific treatments, including alignment strategy, implant selection, and gap balance, for optimal functional and patient outcomes.
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BACKGROUND: The utilization of a different manufacturer for the prosthetic femoral head and the polyethylene insert in dual mobility (DM) for total hip arthroplasty (THA) may be necessary, especially in the revision setting. However, there is no data in the literature about this application. This study evaluated the outcomes of mixed manufacturer components, with the hypothesis that there would be no difference in measured outcomes compared to matched components. METHODS: The DM articulations implanted during THA revision were retrospectively reviewed from 2011 to 2017. The study group was then stratified into 2 cohorts: matching components or mixed components. Of 130 hips included in the study with DM articulations with average follow-up of 7 years, 103 had mixed and 27 had matching manufacturer components. Rates of all cause reoperation and revision, intraprosthetic dislocation, dislocation, and aseptic loosening were compared using Chi-squared and Fisher's exact test; survival analysis was also performed. RESULTS: Matched and mixed manufacturer implants had no significant difference between all cause reoperation (33 versus 25.2%), dislocation (14.8 versus 7.7%), and aseptic loosening (3.7 versus 3.9%), respectively. Higher rates of intraprosthetic dislocation (11 versus 0.97%) were observed in the matching component cohort. Survival analysis showed similar outcomes at 2, 5, and 10 years. CONCLUSIONS: Mixed-component DM articulations show similar results compared to matching components. The off-label use of mixed manufacture DM articulation in THA is a feasible and safe option in the correct patient. Furthermore, when encountering a well-fixed femoral stem or acetabular shell, the use of a mixed component DM articulations may reduce the morbidity for the patient and prevent revision of all components.
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Acetábulo , Artroplastia de Quadril , Prótese de Quadril , Desenho de Prótese , Falha de Prótese , Reoperação , Humanos , Artroplastia de Quadril/instrumentação , Artroplastia de Quadril/métodos , Feminino , Masculino , Estudos Retrospectivos , Reoperação/estatística & dados numéricos , Idoso , Pessoa de Meia-Idade , Acetábulo/cirurgia , Resultado do Tratamento , Polietileno , Idoso de 80 Anos ou mais , Fêmur/cirurgia , Adulto , SeguimentosRESUMO
BACKGROUND: Valgus knee deformity is observed in nearly 10% of patients undergoing total knee arthroplasty (TKA). The degree of polyethylene constraint required to balance a valgus knee remains controversial, and historically, posterior-stabilized (PS) designs have been favored. This study evaluated the survivorship of TKA done in valgus knees based on implant design and specifically compared posterior-stabilized (PS) and ultracongruent (UC) liners. METHODS: A total of 549 primary TKAs performed on valgus knees by fellowship-trained arthroplasty surgeons from 2013 to 2019 were reviewed. Demographics, comorbidities, degrees of preoperative deformity, implants used, and all-cause revisions until final follow-up were recorded. Cox regression analyses evaluated survival to all-cause revision in each cohort. The mean follow-up was 4.9 years (range, 2 to 9). RESULTS: There were 403 UC liners compared to 146 PS liners. There was no difference in patient age (68 versus 67 years; P = .30), body mass index (30.9 versus 30.4; P = .36), or degree of deformity (8.6 versus 8.8 degrees; P = .75) between the cohorts. At final follow-up, there were 5 revisions in the PS cohort (3.4%) versus 11 revisions in the UC cohort (2.7%) (P = .90). The most common reason for revision in both cohorts was periprosthetic joint infection (4 PS; 8 UC). Multivariable regression analyses controlling for age, body mass index, Elixhauser comorbidity score, sex, and degree of deformity demonstrated UC polyethylene liners were not associated with revision (hazard ratio 0.76; 95% confidence interval [CI] 0.26 to 2.21; P = .62). There was no difference in eight-year survivorship to all-cause revision, including aseptic and septic failure. CONCLUSIONS: Alternative polyethylene liners from the historically utilized PS liners for TKA for valgus deformity did not reduce survivorship. With modern polyethylene designs, UC inserts can be utilized for this deformity without increasing the risk of failure.
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Artroplastia do Joelho , Prótese do Joelho , Polietileno , Desenho de Prótese , Falha de Prótese , Reoperação , Humanos , Artroplastia do Joelho/instrumentação , Idoso , Masculino , Feminino , Pessoa de Meia-Idade , Reoperação/estatística & dados numéricos , Articulação do Joelho/cirurgia , Estudos Retrospectivos , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: The clinical impact of the surgical approach in total hip arthroplasty (THA) has been widely reviewed. This study evaluated the total encounter and 90-day costs of THA for 2 surgical approaches (posterior [P] and direct anterior [DA]) in 1 tertiary health system. METHODS: This is a retrospective review of 2,101 THAs (1,092 P and 1,009 DA) by 4 surgeons (2 with the highest volume of DA and P, respectively) from 2017 to 2022 at 1 academic center. Demographics, comorbidities, operative time, length of hospital stay, 90-day hospital returns, and complications were compared. The total encounter cost and 90-day postoperative cost were itemized. Multivariable regression analyses evaluated associations with increased cost at each time point. RESULTS: The DA cohort had a higher median encounter cost ($8,348.66 versus 7,332.42, P < .01), resulting from higher intraoperative (P < .01) and radiology (P < .01) expenses. Regression analyses demonstrated the DA was independently associated with increased encounter costs (odds ratio 1.1; 95% confidence interval 1.1 to 1.1; P < .01). There was a higher incidence of 90-day emergency department visits in the DA cohort (16 versus 12%, P = .02), with a trend toward increased readmissions. There was no difference in 90-day reoperations. Median 90-day cost was higher in the DA cohort ($126.99 versus 0.00, P < .01), and regression analyses demonstrated the DA had an association with increased 90-day cost (odds ratio 2.2; 95% confidence interval 1.5 to 3.0; P < .01). CONCLUSIONS: Despite a younger patient population, the DA was independently associated with increased encounter and 90-day costs in a single academic hospital system. This study may underestimate the cost difference, as capital costs such as specialized tables were not analyzed.
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Artroplastia de Quadril , Tempo de Internação , Humanos , Artroplastia de Quadril/economia , Masculino , Feminino , Estudos Retrospectivos , Pessoa de Meia-Idade , Idoso , Tempo de Internação/economia , Tempo de Internação/estatística & dados numéricos , Duração da Cirurgia , Complicações Pós-Operatórias/economia , Complicações Pós-Operatórias/epidemiologia , Readmissão do Paciente/economia , Readmissão do Paciente/estatística & dados numéricos , AdultoRESUMO
OBJECTIVE: The primary purpose was to determine how trust changes over time when automation reliability increases or decreases. A secondary purpose was to determine how task-specific self-confidence is associated with trust and reliability level. BACKGROUND: Both overtrust and undertrust can be detrimental to system performance; therefore, the temporal dynamics of trust with changing reliability level need to be explored. METHOD: Two experiments used a dominant-color identification task, where automation provided a recommendation to users, with the reliability of the recommendation changing over 300 trials. In Experiment 1, two groups of participants interacted with the system: one group started with a 50% reliable system which increased to 100%, while the other used a system that decreased from 100% to 50%. Experiment 2 included a group where automation reliability increased from 70% to 100%. RESULTS: Trust was initially high in the decreasing group and then declined as reliability level decreased; however, trust also declined in the 50% increasing reliability group. Furthermore, when user self-confidence increased, automation reliability had a greater influence on trust. In Experiment 2, the 70% increasing reliability group showed increased trust in the system. CONCLUSION: Trust does not always track the reliability of automated systems; in particular, it is difficult for trust to recover once the user has interacted with a low reliability system. APPLICATIONS: This study provides initial evidence into the dynamics of trust for automation that gets better over time suggesting that users should only start interacting with automation when it is sufficiently reliable.
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BACKGROUND: Blinatumomab, a bispecific monoclonal antibody construct that enables CD3-positive T cells to recognize and eliminate CD19-positive acute lymphoblastic leukemia (ALL) blasts, was approved for use in patients with relapsed or refractory B-cell precursor ALL on the basis of single-group trials that showed efficacy and manageable toxic effects. METHODS: In this multi-institutional phase 3 trial, we randomly assigned adults with heavily pretreated B-cell precursor ALL, in a 2:1 ratio, to receive either blinatumomab or standard-of-care chemotherapy. The primary end point was overall survival. RESULTS: Of the 405 patients who were randomly assigned to receive blinatumomab (271 patients) or chemotherapy (134 patients), 376 patients received at least one dose. Overall survival was significantly longer in the blinatumomab group than in the chemotherapy group. The median overall survival was 7.7 months in the blinatumomab group and 4.0 months in the chemotherapy group (hazard ratio for death with blinatumomab vs. chemotherapy, 0.71; 95% confidence interval [CI], 0.55 to 0.93; P=0.01). Remission rates within 12 weeks after treatment initiation were significantly higher in the blinatumomab group than in the chemotherapy group, both with respect to complete remission with full hematologic recovery (34% vs. 16%, P<0.001) and with respect to complete remission with full, partial, or incomplete hematologic recovery (44% vs. 25%, P<0.001). Treatment with blinatumomab resulted in a higher rate of event-free survival than that with chemotherapy (6-month estimates, 31% vs. 12%; hazard ratio for an event of relapse after achieving a complete remission with full, partial, or incomplete hematologic recovery, or death, 0.55; 95% CI, 0.43 to 0.71; P<0.001), as well as a longer median duration of remission (7.3 vs. 4.6 months). A total of 24% of the patients in each treatment group underwent allogeneic stem-cell transplantation. Adverse events of grade 3 or higher were reported in 87% of the patients in the blinatumomab group and in 92% of the patients in the chemotherapy group. CONCLUSIONS: Treatment with blinatumomab resulted in significantly longer overall survival than chemotherapy among adult patients with relapsed or refractory B-cell precursor ALL. (Funded by Amgen; TOWER ClinicalTrials.gov number, NCT02013167 .).
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Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Biespecíficos/efeitos adversos , Antineoplásicos/efeitos adversos , Terapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Sepse/induzido quimicamente , Transplante de Células-Tronco , Análise de Sobrevida , Transplante Homólogo , Adulto JovemRESUMO
The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.
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Epitopos Imunodominantes/metabolismo , Imunoterapia Adotiva/métodos , Antígeno MART-1/metabolismo , Melanoma/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Aminoácidos , Apresentação de Antígeno , Sítios de Ligação , Células Cultivadas , Células Clonais , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Ativação Linfocitária , Antígeno MART-1/química , Melanoma/terapia , Peptídeos/química , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/transplanteRESUMO
Human γδ T cells display potent responses to pathogens and malignancies. Of particular interest are those expressing a γδ T-cell receptor (TCR) incorporating TCRδ-chain variable-region-2 [Vδ2(+)], which are activated by pathogen-derived phosphoantigens (pAgs), or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Once activated, Vδ2(+) T cells exhibit multiple effector functions that have made them attractive candidates for immunotherapy. Despite this, clinical trials have reported mixed patient responses, highlighting a need for better understanding of Vδ2(+) T-cell biology. Here, we reveal previously unappreciated functional heterogeneity between the Vδ2(+) T-cell compartments of 63 healthy individuals. In this cohort, we identify distinct "Vδ2 profiles" that are stable over time; that do not correlate with age, gender, or history of phosphoantigen activation; and that develop after leaving the thymus. Multiple analyses suggest these Vδ2 profiles consist of variable proportions of two dominant but contrasting Vδ2(+) T-cell subsets that have divergent transcriptional programs and that display mechanistically distinct cytotoxic potentials. Importantly, an individual's Vδ2 profile predicts defined effector capacities, demonstrated by contrasting mechanisms and efficiencies of killing of a range of tumor cell lines. In short, these data support patient stratification to identify individuals with Vδ2 profiles that have effector mechanisms compatible with tumor killing and suggest that tailored Vδ2-profile-specific activation protocols may maximize the chances of future treatment success.
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Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Adolescente , Adulto , Idoso , Receptor 1 de Quimiocina CX3C/metabolismo , Criança , Pré-Escolar , Citotoxicidade Imunológica , Feminino , Perfilação da Expressão Gênica , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T , Voluntários Saudáveis , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores CCR6/metabolismo , Adulto JovemRESUMO
OBJECTIVES: Suicide is an Aboriginal and Torres Strait Islander (hereafter 'Indigenous') population health issue. Over 2015-2016, the Aboriginal and Torres Strait Islander Suicide Prevention Project (ATSISPEP) aimed to identify success factors in Indigenous suicide prevention. CONCLUSIONS: For non-Indigenous practitioners working with indigenous clients at risk of suicide, ATSISPEP identified important considerations to make treatment more effective. The start is acknowledging the differences in the historical, cultural, political, social and economic experiences of Indigenous peoples, and their greater exposure to trauma, psychological distress and risks to mental health. These mental health difficulties are specific and more prevalent amongst Indigenous peoples and communities due to the ongoing impacts of colonisation in Australia including a range of social determinants impacting on the well-being of Indigenous peoples today. Working effectively with Indigenous clients also includes being able to establish culturally safe work environments, and the ability of non-Indigenous practitioners to work in a culturally competent and trauma-informed manner. There are also considerations regarding time protocols and client follow-up. Further, postvention responses might be required. Supporting selective suicide prevention activity among younger people (and other groups at increased risk) and community-level work is an important complement to working with Indigenous individuals at risk of suicide.
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Assistência à Saúde Culturalmente Competente , Transtorno Depressivo/etnologia , Serviços de Saúde Mental , Havaiano Nativo ou Outro Ilhéu do Pacífico/etnologia , Trauma Psicológico/etnologia , Estresse Psicológico/etnologia , Prevenção do Suicídio , Suicídio/etnologia , Adolescente , Adulto , Austrália/etnologia , Transtorno Depressivo/terapia , Humanos , Trauma Psicológico/terapia , Estresse Psicológico/terapia , Adulto JovemRESUMO
The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.
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Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Membrana Celular/metabolismo , Humanos , Ativação Linfocitária/imunologia , Mutação/genética , Peptídeos/metabolismoRESUMO
Fluorochrome-conjugated peptide-MHC (pMHC) class I multimers are staple components of the immunologist's toolbox, enabling reliable quantification and analysis of Ag-specific CD8(+) T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4(+) T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II-bound peptides, can enhance TCR-pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4(+) T cells, highlighting an unappreciated feature of TCR-pMHC-II interactions.
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Antígenos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Separação Celular/métodos , Antígenos HLA/imunologia , Fragmentos de Peptídeos/metabolismo , Linhagem Celular , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Ativação Linfocitária , Ligação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.
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Citometria de Fluxo/métodos , Imunofluorescência/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Coloração e Rotulagem/métodos , Linfócitos T/imunologia , Anticorpos/química , Anticorpos/imunologia , Células Cultivadas , Corantes Fluorescentes/química , Humanos , Ficocianina/química , Ligação Proteica/imunologia , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T/citologiaRESUMO
BACKGROUND: Patient and consumer access to eHealth information is of crucial importance because of its role in patient-centered medicine and to improve knowledge about general aspects of health and medical topics. OBJECTIVES: The objectives were to analyze and compare eHealth search patterns in a private (United States) and a public (United Kingdom) health care market. METHODS: A new taxonomy of eHealth websites is proposed to organize the largest eHealth websites. An online measurement framework is developed that provides a precise and detailed measurement system. Online panel data are used to accurately track and analyze detailed search behavior across 100 of the largest eHealth websites in the US and UK health care markets. RESULTS: The health, medical, and lifestyle categories account for approximately 90% of online activity, and e-pharmacies, social media, and professional categories account for the remaining 10% of online activity. Overall search penetration of eHealth websites is significantly higher in the private (United States) than the public market (United Kingdom). Almost twice the number of eHealth users in the private market have adopted online search in the health and lifestyle categories and also spend more time per website than those in the public market. The use of medical websites for specific conditions is almost identical in both markets. The allocation of search effort across categories is similar in both the markets. For all categories, the vast majority of eHealth users only access one website within each category. Those that conduct a search of two or more websites display very narrow search patterns. All users spend relatively little time on eHealth, that is, 3-7 minutes per website. CONCLUSIONS: The proposed online measurement framework exploits online panel data to provide a powerful and objective method of analyzing and exploring eHealth behavior. The private health care system does appear to have an influence on eHealth search behavior in terms of search penetration and time spent per website in the health and lifestyle categories. Two explanations are offered: (1) the personal incentive of medical costs in the private market incentivizes users to conduct online search; and (2) health care information is more easily accessible through health care professionals in the United Kingdom compared with the United States. However, the use of medical websites is almost identical, suggesting that patients interested in a specific condition have a motivation to search and evaluate health information, irrespective of the health care market. The relatively low level of search in terms of the number of websites accessed and the average time per website raise important questions about the actual level of patient informedness in both the markets. Areas for future research are outlined.
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Setor de Assistência à Saúde/normas , Informática Médica/métodos , Telemedicina/estatística & dados numéricos , Humanos , Internet , Mídias Sociais , Estados UnidosRESUMO
MHC anchor residue-modified "heteroclitic" peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26-35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to "pull" the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation.
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Alanina/química , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/química , Leucina/química , Antígeno MART-1/química , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Cristalografia por Raios X , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Leucina/genética , Antígeno MART-1/genética , Antígeno MART-1/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
αß T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short "foreign" peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.
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Regiões Determinantes de Complementaridade/metabolismo , Antígenos HLA/metabolismo , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Cristalografia por Raios X , Antígenos HLA/química , Antígenos HLA/genética , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.