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Currently, no disease-modifying therapies are approved for osteoarthritis (OA) use. One obstacle to trial success in this field has been our existing endpoints' limited validity and responsiveness. To overcome this impasse, the Foundation for the NIH OA Biomarkers Consortium is focused on investigating biomarkers for a prognostic context of use for subsequent qualification through regulatory agencies. This narrative review describes this activity and the work underway, focusing on the PROGRESS OA study.
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Osteoartrite , Humanos , Osteoartrite/diagnóstico por imagem , Biomarcadores , PrognósticoRESUMO
OBJECTIVE: To investigate a targeted set of biochemical biomarkers as predictors of clinically relevant osteoarthritis (OA) progression. METHODS: Eighteen biomarkers were measured at baseline, 12â months (M) and 24â M in serum (s) and/or urine (u) of cases (n=194) from the OA initiative cohort with knee OA and radiographic and persistent pain worsening from 24 to 48â M and controls (n=406) not meeting both end point criteria. Primary analyses used multivariable regression models to evaluate the association between biomarkers (baseline and time-integrated concentrations (TICs) over 12 and 24â M, transposed to z values) and case status, adjusted for age, sex, body mass index, race, baseline radiographic joint space width, Kellgren-Lawrence grade, pain and pain medication use. For biomarkers with adjusted p<0.1, the c-statistic (area under the curve (AUC)), net reclassification index and the integrated discrimination improvement index were used to further select for hierarchical multivariable discriminative analysis and to determine the most predictive and parsimonious model. RESULTS: The 24â M TIC of eight biomarkers significantly predicted case status (ORs per 1 SD change in biomarker): sCTXI 1.28, sHA 1.22, sNTXI 1.25, uC2C-HUSA 1.27, uCTXII, 1.37, uNTXI 1.29, uCTXIα 1.32, uCTXIß 1.27. 24 M TIC of uCTXII (1.47-1.72) and uC2C-Human Urine Sandwich Assay (HUSA) (1.36-1.50) both predicted individual group status (pain worsening, joint space loss and their combination). The most predictive and parsimonious combinatorial model for case status consisted of 24 M TIC uCTXII, sHA and sNTXI (AUC 0.667 adjusted). Baseline uCTXII and uCTXIα both significantly predicted case status (OR 1.29 and 1.20, respectively). CONCLUSIONS: Several systemic candidate biomarkers hold promise as predictors of pain and structural worsening of OA.
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Biomarcadores/metabolismo , Osteoartrite do Joelho/diagnóstico , Idoso , Biomarcadores/sangue , Biomarcadores/urina , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico por imagem , Medição da Dor/métodos , Valor Preditivo dos Testes , Prognóstico , Curva ROC , Radiografia , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
One important component in determining the benefits and harms of medical interventions is the use of well-defined and reliable outcome assessments as endpoints in clinical trials. Improving endpoints can better define patient benefits, allowing more accurate assessment of drug efficacy and more informed benefit-vs-risk decisions; another potential plus is facilitating efficient trial design. Since our first report in 2012, 2 Foundation for the National Institutes of Health Biomarkers Consortium Project Teams have continued to develop outcome assessments for potential uses as endpoints in registrational clinical trials of community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections. In addition, the teams have initiated similar work in the indications of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia. This report provides an update on progress to date in these 4 diseases.
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Antibacterianos/uso terapêutico , Ensaios Clínicos como Assunto , Determinação de Ponto Final , Avaliação de Resultados em Cuidados de Saúde , Dermatopatias Bacterianas/tratamento farmacológico , Biomarcadores , Diretrizes para o Planejamento em Saúde , HumanosRESUMO
Despite widespread interest and substantial investment in the adoption of sensor-based digital health technologies (sDHTs) for remote data capture in drug development trials, no drug has been approved based on an sDHT-derived primary endpoint in the United States (US). One reason for this lack of advancement is the complexity of obtaining regulatory endorsement for those endpoints within current US regulatory pathways. The goal of our review is to describe the two choices currently available to pharmaceutical study Sponsors: (i) they may navigate the traditional route of compiling the evidence to support the sDHT-derived endpoint in their investigational new drug (IND) application, requiring specific expertise and substantial resources; or (ii) they may navigate the drug development tool (DDT) pathway with the goal of qualifying their sDHT-derived endpoint as a biomarker or clinical outcome assessment applicable to a broader context of use (COU), either alone or as part of a partnership or consortium. We describe the nuances of each pathway; the evidentiary requirements for supporting an sDHT-derived endpoint and the technology used to capture it; and the impact that an sDHT's regulatory status may have on a Sponsor's decision to use it for data capture. By systematically comparing the IND and DDT pathways, our over-arching goals are to support the increasing deployment of sDHTs within the clinical research setting and help advance regulatory science in the field of digital medicine.
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Background: Digital measures offer an unparalleled opportunity to create a more holistic picture of how people who are patients behave in their real-world environments, thereby establishing a better connection between patients, caregivers, and the clinical evidence used to drive drug development and disease management. Reaching this vision will require achieving a new level of co-creation between the stakeholders who design, develop, use, and make decisions using evidence from digital measures. Summary: In September 2022, the second in a series of meetings hosted by the Swiss Federal Institute of Technology in Zürich, the Foundation for the National Institutes of Health Biomarkers Consortium, and sponsored by Wellcome Trust, entitled "Reverse Engineering of Digital Measures," was held in Zurich, Switzerland, with a broad range of stakeholders sharing their experience across four case studies to examine how patient centricity is essential in shaping development and validation of digital evidence generation tools. Key Messages: In this paper, we discuss progress and the remaining barriers to widespread use of digital measures for evidence generation in clinical development and care delivery. We also present key discussion points and takeaways in order to continue discourse and provide a basis for dissemination and outreach to the wider community and other stakeholders. The work presented here shows us a blueprint for how and why the patient voice can be thoughtfully integrated into digital measure development and that continued multistakeholder engagement is critical for further progress.
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Clinical safety findings remain one of the reasons for attrition of drug candidates during clinical development. Cardiovascular liabilities are not consistently detected in early-stage clinical trials and often become apparent when drugs are administered chronically for extended periods of time. Vital sign data collection outside of the clinic offers an opportunity for deeper physiological characterization of drug candidates and earlier safety signal detection. A working group representing expertise from biopharmaceutical and technology sectors, US Food and Drug Administration (FDA) public-private partnerships, academia, and regulators discussed and presented a remote cardiac monitoring case study at the FNIH Biomarkers Consortium Remote Digital Monitoring for Medical Product Development workshop to examine applicability of the biomarker qualification evidentiary framework by the FDA. This use case examined the components of the framework, including the statement of need, the context of use, the state of the evidence, and the benefit/risk profile. Examination of results from 2 clinical trials deploying 510(k)-cleared devices for remote cardiac data collection demonstrated the need for analytical and clinical validity irrespectively of the regulatory status of a device of interest, emphasizing the importance of data collection method assessment in the context of intended use. Additionally, collection of large amounts of ambulatory data also highlighted the need for new statistical methods and contextual information to enable data interpretation. A wider adoption of this approach for drug development purposes will require collaborations across industry, academia, and regulatory agencies to establish methodologies and supportive data sets to enable data interpretation and decision-making.
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BACKGROUND: Currently, 2 coprimary end points are used by health authorities to determine the effectiveness of therapeutic interventions in patients with Crohn's disease (CD): symptomatic remission (patient-reported outcome assessment) and endoscopic remission (ileocolonoscopy). However, there is lack of accepted biomarkers to facilitate regulatory decision-making in the development of novel therapeutics for the treatment of CD. METHODS: With support from the Helmsley Charitable Trust, Critical Path Institute formed the Crohn's Disease Biomarkers preconsortium (CDBpC) with members from the pharmaceutical industry, academia, and nonprofit organizations to evaluate the CD biomarker landscape. Biomarkers were evaluated based on biological relevance, availability of biomarker assays, and clinical validation data. RESULTS: The CDBpC identified the most critical need as pharmacodynamic/response biomarkers to monitor disease activity in response to therapeutic intervention. Fecal calprotectin (FC) and serum C-reactive protein (CRP) were identified as biomarkers ready for the regulatory qualification process. A number of exploratory biomarkers and potential panels of these biomarkers was also identified for additional development. Given the different factors involved in CD and disease progression, a combination of biomarkers, including inflammatory, tissue injury, genetic, and microbiome-associated biomarkers, will likely have the most utility. CONCLUSIONS: The primary focus of the Inflammatory Bowel Disease Regulatory Science Consortium will be development of exploratory biomarkers and the qualification of FC and CRP for IBD. The Inflammatory Bowel Disease Regulatory Science Consortium, focused on tools to support IBD drug development, will operate in the precompetitive space to share data, biological samples for biomarker testing, and assay information for novel biomarkers.
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Proteína C-Reativa/análise , Tomada de Decisão Clínica/métodos , Doença de Crohn/diagnóstico , Monitoramento de Medicamentos/métodos , Complexo Antígeno L1 Leucocitário/análise , Biomarcadores/análise , Consenso , Doença de Crohn/metabolismo , Doença de Crohn/terapia , Descoberta de Drogas , Fezes/química , Humanos , Reprodutibilidade dos Testes , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Neonatal-onset multisystem inflammatory disease is characterized by fever, urticarial rash, aseptic meningitis, deforming arthropathy, hearing loss, and mental retardation. Many patients have mutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene, encoding cryopyrin, a protein that regulates inflammation. METHODS: We selected 18 patients with neonatal-onset multisystem inflammatory disease (12 with identifiable CIAS1 mutations) to receive anakinra, an interleukin-1-receptor antagonist (1 to 2 mg per kilogram of body weight per day subcutaneously). In 11 patients, anakinra was withdrawn at three months until a flare occurred. The primary end points included changes in scores in a daily diary of symptoms, serum levels of amyloid A and C-reactive protein, and the erythrocyte sedimentation rate from baseline to month 3 and from month 3 until a disease flare. RESULTS: All 18 patients had a rapid response to anakinra, with disappearance of rash. Diary scores improved (P<0.001) and serum amyloid A (from a median of 174 mg to 8 mg per liter), C-reactive protein (from a median of 5.29 mg to 0.34 mg per deciliter), and the erythrocyte sedimentation rate decreased at month 3 (all P<0.001), and remained low at month 6. Magnetic resonance imaging showed improvement in cochlear and leptomeningeal lesions as compared with baseline. Withdrawal of anakinra uniformly resulted in relapse within days; retreatment led to rapid improvement. There were no drug-related serious adverse events. CONCLUSIONS: Daily injections of anakinra markedly improved clinical and laboratory manifestations in patients with neonatal-onset multisystem inflammatory disease, with or without CIAS1 mutations. (ClinicalTrials.gov number, NCT00069329 [ClinicalTrials.gov].).
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Inflamação/tratamento farmacológico , Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/uso terapêutico , Urticária/tratamento farmacológico , Adolescente , Adulto , Proteínas de Transporte/genética , Criança , Pré-Escolar , Feminino , Perda Auditiva/tratamento farmacológico , Humanos , Inflamação/genética , Deficiência Intelectual , Proteína Antagonista do Receptor de Interleucina 1 , Masculino , Meningite/tratamento farmacológico , Mutação , Proteína 3 que Contém Domínio de Pirina da Família NLR , Papiledema/tratamento farmacológico , Sialoglicoproteínas/efeitos adversos , SíndromeRESUMO
Monocyte accumulation in renal allografts is associated with allograft dysfunction. As monocyte influx occurs acutely following reperfusion, we investigated the effect of ischemia-reperfusion injury (IRI) on monocyte colony stimulating factor (m-CSF), a key cytokine in monocyte recruitment. We hypothesized that renal tubule epithelial cells (RTECs) could produce m-CSF in response to IRI, which could in turn promote monocyte activation. Real time PCR was used to measure levels of intragraft m-CSF transcripts in patients during IRI and clinical rejection. Also, m-CSF production by RTECs following IRI simulation in vitro was measured using ELISA. Monocyte expression of CD40 and CD80 was then analyzed using flow cytometry following co-culture with supernatants of RTECs after IRI. Monocyte expression of CD40, CD80 and HLA-DR was then examined following treatment with rh-m-CSF (10, 36, and 100 ng/ml), as was monocyte size and granularity. We found that intragraft m-CSF transcription was significantly increased postreperfusion (P = 0.002) and during clinical rejection (P = 0.002). We also found that RTECs produced m-CSF in response to IRI in vitro (P = 0.036). Monocytes co-cultured with the supernatants of postischemic RTECs became activated as evidenced by increased expression of CD40 and CD80. Also, monocytes treated with recombinant m-CSF assumed an activated phenotype exhibiting increased size, granularity and expression of CD40, CD80, CD86, and HLA-DR, and demonstrating enhanced phagocytic activity. Taken together, we suggest that renal tubular cell derived m-CSF is a stimulus for monocyte activation and may be an important target for control of IRI-associated immune activation.
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Fatores Estimuladores de Colônias/metabolismo , Células Epiteliais/citologia , Transplante de Rim/métodos , Rim/lesões , Rim/patologia , Monócitos/metabolismo , Antígeno B7-1/biossíntese , Antígenos CD40/biossíntese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo/métodos , Antígenos HLA-DR/metabolismo , Humanos , Sistema Imunitário , Isquemia/patologia , Modelos Biológicos , Monócitos/citologia , ReperfusãoRESUMO
The Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium (BC) is a public-private partnership that aims to facilitate drug development with biomarkers across a range of therapeutic areas. The BC is organized to address specific precompetitive biomarker projects, giving participating stakeholders a role in the design and conduct of projects and making the results freely public. Ultimately, the goals of the BC are to accelerate the development of new medicines, inform regulatory decision making, and improve patient care. Here, we describe how the BC works and briefly highlight its accomplishments. The BC has had many notable successful biomarker projects in the past 12 years, including I-SPY2, which has improved clinical trials and biomarker use for breast cancer, and an evidentiary framework for biomarker qualification. Recently, the BC has undergone a strategic expansion of its scope to include related drug development tools along the lines of the Biomarkers, Endpoints, and other Tools (BEST) resource.
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Biomarcadores/química , Desenvolvimento de Medicamentos/legislação & jurisprudência , Descoberta de Drogas/legislação & jurisprudência , National Institutes of Health (U.S.)/legislação & jurisprudência , Tomada de Decisões , Humanos , Parcerias Público-Privadas/legislação & jurisprudência , Estados Unidos , United States Food and Drug Administration/legislação & jurisprudênciaRESUMO
BACKGROUND: Insulinomas are beta-cell tumours characterised by uncontrolled insulin secretion even in the presence of hypoglycaemia. However, the mechanisms allowing such excessive insulin secretion are not known. Insulin secretion can occur only when the beta-cell insulin stores have been replenished by insulin biosynthesis, which is mainly controlled by translation. Such specific translational regulation often involves the 5' untranslated region. We have identified an insulin splice variant in isolated human pancreatic islets of non-diabetic donors that retains 26 bp of intron 1 and thereby changes the 5' untranslated region, but leaves the coding region unchanged. This splice variant has increased translation efficiency in vitro and in vivo compared with native insulin mRNA. However, splice variant expression is less than 1% of native insulin mRNA in normal islets. METHODS: To test whether this splice variant is involved in insulin production by human insulinomas, we extracted RNA from nine laser-captured surgical insulinoma samples and from isolated islets of nine donors who did not have diabetes. We then determined the ratio of splice variant to native insulin mRNA by quantitative real-time RT-PCR. FINDINGS: The mean ratio of the splice variant to native insulin mRNA was increased more than 50-fold in insulinomas compared with normal islets, and this difference was present in all nine human insulinomas. Overexpression of the splice variant therefore seems to be a general characteristic of insulinomas and is estimated to contribute about 90% to insulin synthesis by these tumours. INTERPRETATION: Overexpression of the insulin splice variant with increased translation efficiency in insulinomas might explain how these tumours maintain high levels of insulin synthesis and secretion leading to hyperinsulinaemia-the hallmark of this disease.
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Processamento Alternativo , Insulina/genética , Insulinoma/genética , Neoplasias Pancreáticas/genética , Animais , Linhagem Celular Tumoral , Feminino , Variação Genética , Humanos , Insulina/biossíntese , Insulinoma/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Perioperative lymphocyte depletion induces allograft tolerance in some animal models, but in humans has only been shown to reduce immunosuppressive requirements. Without maintenance immunosuppression, depleted human renal allograft recipients experience rejection characterized by infiltration of the allograft with monocytes and macrophages. T-cell depletion combined with a brief course of deoxyspergualin (DSG), a drug with inhibitory effects on monocytes and macrophages, induces tolerance in nonhuman primates. We therefore performed a trial to determine if lymphocyte depletion with alemtuzumab combined with DSG would induce tolerance in humans. METHODS: Five recipients of live donor kidneys were treated perioperatively with alemtuzumab and DSG and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically, by flow cytometry, and by protocol biopsies analyzed immunohistochemically and with real-time polymerase chain reaction. Results were compared to previously studied patients receiving alemtuzumab alone or standard immunosuppression. RESULTS: Despite profound T-cell depletion and therapeutic DSG dosing, all alemtuzumab/DSG patients developed reversible rejection that was similar in timing, histology, and transcriptional profile to that seen in patients treated with alemtuzumab alone. Chemokine expression was marked prior to and during rejections. CONCLUSIONS: We conclude that treatment with alemtuzumab and DSG does not induce tolerance in humans. Chemokine production may not be adequately suppressed using this approach.
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Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Guanidinas/farmacologia , Imunossupressores/farmacologia , Transplante de Rim/imunologia , Depleção Linfocítica , Tolerância ao Transplante/efeitos dos fármacos , Adulto , Alemtuzumab , Anticorpos Monoclonais Humanizados , Quimiocinas/genética , Quimiocinas/metabolismo , Creatina/sangue , Feminino , Rejeição de Enxerto/metabolismo , Humanos , Rim/citologia , Rim/imunologia , Rim/fisiologia , Linfonodos/citologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linfócitos T/efeitos dos fármacos , Transcrição GênicaAssuntos
Ensaios Clínicos como Assunto/métodos , Infecções por Coronavirus , Monitorização Fisiológica/métodos , Pandemias , Pneumonia Viral , COVID-19 , Ensaios Clínicos como Assunto/legislação & jurisprudência , Descoberta de Drogas , Reposicionamento de Medicamentos , Determinação de Ponto Final , HumanosRESUMO
PURPOSE: We describe the outcome of the Biomarkers Consortium CSF Proteomics Project (where CSF is cerebral spinal fluid), a public-private partnership of government, academia, nonprofit, and industry. The goal of this study was to evaluate a multiplexed MS-based approach for the qualification of candidate Alzheimer's disease (AD) biomarkers using CSF samples from the AD Neuroimaging Initiative. EXPERIMENTAL DESIGN: Reproducibility of sample processing, analytic variability, and ability to detect a variety of analytes of interest were thoroughly investigated. Multiple approaches to statistical analyses assessed whether panel analytes were associated with baseline pathology (mild cognitive impairment (MCI), AD) versus healthy controls or associated with progression for MCI patients, and included (i) univariate association analyses, (ii) univariate prediction models, (iii) exploratory multivariate analyses, and (iv) supervised multivariate analysis. RESULTS: A robust targeted MS-based approach for the qualification of candidate AD biomarkers was developed. The results identified several peptides with potential diagnostic or predictive utility, with the most significant differences observed for the following peptides for differentiating (including peptides from hemoglobin A, hemoglobin B, and superoxide dismutase) or predicting (including peptides from neuronal pentraxin-2, neurosecretory protein VGF (VGF), and secretogranin-2) progression versus nonprogression from MCI to AD. CONCLUSIONS AND CLINICAL RELEVANCE: These data provide potential insights into the biology of CSF in AD and MCI progression and provide a novel tool for AD researchers and clinicians working to improve diagnostic accuracy, evaluation of treatment efficacy, and early diagnosis.
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Doença de Alzheimer/líquido cefalorraquidiano , Bioensaio/métodos , Biomarcadores/líquido cefalorraquidiano , Espectrometria de Massas/métodos , Neuroimagem/métodos , Idoso , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Apolipoproteínas E/líquido cefalorraquidiano , Área Sob a Curva , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/patologia , Progressão da Doença , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Peptídeos/líquido cefalorraquidiano , Peptídeos/química , Análise de Componente Principal , Controle de Qualidade , Reprodutibilidade dos Testes , Estatística como AssuntoRESUMO
As glucose-induced insulin expression is mainly regulated at the translational level, and such regulation often involves the 5'-untranslated region (5'UTR), we examined the human proinsulin gene 5'UTR. RT-PCR and sequencing demonstrated that a proinsulin splice variant (SPV) generated from a cryptic 5'-splice site and retaining the first 26 bp of intron 1 was present in human pancreatic islets from normal donors. The expression of this SPV was metabolically regulated, as shown by quantitative real-time RT-PCR, revealing a more than 10-fold increase in the SPV in isolated human islets incubated at 16.7 mM compared with 1.67 mM glucose. In vitro wheat-germ translation and in vivom transfection studies demonstrated that the altered 5'UTR of the SPV increased translation. The SPV yielded 4-fold more in vitro translated preproinsulin protein than the native proinsulin mRNA, and the SPV 5'UTR inserted upstream from a luciferase reporter gene resulted in a more than 6-fold higher luciferase activity, suggesting enhanced translation in vivo. Retention of the 26 bp changed the proposed secondary RNA structure of the SPV, which may facilitate ribosomal binding and explain the increase in translation efficiency. These results suggest a novel mechanism by which metabolic changes can modulate the expression of 5'UTR SPVs and thereby regulate translation efficiency.
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Processamento Alternativo/genética , Ilhotas Pancreáticas/metabolismo , Proinsulina/genética , Biossíntese de Proteínas/genética , Regiões 5' não Traduzidas/genética , Western Blotting , Células Cultivadas , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Glucose/farmacologia , Humanos , Íntrons/genética , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Proinsulina/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human pancreatic islets are a major focus of diabetes research due to their key role in glucose homeostasis and their potential for transplantation in the treatment of type 1 diabetes. Currently, no comprehensive analysis of baseline or glucose-stimulated islet gene expression is available. Using oligonucleotide microarrays we analyzed isolated intact human islets incubated at low and high glucose. We identified approximately 6000 islet genes, several with clinical implications, as well as a number of glucose-regulated genes. Interestingly, two transforming growth factor beta (TGFbeta) superfamily members were highly regulated by glucose. One of them, PDF, was found to have a very high expression level compared to other TGFbeta superfamily members. Quantitative reverse transcriptase polymerase chain reaction confirmed these results and demonstrated that the highly expressed PDF was approximately 10-fold down- regulated by glucose while other TGFbeta superfamily members and target genes were up-regulated. These results suggest that a highly regulated TGFbeta signaling cascade exists in human islets, and that PDF may play a central role in islet biology. Since TGFbeta is involved in differentiation and immune modulation, this novel pathway may link glucose metabolism, immune response and development of human islets. We report here the first gene expression profile of intact human islets. These and similar analyses will provide better understanding of human islet biology and enhance the development of novel diabetes therapies.
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Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Tiorredoxinas , Fator de Crescimento Transformador beta/metabolismo , Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Neuropeptídeos/genética , Pró-Proteína Convertases , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genéticaRESUMO
Molecular techniques have become a mainstay for most biomedical research. In particular, sensitive methods for gene transcript detection and advanced flow cytometry have been crucial in fostering our understanding of the basic mechanisms promoting allosensitization and adaptive immune regulation. These technologies have been validated in vitro, and in pre-clinical settings, and as such their clinical application is now clearly appropriate. It is becoming increasingly clear that these robust techniques hold much promise to better elucidate human transplant biology, and more importantly, guide clinical decision making with mechanistically-based information. This article will discuss our laboratory's use of several novel technologies, including gene polymorphism analysis, real-time polymerase chain reaction transcript quantification, and multi-color flow cytometry in clinical human renal transplantation. Specific technical methodology will be presented outlining keys for effective clinical application. Clinical correlations will be presented as examples of how these techniques may have clinical relevance. Suggestions for the adaptation of these methods for therapeutic intervention will be given. We propose that clinical transplantation should proceed in close step with modern molecular diagnostics.
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Perfilação da Expressão Gênica/métodos , Transplante de Rim/métodos , Técnicas de Diagnóstico Molecular/métodos , Monitorização Imunológica/métodos , Animais , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/genética , Humanos , Transplante de Rim/tendências , Técnicas de Diagnóstico Molecular/tendências , Monitorização Imunológica/tendênciasRESUMO
BACKGROUND: Lung transplantation is an effective therapy plagued by a high incidence of early graft dysfunction, in part because of reperfusion injury. The optimal preservation solution for lung transplantation is unknown. We performed experiments using an isolated perfused rat lung model to test the effect of lung preservation with three solutions commonly used in clinical practice. METHODS: Lungs were retrieved from Sprague-Dawley rats and flushed with one of three solutions: modified Euro-Collins (MEC), University of Wisconsin (UW), or low potassium dextran and glucose (LPDG), then stored cold for varying periods before reperfusion with Earle's balanced salt solution using the isolated perfused rat lung model. Outcome measures were capillary filtration coefficient (Kfc), wet-to-dry weight ratio, and lung tissue levels of adenine nucleotides and cyclic AMP. RESULTS: All lungs functioned well after 4 hr of storage. By 6 hr, UW-flushed lungs had a lower Kfc than LPDG-flushed lungs. After 8 hr of storage, only UW-flushed lungs had a measurable Kfc. Adenine nucleotide levels were higher in UW-flushed lungs after prolonged storage. Cyclic AMP levels correlated with Kfc in all groups. CONCLUSIONS: Early changes in endothelial permeability seemed to be better attenuated in lungs flushed with UW compared with LPDG or MEC; this was associated with higher amounts of adenine nucleotides. MEC-flushed lungs failed earlier than LPDG-flushed or UW-flushed lungs. The content of the solution may be more important for lung preservation than whether the ionic composition is intracellular or extracellular.
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Nucleotídeos de Adenina/metabolismo , Soluções Hipertônicas/farmacologia , Transplante de Pulmão , Pulmão/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Adenosina/farmacologia , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Alopurinol/farmacologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , AMP Cíclico/metabolismo , Dextranos/farmacologia , Endotélio Vascular/metabolismo , Glucose/farmacologia , Glutationa/farmacologia , Insulina/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Tamanho do Órgão , Rafinose/farmacologia , Ratos , Ratos Sprague-Dawley , ÁguaRESUMO
BACKGROUND: Profound T-cell depletion before allotransplantation with gradual posttransplant T-cell repopulation induces a state of donor-specific immune hyporesponsiveness or tolerance in some animal models. Alemtuzumab (Campath-1H, Millennium Pharmaceuticals, Cambridge, MA) is a humanized CD52-specific monoclonal antibody that produces profound T-cell depletion in humans and reduces the need for maintenance immunosuppression after renal transplantation. We therefore performed a study to determine if pretransplant T-cell depletion with alemtuzumab would induce tolerance in human renal allografts and to evaluate the nature of the alloimmune response in the setting of T-cell depletion. METHODS: Seven nonsensitized recipients of living-donor kidneys were treated perioperatively with alemtuzumab and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically by peripheral flow cytometry, protocol biopsies evaluated immunohistochemically, and real-time polymerase chain reaction-based transcriptional analysis. RESULTS: Lymphocyte depletion was profound in the periphery and secondary lymphoid tissues. All patients developed reversible rejection episodes within the first month that were characterized by predominantly monocytic (not lymphocytic) infiltrates with only rare T cells in the peripheral blood or allograft. These episodes were responsive to treatment with steroids or sirolimus or both. After therapy, patients remained rejection-free on reduced immunosuppression, generally monotherapy sirolimus, despite the recovery of lymphocytes to normal levels. CONCLUSIONS: T-cell depletion alone does not induce tolerance in humans. These data underscore a prominent role for early responding monocytes in human allograft rejection.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Sobrevivência de Enxerto/imunologia , Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Adulto , Alemtuzumab , Anticorpos Monoclonais Humanizados , População Negra , Feminino , Seguimentos , Rejeição de Enxerto/epidemiologia , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Linfonodos/imunologia , Depleção Linfocítica , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Fatores de Tempo , Transplante Homólogo , Estados Unidos , População BrancaRESUMO
BACKGROUND Following allotransplantation, renal ischemia-reperfusion (I/R) injury initiates a series of events that provokes counter-adaptive immunity. Though T cells clearly mediate allospecific immunity, the manner in which reperfusion events augment their activation has not been established. In addition, comprehensive analysis of I/R injury in humans has been limited. METHODS To evaluate the earliest events occurring following allograft reperfusion and gain insight into those factors linking reperfusion to alloimmunity, we examined human renal allografts 30 to 60 minutes postreperfusion (n=10) and compared them with allografts with normal function that had resolved their I/R injury insult (>1 month posttransplant, n=6) and to normal kidneys (living donor kidneys before procurement, n=8). Biopsies were processed both for immunohistochemical analysis as well as for transcript analysis by real-time quantitative polymerase chain reaction (RT-PCR). RESULTS Reperfusion injury was characterized by increased levels of gene transcripts known to be involved in cellular adhesion, chemotaxis, apoptosis, and monocyte recruitment and activation. T-cell-associated transcripts were generally absent. However, recovered allografts exhibited increased levels of T-cell and costimulation-related gene transcripts despite normal allograft function. Consistent with these findings, the immediate postreperfusion state was characterized histologically by tubular injury and monocyte infiltration, while the stable posttransplant state was notable for T-cell infiltration. CONCLUSIONS These data suggest that monocytes and transcripts related to their recruitment dominate the immediate postreperfusion state. This gives way to a T-cell dominant milieu even in grafts selected for their stable function and absence of rejection. These data have implications for understanding the fundamental link between I/R injury and alloimmunity.