RESUMO
Oesophagogastric (OG) cancer is a highly lethal disease requiring novel treatment options. c-MYC and/or HER-2 amplified oesophageal cancer models have demonstrated sensitivity to BTK inhibition with ibrutinib. We evaluated the safety and efficacy of ibrutinib in patients with c-MYC and/or HER2 amplified pre-treated advanced OG cancer. c-MYC and HER2 amplification status were determined by FISH. The primary endpoint was overall response rate (ORR). Secondary endpoints were disease control rate (DC) at 8 weeks, safety, progression-free survival (PFS) and overall survival (OS). Eleven patients were enrolled. Eight patients had c-MYC amplified tumours, six were HER2 amplified and three were c-MYC and HER2 co-amplified. Grade ≥ 3 adverse events were fever, neutropenia, and vomiting. Grade ≥ 3 gastrointestinal haemorrhage occurred in three patients and was fatal in two cases. Among seven evaluable patients, three patients (43%) achieved a best response of SD at 8 weeks. No PR or CR was observed. Disease control was achieved for 32 weeks in one patient with a dual c-MYC and HER2 highly co-amplified tumour. The median PFS and OS were 1.5 (95% CI: 0.8-5.1) and 5.1 (95% CI: 0.8-14.5) months, respectively. Ibrutinib had limited clinical efficacy in patients with c-MYC and/or HER2 amplified OG cancer. Unexpected gastrointestinal bleeding was observed in 3 out of 8 treated patients which was considered a new safety finding for ibrutinib in this population.
Assuntos
Neoplasias Esofágicas , Piperidinas , Adenina/análogos & derivados , Adenina/uso terapêutico , Humanos , Piperidinas/uso terapêutico , Intervalo Livre de ProgressãoRESUMO
The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Choque Térmico/genética , Proteínas Mitocondriais/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Antígeno Nuclear de Célula em Proliferação/genética , Neoplasias da Próstata/genética , Tolerância a Radiação/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Estudos de Casos e Controles , Cromossomos/efeitos da radiação , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/métodos , Prognóstico , Neoplasias da Próstata/sangue , Neoplasias da Próstata/tratamento farmacológico , Doses de Radiação , Tolerância a Radiação/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto JovemRESUMO
BACKGROUND: Screening for prostate cancer with prostate specific antigen and digital rectal examination allows early diagnosis of prostate malignancy but has been associated with poor sensitivity and specificity. There is also a considerable risk of over-diagnosis and over-treatment, which highlights the need for better tools for diagnosis of prostate cancer. This study investigates the potential of high throughput Raman and Fourier Transform Infrared (FTIR) spectroscopy of liquid biopsies for rapid and accurate diagnosis of prostate cancer. METHODS: Blood samples (plasma and lymphocytes) were obtained from healthy control subjects and prostate cancer patients. FTIR and Raman spectra were recorded from plasma samples, while Raman spectra were recorded from the lymphocytes. The acquired spectral data was analysed with various multivariate statistical methods, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and classical least squares (CLS) fitting analysis. RESULTS: Discrimination was observed between the infrared and Raman spectra of plasma and lymphocytes from healthy donors and prostate cancer patients using PCA. In addition, plasma and lymphocytes displayed differentiating signatures in patients exhibiting different Gleason scores. A PLS-DA model was able to discriminate these groups with sensitivity and specificity rates ranging from 90% to 99%. CLS fitting analysis identified key analytes that are involved in the development and progression of prostate cancer. CONCLUSIONS: This technology may have potential as an alternative first stage diagnostic triage for prostate cancer. This technology can be easily adaptable to many other bodily fluids and could be useful for translation of liquid biopsy-based diagnostics into the clinic.
RESUMO
Genetic and epigenetic profile changes associated with individual radiation sensitivity are well documented and have led to enhanced understanding of the mechanisms of the radiation-induced DNA damage response. However, the search continues to identify reliable biomarkers of individual radiation sensitivity. Herein, we report on a multi-biomarker approach using traditional cytogenetic biomarkers, DNA damage biomarkers and transcriptional microRNA (miR) biomarkers coupled with their potential gene targets to identify radiosensitivity in ataxia-telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines (LCL); ATM-proficient cell lines were used as controls. Cells were 0.05 and 0.5 Gy irradiated, using a linear accelerator, with sham-irradiated cells as controls. At 1 h postirradiation, cells were fixed for γ-H2AX analysis as a measurement of DNA damage, and cytogenetic analysis using the G2 chromosomal sensitivity assay, G-banding and FISH techniques. RNA was also isolated for genetic profiling by microRNA (miR) and RT-PCR analysis. A panel of 752 miR were analyzed, and potential target genes, phosphatase and tensin homolog (PTEN) and cyclin D1 (CCND1), were measured. The cytogenetic assays revealed that although the control cell line had functional cell cycle checkpoints, the radiosensitivity of the control and AT cell lines were similar. Analysis of DNA damage in all cell lines, including an additional control cell line, showed elevated γ-H2AX levels for only one AT cell line. Of the 752 miR analyzed, eight miR were upregulated, and six miR were downregulated in the AT cells compared to the control. Upregulated miR-152-3p, miR-24-5p and miR-92-15p and all downregulated miR were indicated as modulators of PTEN and CCDN1. Further measurement of both genes validated their potential role as radiation-response biomarkers. The multi-biomarker approach not only revealed potential candidates for radiation response, but provided additional mechanistic insights into the response in AT-deficient cells.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Ciclina D1/metabolismo , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Linfócitos/citologiaRESUMO
Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)-FTIR and HT-Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.
Assuntos
Hemólise , Plasma , Análise de Fourier , Humanos , Masculino , Análise de Componente Principal , Soro , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral RamanRESUMO
Radiation therapy (RT) is used to treat approximately 50% of all cancer patients. However, RT causes a wide range of adverse late effects that can affect a patient's quality of life. There are currently no predictive assays in clinical use to identify patients at risk of normal tissue radiation toxicity. This study aimed to investigate the potential of Fourier transform infrared (FTIR) spectroscopy for monitoring radiotherapeutic response. Blood plasma was acquired from 53 prostate cancer patients at five different time points: prior to treatment, after hormone treatment, at the end of radiotherapy, two months post radiotherapy and eight months post radiotherapy. FTIR spectra were recorded from plasma samples at all time points and the data was analysed using MATLAB software. Discrimination was observed between spectra recorded at baseline versus follow up time points, as well as between spectra from patients showing minimal and severe acute and late toxicity using principal component analysis. A partial least squares discriminant analysis model achieved sensitivity and specificity rates ranging from 80% to 99%. This technology may have potential to monitor radiotherapeutic response in prostate cancer patients using non-invasive blood plasma samples and could lead to individualised patient radiotherapy.
RESUMO
In advanced radiotherapy, treatment of the tumor with high-intensity modulated fields is balanced with normal tissue sparing. However, the non-target dose delivered to surrounding healthy tissue within the irradiated volume is a potential cause for concern. Whether the effects observed are caused after exposure to out-of-field radiation or bystander effects through neighboring irradiated cells is not fully understood. The goal of this study was to determine the effect of exposure to out-of-field radiation in lymphocyte cell lines and primary blood cells. The role of cellular radiosensitivity in altering bystander responses in out-of-field exposed cells was also investigated. Target cells were positioned in a phantom in the center of the radiation field (in-field dose) and exposed to 2 Gy irradiation. Lymphocyte cell lines (C1, AT3ABR, Jurkat, THP-1, AT2Bi and AT3Bi) and peripheral blood were placed 1 cm away from the radiation field edge (out-of-field dose) and received an average dose of 10.8 ± 4.2 cGy. Double-stranded DNA damage, cell growth and gene expression were measured in the out-of-field cells. Radiosensitive AT3ABR and primary blood cells demonstrated the largest increase in γ-H2AX foci after irradiation. Exposure of normal cells to bystander factors from irradiated radiosensitive cell lines also increased DNA damage. Expression of IL-1, IL-6, TNFα and TGFß after addition of bystander factors from radiosensitive cells showed differential effects in normally responding cells, with some evidence of an adaptive response observed. Exposure to out-of-field radiation induces DNA damage and reduces growth in radiosensitive cells. Bystander factors produced by directly irradiated cells in combination with out-of-field exposure may upregulate pro- and anti-inflammatory genes in responding cells of different radiosensitivities, with the potential of affecting the tumor microenvironment. A greater understanding of the radio-biological response in normal cells outside the primary treatment field would assist in radiation treatment planning and in reducing early and late toxicities.
Assuntos
Efeito Espectador/genética , Efeito Espectador/efeitos da radiação , Citocinas/biossíntese , Dano ao DNA , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Proliferação de Células/efeitos da radiação , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Histonas/metabolismo , Humanos , Células Jurkat , Linfócitos/citologia , Tolerância a Radiação , Microambiente Tumoral/efeitos da radiaçãoRESUMO
PURPOSE: Liquid biopsies are a potentially rich store of biochemical information that can be linked to an individual's response to therapeutic treatments, including radiotherapy, and which may ultimately play a role in the individualization of treatment regimens. Peripheral blood mononuclear cells (PBMCs) can be used not only for the biochemical profiling of the individual, but also, being living cells, can provide insights into the individuals response to ionizing radiation exposure. MATERIALS AND METHODS: The present study attempts to link the biochemical profile of lymphocytes within PBMCs obtained through Raman spectroscopy to in vitro measures of low-dose (<0.5Gy) DNA damage response and cytogenetic metrics of radiosensitivity in a cohort of healthy controls and prostate cancer patients (from CTRIAL-IE(ICORG) 08-17, NCT00951535). All parallel metrics to the Raman spectra of the cells were obtained ex vivo in cycling peripheral blood lymphocytes, with radiosensitivity estimated using the G2 chromosomal assay and DNA damage assessed using γH2AX fluorescence. Spectra from a total of 26 healthy volunteers and 22 prostate cancer patients were obtained. RESULTS: The links between both measures of cellular response to ionizing radiation and the Raman spectra were modeled using partial least squares regression (PLSR) and support-vector regression (SVR). It was found that neither regression approach could predict radiation-induced G2 score well, but could predict γH2AX MFI with the SVR outperforming PLSR, implying a non-linear relationship between spectral measurements and measures of DNA damage. CONCLUSIONS: Raman spectroscopy of PBMCs represents a label-free approach for prediction of DNA damage levels for either prospective or retrospective analysis.
Assuntos
Cromossomos Humanos/genética , Cromossomos Humanos/efeitos da radiação , Dano ao DNA , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Tolerância a Radiação/genética , Análise Espectral Raman , Adulto , Aberrações Cromossômicas/efeitos da radiação , Humanos , Masculino , Neoplasias da Próstata/patologia , Adulto JovemRESUMO
The potential of Raman micro spectroscopy as an in vitro, non-invasive tool for clinical applications has been demonstrated in recent years, specifically for cancer research. To further illustrate its potential as a high content and label free technique, it is important to show its capability to elucidate drug mechanisms of action and cellular resistances. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 24 hr exposure of lung cancer cell lines, A549 and Calu-1, to the commercially available drug, doxorubicin (DOX). Raman spectroscopy, coupled with Confocal Laser Scanning Microscopy and Flow Cytometry, was used to track the DOX mechanism of action, at a subcellular level, and to study the mechanisms of cellular resistance to DOX. Biomarkers related to the drug mechanism of action and cellular resistance to apoptosis, namely reactive oxygen species (ROS) and bcl-2 protein expression, respectively, were also measured and correlated to Raman spectral profiles. Calu-1 cells are shown to exhibit spectroscopic signatures of both direct DNA damage due to intercalation in the nucleus and indirect damage due to oxidative stress in the cytoplasm, whereas the A549 cell line only exhibits signatures of the former mechanism of action. PCA of nucleolar, nuclear and cytoplasmic regions of A549 and Calu-1 with corresponding loadings of PC1 and PC2.
Assuntos
Doxorrubicina/farmacologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Neoplasias Pulmonares , Análise Espectral RamanRESUMO
Extensive research has been undertaken on the examination of tissue biopsies using vibrational spectroscopic techniques. However, fewer studies have focused on less invasive and commonly acquired blood samples. Recent studies have shown the ability of Raman and Fourier transform infrared (FTIR) spectroscopy to discriminate between non-cancer controls and cancer cases using blood serum or plasma. Even though many studies have proposed Raman spectroscopy as a potential diagnostic tool in various cancers, the Raman spectroscopic technique has not been introduced as a routine clinical technology. This is due to multiple drawbacks with the application of the technique, including sample preparation, the requirement for expensive substrates and long acquisition times. The current study aims to overcome these limitations and focuses on the translation of Raman spectroscopy into a high throughput clinical diagnostic tool for prostate cancer. In this study, the effect of different instrumental and sample preparation parameters were investigated, with the aim of identifying a combination that would reduce the overall acquisition time for spectra from peripheral blood plasma, reduce the complexity of sample preparation and retain the classification accuracy from Raman spectroscopic diagnostics. A high throughput (HT) system was developed and Raman spectroscopic measurements were performed on plasma samples from 10 prostate cancer patients and 10 healthy volunteers. The spectra were pre-processed and classified by principal component analysis - linear discriminant analysis (PCA-LDA) in the R environment. Statistically significant differences were observed between Raman spectra of prostate cancer patients and non-cancer controls. The (HT) classification resulted in a sensitivity and specificity of 96.5% and 95% respectively. Overall, this study has overcome some of the limitations associated with clinical translation of Raman spectroscopy. The HT-Raman spectroscopy method developed in this study can be used for rapid and accurate diagnosis of prostate cancer using liquid plasma samples.
Assuntos
Detecção Precoce de Câncer , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Análise Espectral Raman , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Discriminante , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Adulto JovemRESUMO
We have previously developed a robust regimen for tolerance induction in murine models of islet cell transplantation using pre- and posttransplant infusions of donor splenocytes (SPs) treated with a chemical cross-linker ethylcarbodiimide (ECDI). However, the requirement for large numbers of fresh donor SPs for ECDI coupling impairs its clinical feasibility, and additionally, the compatibility of this tolerance regimen with commonly used immunosuppressive drugs is largely unknown. In the current study, we demonstrate that equivalent tolerance efficacy for islet cell transplantation can be successfully achieved not only with a significantly lower dose of ECDI-SPs than originally established but also with culture-expanded donor B-cells or with soluble donor antigens in the form of donor cell lysate, which is ECDI coupled to recipient SPs. We further demonstrate that tolerance induced by donor ECDI-SPs is dependent on a favorable apoptotic-to-necrotic cell ratio post-ECDI coupling and is not affected by a transient course of conventional immunosuppressive drugs including tacrolimus and mycophenolate mofetil. While splenic antigen-presenting cells of the recipient play an important role in mediating the tolerogenic effects of donor ECDI-SPs, splenectomized recipients can be readily tolerized and appear to employ liver Kupffer cells for uptaking and processing of the ECDI-SPs. We conclude that infusion of donor ECDI-SPs is a versatile tolerance strategy that has a high potential for adaptation to clinically feasible regimens for tolerance trials for human islet cell transplantation.
Assuntos
Antígenos/imunologia , Tolerância Imunológica/imunologia , Transplante das Ilhotas Pancreáticas , Animais , Antígeno CD47/metabolismo , Carbodi-Imidas/farmacologia , Extratos Celulares , Congelamento , Humanos , Tolerância Imunológica/efeitos dos fármacos , Imunossupressores/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/citologia , Doadores de Tecidos , Transplante HomólogoRESUMO
Human islet cell transplantation is a promising treatment for type 1 diabetes; however, long-term donor-specific tolerance to islet allografts remains a clinically unmet goal. We have previously shown that recipient infusions of apoptotic donor splenocytes chemically treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (donor ECDI-SP) can mediate long-term acceptance of full major histocompatibility complex (MHC)-mismatched murine islet allografts without the use of immunosuppression. In this report, we investigated the use of poly(lactide-co-glycolide) (PLG) particles in lieu of donor ECDI-SP as a synthetic, cell-free carrier for delivery of donor antigens for the induction of transplant tolerance in full MHC-mismatched murine allogeneic islet transplantation. Infusions of donor antigen-coupled PLG particles (PLG-dAg) mediated tolerance in â¼20% of recipient mice, and the distribution of cellular uptake of PLG-dAg within the spleen was similar to that of donor ECDI-SP. PLG-dAg mediated the contraction of indirectly activated T cells but did not modulate the direct pathway of allorecognition. Combination of PLG-dAg with a short course of low dose immunosuppressant rapamycin at the time of transplant significantly improved the tolerance efficacy to â¼60%. Furthermore, altering the timing of PLG-dAg administration to a schedule that is more feasible for clinical transplantation resulted in equal tolerance efficacy. Thus, the combination therapy of PLG-dAg infusions with peritransplant rapamycin represents a clinically attractive, biomaterials-based and cell-free method for inducing long-term donor-specific tolerance for allogeneic cell transplantation, such as for allogeneic islet transplantation.
Assuntos
Antígenos/administração & dosagem , Portadores de Fármacos/química , Tolerância Imunológica/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas/métodos , Poliglactina 910/química , Animais , Antígenos/uso terapêutico , Etildimetilaminopropil Carbodi-Imida/química , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Masculino , Camundongos Endogâmicos BALB C , Nanopartículas/química , Sirolimo/administração & dosagem , Sirolimo/uso terapêutico , Transplante HomólogoRESUMO
We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid-derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b(+) cells in the spleen that phenotypically resemble monocytic-like (CD11b(+)Ly6C(high)) and granulocytic-like (CD11b(+)Gr1(high)) MDSCs. Both populations suppress T cell proliferation in vitro and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b(+)Gr1(high) cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ-dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction.
Assuntos
Apoptose , Sobrevivência de Enxerto/imunologia , Transplante de Coração , Interferon gama/imunologia , Células Mieloides/imunologia , Baço/imunologia , Baço/transplante , Aloenxertos , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
QUESTION: Is electrical stimulation and splinting more effective than splinting alone for the management of wrist contracture following acquired brain injury? DESIGN: A multi-centre randomised trial with concealed allocation, assessor blinding, and intention-to-treat analysis. PARTICIPANTS: Thirty-six adults with first stroke or traumatic brain injury and mild to moderate wrist flexion contractures. INTERVENTION: The experimental group received electrical stimulation to the wrist and finger extensor muscles for 1 hour a day over 4 weeks while the control group did not. Both groups wore a splint for 12 hours a day during this 4-week period. OUTCOME MEASURES: The primary outcome was passive wrist extension measured with a 3Nm torque and with the fingers in extension. Secondary outcomes included passive wrist extension, wrist and finger extensor strength, wrist flexor spasticity, motor control of the hand, and Global Perceived Effect of Treatment, and perception of treatment credibility. Outcome measures were taken at baseline, at the end of the intervention period (4 weeks), and after a 2-week follow-up period (6 weeks). RESULTS: At 4 and 6 weeks, the mean between-group difference (95% CI) for passive wrist extension was 7 degrees (-2 to 15) and -3 degrees (-13 to 7), respectively. Secondary outcomes were statistically non-significant or were of borderline statistical significance. CONCLUSION: It is not clear whether electrical stimulation and splinting is more effective than splinting alone for the management of wrist contracture after acquired brain injury. Therapists' confidence in the efficacy of electrical stimulation for contracture management is not yet justified.
Assuntos
Lesões Encefálicas/reabilitação , Contratura/reabilitação , Terapia por Estimulação Elétrica/métodos , Modalidades de Fisioterapia , Contenções , Adulto , Idoso , Lesões Encefálicas/complicações , Contratura/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Strategic exposure to donor Ags prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pretransplant infusion of donor splenocytes treated with the chemical cross-linker ethylenecarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allospecificities. To target indirect allospecific T cells, ECDI-SPs induce upregulation of negative, but not positive, costimulatory molecules on recipient splenic CD11c(+) dendritic cells phagocytosing the injected ECDI-SPs. Indirect allospecific T cells activated by such CD11c(+) dendritic cells undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allospecific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4(+)CD25(+)Foxp3(+) T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SP infusions target host allogeneic responses via a multitude of mechanisms, including clonal depletion, anergy, and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.
Assuntos
Carbodi-Imidas/administração & dosagem , Isoantígenos/metabolismo , Transdução de Sinais/imunologia , Baço/imunologia , Baço/transplante , Tolerância ao Transplante/imunologia , Transferência Adotiva/métodos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Reagentes de Ligações Cruzadas/administração & dosagem , Técnicas de Introdução de Genes , Sobrevivência de Enxerto/imunologia , Infusões Intravenosas , Isoantígenos/administração & dosagem , Isoantígenos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fagócitos/imunologia , Fagócitos/metabolismo , Baço/citologiaRESUMO
OBJECTIVE: To examine the migratory properties of cytokine-activated T (Tck) cells. METHODS: Tck cells were generated by culture of peripheral blood T cells in the presence of interleukin-6 (IL-6), tumor necrosis factor α, and IL-2. Changes in cell surface phenotype were analyzed by flow cytometry. Chemotactic responsiveness was measured using in vitro chemotaxis assays and transendothelial migration through human umbilical vein endothelial cell monolayers. Levels of vascular cell adhesion molecule 1 (VCAM-1) were measured by sandwich enzyme-linked immunosorbent assay. RESULTS: Cytokine stimulation up-regulated the expression of chemokine receptors and integrins on Tck cells, including CXCR4, very late activation antigen 4 (VLA-4), and lymphocyte function-associated antigen 1. Increased expression of CXCR4 and VLA-4 integrin resulted in concentration-dependent chemotaxis to their ligands, stromal cell-derived factor 1 (SDF-1) and VCAM-1, which could be selectively blocked using a specific CXCR4 inhibitor and antibodies against VLA-4. Increased expression of VLA-4 also resulted in increased transendothelial migration of Tck cells, which could be abrogated using blocking antibodies against VLA-4. Tck cells also showed an increased chemotactic response to rheumatoid arthritis (RA) fibroblast-like synoviocytes cultured in vitro, which could be blocked using inhibitors against VLA-4 and CXCR4. CONCLUSION: The activated phenotype of Tck cells results in increased migratory responsiveness to SDF-1 and soluble VCAM-1, which are among the chemokines and proteins found elevated in the RA synovial joint environment. Cytokine-dependent activation may contribute to RA pathogenicity by promoting T cell recruitment to and retention in the joint, perpetuating the inflammatory cascade in RA.
Assuntos
Quimiotaxia de Leucócito/imunologia , Receptores CXCR4/metabolismo , Linfócitos T/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfa4beta1/metabolismo , Interleucina-2/farmacologia , Interleucina-6/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Linfócitos T/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
TRPML3 (mucolipin-3) belongs to one of the transient-receptor-potential (TRP) ion channel families. Mutations in the Trpml3 gene cause disorganization of the stereociliary hair bundle, structural aberrations in outer and inner hair cells and stria vascularis defects, leading to deafness in the varitint-waddler (Va) mouse. Here we refined the stereociliary localization of TRPML3 and investigated cochlear hair cell function in varitint-waddler (Va(J)) mice carrying the TRPML3
Assuntos
Células Ciliadas Auditivas/fisiologia , Mutação , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/fisiologia , Animais , Sequência de Bases , Cóclea/fisiopatologia , Primers do DNA/genética , Surdez/genética , Surdez/fisiopatologia , Eletrofisiologia , Heterozigoto , Homozigoto , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Before the introduction of human immunodeficiency virus (HIV) combination assays, serologic diagnosis of HIV infection was performed with assays that detected either antibodies or p24 antigen. Owing to the capability to detect the early appearance of p24 antigen, combination assays that are designed for simultaneous detection of antibodies and antigen can significantly reduce the diagnostic window. STUDY DESIGN AND METHODS: Specificity and sensitivity of a commercially available HIV antigen-antibody combination assay (Abbott PRISM; assay is not licensed by the FDA for use in the United States) were evaluated in a multicenter study by testing volunteer blood donors, hospitalized patients, seroconversion panels, and p24 antigen and HIV antibody subtype panels. Performance data were compared to a commercially available HIV combination assay and the PRISM HIV O Plus assay. RESULTS: Apparent specificity of 99.95 percent was observed in the donor population for the PRISM HIV antigen-antibody combination assay, and better seroconversion sensitivity was demonstrated compared with another combination assay and the PRISM HIV O Plus assay. Analytical HIV antigen detection sensitivity averaged 33 pg per mL on the Agence Française de Sécurité Sanitaire des Produits de Santé (AFSSAPS) panel. Furthermore, comparable antigen sensitivity was demonstrated for 32 HIV-1 group M subtype and group O panels. The PRISM HIV combination assay detected all HIV-1 group M and O and HIV-2 antibody-positive specimens evaluated. CONCLUSIONS: The PRISM HIV antigen-antibody combination assay demonstrated a significant reduction of the window period for diagnosis of HIV infection. The assay demonstrated enhanced specificity and sensitivity along with broad subtype detection. The assay performance represents the "state-of-the art" technology for serologic blood screening of HIV infection.
Assuntos
Seleção do Doador/métodos , Anticorpos Anti-HIV/sangue , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Infecções por HIV/virologia , Humanos , Imunoensaio/métodos , Modelos Biológicos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that can be transmitted by transfusion. The diagnosis of chronic T. cruzi infection is generally made by detecting specific antibodies that bind to parasite antigens. The aim of this study was to assess the sensitivity and specificity of a new serologic assay for antibodies to T. cruzi on a fully automated analyzer (PRISM, Abbott Laboratories). STUDY DESIGN AND METHODS: A prototype chemiluminescent immunoassay based on chimeric recombinant antigens and run on the automated PRISM system was developed for detecting antibodies to T. cruzi in human serum and plasma. Assay specificity was evaluated by testing samples from random blood donors and from a diverse group of specimens from persons with diseases or conditions often associated with false-positive reactions in T. cruzi assays. Sensitivity was determined by testing 377 geographically diverse T. cruzi antibody-positive specimens. RESULTS: Six of 7911 samples (0.08%) from random donors were repeatedly reactive in the prototype PRISM Chagas assay. One of these was reactive in three other tests, including the radioimmune precipitation assay and was presumed to be a true positive. Hence, the specificity was 99.94 percent (7905/7910) in the negative donor group studied. All 377 T. cruzi antibody-positive specimens were positive in the prototype assay and thus the sensitivity was 100 percent. CONCLUSION: The results obtained to date, in terms of sensitivity as well as specificity, strongly suggest that the PRISM Chagas assay should function well as a tool for screening blood for serologic evidence of T. cruzi infection.
Assuntos
Anticorpos Antiprotozoários/química , Doadores de Sangue , Doença de Chagas/sangue , Seleção do Doador , Trypanosoma cruzi , Animais , Anticorpos Antiprotozoários/genética , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/genética , Doença de Chagas/imunologia , Seleção do Doador/métodos , Humanos , Imunoensaio/métodos , Medições Luminescentes/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Trypanosoma cruzi/química , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologiaRESUMO
The molecular mechanisms underlying the specification of sensory organs in the inner ear and the development of hair and supporting cells within these organs are described. The different organs are all derived from a common pro-sensory region, and may be specified by their proximity to the boundaries between compartments - broad domains within the otocyst defined by the asymmetric expression patterns of transcription factors. Activation of Notch may specify the pro-sensory region, and lateral inhibition mediated by Notch signalling influences whether cells of common lineage in a sensory patch differentiate as either hair cells or supporting cells. The transcription factors Math1 and Brn3.1 are required for hair cell differentiation, and supporting cells express negative regulators of neurogenesis, Hes1 and Hes5. Retinoic acid and thyroid hormone influence early aspects and timing of hair cell differentiation, respectively. Development of the hair cell's mechanosensory hair bundle involves interactions between the cytoskeleton, cell-surface adhesion molecules, receptors and associated extracellular matrix.