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Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor ß-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2, with preclinical evidence to support their efficacy in T cell malignancies.
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Neoplasias , Linfócitos T , Humanos , Imunoterapia , Receptores de Antígenos de Linfócitos TRESUMO
There is a lack of awareness of how sleep health and sleep disorders are experienced. Previous research has found that living with narcolepsy has a debilitating impact on several areas of an individual's life alongside significant diagnostic delays. This study uses a phenomenological, qualitative methodology to explore experiences of being diagnosed with and living with narcolepsy. Six women with type 1 narcolepsy participated in semi-structured interviews. Transcripts were analysed using Interpretative Phenomenological Analysis. Capturing the whole illness experience of narcolepsy, our analysis illuminated three superordinate themes; 'minimising, dismissing and downplaying symptoms', 'navigating the winding journey to diagnosis' and 'a different way of living'. Through our analysis, we are able to demonstrate the affective impact lack of awareness of sleep and sleep disorders has; resulting in significant diagnostic delays and a lack of support post-diagnosis. Findings demonstrate a need for greater awareness and increased support.
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BACKGROUND: We used a proliferating ligand (APRIL) to construct a ligand-based third generation chimeric antigen receptor (CAR) able to target two myeloma antigens, B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor. METHODS: The APRIL CAR was evaluated in a Phase 1 clinical trial (NCT03287804, AUTO2) in patients with relapsed, refractory multiple myeloma. Eleven patients received 13 doses, the first 15×106 CARs, and subsequent patients received 75,225,600 and 900×106 CARs in a 3+3 escalation design. RESULTS: The APRIL CAR was well tolerated. Five (45.5%) patients developed Grade 1 cytokine release syndrome and there was no neurotoxicity. However, responses were only observed in 45.5% patients (1×very good partial response, 3×partial response, 1×minimal response). Exploring the mechanistic basis for poor responses, we then compared the APRIL CAR to two other BCMA CARs in a series of in vitro assays, observing reduced interleukin-2 secretion and lack of sustained tumor control by APRIL CAR regardless of transduction method or co-stimulatory domain. There was also impaired interferon signaling of APRIL CAR and no evidence of autoactivation. Thus focusing on APRIL itself, we confirmed similar affinity to BCMA and protein stability in comparison to BCMA CAR binders but reduced binding by cell-expressed APRIL to soluble BCMA and reduced avidity to tumor cells. This indicated either suboptimal folding or stability of membrane-bound APRIL attenuating CAR activation. CONCLUSIONS: The APRIL CAR was well tolerated, but the clinical responses observed in AUTO2 were disappointing. Subsequently, when comparing the APRIL CAR to other BCMA CARs, we observed in vitro functional deficiencies due to reduced target binding by cell-expressed ligand.
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Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Mieloma Múltiplo/tratamento farmacológico , Ligantes , Antígeno de Maturação de Linfócitos B/metabolismo , Antígeno de Maturação de Linfócitos B/uso terapêutico , Linfócitos TRESUMO
Huanglongbing (HLB, citrus greening disease), the most destructive disease affecting citrus production, is primarily linked to the gram-negative, insect-vectored, phloem-inhabiting α-proteobacterium 'Candidatus Liberibacter asiaticus' (CLas). With no effective treatment available, management strategies have largely focused on the use of insecticides in addition to the destruction of infected trees, which are environmentally hazardous and cost-prohibitive for growers, respectively. A major limitation to combating HLB is the inability to isolate CLas in axenic culture, which hinders in vitro studies and creates a need for robust in situ CLas detection and visualization methods. The aim of this study was to investigate the efficacy of a nutritional program-based approach for HLB treatment, and to explore the effectiveness of an enhanced immunodetection method to detect CLas-infected tissues. To achieve this, four different biologically enhanced nutritional programs (bENPs; P1, P2, P3, and P4) were tested on CLas-infected citrus trees. Structured illumination microscopy preceded by a modified immunolabeling process and transmission electron microscopy were used to show treatment-dependent reduction of CLas cells in phloem tissues. No sieve pore plugging was seen in the leaves of P2 trees. This was accompanied by an 80% annual increase in fruit number per tree and 1,503 (611 upregulated and 892 downregulated) differentially expressed genes. These included an MLRQ subunit gene, UDP-glucose transferase, and genes associated with the alpha-amino linolenic acid metabolism pathway in P2 trees. Taken together, the results highlight a major role for bENPs as a viable, sustainable, and cost effective option for HLB management.
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Citrus , Rhizobiaceae , Transcriptoma , Rhizobiaceae/genética , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologia , Citrus/microbiologia , ÁrvoresRESUMO
A displaced medial tibial plateau fracture with central and lateral impaction, but an intact anterolateral cortical rim, is an uncommon variant of bicondylar tibial plateau fracture that presents a number of challenges. Without a lateral metaphyseal fracture line to work through, it is challenging to address central and lateral impaction. Previously published techniques for addressing this fracture pattern describe an intra-articular osteotomy of the lateral plateau to aid visualization and reduction, or use a posterolateral approach to the proximal tibia with or without an osteotomy of the proximal fibula. This study presents a technique which utilizes standard dual incision approaches and does not involve an intra-articular osteotomy of the lateral tibial plateau or a posterolateral approach. A case series was conducted evaluating radiographic and functional outcomes of 8 patients.
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Fraturas da Tíbia , Fraturas do Planalto Tibial , Humanos , Fraturas da Tíbia/cirurgia , Fixação Interna de Fraturas/métodos , Tíbia/cirurgia , Fíbula/cirurgiaRESUMO
γ-Retroviral vectors (γ-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for γ-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced γ-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for γ-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 107-108 TU/mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in product- and process-related impurities. Furthermore, product characterization of clinical-grade γ-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade γ-RV produced by transient transfection and purified by high-speed centrifugation was similar to γ-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using γ-RV to bridge vector supply between early- and late-stage clinical trials.
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Purpose: Endoscopic transsphenoidal surgery (ETSS) is an increasingly utilized approach for resection of pituitary tumors. Prior studies have evaluated preoperative tumor size, location, and extent as prognostic factors for surgical resection. There is little data on the relationship between preoperative pituitary tumor radiographic morphology and surgical outcomes. Study Design: Retrospective longitudinal study. Setting: Single tertiary care institution. Subjects and Methods: Preoperative magnetic resonance imaging and computed tomography scans from patients undergoing ETSS for pituitary tumor resections from 2007 to 2017 were retrospectively evaluated. A neuroradiologist classified these pituitary tumors into six morphologic groups, each defined by volume, dimensions, extension, and shape. Surgical difficulty, rates of incomplete resection, and postoperative complications were then stratified in relation to the morphologic groups. Results: Pituitary tumors from 131 patients were classified from preoperative imaging into six characteristic morphologies: (1) microtumor, (2) round, (3) transverse oblong, (4) superior-inferior oblong, (5) bilobed, and (6) large lobulated. Tumors that were characterized with the large lobulated, bilobed, and transverse oblong morphologies correlated with higher rates of postoperative evidence of residual tumor (70%, 36%, and 47%, respectively, all P < 0.002). Likewise, large lobulated, bilobed, and transverse oblong morphologies were also associated with intraoperative cerebrospinal fluid leaks (70%, 31%, and 35%, respectively, all P < 0.05). Conclusions: We describe a novel descriptive system for the morphology of pituitary tumors that can be determined from preoperative imaging. Different tumor morphologic groups are associated with varying degrees of gross tumor resection, complications, and surgical difficulty. Utilizing pituitary tumor morphology may aid surgeons in planning the extent of resection, need for complex closure, and patient counseling.
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Individuals living with chronic physical health conditions are more likely to be out-of-work than other groups. Often framed as a 'response' to these statistics, many countries have introduced policy instruments for promoting the employment of individuals with chronic conditions. This qualitative study sought to explore the impact of welfare reforms on UK individuals. Employing a phenomenological approach, semi-structured interviews were conducted with five participants living with chronic conditions. Three themes were generated using Interpretative Phenomenological Analysis: 'intersubjective sense making of the condition'; 'battles for control' and 'the fluidity and strengthening of identity'. Implications for further, holistic, policy reform are explored.
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Doença Crônica , Humanos , Pesquisa Qualitativa , Reino UnidoRESUMO
Chimeric antigen receptor (CAR) T cells targeting CD19 or CD22 have shown remarkable activity in B cell acute lymphoblastic leukemia (B-ALL). The major cause of treatment failure is antigen downregulation or loss. Dual antigen targeting could potentially prevent this, but the clinical safety and efficacy of CAR T cells targeting both CD19 and CD22 remain unclear. We conducted a phase 1 trial in pediatric and young adult patients with relapsed or refractory B-ALL (n = 15) to test AUTO3, autologous transduced T cells expressing both anti-CD19 and anti-CD22 CARs (AMELIA trial, EUDRA CT 2016-004680-39). The primary endpoints were the incidence of grade 3-5 toxicity in the dose-limiting toxicity period and the frequency of dose-limiting toxicities. Secondary endpoints included the rate of morphological remission (complete response or complete response with incomplete bone marrow recovery) with minimal residual disease-negative response, as well as the frequency and severity of adverse events, expansion and persistence of AUTO3, duration of B cell aplasia, and overall and event-free survival. The study endpoints were met. AUTO3 showed a favorable safety profile, with no dose-limiting toxicities or cases of AUTO3-related severe cytokine release syndrome or neurotoxicity reported. At 1 month after treatment the remission rate (that is, complete response or complete response with incomplete bone marrow recovery) was 86% (13 of 15 patients). The 1 year overall and event-free survival rates were 60% and 32%, respectively. Relapses were probably due to limited long-term AUTO3 persistence. Strategies to improve CAR T cell persistence are needed to fully realize the potential of dual targeting CAR T cell therapy in B-ALL.
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Antígenos CD19/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos Quiméricos/administração & dosagem , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Adolescente , Adulto , Antígenos CD19/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/tendências , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/tendências , Lactente , Masculino , Pediatria , Intervalo Livre de Progressão , Receptores de Antígenos Quiméricos/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Adulto JovemRESUMO
The reprogramming of a patient's immune system through genetic modification of the T cell compartment with chimeric antigen receptors (CARs) has led to durable remissions in chemotherapy-refractory B cell cancers. Targeting of solid cancers by CAR-T cells is dependent on their infiltration and expansion within the tumor microenvironment, and thus far, fewer clinical responses have been reported. Here, we report a phase 1 study (NCT02761915) in which we treated 12 children with relapsed/refractory neuroblastoma with escalating doses of second-generation GD2-directed CAR-T cells and increasing intensity of preparative lymphodepletion. Overall, no patients had objective clinical response at the evaluation point +28 days after CAR-T cell infusion using standard radiological response criteria. However, of the six patients receiving ≥108/meter2 CAR-T cells after fludarabine/cyclophosphamide conditioning, two experienced grade 2 to 3 cytokine release syndrome, and three demonstrated regression of soft tissue and bone marrow disease. This clinical activity was achieved without on-target off-tumor toxicity. Targeting neuroblastoma with GD2 CAR-T cells appears to be a valid and safe strategy but requires further modification to promote CAR-T cell longevity.
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Neuroblastoma , Receptores de Antígenos Quiméricos , Criança , Humanos , Imunoterapia Adotiva , Recidiva Local de Neoplasia , Neuroblastoma/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T , Microambiente TumoralRESUMO
In August 2017, the USS Bataan received a mass casualty incident (MCI) of 6 foreign special forces operators after a helicopter crash. All 6 patients were medically evacuated successfully to the USS Bataan, and all patients survived and were successfully returned to their allied country. Four of the patients received whole blood with 2 receiving over 10 units of blood or massive transfusions. One patient required 44 units of blood, and at 1 point in his resuscitation, he received 12 units of whole blood every 30 minutes. Due to administrative factors outside of the ship's control, these 6 patients had prolonged stabilization during the MCI. This factor differentiates this MCI on the USS Bataan from previous cases. Internal medicine trained physicians with their expertise in inpatient care, postsurgical management, and critical care were instrumental in sustaining these casualties in this prolonged stabilization environment. In the era of distributed maritime operations, where casualty-receiving ships will experience more geographic and resource isolation, there is a potential for the need for prolonged stabilization above the 6 to 12-hour window typical of role II platforms. The known increase in cardiac and pulmonary morbidity and mortality with medical evacuation delay highlights the importance of internal medicine physicians in the role II setting. It is critical that we emphasize the inpatient and critical care principles of these patients in the prolonged field care environment.
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Incidentes com Feridos em Massa , Aeronaves , Transfusão de Sangue , Humanos , Masculino , Ressuscitação , NaviosRESUMO
Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Diferenciação Celular/genética , Humanos , Neoplasias Pulmonares/genética , MutaçãoRESUMO
Sustained demand for dermatologic care throughout military medicine, in conjunction with increasing dermatologic provider shortages, has led to increase use of teledermatology in military treatment facilities (MTFs). Initially used to aid in the differentiation of suspicious melanocytic lesions, dermoscopy has found increasing clinical utility in an expanding realm of general dermatologic conditions. We demonstrate the use of synchronous teledermoscopy within a remote MTF by repurposing webcam technology already available at most MTFs. Two patients were seen in clinic at a remote naval primary care clinic with limited subspecialties. Once written consent was retrieved, an on-site dermatologist evaluated each patient and performed a history and skin exam with dermoscopy. Synchronous consultations were conducted with the Global Med Cart (GlobalMed(R) Clinical Access Station with TotalExam(R) 3 HDUSB camera), and Cisco webcam video jabber (Cisco TelePresence PrecisionHD USB Camera part number TTC8-03). The patients then underwent individual synchronous teledermatology consultations with an off-site U.S. Navy dermatologist located in the continental United States. The methodology for the consultation involved the use of a standard dermatoscope and jabber webcam. Two synchronous teledermatology consultations were completed successfully on patients in MTFs with limited subspecialty capabilities. Both cases, with two lesions of concern per case, had 100% concordance between the on-site and teleconsulted dermatologist. Through observing inter-rater agreements between the on-site and remote dermatologists, this small study demonstrates a novel application of technology readily available at most MTFs.
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Militares , Dermatologia , Dermoscopia , Humanos , Consulta Remota , Neoplasias Cutâneas , TelemedicinaRESUMO
Dermatomyositis (DM) is a multi-system disease that results in chronic inflammation principally of the skin and striated muscle. Small blood vessel injury in the GI tract has been described in dermatomyositis, manifesting as bleeding, ulceration, pneumatosis intestinalis, and ultimately perforation. Recent histopathological studies have shown deposits in the capillaries of the skin, gastrointestinal tract, and brain of patients with dermatomyositis similar to that found in patients with Degos disease, suggesting these disease processes are closely related or represent varying degrees of severity on the same pathologic spectrum. We report a case of juvenile dermatomyositis (JDM) resembling late-stage Degos disease with gastrointestinal perforations successfully treated with combination rituximab and cyclophosphamide therapy. We systematically reviewed the literature detailing the medical and surgical treatments for gastrointestinal perforation in dermatomyositis, Degos-like dermatomyositis, and Degos disease. In addition to our case, as of October 2019, we identified 36 cases describing gastrointestinal perforation in patients with underlying dermatomyositis, 5 cases of Degos-like dermatomyositis and 17 cases of idiopathic Degos disease. Corticosteroid therapy was used widely for dermatomyositis and Degos-like dermatomyositis, while antiplatelet and anticoagulant medications were chiefly used for patients with idiopathic Degos disease. However, there were no cases that detailed the successful treatment of dermatomyositis or Degos disease with gastrointestinal perforation with rituximab alone or combined with cyclophosphamide. We report that rituximab, in combination with cyclophosphamide, can be used as a novel adjunctive therapy to successfully treat dermatomyositis with Degos-like gastrointestinal perforation.
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Antirreumáticos/uso terapêutico , Dermatomiosite/diagnóstico , Perfuração Intestinal/diagnóstico , Papulose Atrófica Maligna/diagnóstico , Criança , Ciclofosfamida/uso terapêutico , Dermatomiosite/complicações , Dermatomiosite/tratamento farmacológico , Diagnóstico Diferencial , Procedimentos Cirúrgicos do Sistema Digestório , Duodenopatias/diagnóstico , Duodenopatias/terapia , Perfuração Esofágica/diagnóstico , Perfuração Esofágica/terapia , Feminino , Humanos , Perfuração Intestinal/etiologia , Perfuração Intestinal/terapia , Doenças do Jejuno/diagnóstico , Doenças do Jejuno/terapia , Rituximab/uso terapêuticoRESUMO
We investigated and modeled the mesenchymal stromal cell (MSC) niche in adult acute lymphoblastic leukemia (ALL). We used gene expression profiling, cytokine/chemokine quantification, flow cytometry, and a variety of imaging techniques to show that MSCs, directly isolated from the primary bone marrow specimens of patients with ALL, frequently adopted an activated, cancer-associated fibroblast phenotype. Normal, primary human MSCs and the MSC cell line HS27a both were activated de novo, when exposed to the reactive oxygen species (ROS)-inducing chemotherapy agents cytarabine (AraC) and daunorubicin (DNR), a phenomenon blocked by the antioxidant N-acetyl cysteine. Chemotherapy-activated HS27a cells were functionally evaluated in a coculture model with ALL targets. Activated MSCs prevented therapy-induced apoptosis and death in ALL targets, via mitochondrial transfer through tunneling nanotubes (TNTs). Reduction of mitochondrial transfer by selective mitochondrial depletion or interference with TNT formation by microtubule inhibitors, such as vincristine (VCR), prevented the "rescue" function of activated MSCs. Corticosteroids, also a mainstay of ALL therapy, prevented the activation of MSCs. We also demonstrated that AraC (but not VCR) induced activation of MSCs, mitochondrial transfer, and mitochondrial mass increase in a murine NSG model of disseminated SEM cell-derived ALL, wherein CD19+ cells closely associated with nestin+ MSCs after AraC, but not in the other conditions. Our data propose a readily clinically exploitable mechanism for improving treatment of ALL, in which traditional ROS-inducing chemotherapies are often ineffective at eradicating residual disease, despite efficiently killing the bulk population.
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Antineoplásicos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adulto , Idoso , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citarabina/farmacologia , Citarabina/uso terapêutico , Daunorrubicina/farmacologia , Daunorrubicina/uso terapêutico , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Adulto JovemRESUMO
Despite the benefits of chimeric antigen receptor (CAR)-T cell therapies against lymphoid malignancies, responses in solid tumors have been more limited and off-target toxicities have been more marked. Among the possible design limitations of CAR-T cells for cancer are unwanted tonic (antigen-independent) signaling and off-target activation. Efforts to overcome these hurdles have been blunted by a lack of mechanistic understanding. Here, we showed that single-cell analysis with time course mass cytometry provided a rapid means of assessing CAR-T cell activation. We compared signal transduction in expanded T cells to that in T cells transduced to express second-generation CARs and found that cell expansion enhanced the response to stimulation. However, expansion also induced tonic signaling and reduced network plasticity, which were associated with expression of the T cell exhaustion markers PD-1 and TIM-3. Because this was most evident in pathways downstream of CD3ζ, we performed similar analyses on γδT cells that expressed chimeric costimulatory receptors (CCRs) lacking CD3ζ but containing DAP10 stimulatory domains. These CCR-γδT cells did not exhibit tonic signaling but were efficiently activated and mounted cytotoxic responses in the presence of CCR-specific stimuli or cognate leukemic cells. Single-cell signaling analysis enabled detailed characterization of CAR-T and CCR-T cell activation to better understand their functional activities. Furthermore, we demonstrated that CCR-γδT cells may offer the potential to avoid on-target, off-tumor toxicity and allo-reactivity in the context of myeloid malignancies.
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Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos Quiméricos/imunologia , Transdução de Sinais/imunologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica/imunologia , Engenharia Genética , Células HEK293 , Humanos , Ativação Linfocitária/imunologia , Neoplasias/genética , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismoAssuntos
Hanseníase Dimorfa/diagnóstico , Hanseníase Virchowiana/diagnóstico , Tinha Versicolor/diagnóstico , Biópsia , Criança , Diagnóstico Diferencial , Feminino , Humanos , Hansenostáticos/uso terapêutico , Hanseníase Dimorfa/tratamento farmacológico , Hanseníase Dimorfa/patologia , Hanseníase Virchowiana/tratamento farmacológico , Hanseníase Virchowiana/patologia , Pele/patologiaRESUMO
The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.
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Amidas/química , Anticorpos/química , Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Amidas/metabolismo , Anticorpos/metabolismo , Mama/química , Feminino , Corantes Fluorescentes/metabolismo , Humanos , Neoplasias/química , Tonsila Palatina/química , Reprodutibilidade dos TestesRESUMO
Prostate cancer (PCa) remains the most common form of cancer affecting men in the Western Hemisphere. Mortality rate is 130% higher among African-American men (AAM) than Caucasian-American men. As this trend is not new nor changing, there is an urgent need to identify markers with the ability to specifically distinguish aggressive PCa in the context of race. Gene expression patterns have been used as a tool to identify prognostic biomarkers for PCa to help reduce this disparity. Gene expression profiles reveal molecular mechanisms useful in understanding the biologic basis of tumorigenesis. Thus far, gene expression profiling analyses focused on race between AAM and Caucasian-American men (CAM) demonstrated distinct tumor microenvironments in the tumor-adjacent stroma and pathways associated with inflammation, lipid metabolism, and regulation of epithelial-to-mesenchymal transition. Additionally, we and others have established that hypoxia, another component of the tumor microenvironment, can been linked to malignant progression, metastasis, resistance to therapy, and poor clinical outcome in PCa. Gene expression panels, including distinct components related to the biology of PCa in AAM, may increase prognostic accuracy for this ethnic group. Furthermore, reference gene expression patterns, especially in the context of the emerging molecular taxonomy of PCa, would be buttressed by including more AAM in their development to consider the aspects of expression profiles differentially associated with race.