RESUMO
Palmar-plantar erythrodysesthesia (PPE), also known as hand and foot syndrome, is a condition characterized by inflammation-mediated damage to the skin on the palms and soles of the hands and feet. PPE limits the successful therapeutic applications of anticancer drugs. However, identifying this toxicity during preclinical studies is challenging due to the lack of accurate in vitro and in vivo animal-based models. Therefore, there is a need for reliable models that would allow the detection of this toxicity early during the drug development process. Herein, we describe the use of an in vitro skin explant assay to assess traditional DXR, Doxil reference listed drug (RLD) and two generic PEGylated liposomal DXR formulations for their abilities to cause inflammation and skin damage. We demonstrate that the results obtained with the in vitro skin explant assay model for traditional DXR and Doxil correlate with the clinical data.
RESUMO
Alternative 3-dimensional (3D) skin models that replicate in vivo human skin are required to investigate important events during wound healing, such as collective cell migration, epidermal layer formation, dermal substrate formation, re-epithelialisation and collagen production. In this study, a matched human 3D skin equivalent model (3D-SEM) was developed from human skin cells (fibroblast and keratinocytes), characterised using haematoxylin and eosin, immunofluorescence staining and microRNA profiling. The 3D-SEM was then functionally tested for its use in wound healing studies. Mesenchymal stem cells (MSCs) were isolated and characterised according to the criteria stipulated by the International Society for Cell Therapy. Cytokine and growth factor secretions were analysed by enzyme-linked immunosorbent assay. MSC-conditioned medium (MSC-CM) was then tested for wound healing capacity using the developed 3D-SEM at different timepoints i.e., at one, two and four weeks. The constructed 3D-SEM showed consistent development of skin-like structures composed of dermal layers and epidermal layers, with the ability to express epidermal differentiation markers and full stratification. They also showed prolonged longevity in culture media, retaining full differentiation and stratification within the four weeks. MicroRNA profiling revealed a strong correlation in microRNA expression between the developed 3D-SEM and the original native skin (p<0.001; R=0.64). Additionally, MSC-CM significantly enhanced migration, proliferation and differentiation of epidermal cells in the wounded models compared to control models at the different timepoints. In conclusion, in this study, the developed 3D-SEM mimicked native skin at the cellular and molecular levels, and clearly showed the important stages of skin regeneration during the healing process. MSC secretome contains growth factors that play a pivotal role in the healing process and could be used as a therapeutic option to accelerate skin healing.
Assuntos
Células-Tronco Mesenquimais , Cicatrização , Humanos , Meios de Cultivo Condicionados/farmacologia , Cicatrização/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Pele/lesões , Pele/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células CultivadasRESUMO
The United States Senate passed the "FDA Modernization Act 2.0." on September 29, 2022. Although the effectiveness of this Bill, which aims to eliminate the mandatory use of laboratory animals in new drug development, is limited, it represents a significant trend that will change the shape of drug applications in the United States and other countries. However, pharmaceutical companies have not taken major steps towards the complete elimination of animal testing from the standpoint of product safety, where they prioritize patient safety. Nonetheless, society is becoming increasingly opposed to animal testing, and efforts will be made to use fewer animals and conduct fewer animal tests as a natural and reasonable response. These changes eventually alter the shape of new drug applications. Based on the assumption that fewer animal tests will be conducted or fewer animals will be used in testing, this study explored bioinformatics and new technologies as alternatives to compensate for reduced information and provide a picture of how future new drug applications may look. The authors also discuss the directions that pharmaceutical companies and nonclinical contract research organizations should adopt to promote the replacement, reduction, and refinement of animals used in research, teaching, testing, and exhibitions.
RESUMO
Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/imunologia , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Animais , Autofagia/imunologia , Proteínas Relacionadas à Autofagia , Antígeno B7-1/biossíntese , Antígeno B7-2/biossíntese , Antígenos CD40/biossíntese , Proteínas de Transporte/genética , Proliferação de Células , Células Cultivadas , Colite/imunologia , Cisteína Endopeptidases/biossíntese , Modelos Animais de Doenças , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/genética , Proteínas Imediatamente Precoces/biossíntese , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Ativação Linfocitária/imunologia , Lisossomos/patologia , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transplante Homólogo , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
BACKGROUND: The effectiveness of Nicotine Replacement Therapy (NRT) for smoking cessation in pregnancy is limited by inconsistent and incorrect use. This paper describes the development process for "Baby, Me, & NRT", a novel pregnancy-specific intervention aimed at enhancing adherence to NRT. METHODS: An integrated approach to intervention development was used, combining evidence, theory, stakeholders' feedback, and tailoring principles. The process involved six iterative steps: (1) synthesizing relevant published evidence and guidance, (2) collecting primary qualitative data on barriers and facilitators to NRT adherence along with potential intervention design features, (3) identifying relevant behavioral theories and mapping the evidence against these, (4) prioritizing behavioral determinants identified in steps 1 and 2, generating intervention objectives, and identifying behavior change techniques which target the prioritized determinants, (5) consulting with stakeholders on intervention components, key content and tailoring features, and (6) producing a prototype intervention along with implementation guidance. RESULTS: The prototype intervention comprises of a multi-component, 1-month cessation programme, which includes six enhanced behavioral support sessions delivered by a trained advisor, tailored text messages, a website, and an illustrated booklet. It promotes the uptake of high-dose and combination NRT, emphasizes the importance of adherence, addresses motivation to use NRT, proactively helps problem solve NRT use issues, and provides guidance on preventing and managing smoking lapses. CONCLUSION: The development process generated an evidence- and theory-guided intervention, designed with stakeholder input, aimed at improving NRT effectiveness for smoking cessation in pregnancy. The prototype intervention has since been optimized and is being evaluated in a randomized controlled trial.
Assuntos
Terapia Comportamental , Abandono do Hábito de Fumar , Feminino , Humanos , Gravidez , Fumar , Abandono do Hábito de Fumar/métodos , Fumar Tabaco , Dispositivos para o Abandono do Uso de TabacoRESUMO
INTRODUCTION: Improving adherence to nicotine replacement therapy (NRT) in pregnancy may result in higher smoking cessation rates. Informed by the Necessities and Concerns Framework, we developed an intervention targeting pregnancy NRT adherence. To evaluate this, we derived the NRT in pregnancy necessities and concerns questionnaire (NiP-NCQ), which measures perceived need for NRT and concerns about potential consequences. AIMS AND METHODS: Here we describe the development and content validation of NiP-NCQ. From qualitative work, we identified potentially modifiable determinants of pregnancy NRT adherence and classed these as necessity beliefs or concerns. We translated these into draft self-report items and piloted items on 39 pregnant women offered NRT and a prototype NRT adherence intervention, assessing distributions and sensitivity to change. After removing poorly performing items, smoking cessation experts (N = 16) completed an online discriminant content validation (DCV) task to determine whether retained items measure a necessity belief, concern, both, or neither construct. RESULTS: Draft NRT concern items encompassed safety for the baby, side effects, too much or insufficient nicotine, and addictiveness. Draft necessity belief items included perceived need for NRT for short- and longer-term abstinence, and desire to minimize or cope without NRT. Of 22 out of 29 items retained after piloting, four were removed following the DCV task: three were judged to measure neither construct and one possibly both. The final NiP-NCQ comprised nine items per construct (18 total). CONCLUSIONS: The NiP-NCQ measures potentially modifiable determinants of pregnancy NRT adherence within two distinct constructs and may have research and clinical utility for evaluating interventions targeting these. IMPLICATIONS: Poor adherence to NRT in pregnancy may result from low perceived need and concerns about consequences; interventions challenging these beliefs may yield higher smoking cessation rates. To evaluate an NRT adherence intervention informed by the Necessities and Concerns Framework, we developed the NiP-NCQ. Through the content development and refinement processes described in this paper, we derived an evidence-based, 18-item questionnaire measuring two distinct constructs within two nine-item subscales. Higher concerns and lower necessity beliefs indicate more negative NRT beliefs; NiP-NCQ may have research and clinical utility for interventions targeting these.
Assuntos
Abandono do Hábito de Fumar , Gravidez , Feminino , Humanos , Dispositivos para o Abandono do Uso de Tabaco/efeitos adversos , Nicotina/uso terapêutico , Gestantes , AutorrelatoRESUMO
With the reduction or elimination of animal testing, manufacturers are left with limited options, as few robust in vitro tests are available and human studies are costly. Recently, concerns have been raised regarding potential adverse health effects associated with use of WEN by Chaz Dean (WCD) cleansing conditioners. The purpose of this study was to evaluate the immunogenic potential of a WCD hair cleansing conditioner by utilizing a novel in vitro human skin explant test. Peripheral blood mononuclear cells (PBMCs) and human skin biopsies were obtained from healthy volunteers. Monocyte derived dendritic cells (MoDCs) were generated, primed by 0.01% WCD cleansing conditioner exposure for 24 h, co-cultured with autologous lymphocytes for 4 days, and then cultured with skin biopsies for 3 days. The skin biopsies then underwent histopathological evaluation, and T cell proliferation and IFNγ levels were determined. Overall, this study showed that treatment with 0.01% WCD cleansing conditioner resulted in a negative prediction for in vivo immune response. Further, this analysis shows that the skin explant test is a viable alternative to animal testing for complex mixtures or commercially available products.
Assuntos
Cosméticos , Leucócitos Mononucleares , Animais , Cosméticos/toxicidade , Humanos , PeleRESUMO
Regulatory T cells (Treg) attenuate dendritic cell (DC) maturation and stimulatory function. Current knowledge on the functional impact of semimature DC is limited to CD4+ T cell proliferation and cytokine production. Little is known about the molecular basis underpinning the functional effects of Treg-treated DC (Treg-DC). We present novel evidence that Treg-DC skewed CD4+ naive T cell polarization toward a regulatory phenotype and impaired CD8+ T cell allo-reactive responses, including their ability to induce target tissue damage in a unique in vitro human graft-versus-host disease skin explant model. Microarray analysis clustered Treg-DC as a discrete population from mature-DC and immature-DC, with 51 and 93 genes that were significantly over- or underexpressed, respectively, compared with mature-DC. Quantitative real-time PCR analysis revealed an intermediate expression level of CD38, CD83, CD80 and CD86 mRNA in Treg-DC, lower than mature-DC, higher than immature-DC. We also observed an attenuation of NF-κB pathway, an upstream regulator of the aforementioned genes, concomitant with reduced expression of two NF-κB-signaling related genes RELB and NFκBIZ, in the Treg-DC, together with an increased expression of Wnt5a, a negative regulator of DC differentiation. We further confirmed that the Treg-DC-mediated skewed CD4+ naive T cell polarization resulted from decreased IL-12 secretion by Treg-DC, which may be post-transcriptionally modulated by decreased expression of microRNA-155 in Treg-DC. To our knowledge, this is the first study demonstrating a transcriptional modulation of DC function by human Treg, partially via attenuation of the NF-κB signaling pathway and upregulation of Wnt5a, suggesting Treg may interfere with DC reprogramming during maturation, thereby modulating DC function.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Linfócitos T Reguladores/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Ativação Linfocitária/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Transcrição Gênica , Proteína Wnt-5a/imunologia , Proteína Wnt-5a/metabolismoRESUMO
BACKGROUND: Electronic cigarettes (ECs) are increasingly used for reducing or stopping smoking, with some studies showing positive outcomes. However, little is known about views on ECs during pregnancy or postpartum and previous studies have nearly all been conducted in the US and have methodological limitations, such as not distinguishing between smokers and ex/non-smokers. A greater understanding of this topic will help to inform both clinicians and EC interventions. We elicited views and experiences of ECs among UK pregnant or recently pregnant women. METHODS: We conducted semi-structured telephone interviews, using topic guides, with pregnant or recently pregnant women, who were current or recent ex-smokers. To ensure broad views of ECs were obtained, recruitment was from several geographical locations and via various avenues of recruitment. This included stop smoking services, antenatal and health visitor clinics, a pregnancy website and an informal network. Participants were 15 pregnant and 15 postpartum women, including nine current EC users, 11 ex-users, and 10 never-users. Five women who were interviewed in pregnancy were later interviewed in postpartum to explore if their views had changed. Audio data was transcribed verbatim and framework analysis was applied. RESULTS: Five main themes emerged: motivations for use (e.g., for stopping or reducing smoking), social stigma (e.g., avoiding use in public, preferring 'discrete' NRT), using the EC (e.g., mostly used at home); consumer aspects (e.g., limited advice available), and harm perceptions (e.g., viewed as less harmful than smoking; concerns about safety and addiction). CONCLUSIONS: ECs were viewed positively by some pregnant and postpartum women and seen as less harmful than smoking and useful as aids for reducing and stopping smoking. However, due to perceived social stigma, some women feel uncomfortable using ECs in public, especially during pregnancy, and had concerns about safety and nicotine dependence. Health professionals and designers of EC interventions need to provide women with up-to-date and consistent information and advice about safety and dependence, as well as considering the influence of social stigma.
Assuntos
Atitude , Gestantes/psicologia , Vaping , Adulto , Informação de Saúde ao Consumidor , Sistemas Eletrônicos de Liberação de Nicotina , Feminino , Humanos , Entrevistas como Assunto , Parto , Período Pós-Parto , Gravidez , Pesquisa Qualitativa , Abandono do Hábito de Fumar , Estigma Social , Nações Unidas , Vaping/efeitos adversos , Adulto JovemRESUMO
Haematopoietic stem cell transplantation (HSCT) remains the only cure for most haematological malignancies, however, the mortality rate remains high. Complications after HSCT include relapse, graft versus host disease (GvHD), graft rejection and infection. Over the last few years several groups, have demonstrated that non-HLA gene polymorphisms can be predictive of outcome after HSCT. Since the glucocorticoid cortisol is pivotal in the regulation of the immune system, we decided to examine single nucleotide polymorphisms (SNPs; rs6198, rs33388 and rs33389) within the glucocorticoid receptor (GR) and correlate with HSCT outcome. The training set consisted of patients (n = 458) who underwent HSCT for acute leukaemia between 1983 and 2005. In the recipients, the absence of the ACT haplotype and absence of the T allele of rs33388 were associated with decreased OS and the absence of the ACT haplotype, the absence of the T allele of rs33388 and the presence of the ATA haplotype were associated with increased risk of relapse. In addition, the presence of the ACT haplotype in the recipient showed a trend to be associated with increased risk of chronic graft versus host disease (cGvHD). The patients in this cohort received mainly myeloablative conditioning (n = 327). The SNPs in the glucocorticoid receptor were then investigated in a validation set (n = 251) of HSCT patients transplanted for acute leukaemia from 2006. This cohort contained significantly more patients that had received reduced intensity conditioning (RIC). Some of the results could be validated in these patients. However, contrary to the training set, the absence of the haplotype ACT in the donor in this cohort was associated with increased risk of cGvHD. Differences in the conditioning were shown to influence the results. These results are the first to associate GR SNPs with HSCT outcome and demonstrate the inherent problems of replicating SNP association studies in HSCT, due to different pre-transplant regimens.
RESUMO
Alemtuzumab conditioning is highly effective at reducing the incidence of acute and chronic graft-versus-host disease (GVHD) in reduced-intensity fludarabine and melphalan transplantation with cyclosporine monotherapy. Less frequent and lower dose scheduling may be used with sibling donors, but an optimal regimen for matched unrelated donors has not been defined. In this retrospective observational study of 313 patients, the incidence and severity of GVHD was compared in patients receiving 3 different dose schedules: the standard 100-mg regimen (20 mg on days -7 to -3), 60 mg (30 mg on days -4 and -2), or 50 mg (10 mg on days -7 to -3). Patients treated with 100 mg, 60 mg, or 50 mg developed acute GVHD grades I to IV with an incidence of 74%, 65%, and 64%, respectively, whereas 36%, 32%, and 41% developed chronic GHVD. An excess of severe acute grades III/IV GVHD was observed in the 50-mg cohort (15% versus 2% to 6%; P = .016). The relative risk of severe acute grade GVHD remained more than 3-fold higher in the 50-mg cohort compared with the 100-mg cohort after adjustment for differences in HLA match, age, gender mismatch, cytomegalovirus risk, and diagnosis (P = .030). The findings indicate that the 60-mg alemtuzumab schedule was comparable with the 100-mg schedule, but more attenuated schedules may increase the risk of severe grade GVHD.
Assuntos
Alemtuzumab/administração & dosagem , Doença Enxerto-Hospedeiro/tratamento farmacológico , Adulto , Idoso , Aloenxertos/química , Aloenxertos/imunologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/terapia , Humanos , Masculino , Melfalan/uso terapêutico , Pessoa de Meia-Idade , Estudos Retrospectivos , Condicionamento Pré-Transplante/métodos , Doadores não Relacionados , Vidarabina/análogos & derivados , Vidarabina/uso terapêutico , Adulto JovemRESUMO
The workshop on "New Approaches to Investigate Drug-Induced Hypersensitivity" was held on June 5, 2014 at the Foresight Center, University of Liverpool. The aims of the workshop were to (1) discuss our current understanding of the genetic, clinical, and chemical basis of small molecule drug hypersensitivity, (2) highlight the current status of assays that might be developed to predict potential drug immunogenicity, and (3) identify the limitations, knowledge gaps, and challenges that limit the use of these assays and utilize the knowledge gained from the workshop to develop a pathway to establish new and improved assays that better predict drug-induced hypersensitivity reactions during the early stages of drug development. This perspective reviews the clinical and immunological bases of drug hypersensitivity and summarizes various experts' views on the different topics covered during the meeting.
Assuntos
Hipersensibilidade a Drogas , Animais , Bioensaio , Hipersensibilidade a Drogas/genética , Hipersensibilidade a Drogas/imunologia , Indústria Farmacêutica , Predisposição Genética para Doença , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Fenótipo , Fatores de RiscoRESUMO
The cornea is a self-renewing tissue located at the front of the eye. Its transparency is essential for allowing light to focus onto the retina for visual perception. The continuous renewal of corneal epithelium is supported by limbal stem cells (LSCs) which are located in the border region between conjunctiva and cornea known as the limbus. Ex vivo expansion of LSCs has been successfully applied in the last two decades to treat patients with limbal stem cell deficiency (LSCD). Various methods have been used for their expansion, yet the most widely used culture media contains a number of ingredients derived from animal sources which may compromise the safety profile of human LSC transplantation. In this study we sought to understand the role of these components namely adenine, cholera toxin, hydrocortisone and triiodothyronine with the aim of re-defining a safe and GMP compatible minimal media for the ex vivo expansion of LSCs on human amniotic membrane. Our data suggest that all four components play a critical role in maintaining LSC proliferation and promoting LSC self-renewal. However removal of adenine and triiodothyronine had a more profound impact and led to LSC differentiation and loss of viability respectively, suggesting their essential role for ex vivo expansion of LSCs. Replacement of each of the components with GMP-grade reagents resulted in equal growth to non-GMP grade media, however an enhanced differentiation of LSCs was observed, suggesting that additional combinations of GMP grade reagents need to be tested to achieve similar or better level of LSC maintenance in the same manner as the traditional LSC media.
Assuntos
Adenina/metabolismo , Toxina da Cólera/metabolismo , Hidrocortisona/metabolismo , Limbo da Córnea/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Tri-Iodotironina/metabolismo , Cadáver , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Doenças da Córnea/cirurgia , Meios de Cultura , DNA/genética , Humanos , Imuno-Histoquímica , Limbo da Córnea/metabolismo , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
BACKGROUND: In countries where there are large disparities in smoking with persistent high rates among disadvantaged groups, there is a need to ensure that stop smoking services (SSS) reach such smokers. The primary aim of this study was to evaluate the effectiveness of a mobile, drop-in, community-based SSS in reaching more disadvantaged smokers, particularly those from routine and manual (RM) occupation groups, than standard services; secondary aims were to evaluate effectiveness in reaching those who had not previously accessed SSS, triggering unplanned quit behaviour, helping people quit and cost-effectiveness. METHODS: Following a 4-week pilot period, a mobile drop-in SSS was delivered across various public locations in Nottingham City, UK for 6 months, offering behavioural and pharmacological support via one-to-one consultations with trained cessation advisors. Detailed demographic and smoking behaviour data were collected from all clients accessing the mobile SSS, and Nottingham's standard SSS for comparison. RESULTS: Compared with smokers accessing the standard SSS (n=1856), mobile SSS smokers (n=811) were significantly more likely to be from the RM group (33.3% vs 27.2%, p=0.002), and to be first-time SSS users (67.8% vs 59.3%, p<0.001). Nearly 1 in 10 smokers setting a quit date through the mobile SSS had no prior quit intentions. The cost per smoker setting a quit date for the mobile SSS was only slightly higher than the standard SSS (£224 vs £202). CONCLUSIONS: A mobile drop-in SSS is an effective way of reaching more disadvantaged smokers from RM occupations, as well as those who have not previously accessed standard SSS and those without prior quit intentions.
Assuntos
Serviços de Saúde , Abandono do Hábito de Fumar , Adulto , Análise Custo-Benefício , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Encaminhamento e Consulta , Abandono do Hábito de Fumar/economia , Reino UnidoRESUMO
In vivo T cell depletion with 100 mg alemtuzumab prevents graft-versus-host disease (GVHD) in reduced intensity conditioned transplants but is associated with delayed immune reconstitution, a higher risk of infection and relapse. De-escalation studies have shown that a reduced dose of 30 mg is as effective as 100 mg in preventing GVHD in matched related donor (MRD) transplants. Dose reduction in matched unrelated donor (MUD) transplants is feasible but the comparative efficacy of alemtuzumab in this setting is not known and opinions vary widely concerning the optimal level of GVHD prophylaxis that should be achieved. Through retrospective analysis we made an objective comparison of MUD transplants receiving an empirically reduced dose of 60 mg, with MRD transplants receiving a 30 mg dose. We observed proportionate levels of alemtuzumab according to dose but an inverse relationship with body surface area particularly in MRD transplants. MUD transplants experienced more acute and chronic GVHD, higher T cell chimerism, more sustained use of ciclosporin and less need for donor lymphocyte infusion than MRD transplants. Thus, doubling the dose of alemtuzumab to 60 mg did not provide equivalent prevention of GVHD after MUD transplant although there was no difference in non-relapse mortality or survival compared with MRD transplants.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Adulto , Alemtuzumab , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/sangue , Antineoplásicos/uso terapêutico , Ciclosporina/administração & dosagem , Relação Dose-Resposta a Droga , Esquema de Medicação , Avaliação de Medicamentos/métodos , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Neoplasias Hematológicas/sangue , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Teste de Histocompatibilidade , Humanos , Imunossupressores/administração & dosagem , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Quimeras de Transplante , Condicionamento Pré-Transplante/métodos , Doadores não RelacionadosRESUMO
BACKGROUND AIMS: Advanced therapy medicinal products (ATMP) are gene therapy, somatic cell therapy or tissue-engineered products regulated under (EC) No. 1394/2007 to ensure their free movement within the European Union while guaranteeing the highest level of health protection for patients. Academic good manufacturing practice (GMP) centers are major contributors in the development of ATMPs and this study assessed the impact of regulations on them. METHODS: European academic and non-industrial facilities (n = 747) were contacted, and a representative sample of 50 replied to a detailed questionnaire. Experienced centres were further selected in every Member State (MS) for semi-structured interviews. Indicators of ATMP production and development success were statistically assessed, and opinions about directive implementation were documented. RESULTS: Facilities experienced in manufacturing cell therapy transplant products are the most successful in developing ATMPs. New centres lacking this background struggle to enter the field, and there remains a shortage of facilities in academia participating in translational research. This is compounded by heterogeneous implementation of the regulations across MS. CONCLUSIONS: GMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.
Assuntos
Centros Médicos Acadêmicos/estatística & dados numéricos , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética/legislação & jurisprudência , Fidelidade a Diretrizes/estatística & dados numéricos , Pesquisa Translacional Biomédica/estatística & dados numéricos , Animais , Biotecnologia , Comércio , União Europeia , Regulamentação Governamental , HumanosRESUMO
BACKGROUND: Digital image analysis has the potential to address issues surrounding traditional histological techniques including a lack of objectivity and high variability, through the application of quantitative analysis. A key initial step in image analysis is the identification of regions of interest. A widely applied methodology is that of segmentation. This paper proposes the application of image analysis techniques to segment skin tissue with varying degrees of histopathological damage. The segmentation of human tissue is challenging as a consequence of the complexity of the tissue structures and inconsistencies in tissue preparation, hence there is a need for a new robust method with the capability to handle the additional challenges materialising from histopathological damage. METHODS: A new algorithm has been developed which combines enhanced colour information, created following a transformation to the L*a*b* colourspace, with general image intensity information. A colour normalisation step is included to enhance the algorithm's robustness to variations in the lighting and staining of the input images. The resulting optimised image is subjected to thresholding and the segmentation is fine-tuned using a combination of morphological processing and object classification rules. The segmentation algorithm was tested on 40 digital images of haematoxylin & eosin (H&E) stained skin biopsies. Accuracy, sensitivity and specificity of the algorithmic procedure were assessed through the comparison of the proposed methodology against manual methods. RESULTS: Experimental results show the proposed fully automated methodology segments the epidermis with a mean specificity of 97.7%, a mean sensitivity of 89.4% and a mean accuracy of 96.5%. When a simple user interaction step is included, the specificity increases to 98.0%, the sensitivity to 91.0% and the accuracy to 96.8%. The algorithm segments effectively for different severities of tissue damage. CONCLUSIONS: Epidermal segmentation is a crucial first step in a range of applications including melanoma detection and the assessment of histopathological damage in skin. The proposed methodology is able to segment the epidermis with different levels of histological damage. The basic method framework could be applied to segmentation of other epithelial tissues.
Assuntos
Corantes , Amarelo de Eosina-(YS) , Hematoxilina , Pele/patologia , Algoritmos , Humanos , Interpretação de Imagem Assistida por Computador , Melanoma/diagnóstico , Sensibilidade e Especificidade , Pele/citologiaRESUMO
Introduction: Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. Material and Methods: Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Results: Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II-III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. Conclusions: The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation.
RESUMO
Biologics, including monoclonal antibodies (mAb), have proved to be effective and successful therapeutic agents, particularly in the treatment of cancer and immune-inflammatory conditions, as well as allergies and infections. However, their use carries an inherent risk of an immune-mediated adverse drug reaction. In this study, we describe the use of a novel pre-clinical human in vitro skin explant test for predicting skin sensitization and adverse immune reactions. The skin explant test was used to investigate the effects of therapeutic antibodies, which are known to cause a limited reaction in a small number of patients or more severe reactions. MATERIAL AND METHODS: Immune responses were determined by T cell proliferation and multiplex cytokine analysis, as well as histopathological analysis of skin damage (grades I-IV in increasing severity), predicting a negative (grade I) or positive (grade ≥ II) response for an adverse skin sensitization effect. RESULTS: T cell proliferation responses were significantly increased in the positive group (p < 0.004). Multiplex cytokine analysis showed significantly increased levels of IFNγ, TNFα, IL-10, IL-12, IL-13, IL-1ß, and IL-4 in the positive response group compared with the negative response group (p < 0.0001, p < 0.0001, p < 0.002, p < 0.01, p < 0.04, p < 0.006, and p < 0.004, respectively). CONCLUSIONS: Overall, the skin explant test correctly predicted the clinical outcome of 13 out of 16 therapeutic monoclonal antibodies with a correlation coefficient of 0.770 (p = 0.0001). This assay therefore provides a valuable pre-clinical test for predicting adverse immune reactions, including T cell proliferation and cytokine release, both associated with skin sensitization to monoclonal antibodies.
RESUMO
Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1ß on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.