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The Standards Coordinating Body for Gene, Cell, and Regenerative Medicines and Cell-Based Drug Discovery (SCB) supports the development and commercialization of regenerative medicine products by identifying and addressing industry-wide challenges through standards. Through extensive stakeholder engagement, the implementation of rapid microbial testing methods (RMTMs) was identified as a high-priority need that must be addressed to facilitate more timely release of products. Since 2017, SCB has coordinated efforts to develop standards for this area through surveys, weekly meetings, workshops, leadership in working groups and participation in standards development organizations. This article describes the results of these efforts and discusses the current landscape of RMTMs for regenerative medicine products. Based on discussions with stakeholders across the field, an overview of traditional culture-based methods and limitations, alternative microbial testing technologies and current challenges, fit-for-purpose rapid microbial testing and case studies, risk-based strategies for selection of novel rapid microbial test methods and ongoing standards efforts for rapid microbial testing are captured here. To this end, SCB is facilitating several initiatives to address challenges associated with rapid microbial testing for regenerative medicine products. Two documentary standards are under development: an International Organization for Standardization standard to provide the framework for a risk-based approach to selecting fit-for-purpose assays primarily intended for cell and gene therapy products and an ASTM standard guide focused on sampling methods for microbial testing methods in tissue-engineered medical products. Working with the National Institute of Standards and Technology, SCB expects to facilitate the process of developing publicly available microbial materials for inter-laboratory testing. These studies will help collect the data necessary to facilitate validation of novel rapid methods. Finally, SCB has been working to increase awareness of, dialog about and participation in efforts to develop standards in the regenerative medicine field.
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Medicina Regenerativa , Ingeniería de Tejidos , Bioensayo , Estándares de ReferenciaRESUMEN
Ancillary materials (AMs) play a critical role in the manufacture of cell and gene therapies, and best practices for their quality management are the subject of ongoing discussion. Given that the final product cannot be sterilized, AM quality becomes increasingly critical to the clinical advancement of cell and gene therapies. Despite a lack of direct legislative direction regarding AM quality, internationally harmonized guidance is available from several industry-standard bodies that describe the principles and application of a risk-based approach to AM qualification and related supply-chain risk management. According to a best-practice risk-based approach, AMs must be adequately qualified to a degree that reflects the level of risk the material presents to patient safety and the drug product's specification. This general approach can be implemented in different ways, and balancing quality with cost of goods is critical to the cost-effective manufacture of advanced therapy medicinal products. In some cases, it may be preferable or necessary to use AMs that are produced in compliance with current Good Manufacturing Practice. However, developers may be able to suppress manufacturing costs without undermining safety or regulatory compliance in the case that a material presents a lower risk profile. Despite a great deal of attention and interest in the quality of AMs in the cell and gene therapy space, there is still a need for greater harmonization to create a shared understanding of what constitutes a risk-based approach to AM production and sourcing. In this article, we propose a staged approach to AM quality that achieves a balance between the competing demands of risk mitigation and cost of goods containment at the various stages of AM quality development. Our novel, heuristic framework for communication among AM suppliers, users and regulators aims to bring down development and manufacturing costs and lessen the workload around regulatory compliance.
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Servicios Técnicos en Hospital/normas , Servicios Técnicos en Hospital/tendencias , Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Genética , Materiales Manufacturados/normas , Guías de Práctica Clínica como Asunto , Control de Calidad , Servicios Técnicos en Hospital/economía , Tratamiento Basado en Trasplante de Células y Tejidos/economía , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Comercio , Análisis Costo-Beneficio , Utilización de Equipos y Suministros/organización & administración , Utilización de Equipos y Suministros/normas , Terapia Genética/economía , Terapia Genética/métodos , Terapia Genética/normas , Terapia Genética/tendencias , Humanos , Materiales Manufacturados/economía , Materiales Manufacturados/provisión & distribución , Seguridad del Paciente/normas , Guías de Práctica Clínica como Asunto/normas , Estándares de Referencia , Gestión de Riesgos/organización & administración , Gestión de Riesgos/normasRESUMEN
Continued growth in the cell therapy industry and commercialization of cell therapies that successfully advance through clinical trials has led to increased awareness around the need for specialized and complex materials utilized in their manufacture. Ancillary materials (AMs) are components or reagents used during the manufacture of cell therapy products but are not intended to be part of the final products. Commonly, there are limitations in the availability of clinical-grade reagents used as AMs. Furthermore, AMs may affect the efficacy of the cell product and subsequent safety of the cell therapy for the patient. As such, AMs must be carefully selected and appropriately qualified during the cell therapy development process. However, the ongoing evolution of cell therapy research, limited number of clinical trials and registered cell therapy products results in the current absence of specific regulations governing the composition, compliance, and qualification of AMs often leads to confusion by suppliers and users in this field. Here we provide an overview and interpretation of the existing global framework surrounding AM use and investigate some common misunderstandings within the industry, with the aim of facilitating the appropriate selection and qualification of AMs. The key message we wish to emphasize is that in order to most effectively mitigate risk around cell therapy development and patient safety, users must work with their suppliers and regulators to qualify each AM to assess source, purity, identity, safety, and suitability in a given application.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Humanos , Internacionalidad , Control Social Formal , Terminología como AsuntoRESUMEN
A successful potency assay for a cell therapy product (CTP) used in the treatment of ischemic conditions should quantitatively measure relevant biological properties that predict therapeutic activity. This is especially challenging because of numerous degrees of complexity stemming from factors that include a multifactorial complex mechanism of action, cell source, inherent cell characteristics, culture method, administration mode and the in vivo conditions to which the cells are exposed. The expected biological function of a CTP encompasses complex interactions that range from a biochemical, metabolic or immunological activity to structural replacement of damaged tissue or organ. Therefore, the requirements for full characterization of the active substance with respect to biological function could be taxing. Moreover, the specific mechanism of action is often difficult to pinpoint to a specific molecular entity; rather, it is more dependent on the functionality of the cellular components acting in a in a multifactorial fashion. In the case of ischemic conditions, the cell therapy mechanism of action can vary from angiogenesis, vasculogenesis and arteriogenesis that may activate different pathways and clinical outcomes. The CTP cellular attributes with relation to the suggested mechanism of action can be used for the development of quantitative and reproducible analytical potency assays. CTPs selected and released on the basis of such potency assays should have the highest probability of providing meaningful clinical benefit for patients. This White Paper will discuss and give examples for key elements in the development of a potency assay for treatment of ischemic disorders treated by the use of CTPs.
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Bioensayo/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Isquemia/terapia , Humanos , Isquemia/diagnóstico , Neovascularización Fisiológica/fisiologíaRESUMEN
Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.
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Criopreservación , Crioprotectores , Crioprotectores/farmacología , Crioprotectores/metabolismo , Criopreservación/métodos , Congelación , Temperatura , Matriz Extracelular/metabolismoRESUMEN
The Regenerative Medicine Manufacturing Society (RMMS) is the first and only professional society dedicated toward advancing manufacturing solutions for the field of regenerative medicine. RMMS's vision is to provide greater patient access to regenerative medicine therapies through innovative manufacturing solutions. Our mission is to identify unmet needs and gaps in regenerative medicine manufacturing and catalyze the generation of new ideas and solutions by working with private and public stakeholders. We aim to accomplish our mission through outreach and education programs and securing grants for public-private collaborations in regenerative medicine manufacturing. This perspective will cover four impact areas that the society's leadership team has identified as critical: (a) cell manufacturing and scale-up/out, respectively, for allogeneic and autologous cell therapies, (b) standards for regenerative medicine, (c) 3D bioprinting, and (d) artificial intelligence-enabled automation. In addition to covering these areas and ways in which the society intends to advance the field in a collaborative nature, we will also discuss education and training. Education and training is an area that is critical for communicating the current challenges, developing solutions to accelerate the commercialization of the latest technological advances, and growing the workforce in the rapidly expanding sector of regenerative medicine.
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Inteligencia Artificial/normas , Automatización/métodos , Bioimpresión/métodos , Educación/métodos , Impresión Tridimensional/normas , Medicina Regenerativa/métodos , Ingeniería de Tejidos/métodos , Humanos , Resultado del TratamientoRESUMEN
The ability to cryopreserve natural killer (NK) cells has a significant potential in modern cancer immunotherapy. Current cryopreservation protocols cause deterioration in NK cell viability and functionality. This work reports the preservation of human cytokine-activated NK cell viability and function following cryopreservation using a cocktail of biocompatible bioinspired cryoprotectants (i.e., dextran and carboxylated ε-poly-L-lysine). Results demonstrate that the recovered NK cells after cryopreservation and rewarming maintain their viability immediately after thawing at a comparable level to control (dimethyl sulfoxide-based cryopreservation). Although, their viability drops in the first day in culture compared to controls, the cells grow back to a comparable level to controls after 1 week in culture. In addition, the anti-tumor functional activity of recovered NK cells demonstrates higher cytotoxic potency against leukemia cells compared to control. This approach presents a new direction for NK cell preservation, focusing on function and potentially enabling storage and distribution for cancer immunotherapy.
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Tear lipocalin (TL) (approximately 18 kDa), a member of the lipocalin superfamily, has been identified as one of the major proteins present in rabbit lacrimal fluid. The concentration of TL has been found to be decreased in the tears of patients with dry eye disease. Lacrimal gland insufficiency, one of the major causes of dry eye disease, is known to affect mainly postmenopausal women, where there is a significant decrease in the production of androgen and estrogen. These observations suggest that sex hormones might influence dry eye indirectly by regulating the expression of TL. The purpose of this study was to determine: (1) the effect of sexual maturation on the expression of TL; and (2) if the expression of TL is regulated by the estrogen, 17beta-estradiol, and/or the androgen, dihydrotestosterone, in sexually mature female rabbits. Lacrimal fluid (LF) and lacrimal gland soluble fraction (Si) was collected from juvenile (2 kg) and sexually mature (4 kg) male and female New Zealand white (NZW) rabbits. In addition, LF and Si were collected from 4 kg rabbits, 7 days after being either sham operated (control), ovariectomized (OVX), ovariectomized treated with estrogen (OVX+E) or ovariectomized treated with dihydrotestosterone (OVX+DHT). Samples were analyzed for protein levels of TL by SDS-PAGE and Western blotting using a polyclonal rat anti-rabbit TL antibody. Densitometry analysis showed that TL protein levels in both LF and Si increased with age in male and female rabbits. In addition, TL protein levels were significantly higher in the sexually mature 4 kg male compared with the 4 kg female, while no significant difference in TL protein levels were seen among the juvenile male and female rabbits. Furthermore, ovariectomy decreased the protein levels of TL in LF and Si fraction by 50% and 20% respectively, compared with control values. Estrogen treatment increased TL protein levels by 30% and 50% in the LF and Si fraction respectively, compared with the sham operated group. DHT treatment also increased TL protein levels by approximately 150% in both LF and Si fraction compared with control values. These results support the hypothesis that sex hormones influence TL protein levels in rabbit lacrimal glands. The possibility of a role of TL in dry eye needs to be further investigated.
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Regulación de la Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/farmacología , Aparato Lagrimal/metabolismo , Lipocalina 1/metabolismo , Actinas/metabolismo , Envejecimiento/metabolismo , Animales , Western Blotting/métodos , Dihidrotestosterona/farmacología , Electroforesis en Gel de Poliacrilamida/métodos , Estradiol/farmacología , Proteínas del Ojo/metabolismo , Femenino , Aparato Lagrimal/anatomía & histología , Aparato Lagrimal/efectos de los fármacos , Lipocalina 1/genética , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , Conejos , Maduración Sexual/fisiologíaRESUMEN
The therapeutic potential of liposomes can be amplified when combined with biomaterial scaffolds. Such configurations overcome the convergent demands of therapies by enabling enhanced delivery, environmental responsiveness and potency. Liposomes benefit from the increased physical and mechanical strength, favorable rheological properties and natural environment conducive to improved tissue formation that scaffolds provide, while enabling biocompatible delivery of hydrophilic and lipophilic compounds that can be further functionalized to achieve targeted delivery. Topical, ocular, oral, nasal and vaginal applications have been explored using various polymer- or nanofiber-based scaffolds. Mechanistic and rheological findings on complexation between biomaterials, liposomes and cargo have led to multimodal systems with tremendous clinical potential. A review of the key developments in bioengineered liposome-scaffold composites is presented in this manuscript.
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Sistemas de Liberación de Medicamentos , Liposomas , Andamios del Tejido , Materiales Biocompatibles , Interacciones Hidrofóbicas e Hidrofílicas , Nanofibras , PolímerosRESUMEN
Natural killer (NK) cells are a critical part of the innate immune system, and have emerged as attractive targets for immunotherapies for various malignancies. Alongside the need for expansion of NK cells to reach clinically useful numbers, a critical component in the availability of NK cells for allogeneic therapy is cryopreservation. While a continuously-growing cell line such as NK-92 can avoid issues associated with isolating, activating, expanding, and manufacturing large numbers of peripheral blood-derived NKs, cryopreservation of these cells has not made much progress. NK cells are highly sensitive to freezing and thawing, while the use of DMSO during cryopreservation raises serious safety concerns. In this work, we evaluated a number of cryoprotectants that do not contain DMSO for their capacity to cryopreserve NK-92 cells over long-term while retaining their cytotoxic activity and viability, with the aim of identifying potential replacements to DMSO for safe clinical use of these cells.
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Criopreservación/métodos , Crioprotectores/farmacología , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Línea Celular , Citotoxicidad Inmunológica , Dimetilsulfóxido/metabolismo , HumanosRESUMEN
Liposomes were the first nanoscale drug to be approved for clinical use in 1995. Since then, the technology has grown considerably, and pioneering recent work in liposome-based delivery systems has brought about remarkable developments with significant clinical implications. This includes long-circulating liposomes, stimuli-responsive liposomes, nebulized liposomes, elastic liposomes for topical, oral and transdermal delivery and covalent lipid-drug complexes for improved drug plasma membrane crossing and targeting to specific organelles. While the regulatory bodies' opinion on liposomes is well-documented, current guidance that address new delivery systems are not. This review describes, in depth, the current state-of-the-art of these new liposomal delivery systems and provides a critical overview of the current regulatory landscape surrounding commercialization efforts of higher-level complexity systems, the expected requirements and the hurdles faced by companies seeking to bring novel liposome-based systems for clinical use to market.
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Liposomas/administración & dosificación , Liposomas/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , HumanosRESUMEN
Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs). The ADSCs isolated by MMPs displayed essentially identical morphological and phenotypical characteristics to cells isolated by bacterially-derived collagenase I and Liberase™. Samples isolated with MMPs and Liberase™ had comparable levels of CD73, CD90, and CD105. The adipogenic and osteogenic potential of the ADSCs isolated by MMPs was retained as compared to cells isolated with Liberase™. However, ADSCs isolated by Liberase™ displayed 6% contamination with other cells as per negative markers revealed by PE staining, as opposed to<1% for all MMP-treated samples. MMP-based cell isolation may contribute to optimization of transplantation technology.
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Separación Celular/métodos , Metaloproteinasas de la Matriz , Adipogénesis , Tejido Adiposo/citología , Diferenciación Celular , Colagenasas/metabolismo , Endotoxinas/análisis , Activación Enzimática , Citometría de Flujo , Proteínas Ligadas a GPI/metabolismo , Humanos , Inmunofenotipificación , Indicadores y Reactivos , Metaloproteinasa 12 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Células Madre Mesenquimatosas/citología , Osteogénesis , Proteínas Recombinantes/metabolismo , Termolisina/metabolismoRESUMEN
The Cancer Genome Anatomy Project (CGAP) database has thousands of Expressed Sequence Tags encompassing both known and novel genes. Bioinformatics of the CGAP database led to the prediction that Single Minded Gene (sim2) could be specific to colon tumors. The sim2 gene is located in a minimum region of the chromosome 21 often implicated in trisomia called Down's Syndrome Critical Region. To date, the sim proteins have not been shown to be involved in cancer. Intrigued by the possible association of a Down's syndrome-related gene to solid tumors, efforts were undertaken to validate the expression specificity. The sim2 isoform (sim2-short-form, sim2-s) expression was seen in carcinomas of colon, pancreas and prostate, but not in corresponding normal tissues. Stage-specific expression of the sim2-s protein was seen in normal matched paraffin sections of the colon tumors. In a matched set of tissues of Benign Prostatic Hyperplasia (BPH) and prostate carcinomas, sim2-s expression was detected in the BPH. The expression specificity of sim2-s in select solid tumors offers both diagnostic and therapeutic potential and warrants additional study.
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Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Neoplasias Pancreáticas/genética , Neoplasias de la Próstata/genética , Factores de Transcripción/genética , Adenoma/genética , Adenoma/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Biomarcadores de Tumor/biosíntesis , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Biología Computacional/métodos , Bases de Datos de Ácidos Nucleicos , Síndrome de Down/genética , Etiquetas de Secuencia Expresada , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Masculino , Estadificación de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/biosíntesisRESUMEN
The lipocalins are a highly divergent, ubiquitous family of proteins that commonly function in binding lipophilic molecules. Although a specific tear lipocalin is a major component of lacrimal fluid and tears in many mammals, there has been no definitive identification of such a protein in rabbit tears. The goals of this project were to identify the major proteins in rabbit (Oryctolagus cuniculus) lacrimal fluid, so as to determine if they include a lipocalin and, if such a protein is present, to determine its source. Lacrimal fluid was collected from NZW sexually mature female rabbits, and culture medium from rabbit lacrimal gland epithelial (acinar) and interstitial cells was isolated. Proteins from these fluids were separated by SDS-PAGE electrophoresis and analyzed by sequencing the intact proteins and sequencing or mass analysis of fragments derived by trypsin digestion. Proteins of approximately 85 and 67 kDa were identified as rabbit transferrin and serum albumin, respectively, while components of 17 and 7 kDa had N-terminal sequences identical to those of lipophilin CL and AL, respectively. BLAST searches of the nr database with the N-terminal sequence of a protein of 18 kDa did not identify any homologues. However, when used to scan the PROSITE database, it was found to contain a lipocalin signature sequence. It is closely related to two lipocalins previously isolated from rabbit saliva and nasal mucus. Further studies with the N-terminal and internal sequences confirmed that the lacrimal protein is a lipocalin that is truncated at the N-terminus as compared with other tear lipocalins and is more similar to odorant binding proteins from rodents.
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Proteínas Portadoras/metabolismo , Lágrimas/química , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Femenino , Immunoblotting , Aparato Lagrimal/metabolismo , Lipocalina 1 , Datos de Secuencia Molecular , Proteínas de la Mielina/análisis , Proteínas de la Mielina/metabolismo , Proteolípidos/análisis , Proteolípidos/metabolismo , Conejos , Secretoglobinas , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , UteroglobinaRESUMEN
In a previous study, a data mining tool called Digital Differential Display (DDD) from the Cancer Genome Anatomy Project (CGAP) was used to predict solid tumor- and organ-specific genes from the expressed sequence tag (EST) database. To validate the use of bioinformatics approaches in gene discovery, one of the ESTs, which was predicted to be colon tumor-specific, was chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from normal and colon tumor tissues indicated that the EST was specifically expressed in the majority of colon tumors. Expression was also detected in early adenomas. Among other normal tissues, EST expression was detected only in the small intestine. The colon tumor specificity of this EST was inferred from the lack of expression in carcinomas of the breast, lung, ovary, pancreas and prostate. To validate the computational prediction of specificity, a full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to the colon tumors, this gene was termed Colon Carcinoma Related Gene (CCRG). CCRG encodes a novel cysteine-rich motif and a putative signal peptide sequence. Supernatant from COS cells transfected with the CCRG expression vector stimulated proliferation of colon cancer cells. Immunoreactive CCRG was also detected in the paraffin sections of colon tumor samples. CCRG belongs to a new class of growth factors and may be important in the diagnosis and treatment of colon cancers. Identification of CCRG using bioinformatics approaches validates gene discovery using computational approaches.
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Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Biología Computacional , Proteínas de Neoplasias/genética , Secuencia de Aminoácidos , Neoplasias del Colon/química , ADN Complementario , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Proteínas de Neoplasias/análisis , Sistemas de Lectura Abierta/genética , Señales de Clasificación de Proteína/genética , Sensibilidad y EspecificidadRESUMEN
Current red-blood-cell cryopreservation methods utilize bulk volumes, causing cryo-injury of cells, which results in irreversible disruption of cell morphology, mechanics, and function. An innovative approach to preserve human red-blood-cell morphology, mechanics, and function following vitrification in nanoliter volumes is developed using a novel cryo-ink integrated with a bioprinting approach.
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Bioimpresión/métodos , Criopreservación/métodos , Eritrocitos/citología , Eritrocitos/fisiología , Nanotecnología/métodos , Vitrificación , Aminoácidos Diaminos/química , Fenómenos Biomecánicos , Bioimpresión/instrumentación , Criopreservación/instrumentación , Humanos , Tinta , Espacio Intracelular/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Nanotecnología/instrumentación , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Complemento 3b/metabolismoRESUMEN
Increased levels of the matrix metalloproteinases (MMPs)-2 and -9 have been found in tear fluids of patients with dry eye disease, suggesting that these MMPs may be implicated in the pathogenesis of this disease. One of the main causes of dry eye disease is lacrimal gland insufficiency. However, the contribution of the lacrimal gland (LG) to the expression and production of MMP-2 and MMP-9 in tears is not known. Since dry eye disease occurs more frequently in women, sex hormones, especially estrogens, have also been implicated in the pathogenesis of this disease. Estrogens have been shown to regulate the synthesis levels of MMP-2 and MMP-9 in several tissues, Thus, the purpose of these studies was to determine if: (1) rabbit lacrimal glands secrete MMP-2 and MMP-9; (2) MMP-2 and MMP-9 are produced by lacrimal epithelial cells and/or lacrimal lymphocytes; and (3) the expression, activity and level of these enzymes are regulated by sex hormones. Lacrimal epithelial cells (LEC) and lacrimal lymphocytes (LL) from sexually mature New Zealand White female rabbits were isolated, purified and cultured with and without 10(-6)M dihydrotestosterone (DHT) or 10(-6), 10(-8), 10(-9) and 10(-10)M 17beta-estradiol (E2). The culture supernatants were analyzed by zymography and western blotting (WB) using polyclonal anti-human MMP-2 and MMP-9 antibodies. LGs were also collected from rabbits 7 days after being sham-operated, ovariectomized (OVX), OVX treated with 4 mg/kg DHT, and OVX treated with 0.5 mg/kg of E2. LGs were collected and processed for RNA extraction as well as protein determination using WB and immunocytochemistry. The pro-forms of MMP-2 and MMP-9 were detected in primary LEC and LL culture medium by zymography and WB. Pro-MMP-2 and pro-MMP-9 were also detected at the gene and protein levels in the lacrimal glands of all four treatment groups, with the highest levels and gene expression found in the estrogen-treated group. These results suggest that both pro-MMP-2 and pro-MMP-9 are secreted by the lacrimal gland and appear to be up-regulated by estrogen. The role of the lacrimal MMPs in the pathogenesis of dry eye disease needs to be further investigated.