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BACKGROUND AIMS: To explore the long-term safety and benefit of umbilical cord mesenchymal stromal cell (MSCs) plus autologous bone marrow mononuclear cell (aBM-MNC) stem cell transplantation (SCT) without immunotherapy in established type 1 diabetes (T1D). METHODS: In the primary completion of this trial (ClinicalTrials.gov identifier: NCT01374854), the authors randomized patients (n = 21 per group) to either SCT or standard care (control) and previously reported effects on insulin secretion. The authors report about the incidence of chronic diabetes complications (primary endpoint) after 8 years of follow-up. The authors also report on secondary endpoints, safety, islet function and metabolic control. RESULTS: Data were obtained from 14 of 21 patients in the SCT group and 15 of 21 patients in the control group who completed follow-up. At 8 years, the incidence of peripheral neuropathy was 7.1% (one of 14) in the SCT group versus 46.7% (seven of 15) in the control group (P = 0.017). The incidence of diabetic nephropathy was 7.1% (one of 14) in the SCT group versus 40.0% (six of 15) in the control group (P = 0.039). The incidence of retinopathy was 7.1% (one of 14) in the SCT group versus 33.3% (five of 15) in the control group (P = 0.081). Two patients (two of 14, 14.3%) in the SCT group and 11 patients (11 of 15, 73.3%) in the control group developed at least one complication (P = 0.001). One and six patients in the SCT group and control group, respectively, had at least two complications (P = 0.039). No malignancies were reported in the treated group. CONCLUSIONS: Co-transplantation of umbilical cord MSCs and aBM-MNCs in patients with established T1D was associated with reduced incidence of chronic diabetes complications.
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Complicaciones de la Diabetes , Diabetes Mellitus Tipo 1 , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Médula Ósea , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/terapia , Estudios de Seguimiento , Humanos , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Proyectos Piloto , Cordón UmbilicalRESUMEN
BACKGROUND AIMS: Extracellular vesicles (EVs) are being tested for their use as novel therapeutics. However, the optimal source of EVs is currently under investigation. Amniotic fluid (AF) is a natural source of EVs that can be easily obtained for use in regenerative medicine, yet AF-EV characterization has not been fully explored. METHODS: Here the authors demonstrate AF as a rich source of EVs and identify the microRNA and proteomic cargo. Bioinformatics analysis of this cargo revealed multiple pathway targets, including immunomodulatory, anti-inflammatory and free radical scavenging networks. The authors further demonstrated the therapeutic potential of this EV product as a novel preventative agent for bronchopulmonary dysplasia (BPD). RESULTS: Intra-tracheal administration of AF-EVs preserved alveolar development, attenuated vascular remodeling and pulmonary hypertension, decreased lung pro-inflammatory cytokine expression and reduced macrophage infiltration in an experimental BPD model. CONCLUSIONS: The authors' results suggest that AF is a viable biological fluid for EV harvest and that AF-EVs have strong therapeutic potential for pulmonary diseases, such as BPD, warranting further development to transition this novel EV product into the clinic.
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Displasia Broncopulmonar , Vesículas Extracelulares , Líquido Amniótico , Animales , Displasia Broncopulmonar/terapia , Modelos Animales de Enfermedad , Humanos , Recién Nacido , Modelos Teóricos , Proteómica , Ratas Sprague-DawleyRESUMEN
PURPOSE OF REVIEW: Discuss the recent progress on the clinical use of mesenchymal stromal (stem) cells (MSC) in solid organ transplantation (SOT). RECENT FINDINGS: Tissue repair and immunomodulatory properties have been recognized for MSC obtained from different human tissues. MSC-based therapy has been proposed to reduce ischemia-reperfusion injury and to promote immune tolerance. The results of recent clinical trial support the safety and promising effects of autologous and allogeneic MSC in SOT. Collectively, the use of MSC in recipients of living donor kidney transplantation was associated with improved graft function, reduced rejection, ability to omit induction and/or lower maintenance immunosuppression regimen, as well as to treat rejection episodes. SUMMARY: We are living in very exciting times with the implementation of novel clinical trials aimed at establishing safety, feasibility and efficacy of cellular therapies including MSC to improve SOT outcomes. The results of the initial clinical trials support the safety of MSC-based therapy and justifying cautious optimism for the immediate future.
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Células Madre Mesenquimatosas/inmunología , Trasplante de Órganos , Ensayos Clínicos como Asunto , Humanos , Tolerancia Inmunológica/inmunología , Trasplante de Células Madre Mesenquimatosas , Trasplante de Órganos/efectos adversos , Daño por Reperfusión/inmunologíaRESUMEN
CONTEXT: Antibody-based induction therapy plus calcineurin inhibitors (CNIs) reduce acute rejection rates in kidney recipients; however, opportunistic infections and toxic CNI effects remain challenging. Reportedly, mesenchymal stem cells (MSCs) have successfully treated graft-vs-host disease. OBJECTIVE: To assess autologous MSCs as replacement of antibody induction for patients with end-stage renal disease who undergo ABO-compatible, cross-match-negative kidney transplants from a living-related donor. DESIGN, SETTING, AND PATIENTS: One hundred fifty-nine patients were enrolled in this single-site, prospective, open-label, randomized study from February 2008-May 2009, when recruitment was completed. INTERVENTION: Patients were inoculated with marrow-derived autologous MSC (1-2 x 10(6)/kg) at kidney reperfusion and two weeks later. Fifty-three patients received standard-dose and 52 patients received low-dose CNIs (80% of standard); 51 patients in the control group received anti-IL-2 receptor antibody plus standard-dose CNIs. MAIN OUTCOME MEASURES: The primary measure was 1-year incidence of acute rejection and renal function (estimated glomerular filtration rate [eGFR]); the secondary measure was patient and graft survival and incidence of adverse events. RESULTS: Patient and graft survival at 13 to 30 months was similar in all groups. After 6 months, 4 of 53 patients (7.5%) in the autologous MSC plus standard-dose CNI group (95% CI, 0.4%-14.7%; P = .04) and 4 of 52 patients (7.7%) in the low-dose group (95% CI, 0.5%-14.9%; P = .046) compared with 11 of 51 controls (21.6%; 95% CI, 10.5%-32.6%) had biopsy-confirmed acute rejection. None of the patients in either autologous MSC group had glucorticoid-resistant rejection, whereas 4 patients (7.8%) in the control group did (95% CI, 0.6%-15.1%; overall P = .02). Renal function recovered faster among both MSC groups showing increased eGFR levels during the first month after surgery than the control group. Patients receiving standard-dose CNI had a mean difference of 6.2 mL/min per 1.73 m(2) (95% CI, 0.4-11.9; P=.04) and those in the low-dose CNI of 10.0 mL/min per 1.73 m(2) (95% CI, 3.8-16.2; P=.002). Also, during the 1-year follow-up, combined analysis of MSC-treated groups revealed significantly decreased risk of opportunistic infections than the control group (hazard ratio, 0.42; 95% CI, 0.20-0.85, P=.02) CONCLUSION: Among patients undergoing renal transplant, the use of autologous MSCs compared with anti-IL-2 receptor antibody induction therapy resulted in lower incidence of acute rejection, decreased risk of opportunistic infection, and better estimated renal function at 1 year. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00658073.
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Terapia de Inmunosupresión/métodos , Fallo Renal Crónico/cirugía , Trasplante de Riñón/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Adolescente , Adulto , Anticuerpos/uso terapéutico , Formación de Anticuerpos , Inhibidores de la Calcineurina , Femenino , Rechazo de Injerto/prevención & control , Humanos , Riñón/fisiología , Trasplante de Riñón/inmunología , Donadores Vivos , Masculino , Persona de Mediana Edad , Infecciones Oportunistas , Receptores de Interleucina-2/antagonistas & inhibidores , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Post-COVID-19 infection symptoms such as mental fog, tachycardia, and extreme fatigue are just a few of the symptoms wreaking havoc on patients' lives. Patients with long-term symptoms following COVID-19 are being called long haulers. To date, long haulers are receiving little to no guidance from physicians on their lingering COVID-19 symptoms with limited treatment options available. Zofin is an acellular biologic that contains the extracellular vesicle (EV) fraction of human amniotic fluid and is under investigation for use as a COVID-19 therapeutic. We obtained FDA and IRB approval to investigate the therapeutic use of Zofin in a single long hauler patient case experiencing prolonged shortness of breath and respiratory impairment. Administration of the EV product was shown to be safe. Furthermore, demonstrated respiratory improvements through chest X ray images and oxygen saturation measurement. The single patient IND studies were completed without any reported adverse events or safety concerns. Furthermore, these completed studies demonstrate the feasibility and a therapeutic potential of amniotic fluid-derived EVs for COVID-19 long hauler intervention.
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A pandemic brought on by COVID-19 has created a scalable health crisis. The search to help alleviate COVID-19-related complications through therapeutics has become a necessity. Zofin is an investigational, acellular biologic derived from full-term perinatal amniotic fluid that contains extracellular vesicles. Extracellular nanoparticles as such have been studied for their immunomodulatory benefits via cellular therapeutics and, if applied to COVID-19-related inflammation, could benefit patient outcome. Subjects (n = 8) experiencing mild-to-moderate COVID-19 symptoms were treated with the experimental intervention. Complete blood count, complete metabolic panel, inflammatory biomarkers, and absolute lymphocyte counts were recorded prior to and on days 4, 8, 14, 21, and 30 as markers of disease progression. Additionally, chest x-rays were taken of the patients prior to and on days 8 and 30. Patients experienced no serious adverse events. All COVID-19-associated symptoms resolved or became stable with no indication of disease worsening as found by patient and chest x-ray reports. Inflammatory biomarkers (CRP, IL-6, TNF- α ) and absolute lymphocyte counts improved throughout the study period. Findings from a proof-of-concept, expanded access trial for COVID-19 patients prove the acellular biologic is safe and potentially effective to prevent disease progression in a high-risk COVID-19 population with mild-to-moderate symptoms.
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Acute respiratory distress syndrome (ARDS) in COVID-19 is associated with high mortality. Mesenchymal stem cells are known to exert immunomodulatory and anti-inflammatory effects and could yield beneficial effects in COVID-19 ARDS. The objective of this study was to determine safety and explore efficacy of umbilical cord mesenchymal stem cell (UC-MSC) infusions in subjects with COVID-19 ARDS. A double-blind, phase 1/2a, randomized, controlled trial was performed. Randomization and stratification by ARDS severity was used to foster balance among groups. All subjects were analyzed under intention to treat design. Twenty-four subjects were randomized 1:1 to either UC-MSC treatment (n = 12) or the control group (n = 12). Subjects in the UC-MSC treatment group received two intravenous infusions (at day 0 and 3) of 100 ± 20 × 106 UC-MSCs; controls received two infusions of vehicle solution. Both groups received best standard of care. Primary endpoint was safety (adverse events [AEs]) within 6 hours; cardiac arrest or death within 24 hours postinfusion). Secondary endpoints included patient survival at 31 days after the first infusion and time to recovery. No difference was observed between groups in infusion-associated AEs. No serious adverse events (SAEs) were observed related to UC-MSC infusions. UC-MSC infusions in COVID-19 ARDS were found to be safe. Inflammatory cytokines were significantly decreased in UC-MSC-treated subjects at day 6. Treatment was associated with significantly improved patient survival (91% vs 42%, P = .015), SAE-free survival (P = .008), and time to recovery (P = .03). UC-MSC infusions are safe and could be beneficial in treating subjects with COVID-19 ARDS.
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Antiinflamatorios/uso terapéutico , COVID-19/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Citocinas/sangre , Método Doble Ciego , Femenino , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Células Madre Mesenquimatosas , Persona de Mediana Edad , SARS-CoV-2/efectos de los fármacos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Cordón Umbilical/citologíaRESUMEN
BACKGROUND: A physiological hallmark of patients with type 2 diabetes mellitus (T2DM) is ß cell dysfunction. Despite adequate treatment, it is an irreversible process that follows disease progression. Therefore, the development of novel therapies that restore ß cell function is of utmost importance. METHODS: This study aims to unveil the mechanistic action of mesenchymal stem cells (MSCs) by investigating its impact on isolated human T2DM islets ex vivo and in vivo. FINDINGS: We propose that MSCs can attenuate ß cell dysfunction by reversing ß cell dedifferentiation in an IL-1Ra-mediated manner. In response to the elevated expression of proinflammatory cytokines in human T2DM islet cells, we observed that MSCs was activated to secret IL-1R antagonist (IL-1Ra) which acted on the inflammed islets and reversed ß cell dedifferentiation, suggesting a crosstalk between MSCs and human T2DM islets. The co-transplantation of MSCs with human T2DM islets in diabetic SCID mice and intravenous infusion of MSCs in db/db mice revealed the reversal of ß cell dedifferentiation and improved glycaemic control in the latter. INTERPRETATION: This evidence highlights the potential of MSCs in future cell-based therapies regarding the amelioration of ß cell dysfunction.
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Desdiferenciación Celular , Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/patología , Células Madre Mesenquimatosas/metabolismo , Animales , Diabetes Mellitus Tipo 2/terapia , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1beta/metabolismo , Masculino , Trasplante de Células Madre Mesenquimatosas , Ratones SCID , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To determine the safety and effects on insulin secretion of umbilical cord (UC) mesenchymal stromal cells (MSCs) plus autologous bone marrow mononuclear cell (aBM-MNC) stem cell transplantation (SCT) without immunotherapy in established type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: Between January 2009 and December 2010, 42 patients with T1D were randomized (n = 21/group) to either SCT (1.1 × 10(6)/kg UC-MSC, 106.8 × 10(6)/kg aBM-MNC through supraselective pancreatic artery cannulation) or standard care (control). Patients were followed for 1 year at 3-month intervals. The primary end point was C-peptide area under the curve (AUC(C-Pep)) during an oral glucose tolerance test at 1 year. Additional end points were safety and tolerability of the procedure, metabolic control, and quality of life. RESULTS: The treatment was well tolerated. At 1 year, metabolic measures improved in treated patients: AUCC-Pep increased 105.7% (6.6 ± 6.1 to 13.6 ± 8.1 pmol/mL/180 min, P = 0.00012) in 20 of 21 responders, whereas it decreased 7.7% in control subjects (8.4 ± 6.8 to 7.7 ± 4.5 pmol/mL/180 min, P = 0.013 vs. SCT); insulin area under the curve increased 49.3% (1,477.8 ± 1,012.8 to 2,205.5 ± 1,194.0 mmol/mL/180 min, P = 0.01), whereas it decreased 5.7% in control subjects (1,517.7 ± 630.2 to 1,431.7 ± 441.6 mmol/mL/180 min, P = 0.027 vs. SCT). HbA1c decreased 12.6% (8.6 ± 0.81% [70.0 ± 7.1 mmol/mol] to 7.5 ± 1.0% [58.0 ± 8.6 mmol/mol], P < 0.01) in the treated group, whereas it increased 1.2% in the control group (8.7 ± 0.9% [72.0 ± 7.5 mmol/mol] to 8.8 ± 0.9% [73 ± 7.5 mmol/mol], P < 0.01 vs. SCT). Fasting glycemia decreased 24.4% (200.0 ± 51.1 to 151.2 ± 22.1 mg/dL, P < 0.002) and 4.3% in control subjects (192.4 ± 35.3 to 184.2 ± 34.3 mg/dL, P < 0.042). Daily insulin requirements decreased 29.2% in only the treated group (0.9 ± 0.2 to 0.6 ± 0.2 IU/day/kg, P = 0.001), with no change found in control subjects (0.9 ± 0.2 to 0.9 ± 0.2 IU/day/kg, P < 0.01 vs. SCT). CONCLUSIONS: Transplantation of UC-MSC and aBM-MNC was safe and associated with moderate improvement of metabolic measures in patients with established T1D.
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Trasplante de Médula Ósea , Diabetes Mellitus Tipo 1/terapia , Trasplante de Células Madre Mesenquimatosas , Adolescente , Adulto , Péptido C/sangre , Niño , Preescolar , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/metabolismo , Insulina/uso terapéutico , Secreción de Insulina , Masculino , Proyectos Piloto , Calidad de Vida , Trasplante Autólogo , Cordón Umbilical/citología , Adulto JovenRESUMEN
Hyperglycemia and increased insulin requirements are indicators of ongoing islet allograft rejection, but there are no methods to predict or confirm rejection. Elevation of cytotoxic lymphocyte (CL) gene expression in peripheral blood (PB) has been correlated with renal allograft rejection in humans, but no published study has assessed the utility of monitoring these markers as predictors of rejection before the onset of clinical symptoms. We have established quantitative real-time PCR methods to determine the levels of mRNA transcripts for the CL genes granzyme B (GB), perforin, and fas ligand in blood samples from rhesus and cynomolgus monkeys. Four rhesus monkeys with long-term islet allograft function were studied. Antirejection (anti-CD154) therapy was discontinued, and weekly PB samples were obtained to determine whether the levels of mRNA transcripts for CL genes correlated with and/or were predictive of islet allograft rejection, defined as a loss of C-peptide production. For all monkeys, elevation of CL gene expression preceded rejection by 83--197 days, with GB as the best predictor. Elevated mRNA levels were sustained for 2--2.5 months in three of four animals and 1 month in the other, thus suggesting that the testing of these parameters may have practical applications in clinical islet cell transplantation.
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Expresión Génica , Rechazo de Injerto/inmunología , Trasplante de Islotes Pancreáticos , Linfocitos T Citotóxicos/metabolismo , Animales , Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/inmunología , Proteína Ligando Fas , Rechazo de Injerto/prevención & control , Granzimas , Macaca mulatta , Glicoproteínas de Membrana/genética , Perforina , Reacción en Cadena de la Polimerasa/métodos , Proteínas Citotóxicas Formadoras de Poros , ARN Mensajero/análisis , Serina Endopeptidasas/genética , Factores de TiempoRESUMEN
Studies in nonhuman primates have demonstrated that elevation of the cytotoxic lymphocyte (CL) genes granzyme B, perforin, and Fas ligand in peripheral blood precedes islet allograft rejection. The purpose of this study was to determine whether this approach has utility for prediction of human islet allograft loss. We studied 13 patients who had long-term type 1 diabetes and were treated with steroid-free immunosuppression and given sequential islet cell infusions. All recipients became insulin independent, and eight of them experienced deterioration in glycemic control, followed by reinitiation of insulin therapy. Frequent peripheral blood samples were collected to monitor CL gene mRNA levels with real-time PCR. For the eight back-to-insulin patients, there was a clear elevation of CL gene mRNA levels 25-203 days before the onset of frequent hyperglycemia. Granzyme B was the most reliable indicator of ongoing graft loss. Additional correlations with infection were noted; however, evidence of sensitization in antidonor mixed lymphocyte reaction was observed in seven of eight patients who experienced partial graft loss, whereas this was not seen when upregulated CL gene expression was associated with infection. The results suggest that, when taken into consideration with other clinical parameters, elevated CL gene levels may enable prediction of islet allograft loss.
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Rechazo de Injerto/diagnóstico , Trasplante de Islotes Pancreáticos , Glicoproteínas de Membrana/genética , Serina Endopeptidasas/genética , Linfocitos T Citotóxicos/fisiología , Biomarcadores , Proteína Ligando Fas , Expresión Génica , Rechazo de Injerto/inmunología , Rechazo de Injerto/fisiopatología , Supervivencia de Injerto/fisiología , Granzimas , Humanos , Terapia de Inmunosupresión , Infecciones/inmunología , Infecciones/fisiopatología , Prueba de Cultivo Mixto de Linfocitos , Perforina , Proteínas Citotóxicas Formadoras de Poros , Valor Predictivo de las Pruebas , Trasplante HomólogoAsunto(s)
Fibrosis Quística/complicaciones , Trasplante de Islotes Pancreáticos/métodos , Pancreatectomía/métodos , Quiste Pancreático/cirugía , Terapia Combinada , Fibrosis Quística/diagnóstico por imagen , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Quiste Pancreático/complicaciones , Quiste Pancreático/diagnóstico por imagen , Trasplante Autólogo , Adulto JovenRESUMEN
An emergency autologous islet transplant after a traumatic Whipple operation and subsequent total pancreatectomy was performed for a 21-year-old patient who was wounded with multiple abdominal gunshot wounds. After Whipple pancreatectomy, the remnant pancreas (63.5 g), along with other damaged organs, was removed by the surgeons at Walter Reed Army Medical Center (WRAMC) and shipped to Diabetes Research Institute (DRI) for islet isolation. The pancreas was preserved in UW solution for 9.25 h prior to islet isolation. Upon arrival, the organ was visually inspected; the pancreatic head was missing, the rest of the pancreas was damaged and full of blood; the tail looked normal. A 16-gauge catheter was inserted into the main duct and directed towards tail of the pancreas after the dissection of main duct in the midbody of the pancreas. The pancreas was distended with collagenase solution (Roche MTF) through the catheter. During 10 min of intraductal delivery of enzyme, the gland was distended uniformly. No leakage of the solution was observed. The pancreas was transferred to a Ricordi chamber for automated mechanical and enzymatic digestion. Islets were purified using a COBE 2991 cell processor. Islet equivalents (IEQ; 221,250) of 40% purity and 90% viability were recovered during the isolation, which were shipped back to WRAMC and infused by intraportal injection into the patient. Immediate islet function was demonstrated by the rapid elevation of serum C peptide followed by insulin independence with near normal oral glucose tolerance test (OGTT) 1 and 2 months later. It is possible to restore near normal glucose tolerance with autologous islet transplantation after total pancreatectomy even with suboptimal number of islets while confirming that islets processed at a remote site are suitable for transplantation.
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Glucosa/metabolismo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/citología , Heridas por Arma de Fuego/terapia , Adenosina , Alopurinol , Péptido C/sangre , Prueba de Tolerancia a la Glucosa , Glutatión , Humanos , Insulina , Masculino , Soluciones Preservantes de Órganos , Pancreatectomía , Rafinosa , Trasplante Autólogo , Adulto JovenRESUMEN
Recombinant proteins are an important tool for research and therapeutic applications. Therapeutic proteins have been delivered to several cell types and tissues and might be used to improve the outcome of the cell transplantation. Recombinant proteins are propagated in bacteria, which will contaminate them with the lypopolysacharide endotoxin found in the outer bacterial membrane. Endotoxin could interfere with in vitro biological assays and is the major pathological factor, which must be removed or inactivated before in vivo administration. Here we describe a one-step protocol in which the endotoxin activity on recombinant proteins is remarkably reduced by transient exposure to acidic conditions. Maximum endotoxin deactivation occurs at acidic pH below their respective isoelectric point (pI). This method does not require additional protein purification or separation of the protein from the endotoxin fraction. The endotoxin level was measured both in vitro and in vivo. For in vitro assessment we have utilized Limulus Amebocyte Lysate method for in vivo the pyrogenic test. We have tested the above-mentioned method with five different recombinant proteins, including a monoclonal antibody clone 5c8 against CD154 produced by hybridomas. More than 99% of endotoxin was deactivated in all of the proteins; the recovery of the protein after deactivation varied between maximum 72.9% and minimum 46.8%. The anti-CD154 clone 5c8 activity remained unchanged as verified by the measurement of binding capability to activated lymphocytes. Furthermore, the effectiveness of this method was not significantly altered by urea, commonly used in protein purification. This procedure provides a simple and cost-efficient way to reduce the endotoxin activity in antibodies and recombinant proteins.
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Endotoxinas/química , Proteínas Recombinantes/aislamiento & purificación , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Urea/químicaRESUMEN
A sorbent was synthesized and investigated for molecularly imprinted solid phase extraction (MISPE). Molecularly imprinted polymers (MIP) were synthesized via precipitation polymerization procedure, where 4-vinyl pyridine (4-VP) was used as functional monomer and ethylene glycol dimethacrylate (EDMA) as cross-linking agent. The imprinting effect of the MISPE was evaluated by elution experiments. The resulting MISPE showed high extraction selectivity to water-soluble and fat-soluble synthetic colorants. The determination of multi-residue for three kinds of water-soluble and six kinds of fat-soluble synthetic colorants in chilli products was also investigated by HPLC coupled with MISPE. The mean recoveries calculated by solvent calibration curve for water-soluble and fat-soluble synthetic colorants were from 72.1% to 95.6% for chilli spice and 72.1% to 92.3% for chilli powder. The decision limit (CCalpha) and the detection capability (CCbeta) obtained for water-soluble and fat-soluble synthetic colorants were in the range of 1.2-1.6 and 1.9-2.4 microg kg(-1) in chilli spice and chilli powder. The resulting MISPE was successfully used off-line for the determination of nine kinds of synthetic colorants in chilli products.
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Compuestos Azo/análisis , Capsicum/química , Colorantes/análisis , Análisis de los Alimentos/métodos , Impresión Molecular/métodos , Naftoles/análisis , Polivinilos/química , Cromatografía Líquida de Alta Presión/métodos , Colorantes/química , Formiatos/química , Metacrilatos/química , Metanol/química , Polivinilos/síntesis química , Piridinas/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , SolubilidadRESUMEN
A sorbent was synthesized and investigated for molecularly imprinted solid-phase extraction (MISPE). Molecularly imprinted polymers (MIPs) were synthesized via precipitation polymerization procedure, where methacrylic acid (MAA) was used as functional monomer and ethylene glycol dimethacrylate (EDMA) as cross-linking agent. The imprinting effect and selectivity of the MISPE were evaluated by elution experiments. The resulting MISPE showed high extraction selectivity to malachite green, gentian violet and their metabolites, which may be caused by both the ion exchange and the hydrophobic interactions. The determination of multi-residue for malachite green, gentian violet and their metabolites in aquatic products by HPLC coupled with MISPE was also investigated. The mean recoveries calculated by solvent calibration curve for malachite green (MG), gentian violet (GV), leucomalachite green (LMG) and leucogentian violet (LGV) were from 89.8% to 99.1% for grass carp, 90.6% to 101.2% for shrimp and 91.3% to 96.3% for shellfish. The decision limit (CCalpha) and the detection capability (CCbeta) obtained for MG, GV, LMG and LGV were in the range of 0.11-0.14 and 0.19-0.24 microg kg(-1) for grass carp, shrimp and shellfish. The MISPE was successfully used off-line for the determination of MG, GV and their metabolites in aquatic products.