RESUMEN
BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.
Asunto(s)
Interferón alfa-2/farmacología , Interferón-alfa/farmacología , Interferón gamma/farmacología , Monocitos/metabolismo , Polietilenglicoles/farmacología , Animales , Recuento de Células , Muerte Celular/efectos de los fármacos , Separación Celular , Supervivencia Celular/efectos de los fármacos , Criopreservación , Femenino , Humanos , Interferón alfa-2/toxicidad , Interferón-alfa/toxicidad , Interferón gamma/toxicidad , Ratones , Monocitos/efectos de los fármacos , Polietilenglicoles/toxicidad , Estabilidad Proteica/efectos de los fármacos , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/toxicidadRESUMEN
BACKGROUND: Cadmium (Cd) exposure has adverse health effects in children. Diet contributes to Cd exposure, but dietary components could affect body Cd levels. OBJECTIVE: To examine associations between diet and urinary Cd (U-Cd) in children. METHODS: In this cross-sectional study conducted in Montevideo, Uruguay, Cd exposure of 5-8â¯year old children (nâ¯=â¯279 with complete data) was assessed in first morning urine (U-Cd), a marker of long-term exposure, measured by ICP-MS and adjusted for specific gravity. Distribution of U-Cd was (median [5%, 95%]: 0.06 [0.02, 0.17] µg/L); data were natural-log-transformed (ln) for statistical analyses. Serum ferritin (SF), an indicator of iron stores, was measured in fasting samples. Trained nutritionists completed two non-consecutive 24-h dietary recalls with both child and caregiver present. Measures of iron, zinc, calcium and fiber intake, and the consumption of grains, root vegetables, milk, and foods rich in heme iron (white and read meats) and non-heme iron (legumes, spinach, broccoli, tomatoes, dried fruit) were derived. Multivariable ordinary least squares (OLS) and ordinal regressions were used to examine associations among tertiles of water Cd, SF, diet, and U-Cd. OLS models were further stratified by sex. RESULTS: In covariate-adjusted models, SF was not related to ln-U-Cd. Children in highest tertile of iron and zinc intake had lower ln-U-Cd: (-0.23 [-0.42, -0.03]) and (-0.25 [-0.44, -0.05]), respectively, compared to the reference group. Children consuming higher amounts of foods rich in heme iron had slightly lower ln-U-Cd (-0.17 [-0.36, 0.03]). High grain consumption was related to higher ln-U-Cd (0.25 [0.06, 0.45]). CONCLUSIONS: Diets rich in grains were related to higher urinary Cd levels among children living in the context of low Cd pollution. Higher intake of iron and zinc was related to lower Cd levels. Given that urinary Cd is mainly a marker of long-term exposure, these findings should be further corroborated.
Asunto(s)
Cadmio/orina , Dieta , Fibras de la Dieta/análisis , Exposición a Riesgos Ambientales , Contaminantes Ambientales/orina , Hierro de la Dieta/análisis , Zinc/análisis , Niño , Estudios Transversales , Monitoreo del Ambiente , Femenino , Humanos , Masculino , Uruguay , Compuestos de Zinc/análisisRESUMEN
BACKGROUND: Ovarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both cytostatic and cytotoxic properties, while monocytes stimulated with interferons have potent cytotoxic properties. Due to the highly immune suppressive properties of ovarian cancer, ex vivo stimulation of autologous patient monocytes with interferons and infusion of all three agents intraperitoneally (IP) can provide a strong pro-inflammatory environment at the site of disease to kill malignant cells. METHODS: Patient monocytes are isolated through counterflow elutriation and stimulated ex vivo with interferons and infused IP through a semi-permanent catheter. We have designed a standard 3 + 3 dose escalation study to explore the highest tolerated dose of interferons and monocytes infused IP in patients with chemotherapy resistant ovarian cancer. Secondary outcome measurements of changes in the peripheral blood immune compartment and plasma cytokines will be studied for correlations of response. DISCUSSION: We have developed a novel immunotherapy focused on the innate immune system for the treatment of ovarian cancer. We have combined the use of autologous monocytes and interferons alpha and gamma for local-regional administration directly into the peritoneal cavity. This therapy is highly unique in that it is the first study of its type using only components of the innate immune system for the locoregional delivery consisting of autologous monocytes and dual interferons alpha and gamma. Trial Registration ClinicalTrials.gov Identifier: NCT02948426, registered on October 28, 2016. https://clinicaltrials.gov/ct2/show/NCT02948426.
Asunto(s)
Interferón alfa-2/administración & dosificación , Interferón alfa-2/uso terapéutico , Interferón-alfa/administración & dosificación , Interferón-alfa/uso terapéutico , Interferón gamma/administración & dosificación , Interferón gamma/uso terapéutico , Monocitos/metabolismo , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Polietilenglicoles/administración & dosificación , Polietilenglicoles/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inyecciones Intraperitoneales , Interferón alfa-2/farmacología , Interferón-alfa/farmacología , Interferón gamma/farmacología , Monocitos/efectos de los fármacos , Polietilenglicoles/farmacología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Resultado del TratamientoRESUMEN
Lead exposure remains an important public health problem. Contaminated foods may act as a source of lead exposure, while certain nutrients may reduce lead absorption. We examined the cross-sectional associations of dietary patterns and the intake of several nutrients and foods with blood (Pb-B) and urinary (Pb-U) lead concentrations in children (5-8y) from Montevideo, Uruguay. From two 24-hour recalls completed by caregivers, we derived the mean daily intake of select nutrients and food groups (dairy, milk, fruit, root vegetables, foods rich in heme and non-heme iron), as well as "nutrient dense" and "processed" food patterns. Pb-B (n=315) was measured using atomic absorption spectrometry; Pb-U (n=321) using ICP-MS. Pb-U was adjusted for specific gravity and log-transformed to approximate a normal distribution. Iron deficiency (ID) and dietary variables were tested as predictors of Pb-B and log-Pb-U in covariate-adjusted regressions. Median [5%, 95%] Pb-B and Pb-U were 3.8 [0.8-7.8] µg/dL and 1.9 [0.6-5.1] µg/L, respectively; ~25% of Pb-B above current U.S. CDC reference concentration of 5µg/dL. ID was associated with 0.75µg/dL higher Pb-B, compared to non-ID (p<0.05). Consumption of root vegetables was not associated with Pb-B or log-Pb-U. Higher scores on the nutrient-dense pattern were related with higher Pb-Bs, possibly due to consumption of green leafy vegetables. Dietary intake of iron or iron-rich foods was not associated with biomarkers of lead. Conversely, children consuming more calcium, dairy, milk and yogurt had lower Pb-B and log-Pb-U. Our findings appear consistent with existing recommendations on including calcium-rich, but not iron- or vitamin-C-rich foods in the diets of lead-exposed children, especially where the consumption of these foods is low.
Asunto(s)
Dieta , Contaminantes Ambientales/sangre , Contaminantes Ambientales/orina , Plomo/sangre , Plomo/orina , Estado Nutricional , Niño , Ciudades , Productos Lácteos , Monitoreo del Ambiente , Femenino , Frutas , Humanos , Deficiencias de Hierro , Masculino , Uruguay , VerdurasRESUMEN
BACKGROUND: Cell selection is an important part of manufacturing cellular therapies. A new highly automated instrument, the CliniMACS Prodigy (Miltenyi Biotec), was evaluated for the selection of CD34+ cells from mobilized peripheral blood stem cell (PBSC) concentrates using monoclonal antibodies conjugated to paramagnetic particles. STUDY DESIGN AND METHODS: PBSCs were collected by apheresis from 36 healthy subjects given granulocyte-colony-stimulating factor (G-CSF) or G-CSF plus plerixafor. CD34+ cells from 11 PBSC concentrates were isolated with the automated CliniMACS Prodigy and 25 with the semiautomated CliniMACS Plus Instrument. RESULTS: The proportion of CD34+ cells in the selected products obtained with the two instruments was similar: 93.6 ± 2.6% for the automated and 95.7 ± 3.3% for the semiautomated instrument (p > 0.05). The recovery of CD34+ cells from PBSC concentrates was less for the automated than the semiautomated instrument (51.4 ± 8.2% vs. 65.1 ± 15.7%; p = 0.019). The selected products from both instruments contained few and similar quantities of platelets (PLTs) and red blood cells. The depletion of CD3+ cells was less with the automated instrument (4.34 ± 0.2 log depletion vs. 5.20 ± 0.35 log depletion; p < 1 × 10(-6) ). Removal of PLTs from PBSC concentrates by washing was associated with better CD34+ cell recovery. We explored the reasons for lower CD34+ cell recovery by the Prodigy and found that the nonselected cells for the Prodigy contained more PLTs than those for the CliniMACS Plus. CONCLUSIONS: CD34+ cells can be effectively selected from mobilized PBSC concentrates with the CliniMAC Prodigy, but the recovery of CD34+ cells and depletion of CD3+ cells was lower than with the semiautomated CliniMACS Plus Instrument.
Asunto(s)
Eliminación de Componentes Sanguíneos/instrumentación , Eliminación de Componentes Sanguíneos/métodos , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Compuestos Heterocíclicos/administración & dosificación , Antígenos CD34/sangre , Bencilaminas , Ciclamas , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , MasculinoRESUMEN
BACKGROUND: Cryopreservation is often used to store cellular therapies, but little is known about how well CD3+ or CD34+ cells tolerate this process. STUDY DESIGN AND METHODS: Viable CD34+ cell recoveries were analyzed from related and unrelated donor granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell (PBSC) products and viable CD3+ cell recoveries from G-CSF-mobilized and nonmobilized apheresis products from related and unrelated donors. All products were cryopreserved with 5% dimethyl sulfoxide and 6% pentastarch using a controlled-rate freezer and were stored in liquid nitrogen. Related donor products were cryopreserved immediately after collection and unrelated donor products greater than 12 hours postcollection. RESULTS: The postthaw recovery of CD34+ cells from related donor PBSCs was high (n = 86; 97.5 ± 23.1%) and there was no difference in postthaw CD34+ cell recovery from unrelated donor PBSCs (n = 14; 98.8 ± 37.2%; p = 0.863). In related donor lymphocyte products the postthaw CD3+ cell recovery (n = 48; 90.7 ± 21.4%) was greater than that of unrelated donor products (n = 14; 66.6 ± 35.8%; p = 0.00251). All unrelated donor lymphocyte products were from G-CSF-mobilized products, while most related donor lymphocyte products were from nonmobilized products. A comparison of the CD3+ cell recovery from related donor G-CSF-mobilized products (n = 19; 85.0 ± 29.2%) with that of unrelated donor products found no significant difference (p = 0.137). CONCLUSIONS: The postthaw recovery of CD34+ cells was high in both related and unrelated donor products, but the recovery of CD3+ cells in unrelated donor G-CSF-mobilized products was lower. G-CSF-mobilized unrelated donor products may contain fewer CD3+ cells than non-G-CSF-exposed products upon thaw and, when indicated, cell doses should be monitored.
Asunto(s)
Conservación de la Sangre , Criopreservación , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Antígenos CD34/metabolismo , Recuento de Células Sanguíneas , Donantes de Sangre , Recolección de Muestras de Sangre , Complejo CD3/metabolismo , Supervivencia Celular , Células Madre Hematopoyéticas/fisiología , Humanos , Donante no EmparentadoRESUMEN
BACKGROUND: Bone marrow stromal cells (BMSCs) are being used to treat a variety of conditions. For many applications a supply of cryopreserved products that can be used for acute therapy is needed. The establishment of a bank of BMSC products from healthy third party donors is described. METHODS: The recruitment of healthy subjects willing to donate marrow for BMSC production and the Good Manufacturing Practices (GMP) used for assessing potential donors, collecting marrow, culturing BMSCs and BMSC cryopreservation are described. RESULTS: Seventeen subjects were enrolled in our marrow collection protocol for BMSC production. Six of the 17 subjects were found to be ineligible during the donor screening process and one became ill and their donation was cancelled. Approximately 12 ml of marrow was aspirated from one posterior iliac crest of 10 donors; one donor donated twice. The BMSCs were initially cultured in T-75 flasks and then expanded for three passages in multilayer cell factories. The final BMSC product was packaged into units of 100 × 106 viable cells, cryopreserved and stored in a vapor phase liquid nitrogen tank under continuous monitoring. BMSC products meeting all lot release criteria were obtained from 8 of the 11 marrow collections. The rate of growth of the primary cultures was similar for all products except those generated from the two oldest donors. One lot did not meet the criteria for final release; its CD34 antigen expression was greater than the cut off set at 5%. The mean number of BMSC units obtained from each donor was 17 and ranged from 3 to 40. CONCLUSIONS: The production of large numbers of BMSCs from bone marrow aspirates of healthy donors is feasible, but is limited by the high number of donors that did not meet eligibility criteria and products that did not meet lot release criteria.
Asunto(s)
Células de la Médula Ósea/citología , Bancos de Tejidos , Conservación de Tejido/métodos , Adulto , Anciano , Envejecimiento/fisiología , Antígenos CD34/metabolismo , Recuento de Células , Núcleo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Femenino , Salud , Humanos , Masculino , Persona de Mediana Edad , Células del Estroma/citología , Factores de Tiempo , Donantes de Tejidos , Adulto JovenRESUMEN
BACKGROUND: Dendritic cells (DCs) generated ex vivo from peripheral blood monocytes or mobilized CD34+ cells and intended for clinical immunotherapy are typically characterized by morphologic, phenotypic, and functional assays. Assay results are highly dependent on conditions used to prepare the cells, so there is no standard assay battery for clinical DC products. This study evaluated gene expression profiling for characterization of immature DCs prepared from monocytes that had been elutriated from normal donor peripheral blood mononuclear cells (PBMNCs) immediately after collection or after storage at 4 degrees C for 48 hours. STUDY DESIGN AND METHODS: RNA was isolated from fresh and 48-hour-stored PBMNCs, elutriated monocytes, elutriated lymphocytes, and immature DCs from five healthy subjects and was analyzed with a cDNA gene expression microarray with 17,500 genes. RESULTS: Unsupervised hierarchical clustering separated the 40 products into four groups: one with all 10 immature DCs, one with all 10 elutriated lymphocytes, one with 7 PBMNCs, and one with 10 elutriated monocytes and 3 PBMNCs. Within each of the four groups, however, fresh and stored products, or products derived from fresh or stored products, clustered together. Comparison of genes differentially expressed by fresh versus stored products (paired t tests, p < 0.005) found 273 genes that differed between fresh and stored PBMCs, 429 between lymphocytes elutriated from fresh versus stored PBMNCs, 711 between monocytes elutriated from fresh versus stored PBMNCs, and 3 between immature DCs prepared from monocytes elutriated from fresh versus stored PBMCs. CONCLUSIONS: This study demonstrates the potential utility of gene expression profiling for characterization of cell therapy products.
Asunto(s)
Células Dendríticas/metabolismo , Perfilación de la Expresión Génica/métodos , Monocitos/citología , Adulto , Anciano , Análisis por Conglomerados , Células Dendríticas/citología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Mobilized peripheral blood is increasingly used as the source of hematopoietic stem cells for allogeneic transplantation, currently the only curative approach for sickle cell anemia. However, the safety and feasibility of stem cell mobilization in individuals with sickle cell trait (SCT) has not been documented. This study is a prospective controlled trial to evaluate the safety and feasibility of peripheral blood stem cell (PBSC) mobilization in 8 SCT subjects and 8 control subjects matched for age and race. Mobilization with filgrastim 10 microg/kg subcutaneous daily for 5 days was followed by 12-L apheresis on the fifth day. Filgrastim administration was accompanied by similar symptoms in all subjects; no untoward adverse events occurred in either group, including sickle cell crises. CD34+ cell mobilization response was not significantly different between SCT and control subjects. Median CD34+ cell content was also similar in PBSCs collected from SCT versus control subjects, 6.8 versus 3.9 x 10(6) CD34+ cells/70 kg, P =.165. Red cell depletion from SCT products was not possible by using hydroxyethyl starch sedimentation but was achievable with ammonium chloride lysis. There was no evidence of gelling of SCT products after thaw, and no difference in cell recovery was seen among red cell-depleted versus nondepleted products. Cryopreservation in 5% dimethyl sulfoxide/6% pentastarch was associated with superior cell recovery (both SCT and control subjects) compared with 10% dimethyl sulfoxide (P =.001). The study concluded that filgrastim mobilization, large volume apheresis, processing, and cryopreservation appears to be safe in donors with SCT, allowing PBSC use for transplantation in patients with sickle cell anemia.