Abstract
In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby ...patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission.
Streptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 ...(GLP-1) has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of β-cells in damaged islets.
The rise of investigative and commercially available cell therapy products adds a new dynamic to academic medical centers; that is, the management of patient-specific cell products. The scope of cell ...therapy has rapidly expanded beyond in-house collection and infusion of cell products such as bone marrow and peripheral blood transplant. The complexities and volumes of cell therapies are likely to continue to become more demanding. As patient-specific “living drugs,” cell therapy products typically require material collection, product provenance, transportation and maintenance of critical quality attributes, including temperature and expiration dates. These requirements are complicated by variations in product-specific attributes, reporting requirements and interactions with industry not required of typical pharmaceuticals.
To manage these requirements, the authors set out to establish a framework within the Immune, Progenitor and Cell Therapeutics Lab, the Current Good Manufacturing Practice facility responsible for cell manufacturing at Mayo Clinic Rochester housed within the Division of Transfusion Medicine. The authors created a work unit (biopharmaceutical unit) dedicated to addressing the specialized procedures required to properly handle these living drugs from collection to delivery and housing the necessary processes to more easily integrate externally manufactured cell therapies into clinical practice.
The result is a clear set of expectations defined for each step of the process, with logical documentation of critical steps that are concise and easy to follow.
The authors believe this system is scalable for addressing the promised growth of cell therapy products well into the future. Here the authors describe this system and provide a framework that could be used by other centers to manage these important new therapies.
Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet, iPS generation from patients with type 2 diabetes (T2D) has not been ...demonstrated. Here, we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4, SOX2, KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions, reprogrammed cells underwent dedifferentiation with mitochondrial restructuring, induction of endogenous pluripotency genes - including NANOG, LIN28, and TERT, and down-regulation of cytoskeletal, MHC class I- and apoptosis-related genes. Notably, derived iPS clones acquired a rejuvenated state, characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors, including Indolactam V and GLP-1, redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus, in elderly T2D patients, reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications.
To determine biological mechanisms involved in posttransplantation diabetes mellitus caused by the immunosuppressant tacrolimus (FK506).
INS-1 cells and isolated rat islets were incubated with ...vehicle or FK506 and harvested at 24-hr intervals. Cells were assessed for viability, apoptosis, proliferation, cell insulin secretion, and content. Gene expression studies by microarray analysis, quantitative polymerase chain reaction, and motifADE analysis of the microarray data identified potential FK506-mediated pathways and regulatory motifs. Mitochondrial functions, including cell respiration, mitochondrial content, and bioenergetics were assessed.
Cell replication, viability, insulin secretion, oxygen consumption, and mitochondrial content were decreased (P<0.05) 1.2-, 1.27-, 1.77-, 1.32-, and 1.43-fold, respectively, after 48-hr FK506 treatment. Differences increased with time. FK506 (50 ng/mL) and cyclosporine A (800 ng/mL) had comparable effects. FK506 significantly decreased mitochondrial content and mitochondrial bioenergetics and showed a trend toward decreased oxygen consumption in isolated islets. Cell apoptosis and proliferation, mitochondrial DNA copy number, and ATP:ADP ratios were not significantly affected. Pathway analysis of microarray data showed FK506 modification of pathways involving ATP metabolism, membrane trafficking, and cytoskeleton remodeling. PGC1-α mRNA was down-regulated by FK506. MotifADE identified nuclear factor of activated T-cells, an important mediator of β-cell survival and function, as a potential factor mediating both up- and down-regulation of gene expression.
At pharmacologically relevant concentrations, FK506 decreases insulin secretion and reduces mitochondrial density and function without changing apoptosis rates, suggesting that posttransplantation diabetes induced by FK506 may be mediated by its effects on mitochondrial function.
Light chain (AL) amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. Patients with LC amyloid involvement of ...the heart have the worst morbidity and mortality. Current treatments target the plasma cells to reduce further production of amyloid proteins. There is dire need to understand the mechanisms of cardiac tissue damage from amyloid to develop novel therapies. We recently reported that LC soluble and fibrillar species cause apoptosis and inhibit cell growth in human cardiomyocytes. Mesenchymal stromal cells (MSCs) can promote wound healing and tissue remodeling. The objective of this study was to evaluate MSCs to protect cardiomyocytes affected by AL amyloid fibrils.
We used live cell imaging and proteomics to analyze the effect of MSCs in the growth arrest caused by AL amyloid fibrils.
We evaluated the growth of human cardiomyocytes (RFP-AC16 cells) in the presence of cytotoxic LC amyloid fibrils. MSCs reversed the cell growth arrest caused by LC fibrils. We also demonstrated that this effect requires cell contact and may be mediated through paracrine factors modulating cell adhesion and extracellular matrix remodeling. To our knowledge, this is the first report of MSC protection of human cardiomyocytes in amyloid disease.
This important proof of concept study will inform future rational development of MSC therapy in cardiac LC amyloid.
The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells, thereby providing a novel cell-therapy platform for severe degenerative diseases. One ...of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming.
We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones.
Ectopic expression of OCT4, SOX2, KLF4, and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers, including SSEA-4, TRA-1-60, and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes, such as LIN28, TERT, DPPA4, and PODXL, in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation, HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers, including insulin-producing cells through endodermal lineage, verifying the pluripotency of the blood-derived iPSC clones.
Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming, mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation, especially from diabetes patients complicated by slow-healing wounds.
In the Houston, Texas region, groundwater use is
regulated by the Harris-Galveston Subsidence District (District) because of
historical regional subsidence from groundwater development. The District
...regulates groundwater production in the Coastal Lowlands Aquifer System
(CLAS) to mitigate subsidence through the implementation of District
Groundwater Regulatory Plan. The District has successfully reduced
groundwater pumping as a percent of demand regionally while controlling
subsidence through the implementation of alternative water supplies. Aquifer
Storage and Recovery (ASR) is an alternative water supply strategy that
provides a means to store water underground and increase water supply more
cost effectively than traditional storage expansion strategies. Groundwater
users in the District are interested in the many potential benefits of ASR
as a water supply strategy. Little is known about the potential effects on
compaction and land surface subsidence resulting from ASR operations.
Recognizing this, the District funded research on the potential subsidence
risk associated with ASR. Two hypothetical, though representative, ASR
projects were developed and analysed: (1) an industrial ASR project meant to
provide water supply during a drought of record (DOR), and (2) a municipal
ASR project designed to provide an annual municipal summer peaking water
supply. Simulations of groundwater hydraulics and subsidence were performed
at three potential locations within the CLAS to provide insight into
variability associated with location and aquifer depth. Theoretical
simulations confirmed the potential for subsidence associated with the
application of ASR in the CLAS, although operating an ASR for summer peaking
needs has less potential risk of subsidence than the DOR scenario in the
scenarios simulated. The study simulations provide insight into how an ASR
project may be designed and operated to minimize compaction and potential
subsidence. Based on this study, ASR operated to address summer peaking
showed the greatest potential to reduce additional compaction verses
sourcing all water from groundwater. This theoretical study provides a basis
for future research on subsidence associated with ASR and provides a
framework for consideration for the regulation of ASR within the District.
Significant undeveloped brackish groundwater resources
exist within the Coastal Lowlands Aquifer System (Gulf Coast Aquifer System)
near Houston, Texas, USA. As the development of these frontier ...resources is
imminent, an improved understanding of the impact development may have on
the availability of the resource and land subsidence is needed. In this
region, land subsidence is caused by the depressurization of the aquifer and
compaction of the many clay lenses in the subsurface. The Gulf Coast Aquifer
System in the study area includes three primary water bearing units (from
shallow to deep): the Chicot (Pleistocene and Pliocene) and Evangeline
(Pliocene and Miocene) aquifers, and the Jasper aquifer (Miocene). Although
there has been much research and data supporting the causal relation between
water-level decline and subsidence in the areas of fresh groundwater
development, little data exists to inform on the potential subsidence
impacts upon deeper brackish groundwater development. Data were compiled,
and multiple hydrologic parameters were utilized to improve the
understanding of the brackish resources within the study area. Geophysical
logs were compiled and analysed to refine the aquifer stratigraphy,
determine the binary classification of sand and clay, and estimate the
groundwater salinity. These data were used to develop a MODFLOW groundwater
flow model to estimate the risk of compaction and land subsidence upon the
development of brackish zones within the Jasper aquifer. Compiled data
detailing the total clay thickness, clay bed thickness, and clay bed
location were input into the model along with a hypothetical stress to
predict compaction within the Jasper aquifer across the study area while
incorporating the observed heterogeneity in clay properties. Using the
results from the model simulations and two other risk performance measures
(depth of burial and surface flood risk), the total subsidence normalized
risk score was estimated. The results of this study confirm the potential
for compaction in the Jasper aquifer and for land subsidence to occur upon
development. Areas with the highest risk are located in the up-dip, inland
areas, near where the aquifer becomes fresh and is currently used for
municipal supply. The results will inform water managers and planners in the
Houston area on the future availability of brackish groundwater resources.
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