The success of adoptive therapy using chimeric antigen receptor (CAR) expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (Fc?Rs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR, and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble Fc?Rs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid Fc?R binding would improve CAR+ T cell engraftment and anti-tumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)), or incorporated a CH2 deletion (CD19Rch2?). These mutations reduced binding to soluble Fc?Rs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2? T cells exhibited improved persistence and more potent CD19-specific anti-lymphoma efficacy in NSG mice. Together these studies suggest that optimal CAR function may require the elimination of cellular Fc?R interactions to improve T cell persistence and anti-tumor responses.Molecular Therapy (2014); doi:10.1038/mt.2014.208.
JAK/STAT3 is one of the major signaling pathways that is aberrantly activated in ovarian cancer and associated with tumor progression and poor prognosis in ovarian cancer patients. In this study, we evaluated the therapeutic potential of targeting JAK/STAT3 signaling in ovarian cancer using a peritoneal dissemination mouse model. We developed this mouse model by injecting a metastatic human ovarian cancer cell line, SKOV3-M-Luc, into the peritoneal cavity of immunodeficient mice. This model displayed a phenotype similar to late stage ovarian cancer, including extensive peritoneal metastasis and ascites production. The constitutive activation of STAT3 in human ovarian cancer cells appeared to be mediated by an autocrine-cytokine loop involving the IL-6 family of cytokines and JAK1 kinase. shRNA-mediated knockdown of JAK1 or STAT3 in ovarian cancer cells led to reduced tumor growth, decreased peritoneal dissemination and diminished ascites production, suggesting a critical role of STAT3 in ovarian cancer progression. Similar results were obtained when a small-molecule inhibitor (JAKi) of the JAK1 kinase was used to treat ovarian cancer in this model. In addition, we found that the expression level of IL-6 was correlated with activation of STAT3 in ovarian cancer cells both in vitro and in vivo, suggesting a potential application of IL-6 as a biomarker. Altogether, our results demonstrate that targeting JAK1/STAT3, using shRNA knockdown or a small molecule inhibitor, effectively suppressed ovarian tumor progression and, therefore, could be a potential novel therapeutic approach for treating advanced ovarian cancer.
Autism Spectrum Disorder (ASD) has been associated with essential fatty acid (EFA) deficiencies, with some researchers theorising that dysregulation of phospholipid metabolism may form part of the biological basis for ASD. This pilot study compared observable signs of fatty acid status of 19 children with an ASD diagnosis to 23 of their typically developing siblings. A pregnancy, birth and breastfeeding history was also obtained from their parents, which included a measure of infant intake of fatty acid rich colostrum immediately post-partum. When considered within their family group, those infants not breastfed (with colostrum) within the first hour of life and who had a history of fatty acid deficiency symptoms were more likely to have an ASD diagnosis. Other variables such as formula use, duration of breastfeeding, gestational age and Apgar scores were not associated with group membership. The results of this study are consistent with previous research showing a relationship between fatty acid metabolism, breastfeeding and ASD such that early infant feeding practices and the influence this has on the fatty acid metabolism of the child may be a risk factor for ASD.
Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen-binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches. Five loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variants were generated in the mouse VWF complementary DNA. The results of these various assays were consistent, although the magnitude of the effects were different: the gain-of-function (p.L1757A) variant showed consistent enhanced collagen binding whereas the loss-of-function mutants showed variable degrees of functional deficit. We further analyzed the impact of direct platelet-collagen binding by blocking glycoprotein VI (GPVI) and integrin ?2?1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF(-/-) mice whereas ?2?1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF(-/-), GPVI, and ?2?1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects.
Ex vivo gene therapy strategies avoid systemic delivery of viruses thereby mitigating the risk of vector-associated immunogenicity. Previously, we delivered autologous factor VIII (FVIII)-expressing blood outgrowth endothelial cells (BOECs) to hemophilia A mice and showed that these cells remained sequestered within the implanted matrix and provided therapeutic levels of FVIII. Prior to translating this strategy into the canine (c) model of hemophilia A, we increased cFVIII transgene expression by at least 100-fold with the use of the elongation factor 1 alpha (EF1?) promoter and a strong endothelial enhancer element. BOECs isolated from hemophilia A dogs transduced with this lentiviral vector express levels of cFVIII ranging between 1.0 and 1.5 U/mL per 10(6) cells over 24 hours. Autologous BOECs have been implanted into the omentum of 2 normal and 3 hemophilia A dogs. These implanted cells formed new vessels in the omentum. All 3 hemophilia A dogs treated with FVIII-expressing autologous BOECs developed anti-FVIII immunoglobulin G2 antibodies, but in only 2 of the dogs were these antibodies inhibitory. FVIII antigen levels >40% in the absence of FVIII coagulant function were detected in the circulation for up to a year after a single gene therapy treatment, indicating prolonged cellular viability and synthesis of FVIII.
Glioblastoma multiforme (GBM) remains one of the most lethal primary brain tumors despite surgical and therapeutic advancements. Targeted therapies of neoplastic diseases, including GBM, have received a great deal of interest in recent years. A highly studied target of GBM is interleukin-13 receptor ? chain variant 2 (IL13R?2). Targeted therapies against IL13R?2 in GBM include fusion chimera proteins of IL-13 and bacterial toxins, nanoparticles, and oncolytic viruses. In addition, immunotherapies have been developed using monoclonal antibodies and cell-based strategies such as IL13R?2-pulsed dendritic cells and IL13R?2-targeted chimeric antigen receptor-modified T cells. Advanced therapeutic development has led to the completion of phase I clinical trials for chimeric antigen receptor-modified T cells and phase III clinical trials for IL-13-conjugated bacterial toxin, with promising outcomes. Selective expression of IL13R?2 on tumor cells, while absent in the surrounding normal brain tissue, has motivated continued study of IL13R?2 as an important candidate for targeted glioma therapy. Here, we review the preclinical and clinical studies targeting IL13R?2 in GBM and discuss new advances and promising applications.
Adhesion molecule L1-CAM (CD171) was originally reported to be overexpressed on neuroblastoma and to play an important role during tumor progression. More recently, it has been shown to be overexpressed on many other solid tumors such as melanoma and carcinomas of the cervix, ovary, bladder, and others. Thus, there has been a growing interest in using this cell-surface molecule as a target for both antibody-based and cellular-based therapy-our group has previously examined the clinical utility of chimeric antigen receptor (CAR)-redirected cytolytic T cells that specifically target the CE7 epitope of L1-CAM on neuroblastoma patients. Here, we sought to determine whether this CE7 epitope is present on other recently identified L1-CAM tumors and whether it too can be targeted by CAR T cells. Our studies demonstrate that a diverse array of human tumor cell lines and primary solid tumors (ovarian, lung, and renal carcinoma, glioblastoma and neuroblastoma) do express the CE7 epitope and can efficiently stimulate CE7-specific CAR-redirected (CE7R) T-cell lytic activity and secretion of proinflamatory cytokines. L1-CAM was also detected on a limited number of normal tissues; however, L1-CAM expressed on normal human monocytes was not bound by the CE7 mAb nor was it targeted by CE7R T cells, suggesting that the CE7 epitope is more tumor restricted and not expressed on all L1-CAM tissues. Overall, the CE7 epitope of L1-CAM on a variety of tumors may be amenable to targeting by CE7R T cells, making it a promising target for adoptive immunotherapy.
Obesity in Canada is a growing concern, but little is known about the available services for managing obesity in adults. Our objectives were to (a) survey and describe programs dedicated to weight management and (b) evaluate program adherence to established recommendations for care.
New developments in the treatment and management of phenylketonuria (PKU) as well as advances in molecular testing have emerged since the National Institutes of Health 2000 PKU Consensus Statement was released. An NIH State-of-the-Science Conference was convened in 2012 to address new findings, particularly the use of the medication sapropterin to treat some individuals with PKU, and to develop a research agenda. Prior to the 2012 conference, five working groups of experts and public members met over a 1-year period. The working groups addressed the following: long-term outcomes and management across the lifespan; PKU and pregnancy; diet control and management; pharmacologic interventions; and molecular testing, new technologies, and epidemiologic considerations. In a parallel and independent activity, an Evidence-based Practice Center supported by the Agency for Healthcare Research and Quality conducted a systematic review of adjuvant treatments for PKU; its conclusions were presented at the conference. The conference included the findings of the working groups, panel discussions from industry and international perspectives, and presentations on topics such as emerging treatments for PKU, transitioning to adult care, and the U.S. Food and Drug Administration regulatory perspective. Over 85 experts participated in the conference through information gathering and/or as presenters during the conference, and they reached several important conclusions. The most serious neurological impairments in PKU are preventable with current dietary treatment approaches. However, a variety of more subtle physical, cognitive, and behavioral consequences of even well-controlled PKU are now recognized. The best outcomes in maternal PKU occur when blood phenylalanine (Phe) concentrations are maintained between 120 and 360 ?mol/L before and during pregnancy. The dietary management treatment goal for individuals with PKU is a blood Phe concentration between 120 and 360 ?mol/L. The use of genotype information in the newborn period may yield valuable insights about the severity of the condition for infants diagnosed before maximal Phe levels are achieved. While emerging and established genotype-phenotype correlations may transform our understanding of PKU, establishing correlations with intellectual outcomes is more challenging. Regarding the use of sapropterin in PKU, there are significant gaps in predicting response to treatment; at least half of those with PKU will have either minimal or no response. A coordinated approach to PKU treatment improves long-term outcomes for those with PKU and facilitates the conduct of research to improve diagnosis and treatment. New drugs that are safe, efficacious, and impact a larger proportion of individuals with PKU are needed. However, it is imperative that treatment guidelines and the decision processes for determining access to treatments be tied to a solid evidence base with rigorous standards for robust and consistent data collection. The process that preceded the PKU State-of-the-Science Conference, the conference itself, and the identification of a research agenda have facilitated the development of clinical practice guidelines by professional organizations and serve as a model for other inborn errors of metabolism.
In this investigation of effectiveness of an alternative pediculicide dosage form, we recruited 228 children and 50 adult participants from Bedfordshire, UK, to a randomised, controlled, assessor blind trial comparing two insecticide products with mechanical removal of lice as a control group. Participants using insecticide were treated with either the investigative 0.5% phenothrin mousse, for 30 minutes, or 0.2% phenothrin lotion, for 2 hours as the reference product. Both treatments were applied only once, followed by shampoo washing. Those treated by wet-combing with conditioner were combed 4 times over 12 days. Parents/carers carried out the treatments to mimic normal consumer use. The outcome measure was the absence of lice, 14 days after treatment for the insecticides, and up to 14 days after completion of combing. Intention to treat analysis of the outcomes for 275 participants showed success for phenothrin mousse in 21/105 (20.0%), in 23/107 (21.5%) for phenothrin lotion, and in 12/63 (19.1%) for wet-combing. People receiving mousse were 1.07 (95% CI, 0.63 to 1.81) times more likely to still have lice after treatment compared with those treated with lotion. The group of participants who received the wet combing treatment were 1.13 (95% CI, 0.61 to 2.11) times more likely to still have lice after the treatment. None of the treatments was significantly (p < 0.05) more effective than any other. This study was carried out in an area where moderate resistance to phenothrin was demonstrated after the study by using a bioassay. Analysis of post treatment assessments found that failure of insecticides to kill louse eggs had influenced the outcome.
The interleukin-13 receptor alpha2 (IL13R?2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13R?2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13R?2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13R?2 cell surface expression, and thereby increase susceptibility to IL13R?2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13R?2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13R?2, because its expression does not consistently correlate with IL13R?2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13R?2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13R?2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13R?2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13R?2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.
Background. Many families find regular checking of children's heads for head louse infestation too onerous and would prefer to be able to prevent infestation by use of a topical application that deters lice from infesting the head. Identification in the laboratory of a repellent activity for piperonal provided the basis for developing a spray product to repel lice. Methods. A proof of principle field study in Dhaka, Bangladesh, compared the effect of using 2% piperonal spray with that of a placebo in 105 children and adults from three communities with infestation levels close to 100%. All participants were treated for infestation and subsequent incidence of reinfestation monitored daily by investigators. A second randomised, controlled, double blind, study in North London, UK, evaluated the effect of the product in normal use. One hundred and sixty-three children from schools with a high level (20-25%) of infestation were treated and confirmed louse free and randomly divided between 2% piperonal, a placebo spray, and a control group for up to 22 weeks. Parents applied the spray and monitored for infestation. Regular investigator visits confirmed the parental monitoring and replenished supplies of spray. Results. In Dhaka, over 18 days there were only 4 infestations in the piperonal group and 8 in the placebo group. This difference was not significant (p = 0.312). In North London, there were 41 cases of infestation over the course of the study. Although there were fewer infestations in the piperonal group, analysis of time to first infestation showed a no significant (p = 0.4368) difference between groups. Conclusion. Routine use of 2% piperonal spray in communities with a high prevalence of head louse infestation may provide some protection from infestation. However, the difference between use of the product and no active intervention was sufficiently small that regular checking for presence of lice is likely to be a more practical and cost effective approach to prevention of infestation.
Glioblastoma (GBM) is the most common primary brain tumor, accounting for approximately 40% of all central nervous system malignancies. Despite standard treatment consisting of surgical resection, radiotherapy and/or chemotherapy, the prognosis for GBM is poor; with a median survival of 14.6 months. The cancer stem cell or cancer-initiating cell model has provided a new paradigm for understanding development and recurrence of GBM following treatment. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plant, and along with its derivatives, has been shown to exhibit antitumor activity in several cancers. Here, we reported that a novel synthetic Berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of cancer stem-like cells (CSCs) in a time- and dose-dependent manner when the CSCs from four GBM patients (PBT003, PBT008, PBT022, and PBT030) were cultured. These CSCs grew in neurospheres and expressed CD133 and nestin as markers. Treatment with BBMD3 destroyed the neurosphere morphology, and led to the induction of apoptosis in the CSCs. Induction of apoptosis in these CSCs is dependent upon activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). MicroRNA-4284 (miR-4284) was shown to be over-expressed about 4-fold in the CSCs following BBMD3 treatment. Furthermore, transfection of synthetic anti-sense oligonucleotide against human miR-4284 partially blocked the anticancer effects of BBMD3 on the GBM derived CSCs. BBMD3 also increased phosphorylation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in an increase expression of phosphorylated c-Jun and total c-Fos; the major components of transcriptional factor AP-1. The JNK-c-Jun/AP-1 signaling pathway plays an important role in the induction of apoptosis in response to UV irradiation and some drug treatments. Targeting glioblastoma stem-like cells with BBMD3 is therefore novel, and may have promise as an effective therapeutic strategy for treating GBM patients.
CPT-11 (irinotecan) has been investigated as a treatment for malignant brain tumors. However, limitations of CPT-11 therapy include low levels of the drug entering brain tumor sites and systemic toxicities associated with higher doses. Neural stem cells (NSCs) offer a novel way to overcome these obstacles because of their inherent tumor tropism and ability to cross the blood-brain barrier, which enables them to selectively target brain tumor sites. Carboxylesterases (CEs) are enzymes that can convert the prodrug CPT-11 (irinotecan) to its active metabolite SN-38, a potent topoisomerase I inhibitor. We have adenovirally transduced an established clonal human NSC line (HB1.F3.CD) to express a rabbit carboxylesterase (rCE) or a modified human CE (hCE1m6), which are more effective at converting CPT-11 to SN-38 than endogenous human CE. We hypothesized that NSC-mediated CE/CPT-11 therapy would allow tumor-localized production of SN-38 and significantly increase the therapeutic efficacy of irinotecan. Here, we report that transduced NSCs transiently expressed high levels of active CE enzymes, retained their tumor-tropic properties, and mediated an increase in the cytotoxicity of CPT-11 toward glioma cells. CE-expressing NSCs (NSC.CEs), whether administered intracranially or intravenously, delivered CE to orthotopic human glioma xenografts in mice. NSC-delivered CE catalyzed conversion of CPT-11 to SN-38 locally at tumor sites. These studies demonstrate the feasibility of NSC-mediated delivery of CE to glioma and lay the foundation for translational studies of this therapeutic paradigm to improve clinical outcome and quality of life in patients with malignant brain tumors.
Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor ? chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-? signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.
Numerous stem cell-based therapies are currently under clinical investigation, including the use of neural stem cells (NSCs) as delivery vehicles to target therapeutic agents to invasive brain tumors. The ability to monitor the time course, migration, and distribution of stem cells following transplantation into patients would provide critical information for optimizing treatment regimens. No effective cell-tracking methodology has yet garnered clinical acceptance. A highly promising noninvasive method for monitoring NSCs and potentially other cell types in vivo involves preloading them with ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) to enable cell tracking using magnetic resonance imaging (MRI). We report here the preclinical studies that led to U.S. Food and Drug Administration approval for first-in-human investigational use of ferumoxytol to label NSCs prior to transplantation into brain tumor patients, followed by surveillance serial MRI. A combination of heparin, protamine sulfate, and ferumoxytol (HPF) was used to label the NSCs. HPF labeling did not affect cell viability, growth kinetics, or tumor tropism in vitro, and it enabled MRI visualization of NSC distribution within orthotopic glioma xenografts. MRI revealed dynamic in vivo NSC distribution at multiple time points following intracerebral or intravenous injection into glioma-bearing mice that correlated with histological analysis. Preclinical safety/toxicity studies of intracerebrally administered HPF-labeled NSCs in mice were also performed, and they showed no significant clinical or behavioral changes, no neuronal or systemic toxicities, and no abnormal accumulation of iron in the liver or spleen. These studies support the clinical use of ferumoxytol labeling of cells for post-transplant MRI visualization and tracking.
In 2010, the American Heart Association established the concept of ideal cardiovascular health. Nationally representative data estimated that <1% of Americans meet the seven health metrics required for achieving ideal cardiovascular health, with the main challenge residing in meeting the criteria for an ideal Healthy Diet Score. In a cohort of young adults (N=196), we aimed to investigate the prevalence of ideal cardiovascular health and ideal Healthy Diet Score and its association to weight gain over a 4-year follow-up period. Anthropometric measures, blood pressure, and blood samples were taken according to standardized procedures. Dietary intake was measured by a 3-day food diary and verified by a registered dietitian. We observed that only 0.5% of our sample met the criteria for ideal cardiovascular health and only 4.1% met the criteria for an ideal Healthy Diet Score. The components of the Healthy Diet Score with the lowest observance were consumption of fruits and vegetables (9.7%) and whole grains (14.8%). Meeting zero or one out of five of the Healthy Diet Score components was associated with increased risk of weight gain over 4 years compared with meeting at least two components (P=0.03). With the exception of dietary criteria, prevalence was high for achieving ideal levels of the remaining six cardiovascular health metrics. In conclusion, in this sample of young adults, a very low prevalence of ideal overall cardiovascular health was observed, mainly driven by poor dietary habits, and a poor Healthy Diet Score was associated with increased weight gain.
High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)-expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non-tumor-bearing and orthotopic glioma-bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.
Fifty years after the implementation of universal newborn screening programs for phenylketonuria, the first disease identified through newborn screening and considered a success story of newborn screening, a cohort of adults with phenylketonuria treated from birth provides valuable information about effects of long-term treatment for inborn errors of metabolism in general, and phenylketonuria specifically. For phenylketonuria, newborn screening allows early implementation of the phenylalanine-restricted diet, eliminating the severe neurocognitive and neuromotor impairment associated with untreated phenylketonuria. However, executive function impairments and psychiatric problems are frequently reported even for those treated early and continuously with the phenylalanine-restricted diet alone. Moreover, a large percentage of adults with phenylketonuria are reported as lost to follow-up by metabolic clinics. While a group of experts identified by the National Institutes of Health convenes to update treatment guidelines for phenylketonuria, we explore individual patient, social, and economic factors preventing >70% of adult phenylketonuria patients in the United States from accessing treatment. As more conditions are identified through newborn screening, factors affecting access to treatment grow in importance, and we must continue to be vigilant in assessing and addressing factors that affect patient treatment outcomes and not just celebrate amelioration of the most severe manifestations of disease.
Therapeutic responses following adoptive transfer of T cells correlate to levels of long-term T cell persistence. Lymphodepletion and exogenous ?c cytokine administration can improve T cell persistence following adoptive transfer, but their effects are not uniform and toxicities are significant. To overcome these limitations, we designed a chimeric ?c cytokine receptor (C?CR) composed of Interleukin-7 (IL-7) tethered to IL-7R?/CD127 that confers exogenous cytokine independent, cell intrinsic, STAT5 cytokine signals. We additionally show that this design is modular in that the IL-2R?/CD122 cytoplasmic chain can be exchanged for that of IL-7R?/CD127, enhancing Shc activity. When expressed in central memory-derived primary human CD8(+) CTL (T(E/CM)), these C?CRs signal according to their corresponding wild-type counterparts to support exogenous cytokine independent viability and homeostatic proliferation, while retaining full effector function. In vivo studies demonstrate that both C?CR-CD127(+) and C?CR-CD122(+) CD8(+) T((E/CM)) engraft in mice and persist in an absence of exogenous cytokine administration. Engrafted C?CR-CD127(+) CD8(+) T(E/CM) preferentially retain central memory marker expression in vivo demonstrating a dichotomy between CD127 versus CD122 signaling. Together, these results suggest that expression of C?CR in therapeutic T cells may aid in the in vivo persistence of these cells, particularly under conditions of limiting homeostatic cytokines.
A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor ?2 (IL13R?2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13R?2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13R?2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13R?2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13R?2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13R?2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13R?2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13R?2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.
Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.
Developing and implementing a program to introduce clinical nurses to research and evidence-based practice (EBP) should spark interest and participation. In this article, the authors describe and evaluate a staff development initiative not only to introduce the principles of EBP and research but also to give nurses the opportunity to participate in the research process and development of EBP questions.
Dysregulated phospholipid metabolism has been proposed as an underlying biological component of neurodevelopmental disorders such as autistic disorder (AD) and attention-deficit/hyperactivity disorder (ADHD). This review provides an overview of fatty acid and phospholipid metabolism and evidence for phospholipid dysregulation with reference to the membrane hypothesis of schizophrenia. While there is evidence that phospholipid metabolism is at least impaired in individuals with AD, it has not been established whether phospholipid metabolism is implicated in causal, mechanistic or epiphenomenological models. More research is needed to ascertain whether breastfeeding, and specifically, the administration of colostrum or an adequate substitute can play a preventative role by supplying the neonate with essential fatty acids (EFAs) at a critical juncture in their development. Regarding treatment, further clinical trials of EFA supplementation are essential to determine the efficacy of EFAs in reducing AD symptomatology and whether supplementation can serve as a cost-effective and readily available intervention.
Weight gain and appetite regulation are complex interplays between internal and external cues. Our aim was to investigate the association of eating behaviors with ghrelin taking into account lifestyle. We conducted a cross-sectional analysis in a sample of first-year university students at the Université de Sherbrooke. We collected medical history, anthropometric measurements, vital signs, fitness index, and fasting blood samples. Questionnaires included a lifestyle questionnaire and the Three-Factor Eating Questionnaire (TFEQ) estimating dietary restraint, disinhibition and hunger. We recruited 308 participants aged 20.7±3.2 years and a mean BMI of 23.3±3.4 kg/m(2). Hunger score was significantly associated with ghrelin levels (r=0.11, P<0.05). In women, this association was independent of age, BMI, dietary and lifestyle factors (P=0.02). The association between ghrelin level and hunger score was observable in leaner individuals (r=0.28, p<0.0001) but not in heavier individuals (r=-0.08, p=0.34; stratified by BMI < vs > 22.6 kg/m(2)). Restraint (R) and disinhibition (D) were not associated with ghrelin levels. The three eating behaviors demonstrated expected correlations with lifestyle supporting the validity of the TFEQ in this cohort. In conclusion, we demonstrated that ghrelin, a biological marker, is associated with self-reported perception of hunger, independently of anthropometric measures and lifestyle.
This paper highlights ethical dilemmas experienced by researchers when undertaking qualitative inquiry with vulnerable older people in care homes. Scenarios from research that adopted a constructivist approach illustrate a range of ethical dilemmas and propose practical strategies.
Acheron (Achn) is a new member of the Lupus antigen family of RNA binding proteins. Previous studies have shown that Achn controls developmental decisions in neurons and muscle. In the human mammary gland, Achn expression is restricted to ductal myoepithelial cells. Microarray analysis and immunohistochemistry have shown that Achn expression is elevated in some basal-like ductal carcinomas. To study the possible role of Achn in breast cancer, we engineered human MDA-MB-231 cells to stably express enhanced green fluorescent protein-tagged wild-type Achn (AchnWT), as well as Achn lacking either its nuclear localization signal (AchnNLS) or its nuclear export signal (AchnNES). In in vitro assays, AchnWT and AchnNES, but not AchnNLS, enhanced cell proliferation, lamellipodia formation, and invasive activity and drove expression of the elevated expression of the metastasis-associated proteins MMP-9 and VEGF. To determine if Achn could alter the behavior of human breast cancer cells in vivo, Achn-engineered MDA-MB-231 cells were injected into athymic SCID/Beige mice. AchnWT and AchnNES-expressing tumors displayed enhanced angiogenesis and an approximately 5-fold increase in tumor size relative to either control cells or those expressing AchnNLS. These data suggest that Achn enhances human breast tumor growth and vascularization and that this activity is dependent on nuclear localization.
This paper provides a brief overview of the supply of healthcare professionals and some of the factors impacting supply. The demand for healthcare professionals and some of the factors impacting demand are then addressed. Finally, a brief overview of the health reform law recently passed, the Patient Protection and Affordable Care Act, is provided as it pertains to the supply of and demand for health professionals.
The purpose of this paper is to identify practical suggestions that could enable other researchers to consider how quality may be evidenced using constructivist principles including the perspectives of older people and their caregivers.
We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5 of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.
Stimulation of toll-like receptor-9 by CpG oligodeoxynucleotides (CpG-ODN) has been shown to counteract the immunosuppressive microenvironment and to inhibit tumor growth in glioma models. These studies, however, have used high doses of CpG-ODN, which can induce toxicity in a clinical setting. The goal of this study was to evaluate the antitumor efficacy of multiple low-dose intratumoral CpG-ODN in a glioma model.
Glioblastoma is the most common type of primary brain tumor and is rapidly progressive with few treatment options. Here, we report that sorafenib (< or =10 micromol/L) inhibited cell proliferation and induced apoptosis in two established cell lines (U87 and U251) and two primary cultures (PBT015 and PBT022) from human glioblastomas. The effects of sorafenib on these tumor cells were associated with inhibiting phosphorylated signal transducers and activators of transcription 3 (STAT3; Tyr705). Expression of a constitutively activated STAT3 mutant partially blocked the effects of sorafenib, consistent with a role for STAT3 inhibition in the response to sorafenib. Phosphorylated Janus-activated kinase (JAK)1 was inhibited in U87 and U251 cells, whereas phosphorylated JAK2 was inhibited in primary cultures. Sodium vanadate, a general inhibitor of protein tyrosine phosphatases, blocked the inhibition of phosphorylation of STAT3 (Tyr705) induced by sorafenib. These data indicate that the inhibition of STAT3 activity by sorafenib involves both the inhibition of upstream kinases (JAK1 and JAK2) of STAT3 and increased phosphatase activity. Phosphorylation of AKT was also reduced by sorafenib. In contrast, mitogen-activated protein kinases were not consistently inhibited by sorafenib in these cells. Two key cyclins (D and E) and the antiapoptotic protein Mcl-1 were downregulated by sorafenib in both cell lines and primary cultures. Our data suggest that inhibition of STAT3 signaling by sorafenib contributes to growth arrest and induction of apoptosis in glioblastoma cells. These findings provide a rationale for potential treatment of malignant gliomas with sorafenib. Mol Cancer Ther; 9(4); 953-62. (c)2010 AACR.
This study aimed at identifying predictors of success (retention after one year of intervention with ?5% weight loss) in subjects at high risk for type 2 diabetes enrolled in a lifestyle modification program. Fifty-one individuals with BMI ?27kg/m(2) and pre-diabetes or metabolic syndrome were enrolled in an individualized multidisciplinary lifestyle intervention to induce weight loss. Subjects were assessed initially with a 16-item weight-loss readiness tool (WLRT) based on stages of change model; a 6-min walk test; and anthropometric measures. The most significant independent factor associated with no success was a lower result to the question "I am capable of doing more physical activity" (P=0.001). The second significant independent predictor was ?0.5% weight loss 6 weeks after initiating intervention (P=0.01). Excluding subjects with both criteria would have reduced by 52% the number of subjects eligible for the program, decreased the dropout rate from 30% to 17%, and increased the proportion of subjects with ?5% weight loss at one year from 51% to 80%. Importantly, only 4% of subjects would have been falsely identified as non-responders. These results indicate that a practical WLRT, in combination with early weight-loss response, is helpful to identify subjects with greater chances of success to lifestyle intervention.
This article presents findings from a cross-study analysis of social engagement between older people and staff in care homes. The studies found that staff and the culture of the care home were influential in determining the quality and type of relationship between residents and staff. Although a number of factors limited the quality of social interactions between these groups, practices existed that overcame barriers to the development of positive social relationships.
Five diastereomeric polyketide glycosides, roselipins 3A-3E (1-5), have been isolated from the acetone extract of Clonostachys candelabrum on the basis of their positive anthelmintic activity. The structures of these compounds were elucidated by comparison of their NMR and MS data to those of previously reported roselipins and related structures, and were confirmed by 2D-NMR spectral analysis. Known compounds linoleic acid (6) and aurantiogliocladin (7) were also isolated as active anthelmintic components, although much less potent than the roselipins.
Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.
One of the hallmarks of development is that many more cells are produced than are ultimately needed for organogenesis. In the case of striated skeletal muscle, large numbers of myoblasts are generated in the somites and then migrate to take up residence in the limbs and the trunk. A subset of these cells fuses to form multinucleated skeletal muscle fibers, while a second group, known as satellite cells, exits the cell cycle and persists as a pool of lineage-restricted stem cells that can repair damaged muscle. The remaining cells initiate apoptosis and are rapidly lost. Primary myoblasts and established satellite cell lines are powerful tools for dissecting the regulatory events that mediate differentiative decisions and have proven to be important models. As well, muscle diseases represent debilitating and often fatal disorders. This chapter provides a general background for muscle development and then details a variety of assays for monitoring the differentiation and the death of muscle. While some of these methods are specialized to address the phenotypic properties of skeletal muscle, others can be employed with a wide variety of cell types.
Within the literature, the formation of therapeutic relationships between professionals, older people and others significant to them in their lives has been considered as central to current care philosophies. Furthermore, relationships between staff, residents and their families have emerged within the literature as fundamental to the experiences of life within the community of a care home. This paper reports part of a wider study that explored relationships between residents, families and staff. The aim of this paper is to contribute to an understanding of the factors that may be significant in the formation of relationships in care homes, and how this may support the development of community. Three case studies of care homes were undertaken using a constructivist approach. Constructivist methodology seeks to share multiple perceptions between participants with the aim of creating a joint construction. This process supported the development of shared meanings as views and ideas were shared between participants using interviews, participant observation and focus groups. The key factors influencing relationships that emerged were leadership, continuity of staff, personal philosophy of staff and contribution of residents and families. This paper suggests that considering how the style of leadership influences the organisation of care may be a useful starting point in developing community within care homes.
Acheron (Achn), a phylogenetically-conserved member of the Lupus antigen family of RNA binding proteins, was initially identified as a novel cell death-associated gene from the intersegmental muscles of the tobacco hawkmoth Manduca sexta. C(2)C(12) cells are a standard model for the study of myogenesis. When deprived of growth factors, these cells can be induced to: form multinucleated myotubes, arrest as quiescent satellite-like reserve cells, or undergo apoptosis. Achn expression is induced in myoblasts that form myotubes and acts upstream of the muscle specific transcription factor MyoD. Forced expression of ectopic Achn resulted in the formation of larger myotubes and massive reserve cell death relative to controls. Conversely, dominant-negative or antisense Achn blocked myotube formation following loss of growth factors, suggesting that Achn plays an essential, permissive role in myogenesis. Studies in zebrafish embryos support this hypothesis. Reduction of Achn with antisense morpholinos led to muscle fiber loss and an increase in the number of surviving cells in the somites, while ectopic Achn enhanced muscle fiber formation and reduced cell numbers. These results display a crucial evolutionarily conserved role for Achn in myogenesis and suggest that it plays key roles in the processes of differentiation and self-renewal.
The aim of this study was to consider how relationships in care homes influence the experience of older people, their families and staff. The main objective reported in this paper considers how these relationships are developed and the contribution that staff make to this process through the routines of care.
In larvae of the tobacco hawkmoth Manduca sexta, the intersegmental muscles (ISMs) span eight abdominal segments and represent the major muscle group. Following pupation, the ISMs in the first two and last two segments undergo programmed cell death (PCD), while the remaining four segments persist until the time of adult eclosion, when they too undergo PCD. ISM death at adult eclosion is initiated by a decline in the circulating ecdysteroid titer and requires de novo gene expression. In this study we have investigated the hormonal regulation and the patterns of gene expression that accompany both early and late ISM death. We find that distinct endocrine cues regulate these two periods of muscle death. Even though the middle segments of ISMs are exposed to the same endocrine environment as the adjacent cells that die following pupation, they do not express death-associated transcripts until they are specifically signaled to die following adult eclosion. These data indicate that subsets of homologous muscles appear to make segment-specific decisions to couple their endogenous cell death programs to distinctly different developmental cues. Nevertheless, once cell death is initiated, they utilize many of the same molecular components.
Complete leptin deficiency is associated with weight gain and extreme obesity, according to studies of animals and of monogenic obesity in humans. It is still a matter of debate whether relative leptin deficiency plays a physiologic role in adiposity regulation in free-living humans. We hypothesized that leptin levels would be associated with subsequent weight changes in healthy normal-weight young adults. Our prospective cohort involved 150 healthy young adults (114 women and 36 men) followed over their years of study at the Université de Sherbrooke. Anthropometric measurements, fasting blood samples, 3-day food diaries, and a physical activity questionnaire were collected at baseline. Leptin levels were measured with radioimmunoassay. Associations between baseline leptin levels and subsequent anthropometric changes were assessed with multivariable linear regression models to account for adiposity at baseline, food intake, and energy expenditure. Over the 2-year follow-up, changes in body mass index (BMI) ranged from -0.8 to +2.6 kg·m(-2) in men (mean BMI change, +0.6 kg·m(-2)) and from -2.5 to +3.7 kg·m(-2) in women (mean BMI change, +0.1 kg·m(-2)). Lower leptin levels at baseline were associated with a higher risk of weight gain in women (r = -0.24; p = 0.01 for change in BMI) and in men (r = -0.27, p = 0.11), even after accounting for baseline BMI, total daily caloric intake, and energy expenditure (p = 0.02). In the subsample measured at 4 years (n = 63), baseline leptin levels were not associated with 4-year weight changes. Lower leptin levels are associated with a higher risk of weight gain over 2 years in healthy young adults.
Pathotropic neural stem and/or progenitor cells (NSCs) can potentially deliver therapeutic agents to otherwise inaccessible cancers. In glioma, NSCs are found in close contact with tumor cells, raising the possibility that specificity of NSC contact with glioma targets originates in the tumor cells themselves. Alternatively, target preferences may originate, at least in part, in the tumor microenvironment. To better understand mechanisms underlying NSC interactions with glioma cells, we examined NSC-target cell contacts in a highly simplified 3-dimensional peptide hydrogel (Puramatrix) in which cell behaviors can be studied in the relative absence of external cues. HB1.F3 is an immortalized clonal human NSC line extensively characterized in preclinical investigations. To study contact formation between HB1.F3 NSCs and glioma cells, we first examined co-cultures of eGFP-expressing HB1.F3 (HB1.F3.eGFP) NSCs and dsRed-expressing U251 glioma (U251.dsRed) cells. Using confocal microscopy, HB1.F3.eGFP cells were observed contacting or encircling U251.dsRed glioma cells, but never the reverse. Next, examining specificity of these contacts, no significant quantitative differences in either percentages of HB1.F3 NSCs contacting targets, or in the extent of target cell encirclement, were observed when HB1.F3.eGFP cells were presented with various potential target cells (human glioma and breast cancer cell lines, patient-derived brain tumor lines, non-tumor fibroblasts, primary mouse and human astroglial cells, and primary adult and newborn human dermal fibroblasts) except that interactions between HB1.F3 cells did not progress beyond establishing contacts. Finally cytoskeletal mechanisms employed by HB1.F3.eGFP cells varied with the substrate. When migrating in Puramatrix, HB1.F3 NSCs exhibited intermittent process extension followed by soma translocation, while during encirclement their movements were more amoeboid. We conclude that formation of contacts and subsequent encirclement of target cells by HB1.F3 NSCs is an intrinsic property of these NSCs, and that preferential contact formation with tumor cells in vivo must therefore be highly dependent on microenvironmental cues.
A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8 effector T cells derived from CD45RA(-) CD62L(+) central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here, we describe a clinical scale, closed system, immunomagnetic selection method to isolate CD8(+) T(CM) from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14(+), CD45RA(+), and CD4(+) cells from PBMC, and then to positively select CD62L(+) cells. The average purity and yield of CD8(+) CD45RA(-) CD62L TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8(+) T(CM) are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3-6 weeks. The resulting CD8(+) T(CM)-derived effectors are polyclonal, retain expression of CD62L and CD28, exhibit CAR-redirected antitumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified T(CM) cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19(+) B-cell malignancies, and 3 clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.
A major challenge in cancer research field is to define molecular features that distinguish cancer stem cells from normal stem cells. In this study, we compared microRNA (miRNA) expression profiles in human glioblastoma stem cells and normal neural stem cells using combined microarray and deep sequencing analyses. These studies allowed us to identify a set of 10 miRNAs that are considerably up-regulated or down-regulated in glioblastoma stem cells. Among them, 5 miRNAs were further confirmed to have altered expression in three independent lines of glioblastoma stem cells by real-time RT-PCR analysis. Moreover, two of the miRNAs with increased expression in glioblastoma stem cells also exhibited elevated expression in glioblastoma patient tissues examined, while two miRNAs with decreased expression in glioblastoma stem cells displayed reduced expression in tumor tissues. Furthermore, we identified two oncogenes, NRAS and PIM3, as downstream targets of miR-124, one of the down-regulated miRNAs; and a tumor suppressor, CSMD1, as a downstream target of miR-10a and miR-10b, two of the up-regulated miRNAs. In summary, this study led to the identification of a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. Characterizing the role of these miRNAs in glioblastoma stem cells may lead to the development of miRNA-based therapies that specifically target tumor stem cells, but spare normal stem cells.
Tumors exploit immunoregulatory checkpoints that serve to attenuate T cell responses as a means of circumventing immunologic rejection. Programmed death ligand 1 (PD-L1) is a negative regulator of T cell function and is frequently expressed by solid tumors. By engaging programmed death 1 (PD-1) on activated T cells, PD-L1(+) tumors directly render tumor-specific T cells, including adoptively transferred T cells, functionally exhausted. As a strategy to overcome tumor PD-L1 effects on adoptively transferred T cells, we sought to convert PD-1 to a T cell costimulatory receptor by exchanging its transmembrane and cytoplasmic tail with that of CD28. Rather than becoming exhausted upon engagement of PD-L1(+) tumors, we hypothesized that CD8(+) cytotoxic T lymphocytes (CTL) genetically modified to express this PD1:CD28 chimera would exhibit enhanced functional attributes. Here we show that cell surface expressed PD1:CD28 retains the capacity to bind PD-L1 resulting in T cell costimulation as evidenced by increased levels of ERK phosphorylation, augmentation of cytokine secretion, increased proliferative capacity, and enhanced expression of effector molecule Granzyme B. We provide evidence that this chimera could serve as a novel engineering strategy to overcome PD-L1 mediated immunosuppression.
Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment.
The main objective of this study was to determine whether expectations and readiness to modify eating habits and physical activity (PA) level are different between young and older individuals with prediabetes who agreed to participate in a lifestyle modification program.
The study aimed to develop, deliver, and evaluate a training programme in care homes to enhance the quality of care for people living with dementia based on the principles of relationship-centred care expressed through the Senses Framework.
To evaluate IL13R?2 as an immunotherapeutic target for eliminating glioma stem-like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13R?2-specific primary human CD8(+) CTLs (IL13-zetakine(+) CTL) to target this therapeutically resistant glioma subpopulation.
To determine the independent effects of total and each health professionals frequency of contacts, on changes in anthropometric measures and physical capacity following a lifestyle intervention offered by a multi-disciplinary team in adults at high risk for type 2 diabetes.
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