Showing papers in "Science Translational Medicine in 2010"
TL;DR: This study shows that sequencing of maternal plasma DNA provides a way for noninvasive prenatal genome- wide scanning for genetic disorders and suggests the feasibility of using genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasively way.
Abstract: Cell-free fetal DNA is present in the plasma of pregnant women. It consists of short DNA fragments among primarily maternally derived DNA fragments. We sequenced a maternal plasma DNA sample at up to 65-fold genomic coverage. We showed that the entire fetal and maternal genomes were represented in maternal plasma at a constant relative proportion. Plasma DNA molecules showed a predictable fragmentation pattern reminiscent of nuclease-cleaved nucleosomes, with the fetal DNA showing a reduction in a 166-base pair (bp) peak relative to a 143-bp peak, when compared with maternal DNA. We constructed a genome-wide genetic map and determined the mutational status of the fetus from the maternal plasma DNA sequences and from information about the paternal genotype and maternal haplotype. Our study suggests the feasibility of using genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasive way.
957 citations
TL;DR: The results demonstrate that IL-17A participates in psoriasis, rheumatoid arthritis, and uveitis and that the antibody against this cytokine may be an effective therapeutic agent, and warrant larger clinical trials to assess further the safety and efficacy of AIN457.
Abstract: Interleukin-17A (IL-17A) is elaborated by the T helper 17 (T(H)17) subset of T(H) cells and exhibits potent proinflammatory properties in animal models of autoimmunity, including collagen-induced arthritis, experimental autoimmune encephalomyelitis, and experimental autoimmune uveitis. To determine whether IL-17A mediates human inflammatory diseases, we investigated the efficacy and safety of AIN457, a human antibody to IL-17A, in patients with psoriasis, rheumatoid arthritis, and chronic noninfectious uveitis. Patients with chronic plaque-type psoriasis (n = 36), rheumatoid arthritis (n = 52), or chronic noninfectious uveitis (n = 16) were enrolled in clinical trials to evaluate the effects of neutralizing IL-17A by AIN457 at doses of 3 to 10 mg/kg, given intravenously. We evaluated efficacy by measuring the psoriasis area and severity index (PASI), the American College of Rheumatology 20% response (ACR20) for rheumatoid arthritis, or the number of responders for uveitis, as defined by either vision improvement or reduction in ocular inflammation or corticosteroid dose. AIN457 treatment induced clinically relevant responses of variable magnitude in patients suffering from each of these diverse immune-mediated diseases. Variable response rates may be due to heterogeneity in small patient populations, differential pathogenic roles of IL-17A in these diseases, and the different involvement or activation of IL-17A-producing cells. The rates of adverse events, including infections, were similar in the AIN457 and placebo groups. These results support a role for IL-17A in the pathophysiology of diverse inflammatory diseases including psoriasis, rheumatoid arthritis, and noninfectious uveitis.
868 citations
University of Cologne1, University of Zurich2, Cornell University3, University of Bonn4, Max Planck Society5, Peter MacCallum Cancer Centre6, St. Vincent's Health System7, Joseph Fourier University8, Oslo University Hospital9, Schiller International University10, University of Groningen11, VU University Medical Center12, Vanderbilt University13
TL;DR: F focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.
Abstract: Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.
828 citations
Brigham and Women's Hospital1, Harvard University2, Purdue University3, Technical University of Denmark4, State University of New York Upstate Medical University5, Duke University6, University of Würzburg7, Technion – Israel Institute of Technology8, University of Rochester9, Georgetown University Medical Center10, University of Bonn11, University of Miami12, Central University, India13, McLean Hospital14, Mayo Clinic15, University of Sydney16
TL;DR: A genome-wide meta-analysis of gene sets (groups of genes that encode the same biological pathway or process) in 410 samples from patients with symptomatic Parkinson’s and subclinical disease and healthy controls identified 10 gene sets that were all associated with PD.
Abstract: Parkinson’s disease affects 5 million people worldwide, but the molecular mechanisms underlying its pathogenesis are still unclear. Here, we report a genome-wide meta-analysis of gene sets (groups of genes that encode the same biological pathway or process) in 410 samples from patients with symptomatic Parkinson’s and subclinical disease and healthy controls. We analyzed 6.8 million raw data points from nine genome-wide expression studies, and 185 laser-captured human dopaminergic neuron and substantia nigra transcriptomes, followed by two-stage replication on three platforms. We found 10 gene sets with previously unknown associations with Parkinson’s disease. These gene sets pinpoint defects in mitochondrial electron transport, glucose utilization, and glucose sensing and reveal that they occur early in disease pathogenesis. Genes controlling cellular bioenergetics that are expressed in response to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) are underexpressed in Parkinson’s disease patients. Activation of PGC-1α results in increased expression of nuclear-encoded subunits of the mitochondrial respiratory chain and blocks the dopaminergic neuron loss induced by mutant α-synuclein or the pesticide rotenone in cellular disease models. Our systems biology analysis of Parkinson’s disease identifies PGC-1α as a potential therapeutic target for early intervention.
737 citations
TL;DR: Dichloroacetate appears to be safe to give to humans at doses that are required for pyruvate dehydrogenase inhibition, and can be added to a growing group of metabolic modulators that may prove useful in cancer therapy.
Abstract: Solid tumors, including the aggressive primary brain cancer glioblastoma multiforme, develop resistance to cell death, in part as a result of a switch from mitochondrial oxidative phosphorylation to cytoplasmic glycolysis. This metabolic remodeling is accompanied by mitochondrial hyperpolarization. We tested whether the small-molecule and orphan drug dichloroacetate (DCA) can reverse this cancer-specific metabolic and mitochondrial remodeling in glioblastoma. Freshly isolated glioblastomas from 49 patients showed mitochondrial hyperpolarization, which was rapidly reversed by DCA. In a separate experiment with five patients who had glioblastoma, we prospectively secured baseline and serial tumor tissue, developed patient-specific cell lines of glioblastoma and putative glioblastoma stem cells (CD133(+), nestin(+) cells), and treated each patient with oral DCA for up to 15 months. DCA depolarized mitochondria, increased mitochondrial reactive oxygen species, and induced apoptosis in GBM cells, as well as in putative GBM stem cells, both in vitro and in vivo. DCA therapy also inhibited the hypoxia-inducible factor-1alpha, promoted p53 activation, and suppressed angiogenesis both in vivo and in vitro. The dose-limiting toxicity was a dose-dependent, reversible peripheral neuropathy, and there was no hematologic, hepatic, renal, or cardiac toxicity. Indications of clinical efficacy were present at a dose that did not cause peripheral neuropathy and at serum concentrations of DCA sufficient to inhibit the target enzyme of DCA, pyruvate dehydrogenase kinase II, which was highly expressed in all glioblastomas. Metabolic modulation may be a viable therapeutic approach in the treatment of glioblastoma.
673 citations
TL;DR: Calreticulin is identified as the “eat me’ signal on cancer cells that leads to phagocytosis when the counterbalancing “don’t eat me” signal CD47 is blocked and provides a key insight for the therapeutic development of CD47-inhibitory agents.
Abstract: Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.
600 citations
TL;DR: By identifying a drug combination that delays or even combats development of resistance when used as a first-line treatment in clinical trials, these results could ultimately improve the lives of patients with medulloblastoma or other cancers that depend on Smo for their survival.
Abstract: The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.
553 citations
TL;DR: A silicon microfluidic cell-capture technology that, when coupled to an automated imaging system, enables the detection and enumeration of prostate cancer cells fished out from the blood, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor–associated marker.
Abstract: Rarecirculatingtumorcells(CTCs)arepresentinthebloodofpatientswithmetastaticepithelialcancersbuthavebeen difficult to measure routinely. We report a quantitative automated imaging system for analysis of prostate CTCs, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor–associated marker. The specificity of PSA staining enabled optimization of criteria for baseline image intensity, morphometric measurements, and integration of multiple signals in a three-dimensional microfluidic device. In a pilot analysis, we detected CTCs in prostate cancer patients with localized disease, before surgical tumor removal in 8 of 19 (42%) patients (range, 38 to 222 CTCs per milliliter). For 6 of the 8 patients with preoperative CTCs, a precipitous postoperative decline (<24 hours) suggests a short half-life for CTCs in the blood circulation. Other patients had persistent CTCs for up to 3 months after prostate removal, suggesting early but transient disseminated tumor deposits. In patients with metastatic prostate cancer, CTCs were detected in 23 of 36 (64%) cases (range, 14 to 5000 CTCs per milliliter). In previously untreated patients followed longitudinally, the numbers of CTCs declined after the initiation of effective therapy. The prostate cancer– specific TMPRSS2-ERG fusion was detectable in RNA extracted from CTCs from 9 of 20 (45%) patients with metastatic disease, and dual staining of captured CTCs for PSA and the cell division marker Ki67 indicated a broad range for the proportion of proliferating cells among CTCs. This method for analysis of CTCs will facilitate the application of noninvasive tumor sampling to direct targeted therapies in advanced prostate cancer and warrants the initiation of longterm clinical studies to test the importance of CTCs in invasive localized disease.
531 citations
TL;DR: In an arena that takes small steps, PARE offers a leap forward in the clinical management and treatment of solid tumors, revealing true biomarkers that enable monitoring of individual tumor progression, tailoring of response to therapeutic treatment, and identification of residual disease at a level previously undetectable by current methods.
Abstract: Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. The evaluation of patient-specific translocations in leukemias and lymphomas has revolutionized diagnostics for these diseases. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers with massively parallel sequencing revealed an average of nine rearranged sequences (range, 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints was able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.
516 citations
TL;DR: Patients with AIDS have fewer immune cells to defend against microbial invasion through the gut, a critical loss that may be caused by a tryptophan metabolite produced by other immune cells, and IDO1 inhibitors are being tested for their efficacy in interfering with this dangerous depletion of defenses.
Abstract: The pathogenesis of human and simian immunodeficiency viruses is characterized by CD4+ T cell depletion and chronic T cell activation, leading ultimately to AIDS. CD4+ T helper (TH) cells provide protective immunity and immune regulation through different immune cell functional subsets, including TH1, TH2, T regulatory (Treg), and interleukin-17 (IL-17)–secreting TH17 cells. Because IL-17 can enhance host defenses against microbial agents, thus maintaining the integrity of the mucosal barrier, loss of TH17 cells may foster microbial translocation and sustained inflammation. Here, we study HIV-seropositive subjects and find that progressive disease is associated with the loss of TH17 cells and a reciprocal increase in the fraction of the immunosuppressive Treg cells both in peripheral blood and in rectosigmoid biopsies. The loss of TH17/Treg balance is associated with induction of indoleamine 2,3-dioxygenase 1 (IDO1) by myeloid antigen-presenting dendritic cells and with increased plasma concentration of microbial products. In vitro, the loss of TH17/Treg balance is mediated directly by the proximal tryptophan catabolite from IDO metabolism, 3-hydroxyanthranilic acid. We postulate that induction of IDO may represent a critical initiating event that results in inversion of the TH17/Treg balance and in the consequent maintenance of a chronic inflammatory state in progressive HIV disease.
493 citations
TL;DR: Findings in cystic fibrosis pigs that survive long enough to develop human-like lung disease are reported, suggesting that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs.
Abstract: Lung disease causes most of the morbidity and mortality in cystic fibrosis (CF). Understanding the pathogenesis of this disease has been hindered, however, by the lack of an animal model with characteristic features of CF. To overcome this problem, we recently generated pigs with mutated CFTR genes. We now report that, within months of birth, CF pigs spontaneously developed hallmark features of CF lung disease, including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting that the lungs of CF pigs have a host defense defect against a wide spectrum of bacteria. In humans, the temporal and causal relations between inflammation and infection have remained uncertain. To investigate these processes, we studied newborn pigs. Their lungs showed no inflammation but were less often sterile than controls. Moreover, after introduction of bacteria into their lungs, pigs with CF failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. Our finding that pigs with CF have a host defense defect against bacteria within hours of birth provides an opportunity to further investigate CF pathogenesis and to test therapeutic and preventive strategies that could be deployed before secondary consequences develop.
TL;DR: The discovery that a small number of peptides can elicit the disease in patients suggests that a similar approach may be successful in humans as well, and a detailed molecular understanding of the peptides driving the immune response in celiac disease is required.
Abstract: Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4(+ )T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat alpha-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from omega-gliadin (wheat) and C-hordein (barley) but not alpha-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.
TL;DR: This work outlines the fundamental properties of a highly participatory rapid learning system that can be developed in part from meaningful use of electronic health records (EHRs), which will make increasing amounts of medical information available in computable form.
Abstract: We outline the fundamental properties of a highly participatory rapid learning system that can be developed in part from meaningful use of electronic health records (EHRs). Future widespread adoption of EHRs will make increasing amounts of medical information available in computable form. Secured and trusted use of these data, beyond their original purpose of supporting the health care of individual patients, can speed the progression of knowledge from the laboratory bench to the patient’s bedside and provide a cornerstone for health care reform.
TL;DR: The authors show that the T cell repertoire is limited and biased from the time of the original V-D-J rearrangement during cell development, and this may help to understand these illnesses and to look for ways to modify patients’ T cell repertoires.
Abstract: Diversity in T lymphocyte antigen receptors is generated by somatic rearrangement of T cell receptor (TCR) genes and is concentrated within the third complementarity-determining region 3 (CDR3) of each chain of the TCR heterodimer. We sequenced the CDR3 regions from millions of rearranged TCR beta chain genes in naive and memory CD8(+) T cells of seven adults. The CDR3 sequence repertoire realized in each individual is strongly biased toward specific V(beta)-J(beta) pair utilization, dominated by sequences containing few inserted nucleotides, and drawn from a defined subset comprising less than 0.1% of the estimated 5 x 10(11) possible sequences. Surprisingly, the overlap in the naive CD8(+) CDR3 sequence repertoires of any two of the individuals is approximately 7000-fold larger than predicted and appears to be independent of the degree of human leukocyte antigen matching.
TL;DR: A closed-loop control system that uses frequent measurements of BG concentration along with subcutaneous delivery of both the fast-acting insulin analog lispro and glucagon as directed by a computer algorithm, demonstrating the feasibility of safe BG control by a bihormonal artificial endocrine pancreas.
Abstract: Automated control of blood glucose (BG) concentration is a long-sought goal for type 1 diabetes therapy. We have developed a closed-loop control system that uses frequent measurements of BG concentration along with subcutaneous delivery of both the fast-acting insulin analog lispro and glucagon (to imitate normal physiology) as directed by a computer algorithm. The algorithm responded only to BG concentrations and incorporated a pharmacokinetic model for lispro. Eleven subjects with type 1 diabetes and no endogenous insulin secretion were studied in 27-hour experiments, which included three carbohydrate-rich meals. In six subjects, the closedloop system achieved a mean BG concentration of 140 mg/dl, which is below the mean BG concentration target of _154 mg/dl recommended by the American Diabetes Association. There were no instances of treatmentrequiring hypoglycemia. Five other subjects exhibited hypoglycemia that required treatment; however, these individuals had slower lispro absorption kinetics than the six subjects that did not become hypoglycemic. The time-to-peak plasma lispro concentrations of subjects that exhibited hypoglycemia ranged from 71 to 191 min (mean, 117 ± 48 min) versus 56 to 72 min (mean, 64 ± 6 min) in the group that did not become hypoglycemic (aggregate mean of 84 min versus 31 min longer than the algorithm’s assumption of 33 min, P = 0.07). In an additional set of experiments, adjustment of the algorithm’s pharmacokinetic parameters (time-to-peak plasma lispro concentration set to 65 min) prevented hypoglycemia in both groups while achieving an aggregate mean BG concentration of 164 mg/dl. These results demonstrate the feasibility of safe BG control by a bihormonal artificial endocrine pancreas.
TL;DR: Protective effect of this heme-catabolizing enzyme relies on its ability to prevent tissue damage caused by the circulating free heme released from hemoglobin during infection, and it is proposed that targeting freeHeme by HPX might be used therapeutically to treat severe sepsis.
Abstract: Low-grade polymicrobial infection induced by cecal ligation and puncture is lethal in heme oxygenase-1-deficient mice (Hmox1(-/-)), but not in wild-type (Hmox1(+/+)) mice. Here we demonstrate that the protective effect of this heme-catabolizing enzyme relies on its ability to prevent tissue damage caused by the circulating free heme released from hemoglobin during infection. Heme administration after low-grade infection in mice promoted tissue damage and severe sepsis. Free heme contributed to the pathogenesis of severe sepsis irrespective of pathogen load, revealing that it compromised host tolerance to infection. Development of lethal forms of severe sepsis after high-grade infection was associated with reduced serum concentrations of the heme sequestering protein hemopexin (HPX), whereas HPX administration after high-grade infection prevented tissue damage and lethality. Finally, the lethal outcome of septic shock in patients was also associated with reduced HPX serum concentrations. We propose that targeting free heme by HPX might be used therapeutically to treat severe sepsis.
TL;DR: Development of a class of mechanically flexible silicon electronics for multiplexed measurement of signals in an intimate, conformal integrated mode on the dynamic, three-dimensional surfaces of soft tissues in the human body is reported.
Abstract: In all current implantable medical devices such as pacemakers, deep brain stimulators, and epilepsy treatment devices, each electrode is independently connected to separate control systems. The ability of these devices to sample and stimulate tissues is hindered by this configuration and by the rigid, planar nature of the electronics and the electrode-tissue interfaces. Here, we report the development of a class of mechanically flexible silicon electronics for multiplexed measurement of signals in an intimate, conformal integrated mode on the dynamic, three-dimensional surfaces of soft tissues in the human body. We demonstrate this technology in sensor systems composed of 2016 silicon nanomembrane transistors configured to record electrical activity directly from the curved, wet surface of a beating porcine heart in vivo. The devices sample with simultaneous submillimeter and submillisecond resolution through 288 amplified and multiplexed channels. We use this system to map the spread of spontaneous and paced ventricular depolarization in real time, at high resolution, on the epicardial surface in a porcine animal model. This demonstration is one example of many possible uses of this technology in minimally invasive medical devices.
TL;DR: In this paper, the authors identify a primary human LSC gene signature and functional characterization of LSC-specific molecules in vivo in a mouse xenotransplantation model and demonstrate that CD32 and CD25-positive LSCs could initiate acute myeloid leukemia and were cell cycle-quiescent and chemotherapy-resistant in vivo.
Abstract: Human acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). Because these chemotherapy-resistant LSCs are thought to underlie disease relapse, effective therapeutic strategies specifically targeting these cells may be beneficial. Here, we report identification of a primary human LSC gene signature and functional characterization of human LSC-specific molecules in vivo in a mouse xenotransplantation model. In 32 of 61 (53%) patients with AML, either CD32 or CD25 or both were highly expressed in LSCs. CD32- or CD25-positive LSCs could initiate AML and were cell cycle–quiescent and chemotherapy-resistant in vivo. Normal human hematopoietic stem cells depleted of CD32- and CD25-positive cells maintained long-term multilineage hematopoietic reconstitution capacity in vivo, indicating the potential safety of treatments targeting these molecules. In addition to CD32 and CD25, quiescent LSCs within the bone marrow niche also expressed the transcription factor WT1 and the kinase HCK. These molecules are also promising targets for LSC-specific therapy.
TL;DR: It is suggested that cone photoreceptors contribute substantially to nonvisual responses at the beginning of a light exposure and at low irradiances, whereas melanopsin appears to be the primary circadian photopigment in response to long-duration light Exposure and at high irradiances.
Abstract: In humans, modulation of circadian rhythms by light is thought to be mediated primarily by melanopsin-containing retinal ganglion cells, not rods or cones. Melanopsin cells are intrinsically blue light-sensitive but also receive input from visual photoreceptors. We therefore tested in humans whether cone photoreceptors contribute to the regulation of circadian and neuroendocrine light responses. Dose-response curves for melatonin suppression and circadian phase resetting were constructed in subjects exposed to blue (460 nm) or green (555 nm) light near the onset of nocturnal melatonin secretion. At the beginning of the intervention, 555-nm light was equally effective as 460-nm light at suppressing melatonin, suggesting a significant contribution from the three-cone visual system (lambda(max) = 555 nm). During the light exposure, however, the spectral sensitivity to 555-nm light decayed exponentially relative to 460-nm light. For phase-resetting responses, the effects of exposure to low-irradiance 555-nm light were too large relative to 460-nm light to be explained solely by the activation of melanopsin. Our findings suggest that cone photoreceptors contribute substantially to nonvisual responses at the beginning of a light exposure and at low irradiances, whereas melanopsin appears to be the primary circadian photopigment in response to long-duration light exposure and at high irradiances. These results suggest that light therapy for sleep disorders and other indications might be optimized by stimulating both photoreceptor systems.
TL;DR: A step toward workable gene therapy is reported in the form of stable expression of a lentiviral vector encoding anti-HIV RNAs in blood stem cells transplanted into AIDS patients, and cells that survived for long periods of time in patients, although too scarce to cure or even improve their HIV infections.
Abstract: AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell–based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood–derived (CD34 + ) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to ~1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.
TL;DR: Plasma metabolic profiles obtained during exercise provide signatures of exercise performance and cardiovascular disease susceptibility, in addition to highlighting molecular pathways that may modulate the salutary effects of exercise.
Abstract: Exercise provides numerous salutary effects, but our understanding of how these occur is limited. To gain a clearer picture of exercise-induced metabolic responses, we have developed comprehensive plasma metabolite signatures by using mass spectrometry to measure >200 metabolites before and after exercise. We identified plasma indicators of glycogenolysis (glucose-6-phosphate), tricarboxylic acid cycle span 2 expansion (succinate, malate, and fumarate), and lipolysis (glycerol), as well as modulators of insulin sensitivity (niacinamide) and fatty acid oxidation (pantothenic acid). Metabolites that were highly correlated with fitness parameters were found in subjects undergoing acute exercise testing and marathon running and in 302 subjects from a longitudinal cohort study. Exercise-induced increases in glycerol were strongly related to fitness levels in normal individuals and were attenuated in subjects with myocardial ischemia. A combination of metabolites that increased in plasma in response to exercise (glycerol, niacinamide, glucose-6-phosphate, pantothenate, and succinate) up-regulated the expression of nur77, a transcriptional regulator of glucose utilization and lipid metabolism genes in skeletal muscle in vitro. Plasma metabolic profiles obtained during exercise provide signatures of exercise performance and cardiovascular disease susceptibility, in addition to highlighting molecular pathways that may modulate the salutary effects of exercise.
TL;DR: A phase 1 clinical study in which biosynthetic mimics of corneal extracellular matrix were implanted to replace the pathologic anterior cornea of 10 patients who had significant vision loss, with the aim of facilitating endogenous tissue regeneration without the use of human donor tissue.
Abstract: Corneas from human donors are used to replace damaged tissue and treat corneal blindness, but there is a severe worldwide shortage of donor corneas. We conducted a phase 1 clinical study in which biosynthetic mimics of corneal extracellular matrix were implanted to replace the pathologic anterior cornea of 10 patients who had significant vision loss, with the aim of facilitating endogenous tissue regeneration without the use of human donor tissue. The biosynthetic implants remained stably integrated and avascular for 24 months after surgery, without the need for long-term use of the steroid immunosuppression that is required for traditional allotransplantation. Corneal reepithelialization occurred in all patients, although a delay in epithelial closure as a result of the overlying retaining sutures led to early, localized implant thinning and fibrosis in some patients. The tear film was restored, and stromal cells were recruited into the implant in all patients. Nerve regeneration was also observed and touch sensitivity was restored, both to an equal or to a greater degree than is seen with human donor tissue. Vision at 24 months improved from preoperative values in six patients. With further optimization, biosynthetic corneal implants could offer a safe and effective alternative to the implantation of human tissue to help address the current donor cornea shortage.
TL;DR: A modeling approach is used to predict how resistance emerges in hepatitis C virus and suggest that a combination of drugs that can fight three or more mutated viral strains may be needed to cure this disease and constructed a model to study the development of drug-resistant virus during treatment.
Abstract: About 170 million people worldwide are infected with hepatitis C virus (HCV). The current standard therapy leads to sustained viral elimination in only approximately 50% of the treated patients. Telaprevir, an HCV protease inhibitor, has substantial antiviral activity in patients with chronic HCV infection. However, in clinical trials, drug-resistant variants emerge at frequencies of 5 to 20% of the total virus population as early as the second day after the beginning of treatment. Here, using probabilistic and viral dynamic models, we show that such rapid emergence of drug resistance is expected. We calculate that all possible single- and double-mutant viruses preexist before treatment and that one additional mutation is expected to arise during therapy. Examining data from a clinical trial of telaprevir therapy for HCV infection in detail, we show that our model fits the observed dynamics of both drug-sensitive and drug-resistant viruses and argue that therapy with only direct antivirals will require drug combinations that have a genetic barrier of four or more mutations.
TL;DR: It is shown that decreased ferroportin gene expression is associated with a significant reduction in metastasis-free and disease-specific survival that is independent of other breast cancer risk factors and identifies an extremely favorable cohort of breast cancer patients who have a 10-year survival of >90%.
Abstract: Ferroportin and hepcidin are critical proteins for the regulation of systemic iron homeostasis. Ferroportin is the only known mechanism for export of intracellular non–heme-associated iron; its stability is regulated by the hormone hepcidin. Although ferroportin profoundly affects concentrations of intracellular iron in tissues important for systemic iron absorption and trafficking, ferroportin concentrations in breast cancer and their influence on growth and prognosis have not been examined. We demonstrate here that both ferroportin and hepcidin are expressed in cultured human breast epithelial cells and that hepcidin regulates ferroportin in these cells. Further, ferroportin protein is substantially reduced in breast cancer cells compared to nonmalignant breast epithelial cells; ferroportin protein abundance correlates with metabolically available iron. Ferroportin protein is also present in normal human mammary tissue and markedly decreased in breast cancer tissue, with the highest degree of anaplasia associated with lowest ferroportin expression. Transfection of breast cancer cells with ferroportin significantly reduces their growth after orthotopic implantation in the mouse mammary fat pad. Gene expression profiles in breast cancers from >800 women reveal that decreased ferroportin gene expression is associated with a significant reduction in metastasis-free and disease-specific survival that is independent of other breast cancer risk factors. High ferroportin and low hepcidin gene expression identifies an extremely favorable cohort of breast cancer patients who have a 10-year survival of >90%. Ferroportin is a pivotal protein in breast biology and a strong and independent predictor of prognosis in breast cancer.
TL;DR: The authors propose that DRESS is caused by an EBV (or other similar virus)–driven selection of CD8+ T lymphocytes, which in turn inappropriately attack multiple organs, and think that the culprit drugs may trigger activation of the patients’ dormant EBV by an as yet undefined mechanism, possibly directly.
Abstract: Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, drug-induced reaction that involves both the skin and the viscera. Evidence for reactivation of herpes family viruses has been seen in some DRESS patients. To understand the immunological components of DRESS and their relationship to viral reactivation, we prospectively assessed 40 patients exhibiting DRESS in response to carbamazepine, allopurinol, or sulfamethoxazole. Peripheral blood T lymphocytes from the patients were evaluated for phenotype, cytokine secretion, and repertoire of CD4 + and CD8 + and for viral reactivation. We found Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), or HHV-7 reactivation in 76% of the patients. In all patients, circulating CD8 + T lymphocytes were activated, exhibited increased cutaneous homing markers, and secreted large amounts of tumor necrosis factor–a and interferon-g. The production of these cytokines was particularly high in patients with the most severe visceral involvement. In addition, expanded populations of CD8 + T lymphocytes sharing the same T cell receptor repertoire were detected in the blood, skin, liver, and lungs of patients. Nearly half of these expanded blood CD8 + Tl ymphocytes specifically recognized one of several EBV epitopes. Finally, we found that the culprit drugs triggered the production of EBV in patients’ EBV-transformed B lymphocytes. Thus, cutaneous and visceral symptoms of DRESS are mediated by activated CD8 + T lymphocytes, which are largely directed against herpes viruses such as EBV.
TL;DR: Blood concentrations of two related oxysterols molecules were almost 10 times higher in Niemann-Pick C1 patients than in age-matched healthy controls or those with other diseases such as atherosclerosis or diabetes, suggesting that the two oxysterol molecules are accurate diagnostic markers of early clinical disease and can be used not only to monitor disease progression but also to demonstrate drug efficacy.
Abstract: Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1 −/− mouse model and found several cholesterol oxidation products that were elevated in Npc1 −/− mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.
TL;DR: A genome-scale, gene-specific analysis of DNA methylation in the same individuals over a decade apart identifies a personalized epigenomic signature that may correlate with a common genetic trait.
Abstract: The epigenome consists of non–sequence-based modifications, such as DNA methylation, that are heritable during cell division and that may affect normal phenotypes and predisposition to disease. Here, we have performed an unbiased genome-scale analysis of ~4 million CpG sites in 74 individuals with comprehensive array-based relative methylation (CHARM) analysis. We found 227 regions that showed extreme interindividual variability [variably methylated regions (VMRs)] across the genome, which are enriched for developmental genes based on Gene Ontology analysis. Furthermore, half of these VMRs were stable within individuals over an average of 11 years, and these VMRs defined a personalized epigenomic signature. Four of these VMRs showed covariation with body mass index consistently at two study visits and were located in or near genes previously implicated in regulating body weight or diabetes. This work suggests an epigenetic strategy for identifying patients at risk of common disease.
TL;DR: It is asserted that successful studies generally use suitable statistical approaches for biomarker definition and confirm results in independent test sets and a brief set of practical and feasible recommendations are described for investigators to properly identify and qualify proteomic biomarkers, which could also be used as reporting requirements.
Abstract: Clinical proteomics has yielded some early positive results-the identification of potential disease biomarkers-indicating the promise for this analytical approach to improve the current state of the art in clinical practice. However, the inability to verify some candidate molecules in subsequent studies has led to skepticism among many clinicians and regulatory bodies, and it has become evident that commonly encountered shortcomings in fundamental aspects of experimental design mainly during biomarker discovery must be addressed in order to provide robust data. In this Perspective, we assert that successful studies generally use suitable statistical approaches for biomarker definition and confirm results in independent test sets; in addition, we describe a brief set of practical and feasible recommendations that we have developed for investigators to properly identify and qualify proteomic biomarkers, which could also be used as reporting requirements. Such recommendations should help put proteomic biomarker discovery on the solid ground needed for turning the old promise into a new reality.
TL;DR: This Commentary proposes a systems perspective that incorporates a mixed-methods approach to SciTS that is commensurate with the conceptual, methodological, and translational complexities addressed within the SciTS field.
Abstract: This Commentary describes recent research progress and professional developments in the study of scientific teamwork, an area of inquiry termed the “science of team science” (SciTS, pronounced “sahyts”). It proposes a systems perspective that incorporates a mixed-methods approach to SciTS that is commensurate with the conceptual, methodological, and translational complexities addressed within the SciTS field. The theoretically grounded and practically useful framework is intended to integrate existing and future lines of SciTS research to facilitate the field’s evolution as it addresses key challenges spanning macro, meso, and micro levels of analysis.
TL;DR: In this article, the Robo4-dependent signaling pathway was used to strengthen the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm.
Abstract: The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1β and tumor necrosis factor–α, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host’s immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host’s response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host’s innate immune response may provide a therapeutic strategy for treating multiple infectious agents.