Why do those who lose weight eventually gain it back? Rosenbaum and colleagues use functional magnetic resonance imaging to examine the effects of weight loss and leptin on neural activity (page 2583). They found alterations in neural activity in response to visual food cues in regions of the brain known to control regulatory and hedonic aspects of energy intake. These changes in neural activity during maintenance of a reduced weight were due substantially to central leptin deficiency.
Image credit: Photo Researchers Inc. (brain section) and Karen Guth (grapes).
The academic scientific enterprise rewards those with the longest CVs and the most publications. Under pressure to generate voluminous output, scientists often fall prey to double publishing, self plagiarism, and submitting the “minimal publishable unit.” Are these ethical gray areas, or true transgressions?
Recent investigations have suggested that it might be possible to reverse the pathology of pulmonary arterial hypertension (PAH), a disorder that can be rapidly progressive and fatal despite current treatments including i.v. prostacyclin. This review will address the cellular and molecular processes implicated in clinical, genetic, and experimental studies as underlying the pulmonary vascular abnormalities associated with PAH. Emerging treatments are aimed at inducing apoptosis of abnormal vascular cells that obstruct blood flow and at promoting regeneration of “lost” distal vasculature.
Maintenance of weight loss is often unsuccessful because of metabolic adaptations that conserve energy. Studies in rodents suggest that a reduction in leptin level during weight loss signals to the brain to increase feeding and decrease energy expenditure. In this issue of the JCI, Rosenbaum et al. examined this concept in obese patients who lost weight and were maintained at 10% below their initial weight (see the related article beginning on page 2583). Brain activity responses to visual food stimuli were visualized using functional MRI. Leptin levels fell during weight loss and increased brain activity in areas involved in emotional, cognitive, and sensory control of food intake. Restoration of leptin levels maintained weight loss and reversed the changes in brain activity. Thus, leptin is a critical factor linking reduced energy stores to eating behavior. Potentially, leptin therapy could sustain weight loss by overriding the tendency toward energy conservation.
Airway irritants cause a variety of lung pathologies. Two separate studies, the first recently reported in the JCI by Bessac et al. and the second reported by Andrè et al. in the current issue of the JCI (see the related article beginning on page 2574), have identified irritants that activate transient receptor potential cation channel, subfamily A, member 1 (TRPA1) receptors in airway sensory neurons, resulting in neurogenic inflammation and respiratory hypersensitivity. The identification of TRPA1 activation by toxicants from cigarette smoke and polluted air, such as crotonaldehyde, acrolein, and oxidizing agents such as hydrogen peroxide, is an important finding. These two studies enhance our understanding of how pollution and cigarette smoke can damage airway function and will hopefully pave the way for the development of rational alternative therapeutics for such airway injury.
Measles is a highly contagious disease that causes immunosuppression in patients. Measles virus infection has been thought to begin in the respiratory epithelium and then spread to lymphoid tissue. In this issue of the JCI, Leonard et al. provide data to suggest an alternative model of measles virus pathogenesis (see the related article beginning on page 2448). In human primary epithelial cells and rhesus monkeys in vivo, the authors show that initial infection of respiratory epithelium is not necessary for the virus to enter the host but that viral entry into epithelial cells via interaction of the virus with a receptor located on the basolateral side of the epithelium is required for viral shedding into the airway and subsequent transmission.
The ErbB receptors, such as EGFR, have been intensely pursued as targets for cancer therapeutics. However, a large percentage of patients who are initially responsive to ErbB-targeted therapies experience tumor recurrence and become refractory to therapy. In this issue of the JCI, Guix et al. demonstrate that downregulation of IGF-binding protein 3 (IGFBP-3) and -4, the negative regulators of IGF-I receptor signaling, contributes to the resistance of human squamous cell carcinomas to the EGFR inhibitor gefitinib (see the related article beginning on page 2609). Understanding the mechanisms involved in the resistance of some tumors to ErbB-targeted molecules may provide guidelines for developing more efficient therapeutic approaches.
To meet tissue requirements for oxygen, capillaries must be properly distributed without excess or shortage. In this process, tissue oxygen concentration is well known to determine capillary density via the hypoxia-induced cascade, in which HIFs and VEGF play key roles. However, some additional mechanisms modulating this cascade are suggested to be involved in precise capillary network formation. Here, we showed that leukemia inhibitory factor (LIF) was predominantly expressed in developing endothelium, while its receptor was expressed in surrounding cells such as retinal astrocytes. The retinas of Lif–/– mice displayed increased microvessel density accompanied by sustained tip cell activity, due to increased VEGF expression by astrocytes in the vascularized area. Lif–/– mice resisted hyperoxygen insult in the oxygen-induced retinopathy model, whereas they paradoxically had increased numbers of neovascular tufts. In an in vitro study, LIF inhibited hypoxia-induced VEGF expression and proliferation in cultured astrocytes. Lif–/– mice also exhibited similarly increased microvessel density and upregulated VEGF in various tissues outside the retina. Together, these findings suggest that tissues and advancing vasculature communicate to ensure adequate vascularization using LIF as well as oxygen, which suggests a new strategy for antiangiogenic therapy in human diseases such as diabetic retinopathy and cancer.
Hedgehog (HH) signaling has emerged as a critical pathway involved in the pathogenesis of a variety of tumors. As a result, HH antagonists are currently being evaluated as potential anticancer therapeutics. Conversely, activation of HH signaling in the adult heart may be beneficial, as HH agonists have been shown to increase coronary vessel density and improve coronary function after myocardial infarction. To investigate a potential homeostatic role for HH signaling in the adult heart, we ablated endogenous HH signaling in murine myocardial and perivascular smooth muscle cells. HH signaling was required for proangiogenic gene expression and maintenance of the adult coronary vasculature in mice. In the absence of HH signaling, loss of coronary blood vessels led to tissue hypoxia, cardiomyocyte cell death, heart failure, and subsequent lethality. We further showed that HH signaling specifically controlled the survival of small coronary arteries and capillaries. Together, these data demonstrate that HH signaling is essential for cardiac function at the level of the coronary vasculature and caution against the use of HH antagonists in patients with prior or ongoing heart disease.
The proinflammatory cytokine IL-6 seems to have an important role in the intestinal inflammation that characterizes inflammatory bowel diseases (IBDs) such as Crohn disease and ulcerative colitis. However, little is known about the molecular mechanisms regulating IL-6 production in IBD. Here, we assessed the role of the transcriptional regulator IFN regulatory factor–4 (IRF4) in this process. Patients with either Crohn disease or ulcerative colitis exhibited increased IRF4 expression in lamina propria CD3+ T cells as compared with control patients. Consistent with IRF4 having a regulatory function in T cells, in a mouse model of IBD whereby colitis is induced in RAG-deficient mice by transplantation with CD4+CD45RBhi T cells, adoptive transfer of wild-type but not IRF4-deficient T cells resulted in severe colitis. Furthermore, IRF4-deficient mice were protected from T cell–dependent chronic intestinal inflammation in trinitrobenzene sulfonic acid– and oxazolone-induced colitis. In addition, IRF4-deficient mice with induced colitis had reduced mucosal IL-6 production, and IRF4 was required for IL-6 production by mucosal CD90+ T cells, which it protected from apoptosis. Finally, the protective effect of IRF4 deficiency could be abrogated by systemic administration of either recombinant IL-6 or a combination of soluble IL-6 receptor (sIL-6R) plus IL-6 (hyper–IL-6). Taken together, our data identify IRF4 as a key regulator of mucosal IL-6 production in T cell–dependent experimental colitis and suggest that IRF4 might provide a therapeutic target for IBDs.
Cancer is associated with immune deficiency, but the biologic basis of this is poorly defined. Here we demonstrate that impaired actin polymerization results in CD4+ and CD8+ T cells from patients with chronic lymphocytic leukemia (CLL) exhibiting defective immunological synapse formation with APCs. Although this synapse dysfunction was in part a result of the CLL cells having poor APC function, defective actin polymerization was also identified in T cells from patients with CLL. We further demonstrate that, following contact with CLL cells, defects in immune synapse formation were induced in healthy allogeneic T cells. This required direct contact and was inhibited by blocking adhesion molecules on CLL B cells. In T cells from patients with CLL and in T cells from healthy individuals that had been in contact with CLL cells, recruitment of key regulatory proteins to the immune synapse was inhibited. Treatment of autologous T cells and CLL cells with the immunomodulating drug lenalidomide resulted in improved synapse formation. These results define what we believe to be a novel immune dysfunction in T cells from patients with CLL that has implications for both autologous and allogeneic immunotherapy approaches and identifies repair of immune synapse defects as an essential step in improving cancer immunotherapy approaches.
Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.
The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.
Huntingtin interacting protein 1 related (Hip1r) is an F-actin– and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.
Stress-induced analgesia (SIA) is a key component of the defensive behavioral “fight-or-flight” response. Although the neural substrates of SIA are incompletely understood, previous studies have implicated the hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) peptidergic systems in the regulation of SIA. Using immunohistochemistry in brain tissue from wild-type mice, we identified N/OFQ-containing fibers forming synaptic contacts with Hcrt neurons at both the light and electron microscopic levels. Patch clamp recordings in GFP-tagged mouse Hcrt neurons revealed that N/OFQ hyperpolarized, decreased input resistance, and blocked the firing of action potentials in Hcrt neurons. N/OFQ postsynaptic effects were consistent with opening of a G protein–regulated inwardly rectifying K+ (GIRK) channel. N/OFQ also modulated presynaptic release of GABA and glutamate onto Hcrt neurons in mouse hypothalamic slices. Orexin/ataxin-3 mice, in which the Hcrt neurons degenerate, did not exhibit SIA, although analgesia was induced by i.c.v. administration of Hcrt-1. N/OFQ blocked SIA in wild-type mice, while coadministration of Hcrt-1 overcame N/OFQ inhibition of SIA. These results establish what is, to our knowledge, a novel interaction between the N/OFQ and Hcrt systems in which the corticotropin-releasing factor and N/OFQ systems coordinately modulate the Hcrt neurons to regulate SIA.
Murine olfactory ensheathing cells (OECs) promote central nervous system axonal regeneration in models of spinal cord injury. We investigated whether OECs could induce a neuroplastic effect to improve the neurological dysfunction caused by hypoxic/ischemic stress. In this study, human OECs/olfactory nerve fibroblasts (hOECs/ONFs) specifically secreted trophic factors including stromal cell–derived factor–1α (SDF-1α). Rats with intracerebral hOEC/ONF implantation showed more improvement on behavioral measures of neurological deficit following stroke than control rats. [18F]fluoro-2-deoxyglucose PET (FDG-PET) showed increased glucose metabolic activity in the hOEC/ONF-treated group compared with controls. In mice, transplanted hOECs/ONFs and endogenous homing stem cells including intrinsic neural progenitor cells and bone marrow stem cells colocalized with specific neural and vascular markers, indicating stem cell fusion. Both hOECs/ONFs and endogenous homing stem cells enhanced neuroplasticity in the rat and mouse ischemic brain. Upregulation of SDF-1α and CXCR4 in hOECs/ONFs promoted neurite outgrowth of cocultured primary cortical neurons under oxygen glucose deprivation conditions and in stroke animals through upregulation of cellular prion protein (PrPC) expression. Therefore, the upregulation of SDF-1α and the enhancement of CXCR4 and PrPC interaction induced by hOEC/ONF implantation mediated neuroplastic signals in response to hypoxia and ischemia.
Hereditary sensory and autonomic neuropathy type II (HSANII) is an early-onset autosomal recessive disorder characterized by loss of perception to pain, touch, and heat due to a loss of peripheral sensory nerves. Mutations in hereditary sensory neuropathy type II (HSN2), a single-exon ORF originally identified in affected families in Quebec and Newfoundland, Canada, were found to cause HSANII. We report here that HSN2 is a nervous system–specific exon of the with-no-lysine(K)–1 (WNK1) gene. WNK1 mutations have previously been reported to cause pseudohypoaldosteronism type II but have not been studied in the nervous system. Given the high degree of conservation of WNK1 between mice and humans, we characterized the structure and expression patterns of this isoform in mice. Immunodetections indicated that this Wnk1/Hsn2 isoform was expressed in sensory components of the peripheral nervous system and CNS associated with relaying sensory and nociceptive signals, including satellite cells, Schwann cells, and sensory neurons. We also demonstrate that the novel protein product of Wnk1/Hsn2 was more abundant in sensory neurons than motor neurons. The characteristics of WNK1/HSN2 point to a possible role for this gene in the peripheral sensory perception deficits characterizing HSANII.
cGMP-dependent protein kinase II (cGKII; encoded by PRKG2) is a serine/threonine kinase that is critical for skeletal growth in mammals; in mice, cGKII deficiency results in dwarfism. Using radiographic analysis, we determined that this growth defect was a consequence of an elongated growth plate and impaired chondrocyte hypertrophy. To investigate the mechanism of cGKII-mediated chondrocyte hypertrophy, we performed a kinase substrate array and identified glycogen synthase kinase–3β (GSK-3β; encoded by Gsk3b) as a principal phosphorylation target of cGKII. In cultured mouse chondrocytes, phosphorylation-mediated inhibition of GSK-3β was associated with enhanced hypertrophic differentiation. Furthermore, cGKII induction of chondrocyte hypertrophy was suppressed by cotransfection with a phosphorylation-deficient mutant of GSK-3β. Analyses of mice with compound deficiencies in both protein kinases (Prkg2–/–Gsk3b+/–) demonstrated that the growth retardation and elongated growth plate associated with cGKII deficiency were partially rescued by haploinsufficiency of Gsk3b. We found that β-catenin levels decreased in Prkg2–/– mice, while overexpression of cGKII increased the accumulation and transactivation function of β-catenin in mouse chondroprogenitor ATDC5 cells. This effect was blocked by coexpression of phosphorylation-deficient GSK-3β. These data indicate that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3β by cGKII.
Chronic inflammation increases cancer risk. While it is clear that cell signaling elicited by inflammatory cytokines promotes tumor development, the impact of DNA damage production resulting from inflammation-associated reactive oxygen and nitrogen species (RONS) on tumor development has not been directly tested. RONS induce DNA damage that can be recognized by alkyladenine DNA glycosylase (Aag) to initiate base excision repair. Using a mouse model of episodic inflammatory bowel disease by repeated administration of dextran sulfate sodium in the drinking water, we show that Aag-mediated DNA repair prevents colonic epithelial damage and reduces the severity of dextran sulfate sodium–induced colon tumorigenesis. Importantly, DNA base lesions expected to be induced by RONS and recognized by Aag accumulated to higher levels in Aag-deficient animals following stimulation of colonic inflammation. Finally, as a test of the generality of this effect we show that Aag-deficient animals display more severe gastric lesions that are precursors of gastric cancer after chronic infection with Helicobacter pylori. These data demonstrate that the repair of DNA lesions formed by RONS during chronic inflammation is important for protection against colon carcinogenesis.
Diabetes mellitus is the most common and rapidly growing cause of end-stage renal disease in developed countries. A classic hallmark of early diabetes mellitus includes activation of the renin-angiotensin system (RAS), which may lead to hypertension and renal tissue injury, but the mechanism of RAS activation is elusive. Here we identified a paracrine signaling pathway in the kidney in which high levels of glucose directly triggered the release of the prohypertensive hormone renin. The signaling cascade involved the local accumulation of succinate and activation of the kidney-specific G protein–coupled metabolic receptor, GPR91, in the glomerular endothelium as observed in rat, mouse, and rabbit kidney sections. Elements of signal transduction included endothelial Ca2+, the production of NO and prostaglandin (PGE2), and their paracrine actions on adjacent renin-producing cells. This GPR91 signaling cascade may serve to modulate kidney function and help remove metabolic waste products through renal hyperfiltration, and it could also link metabolic diseases, such as diabetes, or metabolic syndrome with RAS overactivation, systemic hypertension, and organ injury.
In rodents and humans, alcohol exposure has been shown to predispose the pancreas to cholinergic or viral induction of pancreatitis. We previously developed a rodent model in which exposure to an ethanol (EtOH) diet, followed by carbachol (Cch) stimulation, redirects exocytosis from the apical to the basolateral plasma membrane of acinar cells, resulting in ectopic zymogen enzyme activation and pancreatitis. This redirection of exocytosis involves a soluble NSF attachment receptor (SNARE) complex consisting of syntaxin-4 and synapse-associated protein of 23 kDa (SNAP-23). Here, we investigated the role of the zymogen granule (ZG) SNARE vesicle-associated membrane protein 8 (VAMP8) in mediating basolateral exocytosis. In WT mice, in vitro EtOH exposure or EtOH diet reduced Cch-stimulated amylase release by redirecting apical exocytosis to the basolateral membrane, leading to alcoholic pancreatitis. Further reduction of zymogen secretion, caused by blockade of both apical and basolateral exocytosis and resulting in a more mild induction of alcoholic pancreatitis, was observed in Vamp8–/– mice in response to these treatments. In addition, although ZGs accumulated in Vamp8–/– acinar cells, ZG-ZG fusions were reduced compared with those in WT acinar cells, as visualized by electron microscopy. This reduction in ZG fusion may account for reduced efficiency of apical exocytosis in Vamp8–/– acini. These findings indicate that VAMP8 is the ZG-SNARE that mediates basolateral exocytosis in alcoholic pancreatitis and that VAMP8 is critical for ZG-ZG homotypic fusion.
Type 2 congenital long QT syndrome (LQT-2) is linked to mutations in the human ether a-go-go–related gene (HERG) and is characterized by rate-corrected QT interval (QTc) prolongation, ventricular arrhythmias, syncope, and sudden death. Recognized triggers of these cardiac events include emotional and acoustic stimuli. Here we investigated the repeated occurrence of fever-induced polymorphic ventricular tachycardia and ventricular fibrillation in 2 LQT-2 patients with A558P missense mutation in HERG. ECG analysis showed increased QTc with fever in both patients. WT, A558P, and WT+A558P HERG were expressed heterologously in HEK293 cells and were studied using biochemical and electrophysiological techniques. A558P proteins showed a trafficking-deficient phenotype. WT+A558P coexpression caused a dominant-negative effect, selectively accelerated the rate of channel inactivation, and reduced the temperature-dependent increase in the WT current. Thus, the WT+A558P current did not increase to the same extent as the WT current, leading to larger current density differences at higher temperatures. A similar temperature-dependent phenotype was seen for coexpression of the trafficking-deficient LQT-2 F640V mutation. We postulate that the weak increase in the HERG current density in WT-mutant coassembled channels contributes to the development of QTc prolongation and arrhythmias at febrile temperatures and suggest that fever is a potential trigger of life-threatening arrhythmias in LQT-2 patients.
Histone deacetylase (HDAC) inhibitors are antitumor agents that also have antiinflammatory properties. However, the mechanisms of their immunomodulatory functions are not known. We investigated the mechanisms of action of 2 HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and ITF 2357, on mouse DC responses. Pretreatment of DCs with HDAC inhibitors significantly reduced TLR-induced secretion of proinflammatory cytokines, suppressed the expression of CD40 and CD80, and reduced the in vitro and in vivo allostimulatory responses induced by the DCs. In addition, injection of DCs treated ex vivo with HDAC inhibitors reduced experimental graft-versus-host disease (GVHD) in a murine allogeneic BM transplantation model. Exposure of DCs to HDAC inhibitors increased expression of indoleamine 2,3-dioxygenase (IDO), a suppressor of DC function. Blockade of IDO in WT DCs with siRNA and with DCs from IDO-deficient animals caused substantial reversal of HDAC inhibition–induced in vitro suppression of DC-stimulated responses. Direct injection of HDAC inhibitors early after allogeneic BM transplantation to chimeric animals whose BM-derived cells lacked IDO failed to protect from GVHD, demonstrating an in vivo functional role for IDO. Together, these data show that HDAC inhibitors regulate multiple DC functions through the induction of IDO and suggest that they may represent a novel class of agents to treat immune-mediated diseases.
Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 α,β-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify α,β-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.
Increased hunger and food intake during attempts to maintain weight loss are a critical problem in clinical management of obesity. To determine whether reduced body weight maintenance is accompanied by leptin-sensitive changes in neural activity in brain regions affecting regulatory and hedonic aspects of energy homeostasis, we examined brain region–specific neural activity elicited by food-related visual cues using functional MRI in 6 inpatient obese subjects. Subjects were assessed at their usual weight and, following stabilization at a 10% reduced body weight, while receiving either twice daily subcutaneous injections of leptin or placebo. Following weight loss, there were predictable changes in neural activity, many of which were reversed by leptin, in brain areas known to be involved in the regulatory, emotional, and cognitive control of food intake. Specifically, following weight loss there were leptin-reversible increases in neural activity in response to visual food cues in the brainstem, culmen, parahippocampal gyrus, inferior and middle frontal gyri, middle temporal gyrus, and lingual gyrus. There were also leptin-reversible decreases in activity in response to food cues in the hypothalamus, cingulate gyrus, and middle frontal gyrus. These data are consistent with a model of the weight-reduced state as one of relative leptin deficiency.
Infantile hemangioma is a benign endothelial tumor composed of disorganized blood vessels. It exhibits a unique life cycle of rapid postnatal growth followed by slow regression to a fibrofatty residuum. Here, we have reported the isolation of multipotential stem cells from hemangioma tissue that give rise to hemangioma-like lesions in immunodeficient mice. Cells were isolated based on expression of the stem cell marker CD133 and expanded from single cells as clonal populations. The CD133-selected cells generated human blood vessels 7 days after implantation in immunodeficient mice. Cell retrieval experiments showed the cells could again form vessels when transplanted into secondary recipients. The human vessels expressed GLUT-1 and merosin, immunodiagnostic markers for infantile hemangioma. Two months after implantation, the number of blood vessels diminished and human adipocytes became evident. Lentiviral expression of GFP was used to confirm that the hemangioma-derived cells formed the blood vessels and adipocytes in the immunodeficient mice. Thus, when transplanted into immunodeficient mice, hemangioma-derived cells recapitulated the unique evolution of infantile hemangioma — the formation of blood vessels followed by involution to fatty tissue. In summary, this study identifies a stem cell as the cellular origin of infantile hemangioma and describes for what we believe is the first time an animal model for this common tumor of infancy.
Recent evidence indicates that small noncoding RNA molecules known as microRNAs (miRNAs) can function as tumor suppressors and oncogenes. Mutation, misexpression, and altered mature miRNA processing are implicated in carcinogenesis and tumor progression. Because SNPs in pre-miRNAs could alter miRNA processing, expression, and/or binding to target mRNA, we conducted a systematic survey of common pre-miRNA SNPs and their surrounding regions and evaluated in detail the association of 4 of these SNPs with the survival of individuals with non–small cell lung cancer (NSCLC). When we assumed that disease susceptibility was inherited as a recessive phenotype, we found that the rs11614913 SNP in hsa-mir-196a2 was associated with survival in individuals with NSCLC. Specifically, survival was significantly decreased in individuals who were homozygous CC at SNP rs11614913. In the genotype-phenotype correlation analysis of 23 human lung cancer tissue samples, rs11614913 CC was associated with a statistically significant increase in mature hsa-mir-196a expression but not with changes in levels of the precursor, suggesting enhanced processing of the pre-miRNA to its mature form. Furthermore, binding assays revealed that the rs11614913 SNP can affect binding of mature hsa-mir-196a2-3p to its target mRNA. Therefore, the rs11614913 SNP in hsa-mir-196a2 may be a prognostic biomarker for NSCLC. Further characterization of miRNA SNPs may open new avenues for the study of cancer and therapeutic interventions.
Although some cancers are initially sensitive to EGFR tyrosine kinase inhibitors (TKIs), resistance invariably develops. We investigated mechanisms of acquired resistance to the EGFR TKI gefitinib by generating gefitinib-resistant (GR) A431 squamous cancer cells. In GR cells, gefitinib reduced phosphorylation of EGFR, ErbB-3, and Erk but not Akt. These cells also showed hyperphosphorylation of the IGFI receptor (IGFIR) and constitutive association of IRS-1 with PI3K. Inhibition of IGFIR signaling disrupted the association of IRS-1 with PI3K and restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit GR cell growth. Gene expression analyses revealed that GR cells exhibited markedly reduced IGF-binding protein 3 (IGFBP-3) and IGFBP-4 RNA. Addition of recombinant IGFBP-3 restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit cell growth. Finally, gefitinib treatment of mice with A431 xenografts in combination with an IGFIR-specific monoclonal antibody prevented tumor recurrence, whereas each drug given alone was unable to do so. These data suggest that loss of expression of IGFBPs in tumor cells treated with EGFR TKIs derepresses IGFIR signaling, which in turn mediates resistance to EGFR antagonists. Moreover, combined therapeutic inhibition of EGFR and IGFIR may abrogate this acquired mechanism of drug resistance and is thus worthy of prospective clinical investigation.
Identifying the genetic variants that regulate fasting glucose concentrations may further our understanding of the pathogenesis of diabetes. We therefore investigated the association of fasting glucose levels with SNPs in 2 genome-wide scans including a total of 5,088 nondiabetic individuals from Finland and Sardinia. We found a significant association between the SNP rs563694 and fasting glucose concentrations (P = 3.5 × 10–7). This association was further investigated in an additional 18,436 nondiabetic individuals of mixed European descent from 7 different studies. The combined P value for association in these follow-up samples was 6.9 × 10–26, and combining results from all studies resulted in an overall P value for association of 6.4 × 10–33. Across these studies, fasting glucose concentrations increased 0.01–0.16 mM with each copy of the major allele, accounting for approximately 1% of the total variation in fasting glucose. The rs563694 SNP is located between the genes glucose-6-phosphatase catalytic subunit 2 (G6PC2) and ATP-binding cassette, subfamily B (MDR/TAP), member 11 (ABCB11). Our results in combination with data reported in the literature suggest that G6PC2, a glucose-6-phosphatase almost exclusively expressed in pancreatic islet cells, may underlie variation in fasting glucose, though it is possible that ABCB11, which is expressed primarily in liver, may also contribute to such variation.
Dysfunctional Tregs have been identified in individuals with psoriasis. However, their role in the pathogenesis of the disease remains unclear. Here we explored the effect of diminished CD18 (β2 integrin) expression on the function of CD4+CD25+CD127– Tregs using the Cd18 hypomorphic (Cd18hypo) PL/J mouse model of psoriasis that closely resembles the human disease. We found that reduced CD18 expression impaired cell-cell contact between Tregs and DCs. This led to dysfunctional Tregs, which both failed to suppress the pathogenic T cells and promoted the onset and severity of the disease. This failure was TGF-β–dependent, as Tregs derived from Cd18hypo PL/J mice had diminished TGF-β1 expression. Adoptive transfer of Tregs expressing wild-type levels of CD18 into affected Cd18hypo PL/J mice resulted in a substantial improvement of the psoriasiform skin disease, which did not occur upon coinjection of the cells with TGF-β–specific neutralizing antibody. Our data indicate a primary dysfunction of Cd18hypo Tregs, allowing subsequent hyperproliferation of pathogenic T cells in the Cd18hypo PL/J mouse model of psoriasis. This study may provide a step forward in our understanding of the unique role of CD18 expression levels in avoiding autoimmunity.
Adipose tissue inflammation is a characteristic of obesity. However, the mechanisms that regulate this inflammatory response and link adipose inflammation to systemic metabolic consequences are not fully understood. In this study, we have taken advantage of the highly restricted coexpression of adipocyte/macrophage fatty acid–binding proteins (FABPs) aP2 (FABP4) and mal1 (FABP5) to examine the contribution of these lipid chaperones in macrophages and adipocytes to local and systemic inflammation and metabolic homeostasis in mice. Deletion of FABPs in adipocytes resulted in reduced expression of inflammatory cytokines in macrophages, whereas the same deletion in macrophages led to enhanced insulin signaling and glucose uptake in adipocytes. Using radiation chimerism through bone marrow transplantation, we generated mice with FABP deficiency in bone marrow and stroma-derived elements in vivo and studied the impact of each cellular target on local and systemic insulin action and glucose metabolism in dietary obesity. The results of these experiments indicated that neither macrophages nor adipocytes individually could account for the total impact of FABPs on systemic metabolism and suggest that interactions between these 2 cell types, particularly in adipose tissue, are critical for the inflammatory basis of metabolic deterioration.
Disablement of cell death programs in cancer cells contributes to drug resistance and in some cases has been associated with altered translational control. As eukaryotic translation initiation factor 4E (eIF4E) cooperates with c-Myc during lymphomagenesis, induces drug resistance, and is a genetic modifier of the rapamycin response, we have investigated the effect of dysregulation of the ribosome recruitment phase of translation initiation on tumor progression and chemosensitivity. eIF4E is a subunit of eIF4F, a complex that stimulates ribosome recruitment during translation initiation by delivering the DEAD-box RNA helicase eIF4A to the 5′ end of mRNAs. eIF4A is thought to prepare a ribosome landing pad on mRNA templates for incoming 40S ribosomes (and associated factors). Using small molecule screening, we found that cyclopenta[b]benzofuran flavaglines, a class of natural products, modulate eIF4A activity and inhibit translation initiation. One member of this class of compounds, silvestrol, was able to enhance chemosensitivity in a mouse lymphoma model in which carcinogenesis is driven by phosphatase and tensin homolog (PTEN) inactivation or elevated eIF4E levels. These results establish that targeting translation initiation can restore drug sensitivity in vivo and provide an approach to modulating chemosensitivity.
HIV infiltrates the CNS soon after an individual has become infected with the virus, and can cause dementia and encephalitis in late-stage disease. Here, a global metabolomics approach was used to find and identify metabolites differentially regulated in the cerebrospinal fluid (CSF) of rhesus macaques with SIV-induced CNS disease, as we hypothesized that this might provide biomarkers of virus-induced CNS damage. The screening platform used a non-targeted, mass-based metabolomics approach beginning with capillary reverse phase chromatography and electrospray ionization with accurate mass determination, followed by novel, nonlinear data alignment and online database screening to identify metabolites. CSF was compared before and after viral infection. Significant changes in the metabolome specific to SIV-induced encephalitis were observed. Metabolites that were increased during infection-induced encephalitis included carnitine, acyl-carnitines, fatty acids, and phospholipid molecules. The elevation in free fatty acids and lysophospholipids correlated with increased expression of specific phospholipases in the brains of animals with encephalitis. One of these, a phospholipase A2 isoenzyme, is capable of releasing a number of the fatty acids identified. It was expressed in different areas of the brain in conjunction with glial activation, rather than linked to regions of SIV infection and inflammation, indicating widespread alterations in infected brains. The identification of specific metabolites as well as mechanisms of their increase illustrates the potential of mass-based metabolomics to address problems in CNS biochemistry and neurovirology, as well as neurodegenerative diseases.
Copyright © 2014 American Society for Clinical Investigation