Cells live in changing, dynamic environments. To understand cellular decision-making, we must therefore understand how fluctuating inputs are processed by noisy biomolecular networks. Here we present a general methodology for analyzing the fidelity with which different statistics of a fluctuating input are represented, or encoded, in the output of a signaling system over time. We identify two orthogonal sources of error that corrupt perfect representation of the signal: dynamical error, which occurs when the network responds on average to other features of the input trajectory as well as to the signal of interest, and mechanistic error, which occurs because biochemical reactions comprising the signaling mechanism are stochastic. Trade-offs between these two errors can determine the system's fidelity. By developing mathematical approaches to derive dynamics conditional on input trajectories we can show, for example, that increased biochemical noise (mechanistic error) can improve fidelity and that both negative and positive feedback degrade fidelity, for standard models of genetic autoregulation. For a group of cells, the fidelity of the collective output exceeds that of an individual cell and negative feedback then typically becomes beneficial. We can also predict the dynamic signal for which a given system has highest fidelity and, conversely, how to modify the network design to maximize fidelity for a given dynamic signal. Our approach is general, has applications to both systems and synthetic biology, and will help underpin studies of cellular behavior in natural, dynamic environments. Cells do not live in constant conditions, but in environments that change over time. To adapt to their surroundings, cells must therefore sense fluctuating concentrations and ‘interpret’ the state of their environment to see whether, for example, a change in the pattern of gene expression is needed. This task is achieved via the noisy computations of biomolecular networks. But what levels of signaling fidelity can be achieved and how are dynamic signals encoded in the network's outputs? Here we present a general technique for analyzing such questions. We identify two sources of signaling error: dynamic error, which occurs when the network responds to features of the input other than the signal of interest; and mechanistic error, which arises because of the inevitable stochasticity of biochemical reactions. We show analytically that increased biochemical noise can sometimes improve fidelity and that, for genetic autoregulation, feedback can be deleterious. Our approach also allows us to predict the dynamic signal for which a given signaling network has highest fidelity and to design networks to maximize fidelity for a given signal. We thus propose a new way to analyze the flow of information in signaling networks, particularly for the dynamic environments expected in nature. Cells are continuously challenged by extra- and intracellular fluctuations, or ‘noise’, signal of interest, For cellular signaling to be effective, it should maintain sufficient fidelity. We wish to quantify the extent to which the current output of an intracellular biochemical network, ng input . This siWe will assume that the conditional mean, Many response functions, The difference between the conditional expectations We distinguish between two types of error that reduce fidelity between becomes significant when the response time of the signaling network is comparable to or longer than the timescale on which the signal of interest, For example, if the signal of interest is the current value of the input, he input . Since  We can write the dynamical error asIf fluctuations in is generated by the inherent stochasticity of the biochemical reactions that comprise the signaling network. We define mechanistic error as the deviation of the current value of the output from its average value given a particular history of the input:asticity . The magMechanistic error is related to intrinsic noise. Intrinsic variation measures the expected variation in fidelity error, We then define the We can decompose the output relation . The varic error .To compare signaling by different biochemical mechanisms, we normalize Comparing biochemical systems using the fidelity measure is equivalent to comparison based on the magnitude of the fidelity error, When Whenever Suppose that the input process ty error , becauseAn exact analytical method, applicable to linear cascades and feedforward loops, based on the observation that moments calculated from a chemical master equation with propensities that are the appropriate functions of time are conditional moments, where the conditioning is on the history of the inputs at time A Langevin method that can include non-linearities, requires stationary dynamics, and whose accuracy as an approximation improves as typical numbers of molecules grow.A numerical method, applicable to arbitrary biomolecular networks and signals of interest—based on a modification of the Gillespie algorithm allowing time-varying, stochastic propensities—that uses a ‘conjugate’ reporter to estimate the mechanistic error We note that our methods require that the inputs can be modeled as exogenous processes that are unaffected by interactions with the biochemistry of the signaling network  and absolute dynamical error (faster response times), negative feedback also decreases the dynamic range of the output. The fidelity therefore does not improve because the output distributions corresponding to each value of together . PositivTo explore what happens when the effect of feedback on the dynamic range is directly controlled, we investigated the effect of varying For static signals, we again find the fidelity almost always decreases with increasing negative feedback strength, et al. indeed found that information capacity increased significantly compared to that of a single cell When a physiological response corresponds to the average output of multiple cells, the magnitude of the mechanistic error is that for a single cell divided by the number of cells in the group . This reduction arises because the magnitude of the mechanistic error is now the variance of the average mechanistic error of the cells in the group. The dynamical error, Although negative feedback reduces relative dynamical error, it increases relative mechanistic error in individual cells. At the level of the collective response of multiple cells, the deleterious effect on mechanistic error is attentuated . Using aOur framework naturally adapts to the scenario of controlling a network output to approach a desired ‘target’ response when, for example, the cell's environment changes. Combined with model search procedures for synthetic design Cells use the information conveyed by signaling networks to regulate their behavior and make decisions. Not all features of the input trajectory will, however, be relevant for a particular decision, and we define the fidelity between the output of the network and a signal of interest, Using an orthogonal decomposition of the network's output into the faithfully transformed signal and error terms, we are able to identify two sources of error – dynamical and mechanistic. We assume the transformed signal, To apply our approach experimentally, we can use microfluidic technology to expose cells to the same controlled but time-varying input in the medium Our analysis is complementary to one based on information theory and the entire distribution of input and output The environments in which cells live are inherently dynamic and noisy. Here we have developed mathematical techniques to quantify how cells interpret and respond to fluctuating signals given their stochastic biochemistry. Our approach is general and will help underpin studies of cellular behavior in natural, dynamic environments.Define Text S1The complete supporting information is provided as (PDF)Click here for additional data file.


Cortical LFPs seem to mainly stem from synaptic inputs, but the net LFP signal from several contributing laminar populations is difficult to assess, as the individual contributions will depend on their locations, the morphologies of the postsynaptic neurons, the spatial distribution of active synapses, and the level of correlations in synaptic inputs [2 patch of cat V1, and we incorporate spatially specific pre- to post-synaptic inter- and intra-layer connectivity constrained by experimental observations [http://www.nest-initiative.org), while simulations of LFPs from morphologically realistic neurons used LFPy (compneuro.umb.no/LFPy) along with NEURON [While recordings of extracellular potentials (EP) remain a common method for experimentally measuring neural activity, the interpretation of the low-frequency part, the c inputs . While mc inputs , multicoc inputs . Here wervations  using rervations . Our hybh NEURON .


Obstructive sleep apnea (OSA), characterized by recurrent upper airway (UA) collapse during sleep, is associated with significant morbidity and disorders. Polysomnogram is employed in the evaluation of OSA and apnea-hypopnea number per hour reflects severity. For normal breathing, it is essential that the collapsible UA is patent. However, obstruction of the UA is quite common in adults and infants. Normally, important reflex mechanisms defend against the UA collapse. The muscle activity of UA dilators, including the genioglossus, tensor palatini (TP), and pharyngeal constrictors, is due to the integrated mechanism of afferent sensory input → to motor function. Snoring is harsh breathing to prevent UA obstruction. Unfortunately, snoring vibrations, pharyngeal suction collapse, negative pressure, and hypoxia cause pathological perturbations including dysfunctional UA afferent sensory activity. The current paper posits that peripheral sensory stimulation paradigm, which has been shown to be efficacious in improving several neurological conditions, could be an important therapeutic strategy in OSA also. Obstructive sleep apnea (OSA) is characterized by recurrent upper airway (UA) obstruction, hypoventilation, hypoxia, and hypoxemia. Two cardinal features of OSA are (1) decrease in UA dilator muscles' force during sleep and (2) narrowed cross-sectional pharyngeal lumen that presents a significant mechanical constraint to normal ventilation. During UA collapse, ventilation is compromised (hypopnea) or absent (apnea), and hypoxia and hypercapnia develop. These blood gas changes increase respiratory drive until the UA reopens, at which time ventilation increases to reverse the blood gas abnormalities. This scenario reflects a dysfunctional genioglossus (a major UA dilator muscle) activity during sleep, in conjunction with a hypotonic pharynx that collapses. ∼4% of men and ∼2% of women and is strongly linked to obesity. Owing to recurrent UA obstruction and hypoventilation, clinical consequences due to OSA  are several, ranging from—cardiovascular disease , stroke, metabolic derangement, respiratory failure, and cognitive dysfunction . Rec. Rec∼4% Moving a muscle involves neuromuscular communication. The response to contract by a muscle originates with sensory activation-simply put as in stepping on sharp object or touching a hot cup. The brain then sends a message to the muscle to respond. This pathway involves the afferent sensory limb to the brain and the motor nerve limb to the muscle. This well-characterized sensory-motor reflex pathway for muscle contraction is universal-operating in normal UA dilator musculature as well; however, this particular bidirectional loop is dysfunctional in OSA. Consequently, the compromised afferent input in conjunction with dysfunctional motor activity of dilator muscles influence ventilation causing hypopnea/apnea. Considerable research has focused on issues pertaining to sensory-motor integration . Recent Pharynx and genioglossus are striated muscles; they undergo atrophy and degeneration when subjected to disuse and diseases. They are not only motor organs, but also the regions where a variety of sensory receptors are located. The sensory receptors are located in the mucosa which lines the genioglossus and the UA. The mucosal Proprioceptors transmit touch, temperature, pressure, and pain sensations. Sensory responses are also elicited by air stimuli; the airflow receptors respond to the air puff and cold or warm air humidified by the nose. The sensory receptors respond to the respiratory movements and changes in size and volume as well. Thus any stretch or mechanical alterations would provide important sensory feedback; the perception and response to the above stimuli by the CNS conceivably provide required motor efferent response, essential for various appropriate UA neurophysiological functions. The tactile and chemoreceptors, though not very active during food propulsion, profoundly affect ventilation when stimulated. These receptors provide dynamic sensory feedback. The UA sensory receptors play an important role in maintaining UA patency and contribute to arousal. An impairment of UA sensory function could therefore predispose to UA obstruction during sleep. However, skeletal muscles possess a remarkable capacity of regeneration, mutability, and recovery following injury, disuse, and denervation. There are copious data, particularly in stroke field, demonstrating that physiological levels of electrical stimulation reduce muscular atrophy and dysfunction. However, owing to anatomical location and low approval by OSA patient, pharynx and genioglossus are less suitable to regular electrical stimulation. Since C-PAP is not curative, the challenge in OSA is to develop strategies to prevent and reverse the fundamental pathology and progressive UA deterioration, without side effects of drugs, and has maximal patient approval and cooperation. The current paradigm presents such a therapeutic modality.An area of great interest and importance in nervous system mechanisms is in the generation of adequate sensory stimulation through somatosensory receptors, thus enhancing associated relevant motor functions. Several workers have successfully utilized mechanical stimulation to the soles in order to stimulate specific muscles, resulting in limb muscle contraction, thus showing that controlled sensory stimulation may be used to “drive and upregulate" muscle activation and to maintain their functional integrity. Appropriate sensory stimulation has been shown to help stem the neurophysiological processes associated with muscle unloading in no-gravity flight, and in neurogenic muscular diseases. Similarly, intraoral, tongue, and pharyngeal sensory stimulation in OSA patients may conceivably ameliorate pharyngeal and genioglossal motor dysfunction and attenuate pharyngeal collapse. Sensory input is critical to swallowing—a function that involves intraoral and pharyngeal muscles. Stimulation of sensory receptors participates in initiating bolus preparation of food, appropriate positioning of oral structures, modulation of the strength, velocity, and timing of muscle contractions, and finally facilitating the deglutination. In the case of UA, its muscle activity is stimulated by changes in transmural pressure; any negative transmural pressure, across the wall, therefore, is an important sensory signal, indicating that the UA is narrowed or obstructed. Thus UA negative pressure (UANP), detected by mechanoreceptors , evokes an efferent motor reflex response that increases UA muscle activity, and stabilizes the airway –8. Moreo+T helper lymphocytes. The inflammatory infiltrate produces proinflammatory cytokines in response to muscle atrophy/degeneration. This triggers a cascade of inflammatory cell activation and migration into the affected muscles; the muscle undergoes further degeneration, weakness, and floppiness, due to proinflammatory cytokines, oxygen-free radicals, and nitric oxide [OSA patients snore and suffer from repeated forceful suction collapse of the pharynx and vigorous snoring-related vibration. These factors are traumatic to the UA mucosa and produce inflammation, edema, disturbed sensory function, and neurogenic damage. Habitual snoring, therefore, is considered to be a prelude to OSA. There is copious evidence of denervation of UA muscles in OSA patients. Various studies have documented demyelination , muscle ic oxide –27. The mucosal morphology of pharynx, uvula, and soft palate, has been characterized in OSA patients. The UA mucosa in OSA patients and snorers is described as inflammatory, edematous, and hyperplastic. Uvulas obtained from uvulopalatopharyngoplasty (UPPP) patients with moderate OSA showed a significantly higher number of leukocytes in the lamina propria of their uvular mucosa than in the controls (179 versus control 71). The thickness of the lamina propria  also increases significantly in OSA patients (0.99 mm versus control 0.27 mm). Hence inflammation, characterized by plasma cell infiltration and interstitial edema, present in the uvula mucosa of patients with moderate OSA, clearly suggests soft palate inflammation, contributing to UA occlusion in OSA patients . Morphom∼61% of OSA subjects possess abnormal laryngeal sensation also, showing that mucosal sensory function is impaired at multiple UA foci in OSA [Mucosal sensory dysfunction is present at multiple UA sites in patients with OSA. Utilizing air-pressure pulses endoscopic testing, a significant impairment in sensory detection thresholds in oropharynx, velopharynx, hypopharynx, and larynx (aryepiglottic eminence) has been determined. Further, i in OSA . Determii in OSA . The twoi in OSA , 31. All The decreased sensory afferent input, a narrower pharyngeal lumen, and negative pressure may all enhance the efferent motor output to the hyomandibular muscles as a compensatory mechanism to balance the dilator genioglossal dysfunction; this would be an effort to overcome hypoventilation as well as impending pharyngeal collapse. Thus, all dilators including the anterior hyoid muscles in conjunction with the genioglossus and tensor veli palitini –34 may f It is well established that skeletal muscle fibers are versatile and inherently mutable; they may undergo transformation in response to appropriate stimuli. They may shift from being slow-twitch oxidative Type I to fast-twitch glycolytic Type II fibers, or vice versa. While the former fiber type is more suited to sustained contractility owing to their inherent fatigue resistance, the fast-twitch Type II fibers, although generate increased force as brief bursts, are more prone to fatigue. An increase in fast-twitch type II fibers was shown in the genioglossus muscle of OSA patients ; this sh Aging-related disabilities and diseases are caused by increasing human longevity. Aging causes a general decrease in a variety of functional loops subserving somatosensory, special sensory, perceptual and cognitive operations. Losses in various sensory modalities include vision, auditory, gustatory, olfactory, vestibular, and indeed somatosensory. Over 35% of elderly have hearing deficits. About 11% elderly have noncorrectable visual impairment. In excess of 50% of elderly possess olfactory impairment. The consequence of intrinsic aging and extrinsic environmental insult results in progressive atrophy of dermal and epidermal sensory structures of the tongue, thus compromising the gustatory sense; this has been abundantly documented in geriatric population. Similarly, UA sensory decline occurring in the UA mucosal sensory receptors, as pointed out above, in over 10% of healthy elderly population, comprises part of a global sensory decline in aging , 46. Normal breathing is closely linked with patency of UA. The UA dilator genioglossus muscle plays an essential role in maintaining UA patency. Incidence of sleep-disordered breathing increasing with age and correlated with UA collapsibility, has been documented in old animals also , 42. In  OSA is a specific entity and the apnea-hypopnea index (AHI) defines the degree of severity of this disorder. About 10% of elderly population has an AHI of ≥10. 152 “healthy older adult volunteers without any disease”, (Mean age of 66) except for “Undiagnosed sleep-related breathing disorders (SRBD)” plus an absence of subjective daytime sleepiness, and an AHI of 21 (±15) for males, and 16.7 (±12.9) for females, respectively, were studied in a french study . Whole bAdvances in brain imaging have given many insights into the cortical and subcortical control of human UA and pharyngeal function. UA patency represents a complex coordinated process that is dependent on sensory feedback –60. The Several studies have examined the effect of oropharyngeal stimulation utilizing either thermal or taste stimuli to trigger a more rapid response of pharynx. Cold stimulation of the anterior faucial pillars (AFP) before swallowing hastened the onset of the pharyngeal swallowing phase and reduced the swallowing latency , 65, 66.per se is an essential prerequisite for the development of OSA [OSA is a state-related disorder, and the most fundamental neural event that initiates OSA is the closure of the pharyngeal airway during sleep. UA dilator muscle activity maintains a patent airway during wakefulness; however, with sleep onset, this neural compensation is reduced , thus set of OSA , 73. NeuThe UA is most vulnerable behind the tongue base and the soft palate that lack skeletal support. It is kept patent by a number of factors notably the motor activation of the dilator muscles. Sleep onset is characterized by ventilation decreases, and several studies have confirmed that the UA of OSA patients is more collapsible than the UA of nonapneic control subjects . At sleeUA patency is a function of neural control of dilator muscles; UA collapse involves not only increased intrapharyngeal negative pressure, but dysfunctional receptor types, including vagal pulmonary receptors, UA mechanoreceptors, other somatosensory receptors, and olfactory and gustatory receptors. Edema and inflammation of pharyngeal tissues, due to snoring plus suction collapse, might cause dysfunction of the above-mentioned receptors  and cause narrowing of the UA. UA collapsibility in OSA patients has been shown to correlate with changes in tonic and phasic contraction of UA dilator muscles, that is, TP and GG, respectively. Decreases in GG (EMG) activation occurs during REM in normal subjects , in OSA Sensory input is crucial to the initiation and modulation of several pharyngeal functions, and integration of diverse cortical and brainstem networks that underpin sensory-motor function in the UA. The oral cavity and pharynx are functionally integrated regions involved in complex motor responses that include feeding, chewing, swallowing, speech, and respiration. A variety of sensory receptors that innervate these two regions provide the link in reflexes that control muscles of the UA and upper gastrointestinal tract. The quintessential reflexes affect the neural motor control in general , 91 and Several interesting studies have determined effects of functional oropharyngeal sensory disruption on the pharyngeal motor function , that is, with and without topical oropharyngeal anesthesia . Unlike the normal bilateral activation of the primary sensorimotor cortex, oropharyngeal anesthesia led to a pronounced decrease of both sensory and motor activation. This showed that a short-term decrease in oropharyngeal sensory input impedes the control of pharyngeal function , 59. TheThe primary olfactory pathway is one of the most unique and plastic in the nervous system with neurogenesis and constant turnover of new sensory neurons. The olfactory receptors are known to turnover approximately every 40 days. New sensory neurons in the olfactory epithelium and new interneurons in the olfactory bulb (OB) are added throughout adult life of humans . This results in continuous remodeling of neuronal circuits and synapses in the OB throughout life. In humans, the trigeminal and olfactory nerves share overlapping innervation areas in the nasal cavity and function interactively. Odors perceived through the nose are called “orthonasally” perceived, whereas odors perceived through the mouth are labelled “retronasally” perceived. The latter can be used in older individuals, anosmics, and those with chronic nasal obstruction. Both routes of delivery produce similar brain activation of several brain areas, notably the amygdala and the insula; both reciprocally project to NTS and the dorsal motor nucleus –112. It The pharyngeal UA afferent inputs, mediated by the trigeminal (V), facial (V11), glossopharyngeal (IX), and vagus (X) cranial nerves, terminate in the nucleus of the Solitary Tract (NTS) and the trigeminal nucleus . The NTSOSA is characterized by sensory receptor dysfunction, which adversely impacts several brainstem nuclei including trigeminal, NTS, and hypoglossal. Conceivably, all receptors may not become dysfunctional in all components of the UA, and the OSA patients do possess functional sensory-motor reflex and afferent discharges found in arousal and wakefulness. Consequently, a combination of diurnal sensory stimulation protocols encompassing olfactory, gustatory, and different types of somatosensory  need to abe applied, in a unisensory  and multisensory  manner; conceivably this should strengthen and upregulate the diverse sensory receptors, enhance peripheral and central neuronal excitability/synaptic transmission, causing an increase in genioglossal function. Several studies described above show that the multisensory stimulation is more effective than just the unimodal.There is a remarkable degree of plasticity in primary motor cortex (M1) and the somatosensory cortex, as abundantly documented by the whisker barrel cortex studies. Some plastic changes occur very early; for example, M1 neurons in primates can be activated by a tactile and proprioceptive input with only a short latency . InteresDespite an extraordinary degree of research in understanding OSA, we still lack a satisfactory insight into the early stages of neuropathogenesis of this common condition. The quest for testable hypotheses is ongoing, and efforts to ameliorate pathogenetic stigmata persist. The present paper seeks to highlight the importance of damage in peripheral sensory elements that cause dysfunction in the afferent portion of the “sensory → motor” loop; the resulting dysfunctional motor efferent responses, may be an equally critical factor in the genesis of recurrent UA collapse—causing repeated hypoxic, hypoxemic, and hypercapnic events in OSA. The logical and pragmatic approach, therefore, is to upregulate the dysfunctional sensory receptors, via sensory stimulation, preferably in a natural, physiological, nonpharmacologic, and noninvasive manner from the periphery , in order to stimulate motor output limb and ameliorate the pathology of dilator muscles, including genioglossus and TP. Such ongoing upregulation of sensory → motor loop should regenerate the dysfunctional receptors and enhance the activity of those mucosal receptors that are intact; this conceivably should strengthen UA muscles, decrease mechanical load, and produce nocturnal UA patency through contractility of the dilators' aerobic tonic type 1, tonic-phasic mainly oxidative type 2, and mainly phasic glycolytic type 2 muscle fibers. Further research is critically important focusing on harnessing the role of disparate multimodal afferent signals encompassing somatosensory, olfactory, and gustatory modalities in upregulating key brainstem nuclei, and mediating activation of the motor pathway in possible amelioration of OSA.


We describe a patient with palmar-divergent dislocation of the scaphoid and lunate. After successful closed reduction, the scapholunate and lunotriquetral ligaments were sutured through the dorsal approach, and the anterior capsule was sutured through the palmar approach. The scapholunate and lunotriquetral joints were fixed with Kirschner wires for 7 weeks. At the 1-year follow-up, magnetic resonance imaging showed no evidence of avascular necrosis of the scaphoid or lunate, and radiographs showed no evidence of the dorsal and volar intercalated segment instability patterns associated with carpal instability. However, flexion of the scaphoid and a break in Gilula’s line remained. To our knowledge, this is the first report showing treatment of palmar-divergent dislocation of the scaphoid and lunate by suturing the carpal interosseous ligaments. Simultaneous palmar dislocation of the scaphoid and lunate is rare –7 and haA 46-year-old man who fell from a height of 1.5 m onto his left hand was brought to the emergency center of our hospital and underwent a medical examination. Radiography of the wrist revealed palmar-divergent dislocation of the scaphoid and lunate Fig. . A shortThe patient provided written informed consent prior to inclusion in this study, which was authorized by the local ethics committee and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki as revised in 2000.As palmar-divergent dislocation of the scaphoid and lunate is rare, its optimal treatment remains unclear. In previous reports, two patients were treated by open reduction and cast immobilization , 2, two Among the methods recommended to repair, the anterior and posterior ligaments on both sides of the lunate are combined palmar and dorsal approaches , and opeThe injury to our patient may have been accompanied by avascular necrosis of the scaphoid and lunate . PRC on


Sas4 gene , which is essential for centriole duplication. Sas4 mutant embryos lack cilia and centrosomes, but survive to e9.0. Shh signaling is blocked in Sas4 mutants, as expected because of the absence of cilia, but we have not detected defects in Wnt signaling in the mutants. Sas4 mutants die at an early stage of development than embryos that lack cilia, indicating that the centriole has functions in addition to templating cilia. Sas4 mutants show a high rate of apoptosis, and the early lethality of Sas4 embryos is rescued by removal of p53. We are currently investigating the p53-dependent pathways activated in Sas4 mutants that lead to cell death. Because human genetic diseases that disrupt the centrosome cause microcephaly, we are comparing the roles of cilia and centrosomes in patterning and cell behavior in the developing brain. These experiments will define the roles of cilia and centrosomes in the control asymmetric cell division and migration in the cortex.Work in the past decade has revealed that cilia and centrosomes, specialized microtubule-based organelles, can act as centers for developmental signaling pathways. It is now well-established that Sonic hedgehog (Shh) signaling depends on the primary cilium. Cilia are templated by the basal body, a specialized centrosomes, and it has been suggested that centrosomes may act as signaling centers important in the Wnt and other signaling pathways. To test the role of centrosomes in mouse development, we are analyzing the phenotypes of embryos that lack the


Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways. Colon and rectal cancer , the third most common cancer worldwide, has become one of the leading causes of death from cancers Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid extracted from medical herbs and edible plants, exerts a wide range of biological activities, including hepatoprotective 2, is known to be involved in multiple pathophysiological processes, including inﬂammation and tumorigenesis The enzyme cyclooxygenase-2 (COX-2), responsible for the catalysis of the conversion of arachidonic acid to prostaglandins and thromboxane AHere, we evaluated the effect of UA on colon cancer cell proliferation, migration, and apoptosis in colon cancer cells and analyzed its regulation on the key proteins of some signaling pathways involved in cell proliferation, migration and apoptosis. The results showed the anti-proliferation, anti-migration and pro-apoptotic effects of UA in colon cancer cells were mediated through simultaneous modulation of multiple signaling pathways, including MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2 and cytochrome c/caspase-dependent pathways. Our study not only uncovers the underlying molecular mechanisms of UA action in human colon cancer cells, but also suggests the great potential of UA in human colorectal cancer prevention and treatment.We first quantitatively analyzed the effect of UA on cell proliferation at 48 hours after treatment in SW480 and LoVo cells by a MTT assay. Treatment with UA at the dose of 10 µM to 60 µM significantly inhibited cell viability in a concentration-dependent manner, resulting in a 20% to an 83% inhibition in SW480 cells and a 5% to 55% inhibition in LoVo cells, respectively . We alsoWe next analyzed the UA-mediated changes in cell morphology and spreading in SW480 and LoVo cells. Treatment of cells with DMSO, a vehicle control, formed a cell layer, and more spreading, filopodia and blebbing were observed. By contrast,UA treatment caused a marked reduction of cell-to-cell contact, and had lower spreading with fewer formation of filopodia in both SW480 and LoVo cells, and induced membrane blebbing in cell treated with UA .We also evaluated the effect of UA on cell proliferation in human normal cell lines CCD841 and LO2. Treatment with UA at the doses ≤20 µM did not inhibit cell viability in both normal cell lines, demonstrating the potential specificity of UA in inhibiting tumor cell proliferation at the dose ≤20 µM.We next examined the effect of UA on cell migration by employing a scratch assay. Consistent with the data from cell proliferation and clonogenicity inhibition, treatment with UA at 5 µM effectively inhibited cell migration in both SW480 and LoVo cells . The parMMP9 and CDH1 are two key molecules involved in the cell invasion and migration-related signaling pathway The PI3K/Akt and MAPK signaling pathways play critical roles in controlling cell proliferation and tumorigenesis. To evaluate whether UA is capable of modulating the PI3K/Akt and MAPK signaling to lead to inhibition of cell proliferation, we examined the effects of UA on phosphorylation of the signaling molecules in these two pathways in SW480 cells by Western blot analysis. Treatment with UA at 20 µM considerably inhibited the expression of phosphorylated Akt and mTOR protein, but increased phosphorylated PTEN expression . We alsoTo further confirm the involvement of the Akt and ERK signaling pathways in the UA-mediated inhibition of cell proliferation, we next analyzed the effects of the Akt or ERK-selective inhibitor on UA-mediated proliferation inhibition in SW480 cells. Pretreatment of cells with PI3K/Akt inhibitor LY294002 (LY) at 5µM  or ERK ICOX-2 expression has been shown to upregulate the EGFR, PI3K/Akt and ERK signaling, thereby induce tumor cell proliferation, migration and invasion PGE2 is a downstream product of COX-2, and it is synthesized via the cyclooxygenase and prostaglandin synthase pathways. We treated SW480 and LoVo cells with UA at 20 µM and determined the effect of UA on PGE2 production. The results showed that UA treatment significantly reduced PGE2 production in both cells .To further confirm that the UA-mediated inhibition of cell proliferation is through regulation of COX-2 signaling in colon cancer cells, we pretreated SW480 cells with celecoxib , a COX-2-selective inhibitor, and tested the effect of UA on the celecoxib-mediated inhibition of cell proliferation. As shown in The expression of COX-2 is regulated by the translocation and interaction of transactivator NF-κB and coactivator p300 in tumor cell nuclei. We next performed immunofluorescence assay to evaluate the effect of UA on nuclear localization and interaction of the NF-κB and p300 in colon cancer SW480 and LoVo cells. We detected the constitutive translocation of p50 and p65 NF-κB and p300 to cell nuclei  and the The p300-mediated acetylation of transactivators and their binding to COX-2 gene promote play crucial roles in COX-2 expression. We next determined the effect of UA on p300-mediated acetylation of transactivators NF-κB and CREB2 in colon cancer SW480 cells. Treatment with UA at 20 µM in the p300-transfected SW480 cells markedly inhibited the acetylated levels of p50/p65 and CREB-2 proteins , whereasTo verify the role of UA in regulating the p300 signaling in colon cancer cells, we pretreated with roscovitine, an inhibitor of p300 , or tranWe also examined the effect of UA on cell apoptosis in colon cancer cells by an Annexin-V staining-based FACS assay at 48 h after treatment. Treatment of cells with UA at 20 µM and 40 µM led to a dose-dependent induction of the positive apoptotic cells , leadingCytochrome c (Cyto-c) is the upstream molecule of the caspase-dependent apoptosis pathway. We next performed immunofluorescence imaging (IFI) analysis to monitor changes in the subcellular localization of Cyto-c in the UA-treated SW480 and LoVo cells to determine whether UA could trigger Cyto-c release. Treatment with UA (20 µM) effectively promoted the release of Cyto-c from the inter-mitochondrial space into the cytosol . These rWe also examined the effect of inhibition of p300 on apoptosis induced by UA. The SW480 cells were transfected with a p300 specific siRNA (si-p300) and then treated with UA (20 µM), and the effect of inhibition of p300 on UA-mediated apoptosis were analyzed. The results showed that knockdown of p300 expression by si-p300 markedly enhanced the apoptosis induction mediated by UA , confirmIn this study, we analyzed the response of human colon cancer cells to UA treatment. UA significantly inhibited cell proliferation, migration and induced apoptosis in a dose-dependent manner. We showed that UA induced cytochrome c release, thereby caused caspase activation and apoptosis. UA also regulated the expression of MMP9 and CDH1 genes and inhibited the phosphorylation of Akt and ERK proteins, leading to an inactivation of the cell migration and proliferation-related signaling pathways. Our study also showed that UA suppressed COX-2 expression and PGE2 production. Furthermore, we showed that the UA-mediated suppression of COX-2 expression and cell proliferation is mediated by simultaneous modulation of the p300, NF-κB, and CREB2 signaling. UA inhibited p300-mediated acetylation of NF-κB and CREB2, and also promoted translocation of the NF-κB p65 and p300 from cell nuclei to cytoplasm. To our knowledge, this is the first report that demonstrated that UA simultaneously targets the multiple signaling pathways to inhibit colon cancer cell proliferation and induce apoptosis.UA has been intensively studied in the past; mainly as an anti-cancer compound and for its cardiovascular protective properties. The controversy of reports suggests anti-angiogenic and cytotoxic effects of UA on one side and cardiovascular and endothelial protective effects on the other side. Currently, there is insufficient evidence to recommend an ideal human dose. Animal studies suggest benefit use between 0.05–0.2% of the rat diet as UA. Assuming a range of 10–40 mg/kg bodyweight, the benefits in rat studies associated with UA are about equal to a human dose of 1.6–6.4 mg/kg bodyweight (110–440 mg for a 150 lb person).The side effects of UA from mainly include two aspects: male fertility and DNA damage. Previous study has revealed that UA has the potential of sperm motility inhibition and can serve as a topical vaginal contraceptive The expression of COX-2 plays a key role in human cancers. COX-2 expression and PGE2 production have been shown to upregulate the PI3K/Akt and ERK signaling, thereby induced angiogenesis, cell proliferation, migration and invasion Previous studies showed that UA induces apoptosis in human cancers We also found that UA targeted the Akt and ERK-dependent signaling pathways to inhibit colon cancer cell proliferation. The phosphorylated Akt and ERK are the mediators of cell proliferation and survival and clinical response to tyrosine kinase inhibitors (TKI), and a reduction in phosphorylated Akt and ERK expression is an important event in sensitizing tumor cells to TKI treatment. UA inhibited the phosphorylation of Akt and ERK proteins, resulting in an inactivation of the cell growth and survival-related Akt/ERK signaling pathway. Our findings have important implications in exploring novel therapeutic strategies for colon cancer.In summary, we demonstrated that UA inhibited cell proliferation and migration and induced apoptosis in colon cancer cells by simultaneously modulating the MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase-dependent signaling pathways . Our fin2 at 37°C. In all experiments, cells with 60% confluence were tested. Ursolic acid (UA), celecoxib and roscovitine were purchased from . A 100 mM solution of drug was prepared in dimethyl sulfoxide (DMSO), stored as small aliquots at −20°C and then diluted as needed in cell culture medium.Human colon cancer cell lines  were obtained from the American Type Culture Collection  and cultured in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin, and maintained in an incubator with a humidified atmosphere of 95% air and 5% COCell viability was determined by MTT assay . Briefly, cells plated in 96-well plates (3000 cells/well) were treated with UA at various doses. At 48 h after treatment, cell viability was determined.Cells plated in 6-well plates were treated with UA. After 24 h, cells were washed with PBS and trypsinized. Then cells were seeded at 100 cells/well in 6-well plates, and dispersed evenly by slightly shaking the dishes and incubated at 37°C with 5% of CO2 for 21 days. The medium was discarded and the cells were carefully washed with PBS twice. After being fixed with 4% paraformaldehyde for 15 min, the cells were stained with 0.1% crystal violet for 15 min before washing with tap water and air-drying. The clones with more than 50 cells were counted with an ordinary optical microscope. The clone formation rate was calculated with the following formula: Plate clone formation inhibitory ratio = (number of clones treated with UA/number of cells inoculated)×100%.2 incubator. After 56 h, medium was replaced with phosphate-buffered saline (PBS) buffer, the wound gap was observed and cells were photographed using an Olympus microscope fitted with digital camera.Scratch assay was performed to detect cell migration. The cells were grown to full confluency in six-well plates and incubated overnight in starvation medium. Cell monolayers were wounded with a sterile 100 µL pipette tip, washed with starvation medium to remove detached cells from the plates. Cells were treated with the indicated doses of UA in medium and kept in a CO5 cells/ml) were stained with AnnexinV-FITC using an Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit , placed on room temperature for 15 min in the dark, and then analyzed by flow cytometry . Apoptosis was calculated in terms of the AnnexinV-positive in cells. Cell cycle analysis was performed using PI. The transfected cells were harvested with trypsinization, fixed with cold 70% ethanol at 4°C for 12 hours. The staining was performed according to the producer’s manual. Raw data were analyzed using CXP Software and Multicycle for Windows (Beckman Coulter).The UA-treated cells (1×10Cell lysates were separated by electrophoresis in a 10% sodium dodecyl sulphate-polyacrylamide gradient minigel (SDS-PAGE) and electrophoretically transferred to a PVDF membrane. Western blots were probed with specific antibodies against phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-mTOR, mTOR, phospho-PTEN, PTEN, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2 , GAPDH, PARP, Caspase-3/9, COX-2, p300, NF-κB p50 and p65, CREB-2 and cytochrome c . The protein bands were detected by enhanced chemiluminescence (Pierce).5′- GGGACGGCAATGCTGAT-3′ and 5′-CGCCACGAGGAACAAACT -3′; CDH1-5′-CGCATTGCCACATACACTCT-3′ and 5′CGGGCTTGTTGTCATTCTG -3′; COX-2 5′-TCACAGGCTTCCATTGACCAG-3′ and 5′-CCGAGGCTTTTCTACCAGA-3′, GAPDH,5′- GCACCGTCAAGGCTGAGAAC -3′ and 5′- TGGTGAAGACGCCAGTGGA -3′. The reaction was done in a total volume of 50 µl, with 40 ng of cDNA and 2µl of each primer in 1 * SYBR® Premix. Quantitative data was analyzed by the ratio between the value of MMP9, CDH1, COX-2 and GAPDH. Conditions for all PCRs were optimized for a 50-µl reaction using the following 40 cycle program: step1∶95°C for 30 sec repeat 1 cycle; step2∶95°C for 5 sec, and 60°C for 30 sec repeat 40 cycles. All samples were amplified simultaneously in triplicate in one assay-run. GAPDH was included in each reaction as an internal standard and relative quantitative gene expression was calculated.Total RNA was isolated using TRIZOL reagent , and DNase I-treated RNA (2µg) was used for first-strand cDNA synthesis. Quantitative real-time PCR was done in an ABI PRISM 7900 HT sequence detection system with the preset PCR program, using SYBR® Premix Ex TaqTM II (TAKARA code DERR081A). The primer sequences used in real-time PCR were: MMP9, Total cellular RNA was isolated using the Trizol® reagent, according to the manufacturer’s protocol . Total RNA (2.5 µg) was reverse-transcribed by using the Superscript™-III kit . PCR analysis was performed on aliquots of the cDNA preparations to detect gene expression. PCR conditions were 4 min at 94°C followed by 35 cycles (25 for GAPDH): 30 seconds at 94°C, 30 seconds at 60°C, and 1 min at 72°C,The transfection of p300-overexpressing vector or p300 siRNA  was performed by Lipofectamine 2000 reagent according to the manufacturer’s protocol .Cells grown on chamber slides were washed in phosphate-buffered saline and fixed for 15 min at room temperature with 4% paraformaldehyde. The samples were pretreated with 10% bovine serum albumin (BSA) in PBS for 30 min. Antibodies against p300, p50, p65 or cytochrome c in the blocking solution were added to the sample and incubated for overnight at 4°C. Nonimmune rabbit IgG and mouse IgG were included as controls. Following five 5-min washes with PBS, fluorescein isothiocyanate- and rhodamineconjugated secondary antibodies were added in blocking solutions and incubated for 1 hour. After five additional 5-min washes, samples were examined with a Zeiss LSM700 confocal microscope, and images were processed with Zen 2010 software. More than 100 cells were inspected per experiment, and cells with typical morphology were presented.Cells were treated with UA for 24 h. The nuclear extracts prepared from the treated cells were immunoprecipitated with a specific antibodys against acetyl-lysine and the immunoprecipitate was pulled down with protein A/G agarose beads . After extensive washing, proteins were separated in a 10% SDS-PAGE system and the acetylated p50, p65 or CREB2 was detected with a monoclonal antibody against p50, p65 or CREB2 .t test were used to compare the values of the test and control samples. P<0.05 was considered to a statistically significant difference. SPSS6.0 software was used for all statistical analysis. The significance was evaluated by the paired t test. All the experiments were done three times, and mean values and standard deviation were calculated.Analysis of variance and Student’s


Human T cell lymphotropic virus type 1 (HTLV-1) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. HTLV-1 basic leucine zipper factor (HBZ) gene has a critical role in HTLV-1 infectivity and the development of ATL and HAM/TSP. However, little is known about the immune response against HBZ in HTLV-1-infected individuals. In this study, we examined antibody responses against HBZ in serum/plasma samples from 436 subjects including HTLV-1 seronegative donors, asymptomatic carriers (AC), ATL, and HAM/TSP patients using the luciferase immunoprecipitation system.+ T cell activation in HAM/TSP patients. Moreover, HBZ-specific antibody inhibited lymphoproliferation in the PBMC of HAM/TSP patients.Immunoreactivity against HBZ was detected in subsets of all HTLV-1-infected individuals but the test did not discriminate between AC, ATL and HAM/TSP. However, the frequency of detection of HBZ-specific antibodies in the serum of ATL patients with the chronic subtype was higher than in ATL patients with the lymphomatous subtype. Antibody responses against HBZ were also detected in cerebrospinal fluid of HAM/TSP patients with anti-HBZ in serum. Antibody responses against HBZ did not correlate with proviral load and HBZ mRNA expression in HAM/TSP patients, but the presence of an HBZ-specific response was associated with reduced CD4This is the first report demonstrating humoral immune response against HBZ associated with HTLV-I infection. Thus, a humoral immune response against HBZ might play a role in HTLV-1 infection. HAM/T T cells ,6. High  T cells -9, and r T cells . While t T cells . Therefoin vivo studies also demonstrated that HBZ expression enhanced HTLV-1 infectivity, T cell proliferation and lymphoma [Despite strong HTLV-1-specific immune responses, HTLV-1 proviral loads are significantly elevated in HAM/TSP patients compared to AC . Increaslymphoma -26. Furtlymphoma . Since t+ T cells are detected in AC and HAM/TSP patients, and HBZ-specific CTL clones were able to lyse naturally infected cells isolated from AC and HAM/TSP patients, but not ATL patients [Recent evidence has shown that HBZ is an immunogenic protein recognized by HBZ-specific CTL clones ,29. HBZ-patients ,29. Desppatients ,31. Sincpatients ,31. HereThe demographic characteristics of the study groups are summarized in Table Antibody responses for HBZ were analyzed in the separate groups of Jamaican and NIH subjects. There were no significant differences in frequency or magnitude of anti-HBZ antibody responses in serum/plasma between Jamaican and NIH subjects (data not shown) so they were combined in the remaining analyses, yielding a total of 436 serum/plasma samples obtained from ND, AC, ATL patients and HAM/TSP patients. Strong mean antibody levels against HBZ were detected in the HTLV-1-infected groups, including AC, ATL patients and HAM/TSP patients, compared to the ND group Figure . AlthougSince HBZ has been suggested to play a role in ATL, and ATL patients can be divided into the predominant clinically unique subtypes , it was of interest to determine if serum anti-HBZ responses could discriminate among these subtypes. The mean anti-HBZ antibody levels were not significantly different among the ATL subtypes Figure C. HoweveSince HBZ-specific immunoreactivity was detected in a subset of HTLV-1 infected subjects, we asked whether there is any relationship between HBZ-specific immunoreactivity and other immunological and virological markers of HTLV-1 infection, such as antibody responses to other HTLV-1 proteins and HTLV-1 viral gene expression. Antibody responses against Gag, Env or Tax did not differ significantly between HTLV-1-infected individuals with or without HBZ-specific immunoreactivity Figure A-C. We aSince strong antibody responses against HTLV-1 antigens have been reported in both serum and CSF of HAM/TSP patients ,32, we aRenilla luciferase (Ruc) fusion protein (61 kDa) was detected in nuclear protein extract of HTLV-1 infected cell lines (MT-2 and HUT102) and HBZ/pRen2 transfected 293T cells, respectively, using anti-HBZ (+) B cell culture supernatant. Rabbit anti-HBZ serum was used as a positive control and also reacted with the HBZ protein and HBZ-Ruc fusion protein similar to the anti-HBZ (+) B cell culture supernatant . It remains to be defined how HBZ-specific antibody inhibits CD8+ T cell proliferation in HAM/TSP patients. However, since humoral immunity is not limited to extracellular viral recognition but can neutralize a virus even within the cytosol of infected cells [+ T cell proliferation because this subset exhibits less spontaneous proliferation than CD8+ cells, the presence of antibody responses against HBZ was associated with less CD4+ T cell activation (frequency of CD25+ T cell subset) in HAM/TSP patients with anti-HBZ antibody responses. In CD8+ T cells, the frequency of CD25+ cells was also less in HAM/TSP patients with anti-HBZ antibody responses and approached statistical significance. These results demonstrated that HBZ-specific antibody responses have potentially beneficial roles in suppressing T cell activation in HAM/TSP patients. Since T cell activation is regulated through intricate molecular and immunological signaling networks, it will be of interest to determine how HBZ-specific antibody suppresses T cell activation of HAM/TSP patients.Lastly, to further confirm the presence and the function of HBZ-specific antibody, we generated memory B cells producing HBZ-specific antibody from HAM/TSP patients with antibody response against HBZ. Since antigen-specific human memory B cells circulate at very low frequencies in peripheral blood, many researchers have relied on expansion and conversion of memory B cells into antibody-secreting cells by in vitro culture or development of alternative strategies -52. In tpatients  and HBZ ed cells , HBZ-speIn summary, this is the first report demonstrating the presence of a humoral immune response to HBZ in the context of HTLV-1-infection. Characterization of the immune response against each of the HTLV-1 viral antigens will further improve our knowledge of virus-host interactions and the pathogenesis of HTLV-1-related disorders.The subjects for the present analysis were participants in research studies conducted at the National Institute of Neurological Disorders and Stroke (NINDS) at NIH, or the University of the West Indies (UWI), Kingston, Jamaica in collaboration with the National Cancer Institute (NCI), Bethesda, MD. Informed consent was written and obtained from each subject in accordance with the Declaration of Helsinki.NIH subjects: Serum samples were obtained from a total of 92 subjects, including 25 HTLV-1-seronegative donors (NDs), 12 ACs and 55 HAM/TSP patients. CSF samples were obtained from 5 HAM/TSP patients. The study was reviewed and approved by the National Institute of Neurological Disorders and Stroke Institutional Review Board.Jamaican subjects: All serum and plasma samples from study subjects were previously tested for HTLV by ELISA  or EIA ; seropositive samples were previously tested by Western blot . Serum/plasma samples were obtained from a total of 344 subjects, including 73 NDs, 133 HTLV-1 seropositive ACs, 89 ATL patients and 49 HAM/TSP patients. The NDs and ACs were selected from participants in a nested case–control study of risk factors for HTLV-1 seropositivity conducted among foodhandlers from Kingston and Clarendon parishes in 1987–1988 . SamplesRenilla luciferase expression vector, and generated as previously described [HTLV-1 cDNA clones for Gag, Env and Tax were inserted into pREN2, a mammalian escribed . A HTLV-escribed . The LIPescribed . Serum, HTLV-1 proviral DNA load was measured using Viia™ 7 Real-Time PCR system  as previously described . DNA wasTotal RNAs were extracted from PBMCs of HAM/TSP patients by RNeasy® Mini Kit (QIAGEN), according to the manufacture’s instruction. 85 ng of total RNA was used to be converted into cDNA and amplified in a one step reaction using TaqMan® RNA-to-Ct™ 1-Step Kit (Applied Biosystems) according to the manufacturer’s instructions. The sequences of primers and probe for HBZ mRNA detection were as follows: (forward) 5′-aga acg cga ctc aac cgg-3′, (reverse) 5′-tga cac agg caa gca tcg a-3′ and (probe) 5′-tgg atg gcg gcc tca ggg ct-3′. As the probe for HBZ surrounded the splice junction site of HBZ mRNA, this method detected only the spliced form of HBZ. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was detected as an endogenous control. The HTLV-1-infected cell line MT-2 was used as a calibrator sample and the level of HBZ mRNA expression was then calculated using the comparative CT method on ViiA™ 7 software (Applied Biosystems).+ memory B cells were isolated from PBMCs using IgG+ memory B cells isolation kit . The memory B cells were seeded at 50 cells per wells in 96 U-bottom microplates in complete medium containing 2 ng of ODN 2006  in the presence of EBV (30% supernatant of B95.8 cells) and irradiated allogeneic mononuclear cells . After culture for two weeks, the production of HTLV-1-specific antibodies was screened in the culture supernatants of immortalized memory B cells using LIPS assay. The culture supernatants of memory B cells producing anti-HBZ were collected and stored at −807°C until use. After desalting, HBZ-specific IgG was isolated using HiTrap protein G columns  and concentrated by Amicon® Ultra centrifugal filters .IgGThe production of HBZ-specific antibody from memory B cell culture was further determined by western blot. The nuclear proteins were extracted from HTLV-1-infected (MT-2 and HUT102) and uninfected cell lines (Jurkat and MOLT-3), 293T cells and HBZ/pRen2-transfected 293T cells using Nuclear extract kit . Protein concentration was determined using Quick Start Bradford Protein Assay . From each protein sample, 50 μg was electrophoresed through a NuPAGE® 4-12% Bis–Tris gel (Invitrogen). The gel was transferred to a nitrocellurose membrane (Invitrogen). After blocking with 3% BSA in TBS, the membrane was probed with B cell culture supernatant or rabbit anti-HBZ serum as positive control, and then probed with horseradish peroxidase-conjugated goat anti-human IgG . The membrane was visualized by chemiluminescence using SuperSignal® West Pico Chemiluminescent substrate  and analyzed the profile on Kodak digital science™ 1D image analysis software . The intensity of HBZ proteins detected by B cell culture supernatant or rabbit anti-HBZ serum was normalized by the intensity of β-actin.For analysis of peripheral blood lymphocyte populations, patients’ PBMCs were stained with CD3, CD4, CD8 and CD25 . Flow cytometric analysis was performed using a LSR II (BD Biosciences). The data were analyzed using FlowJo software .5 cells/well into 96 U-bottom microplates with 1 μg/ml of HBZ-specific IgG or human IgG as control. After culture for 6 days, the cells were stained with antibodies against CD3, CD4 and CD8 . The data were acquired on an LSRII flow cytometer (BD Biosciences).PBMCs were labeled with CFSE  according to the manufacturer’s instruction, and plated at 2×10+CD25+ and CD8+CD25+ T cells of NDs, HAM/TSP patients with and without antibody responses for HBZ. Still within Prism, the Chi-Square Test was used to compare the gender and racial distributions among the groups, numbers of anti-HBZ positive and anti-HBZ negative subjects between study groups and separately again to compare numbers of anti-HBZ positive and anti-HBZ negative patients between ATL subtypes. Again in Prism, Wilcoxson matched-pairs signed rank test was used to evaluate the inhibitory effects of HBZ-specific antibody on spontaneous lymphoproliferation in PBMCs of HAM/TSP patients. Lastly in Prism, Spearman’s correlation was used to evaluate both the association between anti-HBZ antibody levels and proviral DNA loads and the association between anti-HBZ antibody levels and HBZ mRNA expression. Using the statistical programming language “R” (http://www.r-project.org/), anti-HBZ data was log (base = 2) transformed then fit via the Four-Way Analysis of Variance (ANOVA) model with interactions; using race, gender, age and patient study group as the factors. Per race, patients were coded “Caucasian-descent”, “African-descent”, or “other”. Per gender, subjects were coded “male” or “female”. Per age, subjects were coded by quartiles as “young”, “young to middle-age”, “middle-age to senior”, or “senior”. Post-hoc testing was accomplished using the Tukey’s Honest Significant Difference method.Race, gender and age information for each patient were organized into matrix form with the anti-HBZ data generated for each patient. Using Prism (GraphPad software), the Mann–Whitney Test was used to compare: the age distributions among the groups, anti-HBZ antibody levels between patient study groups , anti-HBZ antibody levels between ATL subtypes , anti-Gag, anti-Env and anti-Tax antibody levels between anti-HBZ positive and anti-HBZ negative groups, proviral DNA loads and HBZ mRNA expression between anti-HBZ positive and anti-HBZ negative groups, and frequencies of CD4The authors declare no competing financial interests. The views expressed are those of the authors and not necessarily those of the US Department of Health and Human Services, the NIH, or the FDA. Dr. Maloney’s contributions derived from her former affiliation with the National Cancer Institute, and not her current affiliation with the Food and Drug Administration.YE-A performed most of the experimental work, statistical analysis and contributed to paper writing. AA performed the experimental work and contributed to paper writing. RM coordinated clinical work, analyzed gene expressions and contributed to discussion and paper writing. IB generated the HBZ/pRen2 plasmid and contributed to paper writing. KRJ performed statistical analysis and contributed to paper writing. PLG provided rabbit anti-HBZ sera and contributed to paper writing. EMM coordinated the identification of the subjects for this analysis and arranged for the selection of serum/plasma samples and contributed to discussion and paper writing. SJ supervised the project and contributed to discussion and writing. All authors read and approved the final manuscript.


However, HBZ is the only gene that remains intact and is consistently expressed in all ATL cases, while the tax gene is frequently inactivated by epigenetic modifications or deletion of the 5’LTR. HBZ gene promotes the proliferation of ATL cells through its mRNA form. Moreover, HBZ induces T-cell lymphoma and systemic inflammation in vivo. HBZ fulfills its functions mainly through regulating HTLV-1 5’LTR transcription and modulating a variety of cellular signaling pathways which are related with cell growth, immune response, and T-cell differentiation. Taken together, the multiple functions of HBZ render its predominant function in leukemogenesis of ATL.Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). The minus strand of HTLV-1 provirus encodes a bZIP protein donated as HTLV-1 bZIP factor (HBZ). Among the HTLV-1 regulatory and accessory genes, the  The infection of HTLV-1 causes adult T-cell leukemia (ATL) and chronic inflammatory diseases including HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis TSP; . HTLV-1  Figure . The chator HBZ; . HBZ wastor HBZ; . We fountor HBZ; , and nonprovirus . In addiTL cells . Thus, tHBZ gene have been revealed: a spliced form (sHBZ) and an unspliced form , which acts as an enhancer of viral transcription. Tax and CREB can activate transcription of the HBZ gene through 3′ LTR, meanwhile, Tax regulated antisense promoter activity is influenced by the integration site. However, Tax induced 3′ LTR activation is not as significant as it did on the transcription of the HTLV-1 plus strand , central domain (CD), and basic ZIP domain (bZIP) in the C-terminal  deletion of HTLV-1 5′ LTR, (2) hypemethylation of 5′ LTR, and (3) non-sense mutation, deletion, and insertion of tax gene  functions as a dimer comprised of bZIP proteins of Jun family , Fos family , and others. HBZ interacted with c-Jun/JunB and repressed c-Jun/JunB mediated signaling by abrogating its DNA-binding capacity. HBZ induced the degradation of c-Jun protein through a proteasome-dependent pathway, while it is in a ubiquitin-independent manner . In addiIn ATL, Tax-mediated nuclear factor-κB (NF-κB) activation plays an important role in the proliferation of ATL cells and in the transforming activity of HTLV-1 . ActivatRecent studies suggest that the classical NF-κB pathway is mostly involved in innate immunity and inflammatory responses. The inhibition of NF-κB pathway is common among different viruses, suggesting that these activities are important for viral survival and infection. HTLV-1 might facilitate escape from the host immune attack by suppressing the classical NF-κB pathway by HBZ.+CD25+ phenotype, have the same markers as those of natural regulatory T cells  and Listeria monocytogenes (LM) than non-Tg mice . Since PBM is important for the transforming activity, such differences between Tax1 and Tax2 may responsible for the different pathogenicities of HTLV-1 and HTLV-2 (Figure sHBZ gene, APH-2 mRNA is transcribed from the 3′ LTR, and spliced and polyadenylated. APH-2 does not contain bZIP domain but it still could interact with CREB and suppresses Tax2-dependent LTR activation. In addition, APH-2 did not colocalize with the nucleolus, although in non-T and T cells, it did have nuclear localization. Importantly, APH-2 was correlated with proviral load, while it does not promote cell proliferation and does not cause lymphocytosis.There are four kinds of HTLVs have been identified, and their oncogenic property is different. HTLV-1 mainly infected CD4d HTLV-2 . It was  Figure . Like thFigure  But, they retained the capacity to inhibit LTR activation that mediated by their respective Tax proteins. This study demonstrated a nuclear-restricted pattern for APH-4 while APH-3 was localized both in the cytoplasm and in the nucleus.Recent reports showed that HTLV-3 and HTLV-4 produce a spliced and polyadenylated antisense transcript . These tFigure in vivo.A naturally occurring antisense RNA was identified in the cell lines infected with HIV-1, and encodes an ASP with an apparent molecular mass of 19 kDa Figure . AntibodHBZ gene give new ideas into the mechanisms of oncogenesis. HBZ is the only viral gene that is expressed in all ATL cases.Except the HBZ gene, non-sense mutations of other viral genes are detected before integration of provirus. Importantly, HBZ maintains the cell survival when Tax is silenced. These data collectively indicate that HBZ protein has predominate function on the oncogenesis of HTLV-1. Further studies are needed to clarify the oncogenic function of HBZ and develop therapy targeted against HBZ based on the growing knowledge of HTLV-1 molecular biology.Intensive studies on the leukemogenesis of ATL since the discovery of HTLV-1 have revealed some molecular mechanisms by which viral proteins induce viral replication and cellular transformation. However, the precise mechanisms of oncogenesis by HTLV-1 remain to be clarified. Recent progresses on the researches of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human T-lymphotropic virus 1 (HTLV-1) was the first human retrovirus to be discovered and is the causative agent of adult T-cell leukemia/lymphoma (ATL) and the neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The importance of microRNA (miRNA) in the replicative cycle of several other viruses, as well as in the progression of associated pathologies, has been well established in the past decade. Moreover, involvement of miRNA alteration in the HTLV-1 life cycle, and in the progression of its related oncogenic and neurodegenerative diseases, has recently come to light. Several HTLV-1 derived proteins alter transcription factor functionalities, interact with chromatin remodelers, or manipulate components of the RNA interference (RNAi) machinery, thereby establishing various routes by which miRNA expression can be up- or down-regulated in the host cell. Furthermore, the mechanism of action through which dysregulation of host miRNAs affects HTLV-1 infected cells can vary substantially and include mRNA silencing via the RNA-induced silencing complex (RISC), transcriptional gene silencing, inhibition of RNAi components, and chromatin remodeling. These miRNA-induced changes can lead to increased cell survival, invasiveness, proliferation, and differentiation, as well as allow for viral latency. While many recent studies have successfully implicated miRNAs in the life cycle and pathogenesis of HTLV-1 infections, there are still significant outstanding questions to be addressed. Here we will review recent discoveries elucidating HTLV-1 mediated manipulation of host cell miRNA profiles and examine the impact on pathogenesis, as well as explore future lines of inquiry that could increase understanding in this field of study. Human T-lymphotropic virus 1 (HTLV-1) was discovered in the early 1980s by two-independent groups working in the United States   complex and be guided to complementary regions in the chromosomal DNA  of at least 45-bp in length  has been observed , histone methyltransferases (HMTs), and ATP-dependent chromatin remodelers, all of which are manipulated by viral proteins to ensure a favorable transcriptional state. The interaction of the HTLV-1 viral protein Tax with these cellular enzymes, as well as transcription factors, results in the activation of the viral promoter and the production of viral transcripts. Similarly, the viral manipulation of chromatin remodeling enzymes could also alter transcription of cellular genes including those encoding miRNAs. Additionally, due to the ability of Tax to manipulate chromatin structure and the innate host cellular defense mechanism of RNAi to regulate pathogen gene expression, there is most likely interplay between these two competing mechanisms. Here, we also introduce the reciprocal interaction of HTLV-1-induced epigenetic regulation and chromatin remodeling with the host cellular RNAi response.cis-acting replication element (CRE) enhancer sequences on the viral promoter, which is a critical step in viral transactivation and in the formation of the pre-initiation complex  by interacting with Tax-responsive elements (TREs) in the U3 region of the LTR. Instead of binding to DNA directly, Tax induces transcription of TREs, catalyzes post-translational modifications (PTMs) of TRE-binding factors, and forms complexes with transcription factors. Tax interacts with numerous important transcription factors and cellular kinases but of these CRE Binding Protein (CREB) is key to viral transcription . Conversely, histone methylation can also be an indicator of transcriptional activation, such as methylation of H3 at residue K4 (H3K4)  are powerful molecular motors that use energy derived from ATP hydrolysis to disrupt histone/DNA interactions, thereby exposing nucleosomal DNA  or a latent infection (insertion into a heterochromatic region). It has recently been shown that the RNAi machinery in coordination with endogenous miRNAs or exogenous siRNA can play a role in chromatin structure and reorganization, especially in the context of cancer and viral infections. As will be discussed in detail later, TGS can occur through RNA Pol II in an RNAi-dependent manner by recruitment of epigenetic modifiers  occurring through RNA complementary to gene promoters was first observed and examined in detail in plant species starting around 2000  experiments showed enrichment of H3K9me2 at the target promoter of the siRNA, as well as in proximal flanking DNA up to 300-bp down-stream of the target promoter. Furthermore, this boost in the H3K9me2 epigenetic marker was found to increase over a 24 h period post-transfection. Moreover, Ago1 was also enriched at the target promoter and flanking DNA but, unlike the H3K9me2 enrichment, the Ago1 enrichment was transient, peaking at 6 h post-transfection and then rapidly decreasing over the following 18 h. It was also found that the siRNA mediated knockdown of Ago1 disrupted both Ago1 and H3K9me2 enrichment at the target promoter following siRNA transfection indicating the role of Ago1 in recruiting siRNA and/or the necessary HMTs to the promoter. The involvement of Ago1, as well as Ago2, in mammalian TGS was also found to be required for the formation of the RITS complex in other TGS studies , and the tumor suppressor p53 , NF-κB, Cyclins D2 and D3, mitotic checkpoint regulators (MAD1), cyclin-dependent kinases (CDKs), the CDK inhibitors p16in vitro and in vivo in the absence of other viral factors and, thus, has become the focus of much research into HTLV-1-mediated oncogenesis , is also a target of miR-93  is up-regulated by the interaction of Tax with the anaphase-promoting complex/cyclosome (APC/C) consequently causing a phenomenon known as Tax-induced rapid cellular senescence (Tax-IRS). Tax-IRS causes newly infected cells to rapidly progress through the cell cycle and ultimately undergo premature apoptosis. However, HTLV-1 transformed cells experience cell cycle arrest despite continued expression of Tax (Kuo and Giam, One key finding was the up-regulation of miR-93 in HTLV-1 infected cells. A number of groups have reported increased expression of miR-93 in a variety of tumors, as well as in HTLV-1 infected cell lines and ATL patient samples, indicating that this miRNA plays a significant role in cellular transformation (Yanaihara et al., in vitro models and clinical samples from patients with varying carcinomas (Yao et al., in vitro and in vivo gastric carcinoma models increased cell adhesion and invasion, with increased metastasis to lymph nodes (Li et al., in vitro model cell lines to cells infected in highly complex in vivo systems. In order to determine the true mechanism of action with regards to miR-223 in HTLV-1 infected patients, more studies are required in the near future.Yet another host miRNA, miR-223, was shown to be up-regulated in HTLV-1 infected cells from ATL patients (Bellon et al., in vitro HTLV-1 infected cell lines and in vivo cells from ATL patients (Bellon et al., Additionally, up-regulation of miR-143-p3 has also been observed in both The host miRNA, miR-155, has also been identified as important to HTLV-1 mediated cellular transformation. Increased expression of miR-155 in transformed cells is not surprising as it has been observed in other oncogenic viruses, such as EBV (Metzler et al., The maximal level of NF-κB activation by Tax provides the beginning to what could prove to be another fascinating phenomenon in miRNA mediated oncogenesis as miR-146a and miR-21 are both regulated by the NF-κB pathway and are reportedly up-regulated in HTLV-1 infected cells. The up-regulation of miR-146a and miR-21 is likely important in the oncogenesis of HTLV-1 infected cells as these miRNAs have been shown to elicit anti-apoptotic functionality and are over-expressed in a number of human cancers (He et al., As described earlier, transcription of the HTLV-1 viral genome is largely carried out by the formation of the Tax/CREB/CBP/p300 complex which binds to the TRE-1. For Tax activated transcription to proceed, Tax must dimerize and recruit the CREB dimer, which interacts directly with the viral CRE in the TRE-1. Following the formation of the Tax/CREB complex, CBP/p300 is recruited in order to acetylate the histones of proximal nucleosomes and, thus, promote a chromatin remodeling event that results in an euchromatic state. This open chromatin structure provided by CBP/p300 is vital for transcription to proceed.Recent work by Rahman et al. has provided evidence that Tax down-regulated miRNAs that target p300 (Rahman et al., The studies reviewed here indicate that Tax-mediated regulation of miRNA expression is not simply a down-stream by-product of NF-κB or any other transcription factor activation by Tax. Rather it implicates Tax in a direct role in regulation of cellular miRNA. Even more compelling is the notion that this could provide an explanation for the low levels of Tax expression seen in older transformed cells as compared to the strong Tax expression observed in newly transformed cells. Further research into the Tax and miRNA interplay is necessary to fully elucidate the underlying molecular mechanisms; however, the data to date indicates that Tax is required to induce cellular transformation by initiating a cascade effect via miRNA dysregulation. The host cell miRNA dysregulation results in cellular immortalization and immune evasion, while still maintaining viral transcription. Furthermore, once cellular immortalization is established through the miRNA profile alteration, Tax is not required for transformation maintenance as it is difficult, if not impossible, for the cell to correct the imbalance Jeang, . The rolin vitro and ex vivo cultures of HTLV-1 infected cells (Moens et al., In addition to inducing oncogenesis, HTLV-1 up-regulated miRNAs may also contribute to the development of the other major clinical manifestation of this virus, HAM/TSP Table . This coAnother miRNA that is likely involved in the HAM/TSP pathogenesis is miR-132. Specifically, miR-132 was shown to be down-regulated in HTLV-1 infected cell lines, samples from ATL patients, and Tax transfected cell lines (Bellon et al., The manipulation of host miRNAs and RNAi machinery, as well as encoding of viral miRNAs by human pathogenic viruses has only recently come to light. Due to the diverse cellular pathways controlled by miRNAs, the hijacking of the RNAi molecular machinery and alteration of host miRNA profiles represents a significant and diverse mechanism by which viruses can progress through their life cycle and effect varied disease states in the host organism. Here, we examined recent findings that show the human retrovirus, HTLV-1, does indeed alter the miRNA profiles of infected cells which contributes to cellular transformation and leads to the associated ATL and HAM/TSP disease states. Furthermore, we show that alteration of chromatin by viral proteins and host cell miRNAs can contribute to the dysregulation host cell miRNA expression and likely serves as a key mechanism by which the virus manipulates host miRNA profiles. While recent discoveries validate the importance of HTLV-1 mediated variation in miRNA levels, there is still much to be elucidated in this novel field of study. Moreover, a better understanding of the molecular mechanisms by which this viral manipulation occurs will aid in the identification of potential points of therapeutic intervention that could help eradicate this pathogen from infected individuals.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thymic stromal lymphopoietin (TSLP) and eosinophils are prominent components of allergic inflammation. Therefore, we sought to determine whether TSLP could activate eosinophils, focusing on measuring the regulation of TSLPR expression on eosinophils and degranulation in response to TSLP, as well as other eosinophil activation responses.Eosinophil mRNA expression of TSLPR and IL-7Rα was examined by real-time quantitative PCR of human eosinophils treated with TNFα and IL-5 family cytokines, and TSLPR surface expression on eosinophils was analyzed by flow cytometry. Eosinophils were stimulated with TSLP (with and without pre-activation with TNFα and IL-3) and evaluated for release of eosinophil derived neurotoxin (EDN), phosphorylation of STAT5, and survival by trypan blue exclusion. A blocking antibody for TSLPR was used to confirm the specificity of TSLP mediated signaling on eosinophil degranulation.Eosinophil expression of cell surface TSLPR and TSLPR mRNA was upregulated by stimulation with TNFα and IL-3. TSLP stimulation resulted in release of EDN, phosphorylation of STAT5 as well as promotion of viability and survival. TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to TSLPR. Pre-activation of eosinophils with TNFα and IL-3 promoted eosinophil degranulation at lower concentrations of TSLP stimulation.This study demonstrates that eosinophils are activated by TSLP and that eosinophil degranulation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation, indicating that the eosinophil has the capacity to participate in TSLP-driven allergic responses. Thymic stromal lymphopoietin (TSLP) is a cytokine which plays a key role in allergic diseases such as asthma, atopic dermatitis, allergic rhinitis, nasal polyposis, and chronic allergic keratoconjunctivis -5. TSLP A role for TSLP in allergic diseases was initially attributed to its ability to promote TH2 differentiation through a dendritic cell-mediated pathway ,8. SubseAllergic diseases are typically characterized by eosinophilia, both in local tissues and peripheral circulation. In many cases, eosinophils and their granule-associated basic proteins  are associated with disease severity, yet specific mechanisms promoting eosinophil degranulation are not completely understood .Eosinophils have been shown to express IL-7Rα ,15. A rePeripheral blood was obtained from normal or allergic and/or asthmatic donors ranging in age from 18 to 55 years. Informed consent was obtained before participation and the study was approved by the University of Wisconsin Health Sciences Institutional Review Board, Protocol Number H 2008–0096.Eosinophils were isolated from heparinized blood using magnetic bead negative selection, as described previously . Briefly6/ml) were incubated with either media alone or TNFα and/or IL-3  for 24 h at 37°C. Total RNA was extracted from 1 × 106 eosinophils using the RNeasy mini kit  and reverse transcribed using 400 U of Super Script III reverse transcriptase  for 60 min at 37°C in the presence of random hexamer primers . Real-time quantitative PCR was performed in the Applied Biosystems 7500 sequence detector using human TSLPR-specific primers  or human IL-7Rα specific primers  and TaqMan probes (Applied Biosystems). Based on its similar transcription efficiency to the receptor target gene and its consistent expression among treatment groups, the reference gene, β-glucuronidase (GUS) was chosen to normalize the samples. The efficiency of the Taqman assay was determined by assaying serial 10-fold dilutions of target cDNA. All samples were amplified in duplicate, and the mean was obtained for further calculations. The data are expressed as fold inductions using the comparative cycle threshold (Ct) method in which ΔCt = Ct of the chemokine gene minus Ct of GUS; ΔΔCt = ΔCt of activated cells at the indicated time points minus ΔCt of untreated cells. In one subject, the IL-7Rα mRNA quantitative real time PCR did not meet our minimum cycle threshold value criterion and was excluded from our data analysis.Purified eosinophils (1 × 106/ml) were pre-activated with TNFα and IL-3 (10 ng/ml each) or media alone for 16 h at 37°C. CD3+ T cells were activated with anti-CD3/CD28 beads  for 72 h at 37°C. Prior to staining, cells were resuspended in HBSS-BAP  and non-specific binding of IgG was blocked with normal goat IgG, followed by neutralization of endogenous biotin with streptavidin . After staining with goat anti-TSLPR (R&D Systems), a biotinylated rabbit anti-goat IgG secondary antibody  was used and detected using streptavidin conjugated to phycoerythrin . Propidium iodide was added to each tube to determine viability. Detection of phospho-STAT5 was performed using intracellular flow cytometric analysis. Eosinophils were incubated with TSLP , IL-5 (1 ng/ml), or GM-CSF (1 ng/ml) for 15 min, fixed with 2% paraformaldehyde, permeabilized with 90% methanol, and stained with anti-phospho-STAT5-PE  or mouse IgG isotype control. Data was acquired using a FACSCalibur™ (BD Bio-sciences) based on gating of viable cells only  and was analyzed using WinMDI .Purified eosinophils (1 × 106/ml) were incubated with either TSLP (0.2–2.0 μg/ml), FMLP (10−7 M), IL-5 (1 ng/ml) or buffer on 96-well tissue culture plates in 200 μl of HBSS + 0.03% gelatin/well for 4 h at 37°C. In blocking experiments, eosinophils were pre-incubated with goat anti-TSLPR antibody  or goat IgG control prior to treatment as above. In the pre-activation assays, eosinophils were incubated overnight at 37°C with a combination of TNFα and IL-3  and washed prior to challenge with TSLP. Non-preactivated cells from the same donor were challenged separately for TSLP-stimulated EDN release as described above on the day of purification. Total EDN values were determined by lysis of eosinophils with 0.1% Triton X-100. EDN was measured by ELISA .Eosinophils (1 × 106/ml) were suspended in RPMI 1640 supplemented with 5% Fetal Bovine Serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 10 mM HEPES and incubated with either IL-5 (0.1 ng/ml), TSLP (0.0625–1 μg/ml) or media for 48 h at 37°C. Viability was evaluated using trypan blue exclusion and the cells were counted using a hemacytometer. Percent survival was determined by dividing the number of viable cells at a given incubation time by the original number of viable cells placed into the well at time zero. Percent viability was determined by dividing the number of viable cells at a given incubation time by the total number of cells  in the well at the given time [Purified eosinophils (1–1.5 × 10ven time .6/ml) were pre-activated with TNFα and IL-3  or media alone for 24 h at 37°C. Eosinophils were washed and resuspended in HBSS with 0.1% gelatin, incubated at 37°C with 1.2 mg/mL ferro-cytochrome C  and stimulated with either buffer, 1 μg/mL TSLP, 1μM FMLP or 1 ng/ml phorbol myristate acetate , as previously described [6 cells, calculated as previously described [Purified eosinophils (1 × 10escribed . Superoxescribed .6/ml) were pre-activated with TNFα and IL-3  or media alone for 24 h at 37°C. Eosinophils were washed and resuspended in HBSS with 0.1% gelatin and cultured in the upper compartment of 24-well 5.0 μm Transwell plates  as previously described [Purified eosinophils . A probability of less than 0.05 was considered statistically significant. Cytokine synergistic interaction was evaluated by comparing the calculated additivity  with the experimental additivity (response to simultaneous stimulation with TNFα and IL-3) by a paired −7 M) as positive controls. Stimulation of eosinophils with TSLP (1 μg/ml) significantly increased release of EDN relative to unstimulated eosinophils  and with IL-5 10 ng/ml) and FMLP  and enhanced survival at 125 ng/ml and above (p < 0.05). IL-5 stimulation (10 ng/mL) was used as a positive control .Stimulation with TSLP promoted both eosinophil viability and survival. As shown in Figure In eosinophils, STAT5 activation has been shown to enhance survival . In otheWe sought to determine whether upregulation of TSLPR might enhance activation and decrease the concentration of TSLP required. Expression of TSLPR mRNA was examined in both untreated and activated eosinophils. For activation of eosinophils, we focused on cytokines that are typically expressed in allergic inflammation including the proinflammatory cytokine, TNFα, and the IL-5 family cytokine, IL-3 . The results of the quantitative real-time PCR are shown in Figure Time course 3–48 h) and dose response (0.1–10 ng/ml) experiments were also conducted . CD3+ T cells were incubated for 72 h with anti-CD3/CD28 coated beads, previously shown to upregulate TSLPR on T cells, as a positive control . In FiguSince stimulation with TNFα and IL-3 upregulated TSLPR, we examined whether pre-activation would decrease the threshold for TSLP mediated degranulation. As TNFα and IL-3 activation of eosinophils also causes dose dependent EDN release (data not shown) we used the lowest concentrations of TNFα and IL-3 that resulted in upregulation of TSLP mRNA while minimizing background EDN release. Figure We have demonstrated for the first time that eosinophils respond directly to TSLP with degranulation (release of EDN) and activation of STAT5. Furthermore, we have shown that eosinophil expression of both TSLPR mRNA and surface protein can be markedly upregulated by a combination of TNFα and IL-3, cytokines present in allergic inflammation. More importantly, we went on to demonstrate that upregulation of eosinophil surface expression of TSLPR significantly enhanced sensitivity of eosinophils to TSLP-mediated degranulation. While we were also interested in whether TNFα and IL-3 pre-activation would enhance TSLP activation of eosinophil survival and STAT5 phosphorylation, we were unable to evaluate these functions as IL-3 stimulation of eosinophils promotes both, survival and STAT5 phosphorylation .Interestingly, while TNFα and IL-3 had a profound effect on upregulation of TSLPR mRNA, they did very little to modulate expression of mRNA for IL-7Rα. This suggests that, while both the TSLPR and IL-7Rα are required for high affinity binding and signaling via TSLP, TSLPR could be regulated independently from IL-7Rα resulting in expression of TSLPR that is not coupled with the heterodimeric complex required for signaling.in vivo microenvironment during allergic inflammation prime eosinophils to respond to lower concentrations of TSLP. This is supported by our observation that pre-activation of eosinophils with TNFα and IL-3 enhances both TSLPR expression and sensitivity to TSLP stimulation in vitro, suggesting that TSLP responses may be reserved for eosinophils in an inflammatory environment. Another possibility is that eosinophil responses to TSLP are biologically programmed to be more conserved and less promiscuous, reserved for responses to a bolus of TSLP released directly to tissue eosinophils in the activated epithelium.It is notable that the concentration of TSLP (0.5–1.0 μg/ml) required for activation of eosinophil degranulation (in the absence of pre-activation) is greater than the concentrations of GM-CSF and IL-5 (10 ng/ml) required to promote comparable degranulation responses. However, the activation of the IL-7 receptor complex on eosinophils (which shares IL-7Rα with the TSLPR) similarly required high concentrations of IL-7, 50 nM or 0.9 μg/ml . One posin vitro priming of blood eosinophils with IL-5 family cytokines can result in eosinophils with a similar phenotype to airway eosinophils [In asthma and allergic diseases, eosinophil recruitment, survival, and cytotoxic effector functions are largely regulated by IL-5 family cytokines . Besides direct effects on eosinophil activation, these cytokines enhance eosinophil responsiveness to second stimuli, such as chemokines and FMLP . The mecinophils . Our stuOur findings differ from a recently published study, using lower concentrations of TSLP (and ECP as a measure of degranulation) which reported that stimulation of eosinophils with TSLP was unable to promote eosinophil degranulation and phosphorylation of STAT5 . These dStudies using various genetically deficient mice have established the requirement for eosinophils in both local skin and systemic inflammatory responses to intradermally administered TSLP . These sOur studies demonstrate that TSLP, previously thought to preferentially target dendritic cell and T cell interactions, can also promote eosinophil activation and degranulation, and that eosinophil activation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation. It is difficult to predict the role of TSLP activation of eosinophils in allergic diseases, when so many other factors are present that could similarly promote eosinophil degranulation and activation. Activation of eosinophils via receptors for cytokines, immunoglobulins, and complement can lead to the secretion of an array of proinflammatory cytokines, growth factors, chemokines and lipid mediators. Rather than TSLP being singularly important in eosinophil activation, TSLP may be involved in altering eosinophil responses in the presence of other activating influences. However, it is clear from our data and other recent studies that the eosinophil has the capacity to participate in TSLP-driven allergic responses.CD, Cluster of differentiation; CRLF2, Cytokine receptor-like factor 2; Ct, Cycle threshold; EDN, Eosinophil derived neurotoxin; ELISA, Enzyme-linked immunosorbent assay; ERK, Extracellular-signal-regulated kinase; FMLP, N-Formylmethionyl-leucyl-phenylalanine; GM-CSF, Granulocyte-macrophage colony-stimulating factor; GUS, β-glucuronidase; HBSS, Hanks buffered salt solution; HBSS-BAP, Hanks buffered salt solution 1 g/L bovine serum albumin, 0.5 g/L sodium azide, and 18 mg/L phenylmethyl sulfonyl fluoride; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Ig, Immunoglobulin; IL, Interleukin; IL-7Rα, Interleukin 7 receptor alpha; Lyn, Member of the Src family of protein tyrosine kinases; MFI, Mean fluorescence intensity; mRNA, Messenger ribonucleic acid; PBMC, Peripheral blood mononuclear cells; PCR, Polymerase chain reaction; PE, Phycoerythrin; PMA, Phorbol myristate acetate; RPMI, Roswell park memorial institute medium; STAT, Signal transducers and activators of transcription; TNFα, Tumor necrosis factor alpha; TSLP, Thymic stromal lymphopoietin; TSLPR, Thymic stromal lymphopoietin receptor.The authors declare that there are no competing interests.EBC conceived of the study, participated in its design, carried out flow cytometry and eosinophil functional studies and analyzed results. JLS participated in the study design, coordination, carried out eosinophil functional studies, and formatted the final version of the manuscript and figures for submission. EAS, KEF carried out RNA and eosinophil functional studies, and analyzed results. SKM conceived of the study and participated in its design. All authors were involved in drafting or critically revising the manuscript and approved the final version.