Acute Respiratory Distress Syndrome (ARDS) is a common entity in critical care. ARDS is associated with many diagnoses, including trauma and sepsis, can lead to multiple organ failure and has high mortality. The present article is a narrative review of the literature on ARDS, including ARDS pathophysiology and therapeutic options currently being evaluated or in use in clinical practice. The literature review covers relevant publications until January 2011. Recent developments in the therapeutic approach to ARDS include refinements of mechanical ventilatory support with emphasis on protective lung ventilation using low tidal volumes, increased PEEP with use of recruitment maneuvers to promote reopening of collapsed lung alveoli, prone position as rescue therapy for severe hypoxemia, and high frequency ventilation. Supportive measures in the management of ARDS include attention to fluid balance, restrictive transfusion strategies, and minimization of sedatives and neuromuscular blocking agents. Inhaled bronchodilators such as inhaled nitric oxide and prostaglandins confer short term improvement without proven effect on survival, but are currently used in many centers. Use of corticosteroids is also important, and appropriate timely use may reduce mortality. Finally, extra corporeal oxygenation methods are very useful as rescue therapy in patients with intractable hypoxemia, even though a survival benefit has not, to this date been demonstrated. Despite intense ongoing research on the pathophysiology and treatment of ARDS, mortality remains high. Many pharmacologic and supportive strategies have shown promising results, but data from large randomized clinical trials are needed to fully evaluate the true effectiveness of these therapies.ARDS; Pathophysiology; Treatment In 1821, Laennec described a new syndrome characterized by pulmonary edema without heart failure [In 1994, the American-European Consensus Conference on ARDS established criteria for the diagnosis of ARDS. These criteria include acute onset, bilateral lung infiltrates, no evidence of elevated left atrial pressure and arterial oxygen tension to inspired oxygen fraction (PaO2/FiO2) less than 200. The diagnosis of ARDS requires all these features. However, as these clinical criteria do not always correlate well with diffuse alveolar damage, which is the typical pathologic ARDS feature, ARDS remains a syndrome associated with multiple diagnoses , rather Despite substantial progress in understanding ARDS pathophysiology, ARDS remains a major clinical problem, and mortality is still as high as 40 - 46%. The incidence of ARDS in the Intensive Care Unit (ICU) ranges between 4% to 9% depending on patient age and study population [Increased capillary permeability is the hallmark of ARDS. Damage of the capillary endothelium and alveolar epithelium in correlation to impaired fluid remove from the alveolar space result in accumulation of protein-rich fluid inside the alveoli, thereby producing diffuse alveolar damage, with release of pro-inflammatory cytokines, such as Tumor Necrosis Factor (TNF), IL-1 and IL-6 . NeutropInflammation due to neutrophil activation is key in the pathogenesis of ARDS. Fundamental transcription abnormalities, involving NF-kappa B that is required for transcription of genes for many pro-inflammatory mediators, are present in the lungs of ARDS patients . In addiComputed Tomography studies in the 1980s helped us understand the pathophysiologic alterations in the lungs of ARDS patients . In addiPulmonary hypertension (PH) is widely recognized as a characteristic feature of ARDS . PH etioThe initial phase of fluid accumulation is followed by a proliferation phase characterized by resolution of pulmonary edema, proliferation of type II alveolar cells, fibroblasts and myofibroblasts, and new matrix deposition. This phase starts early (within 72 h) in ARDS, and lasts for more than 7 days. Factors influencing the progression to fibro-proliferation vs. resolution and reconstitution of normal pulmonary parenchymal architecture are poorly understood , but patMultiple Organ Failure (MOF) is the leading cause of death in ARDS, but the pathophysiologic link between ARDS and MOF is not well defined . HoweverImproved understanding of ARDS pathophysiology and advances in technology have introduced new treatments and improved therapeutic strategies. The following paragraphs discuss recent developments in the therapeutic approach to ARDS.The concept of ”baby lung“ was introduced in 1980s by Gattinoni et al and generated interest in the use of low tidal volume ventilation as therapeutic strategy in ARDS. Several animal studies showed that ventilation with large tidal volumes and high inspiratory pressures resulted in development of hyaline membranes and inflammatory infiltrates in the lungs, and development of respiratory failure .In the late 1990s four randomized controlled trials (RCTs) evaluated the potential benefit of low tidal volume ventilation in ARDS . Althoug2O as an acceptable cut-off.Because of conflicting results from these studies, the National Heart Lung and Blood Institute ARDS Network conducted a multicenter RCT on 861 ARDS patients , compariLow tidal volume ventilation is generally well tolerated and it has not been associated with clinically important adverse outcomes, except for hypercapnic respiratory acidosis in some patients. In conclusion, hypercapnia and respiratory acidosis are expected consequences of low tidal volume ventilation. However, there is no evidence that hypercapnia is harmful in ARDS patients, and it may in fact confer some protection against ventilator-associated lung injury.PEEP is an essential component of mechanical ventilation for patients with ARDS, as it was early observed that PEEP greatly improves oxygenation in ARDS patients. High PEEP levels may open collapsed alveoli and decrease intrapulmonary shunt. Additionally, ventilation-induced alveolar injury is reduced by decreasing alveolar over-distention, because the volume of each subsequent tidal breath is shared by more open alveoli . On the Three RCTs have evaluated modest vs. high levels of PEEP in patients with ARDS. The NHLB ARDS Network conducted the ALVEOLI trial  . This stIn conclusion, based on published data, high levels of PEEP do not seem to confer any benefit with regards to mortality in ARDS. Because ARDS patients are a heterogeneous population, the apparent absence of benefit from high levels of PEEP could be due to the beneficial effects of high PEEP in some ARDS patients being offset by detrimental effects in other patients . HoweverA recruitment maneuver is a transient increase of trans-pulmonary pressure intended to promote reopening of collapsed alveoli . TechniqProne positioning has been used in ARDS for over 30 years. In 1976 Piehl et al. reported improved oxygenation in ARDS patients when they were turned to the prone position . Since tFour RCTs have investigated the effect of prone positioning on outcome. The first trial by the Prone-Supine Study group randomized 304 patients with a wide range of severity of acute lung injury .PatientsIn conclusion, existing data do not support routine use of the prone position in ARDS. However, because all published studies have shown improved oxygenation, prone positioning is an attractive rescue treatment for ARDS patients with severe hypoxemia, even though a survival benefit has never been demonstrated.The idea of high frequency ventilation (HFV) is to provide tidal volumes below that of anatomic dead space at high frequency (> 60 breaths per minute). Compared to conventional mechanical ventilation, mean airway pressure is higher . Two stuIn HFOV very small tidal volumes (1 - 4 ml/kg) are delivered at high frequency (3 - 15 Hz) by an oscillatory pump . HoweverEarly data indicate that increased fluid administration is correlated with worse outcome in ARDS, whereas negative fluid balance was associated with better outcome . The ARDAllogenic blood transfusion is associated with detrimental immuno-modulatory effects that may result in ARDS . ConsequSeveral studies have shown that newer ventilation strategies using low tidal volume and high levels of PEEP do not require high doses of sedation . FurtherAdministration of neuromuscular blocking agents (NMBA) in addition to sedation can result in improvement in severe hypoxemia, because paralysis improves patient-ventilator synchronism and reduces oxygen consumption . Muscle Two RCTs ,58 and aBecause diffuse pulmonary vasoconstriction is part of ARDS pathophysiology, selective vasodilatation of well ventilated areas seems an attractive method to improve gas exchange in ARDS patients.Inhaled nitric oxide (iNO) causes vasodilation of ventilated lung units and redistribution of pulmonary blood flow away from non-ventilated lung areas, without adverse systemic hemodynamic effects. Four RCTs evaluated the effects of iNO and showed transient improvement in oxygenation , but no Inhaled prostacycline is another selective pulmonary vasodilator. Importantly, liposomal PGE1 influences neutrophil function and decreases neutrophil accumulation and lung leak. Although inhaled prostacycline improves oxygenation, it has not been shown to reduce duration of mechanical ventilation or mortality in ARDS patients . DespiteVasoconstrictors can improve oxygenation in ARDS patients by decreasing intrapulmonary shunt. Phenylephrine  and almiThe interaction between nuclear factor-kappa B (NF-kB) and glucocorticoid receptor alpha (GRa) is a key mechanism regulating the progression of systemic and pulmonary inflammation in ARDS . The abiTiming of corticosteroid administration and duration of therapy may be important, and should be taken into consideration. A RCT conducted by the ARDS Network, randomized 180 patients with persistent (> 7 days) ARDS, to receive methylprednisolone (2 mg/kg/d) or placebo for 21 days, and showed improved oxygenation and more ventilator-free days in the methylprednisolone group, but no significant improvement in mortality . AnotherConflicting data exist concerning the correlation between corticosteroids and ICU neuromyopathy. A sub-analysis of study survivors did not show any association between randomization to corticosteroids and increased risk of neuromyopathy . In concSeveral other anti-inflammatory factors like Ibuprofen , ketoconThere are substantial evidences that b2-agonists may play a potential role in the treatment of patients with ARDS. B2 agonists have been found to have anti-inflammatory effects by direct influence on neutrophil function and by reducing the secretion of several pro-inflammatory cytokines. Additionally, b2-agonists can reduce the endothelial permeability and stimulate the fluid clearance from the lungs . In a smSeveral other pharmacological agents, including glutathione lisofylline , N-AcetyExtracorporeal membrane oxygenation (ECMO) has been studied since 1970s as a method for supporting gas exchange in patients failing conventional ventilation . A RCT cIn a more recent study 180 patients with severe ARDS were randomized to support by ECMO vs. conventional treatments. This study showed significantly improved survival  at 6 months in the ECMO group . AlthougExtracorporeal CO2 removal (ECCO2R) is an alternative device that uses veno-venous circuit for removal of CO2 at much lower extracorporeal flow rates compared to ECMO. A RCT conducted before the year 2000 used ECCO2R and showed no effect on mortality . In anotConflicting data exist about the evolution ARDS mortality over time. A meta-analysis by Phua et al did not find any mortality reduction in recent years , whereas


A 64-year-old male who had a macula-on superior bullous retinal detachment in OD underwent scleral buckling, 20-gauge-pars plana vitrectomy, internal drainage of subretinal fluid with perfluorocarbon fluid, 360° endolaser and perflourocarbon-fluid-air exchange surgery. Patient sat upright immediately after the surgery for the night. At the first postoperative morning although the retina was attached, there was a macular fold extending toward the temporal retinal periphery. Patient denied further surgery. During the follow-up, retinal fold gradually became less visible and it could be noticeable only by fundus autoflorescence imaging at the sixth postoperative year with a subtle epiretinal membrane formation on the optical coherence tomographic section. Retinal fold formation is a rare complication of retinal detachment (RD) surgery and has been described following intravitreal gas injection both with and without scleral buckling elements and with or without vitrectomy. It is thought to result from residual subretinal fluid being sequestered after the retinal break has been closed by intravitreal gas tamponade allowing subretinal fluid to accumulate in a gravity dependent position at the margin of attached and detached retina . It may We hereby report a case with macular fold that was developed following a successful scleral buckling surgery together with vitrectomy and fluid-air exchange for a bullous macula-on superior rhegmatogenous retinal detachment and describe its natural course over a six-year period.A 64-year-old otherwise healthy man presented with a visual loss in his right eye of three days duration. Slit-lamp examination and intraocular pressure were normal with a clear lens in both eyes. On funduscopy, there was a macula-on superior bullous RD associated with four adjacent tears located at the superior quadrant in OD. The patient underwent encircling with an additional silicone tire extending from 10 to 2 hours in association with 20-gauge-pars plana vitrectomy, internal drainage of subretinal fluid (SRF) using perfluorocarbon liquid, 360° endolaser photocoagulation, and perflourocarbon-fluid-air exchange. Patient sat upright immediately after the surgery for the night. At the first postoperative morning, the retina was attached and the 2/3 of vitreous cavity was full of air. However, there was a significant macular fold extending toward the temporal retinal periphery. The patient was advised to take facedown position for the next five days. Two weeks later, macular fold remained almost nearly unchanged Figures .Surgical option was discussed with the patient, and no further ocular surgery was carried out upon his denial. Visual acuity was 20/200 in his right eye. Nine months later, his visual acuity remained unchanged, and significant nuclear sclerosis was noted. However retinal fold looked less prominent .He subsequently underwent an uncomplicated phacoemulsification and intraocular lens implantation at the 9th month post vitreoretinal surgery. At the third postoperative year his visual acuity was 20/100 and the fold looked almost gone .At the sixth postoperative year, the fold could be noticeable only by fundus autoflorescence imaging with slight epiretinal membrane formation on the optical coherence tomographic section. However his visual acuity remained the same Figures .There are very few reports about the macular fold formation after retinal detachment surgery . One pubSeveral risk factors were characterized: use of an intraocular gas tamponade, recent onset of retinal detachment and superior bullous detachment, large and circumferential buckle, external drainage of SRF, incomplete internal drainage of SRF in particular when performing primary vitrectomy, retinal detachment running through the fovea, and slippage of the retina following a retinal detachment with a giant tear –5.Outcome varies among the cases. Complete regression of the retinal fold with full restoration of visual function, partial flattening with minimal or moderate improvement of visual function, or severe permanent structural damage are all possible consequences if the fold is left untreated , 5–8. InIn light of our case, we once again pointed out strongly the importance of immediate facedown position following any vitreoretinal surgery with air gas injection for rhegmatogenous retinal detachment to prevent macular fold formation in order not to endanger good visual outcome.


The prevalence of chronic heart failure is still increasing making it a major health issue in the 21st century. Tremendous evidence has emerged over the past decades that heart failure is associated with a wide array of mechanisms subsumed under the term “inflammation”. Based on the great success of immuno-suppressive treatments in auto-immunity and transplantation, clinical trials were launched targeting inflammatory mediators in patients with chronic heart failure. However, they widely lacked positive outcomes. The failure of the initial study program directed against tumor necrosis factor-α led to the search for alternative therapeutic targets involving a broader spectrum of mechanisms besides cytokines. We here provide an overview of the current knowledge on immune activation in chronic heart failure of different etiologies, summarize clinical studies in the field, address unresolved key questions, and highlight some promising novel therapeutic targets for clinical trials from a translational basic science and clinical perspective. Congestive heart failure (CHF) is a leading cause for both hospitalization and death in the western world. Its prevalence is rather increasing with the broad implementation of standardized evidence-based treatment algorithms for heart failure. The heart failure syndrome is characterized by impaired systolic and/or diastolic function and various clinical signs such as fatigue, dyspnea, fluid retention, and cachexia. Besides symptomatic treatment to relieve these symptoms several classes of drugs improve the prognosis of patients with chronic heart failure. These agents target the so-called neurohumoral activation process that comes along with the evolution of the heart failure syndrome irrespective of the underlying pathophysiology. Inhibition of the activity of the renin–angiotensin–aldosterone system and β-adrenergic signaling are well-established treatment principles which are incorporated in current guidelines . HoweverAn inflammatory activation in CHF patients has long been recognized. Indeed, immune mechanisms modulate interstitial fibrosis, cardiomyocyte apoptosis, and hypertrophy, all of which are central processes leading to maladaptive remodeling in response to a variety of stimuli Fig. . EspeciaFirst, systemic cytokines came into focus, which have been monitored in several clinical trials , 112. ThMeanwhile, basic science and translational clinical research gathered a lot of mechanistic information on new players in the field of inflammation which constitute very promising future targets for therapy. Therefore, a recent scientific statement from the European Society of Cardiology defined key questions and requirements for future studies in the enormously expanding field .The current review will provide the reader with an elected overview of experimental and clinical evidence for immune activation in chronic heart failure, summarize key clinical trials addressing the immune system, and provide an outlook for several new promising clinical targets.Different etiologies like coronary artery disease, hypertension, infection, etc., lead to heart failure. Therefore, one of the key questions in the field is whether activation of immunity in humans and animal models of heart failure is independent of the etiology or if there are disease specific mechanisms. As it turns out, both are the case.Different heart failure models indicate a role of innate immunity independent of heart failure etiology. Innate immunity is activated in the myocardium early by recognition of rather unspecific stimuli, summarized as danger-associated molecular patterns . This foIt is likely that inflammation is also initiated in human myocardium by innate recognition of pathogen-associated molecular pattern even well before the heart failure becomes symptomatic/diagnosed. However, clinical data to corroborate findings made in animal studies are widely limited to the demonstration of increased circulating levels of soluble mediators, mainly cytokines , in a vaFor heart failure with reduced ejection fraction due to different clinical etiologies there is good evidence from both experimental and clinical studies that pro-inflammatory cytokines are locally expressed in myocardium. For example, TNF-α has been mechanistically related to more or less all etiologies of systolic heart failure. Besides its direct effect on myocardial contractile function , 155, TNNevertheless, there are also distinct differences in the activation of the immune system in different heart failure etiologies:Heart failure due to large myocardial infarction is the most prevalent cause of systolic heart failure, so the majority of small animal studies for heart failure were conducted in the permanent myocardial infarction model. Besides early infiltration of leukocytes and local expression of cytokines and chemokines, there is ongoing inflammation that extends to the remote myocardium and is a part of the underlying pathophysiology of left ventricular remodeling. A detailed review of all the processes involved in myocardial remodeling is beyond the scope of the present article and was summarized recently . InflammMost animal studies are performed in relatively acute settings with follow-up of no longer than 8 weeks. Especially experiments with genetically altered mice often do not really allow distinguishing the effect on initial myocardial injury and wound healing, when the most pronounced inflammatory activation takes place, from chronic remodeling of the myocardium. Therefore, therapeutic approaches based on such animal experiments rather reflect the clinical situation weeks to months after a myocardial infarction when mechanisms, such as infarct expansion are still operative and do not easily allow transfer of the findings to the situation of chronic, non-ischemic, systolic heart failure.In contrast to the broad evidence from small animal studies, due to the limited availability of myocardial specimens clinical evidence for immune activation in chronic ischemic heart failure mainly comes from analysis of cytokines and cytokine receptors in peripheral blood  which fiThe threshold of >14 leukocytes/mm² is widely accepted for the definition of myocardial inflammation . EndomyoIt is further unclear whether, in analogy to mouse models of auto-immune myocarditis induced by vaccination against myocardial proteins, auto-immune myocarditis develops in patients without previous infection of the myocardium. Data from the TIMIC trial  demonstr+ T-cells, especially modulate collagen deposition in myocardium.Chronic pressure overload also leads to the expression of chemokines, cytokines, and infiltration of monocytes in the myocardium. Most evidence comes from animal models of transverse aortic constriction. There is a well-described pathophysiological link between inflammation and fibrosis , 83, wheAn expression of pro-inflammatory cytokines was also described in myocardium from patients with aortic stenosis . Here, tInflammation in heart failure with preserved ejection fraction without overt hypertrophy has been less well characterized. In patients with coronary artery disease and diabetes, a small study found an association between pro-inflammatory serum cytokines and echocardiographic evidence of diastolic dysfunction .In sum, clinical evidence for the implication of inflammation in the development of myocardial hypertrophy and diastolic dysfunction is sparse, although experimental studies suggest a role especially for lymphocytes and macrophages.More and more evidence is accumulating that obesity is associated with a systemic inflammatory response that is characterized by endothelial cell dysfunction, oxidative stress, and the activation of leukocytes . The infThe amount of epicardial fat was often correlated with the severity of hypertrophy and cardiac dysfunction as evidenced mainly by non-invasive imaging with its inherent limitations in obese humans. Especially the adipocytokine adiponectin, which is decreased in patients with obesity-linked diseases, prevents left ventricular hypertrophy. Studies in adiponectin deficient mice revealed increased afterload-induced hypertrophy due to impaired activation of the activated protein kinase AMP . HoweverThe best studied pro-inflammatory cytokine in the scenario of heart failure is TNF-α , 102. ClBesides its immediate effects on the contractile performance of the myocardium, there is also good experimental evidence for a causal pathophysiological role of TNF-α for the progression of myocardial remodeling leading to chronic heart failure. Relevant mechanism demonstrated in experimental studies are induction of cardiomyocyte hypertrophy , inductiBased on these extensive animal data indicating a causal link between TNF-α and cardiac dysfunction in both the acute and chronic setting, clinical trials were launched which tested compounds initially developed for binding of TNF-α in rheumatic diseases. The compound etanercept consists of two human TNF-α receptor fragments linked to the Fc portion of immunoglobulin G. In a phase I study, etanercept single administration was well tolerated and improved ejection fraction in chronic heart failure patients . EtanercInfliximab is a chimeric antibody binding soluble and membrane-bound TNF-α and prevents it from binding to its receptors. In a smaller phase II trial (ATTACH), NYHA III–IV patients treated with lower doses of infliximab demonstrated an increase in ejection fraction, but there was an increased risk of hospitalization and death in a high dose group .Besides dosing and timing issues, two main points have been worked out as possible explanations for the disillusioning results . First, The disappointing results of the anti-TNF-α studies in CHF patients dampened the enthusiasm in the field and supported a common belief that the redundancy of the immune system precludes success of specific cytokine-targeting strategies. Thus, although other pro-inflammatory cytokines were demonstrated to be involved in cardiac injury and significantly correlated with clinical status and prognosis of patients with chronic heart failure , especiaOne of the first randomized trials on immuno-modulation was conducted years before the event of the anti-TNF-α trials. Glucosteroids were tested in a number of small studies before the advent of current reperfusion therapy. A number of rather small studies produced controversial results when patients were treated in the setting of an acute myocardial infarction to limit infarct size and improve wound healing . Later oThalidomide was initially believed to act as an anti-TNF-α agent. In a small pilot uncontrolled trial, thalidomide rather increased plasma levels of TNF-α in patients with chronic heart failure, but improved left ventricular function . ThalidoStatins are well known for their “pleiotropic” effects besides cholesterol lowering. Immuno-modulatory and anti-inflammatory effects have been described which in part, inter alia, relate to attenuation of T-cell activation by repression of MHC-II expression . RosuvasPentoxifylline is a phosphodiesterase inhibitor which is approved for the treatment of peripheral vascular disease. The initial rationale for using pentoxifylline was due to its ability to inhibit TNF-α transcription. Yet, not all studies could link improvements in clinical outcome to a concordant effect on TNF-α modulation .In several clinical trials, pentoxifylline therapy was reported to reduce circulating levels of C-reactive protein, FASL, and TNF-α in heart failure patients. In those small heart failure trials, pentoxifylline also improved ejection fraction and functional outcome , 124. HoOxidative stress is believed to play a causative role in the evolution of the heart failure syndrome mainly on the basis of experimental studies. On the cellular level, reactive oxygen species can activate multiple signaling pathways, including MAP kinases and nuclear factor-κB . This inAnother small double-blinded controlled randomized trial aimed to determine whether vitamin E supplementation of patients with advanced heart failure (NYHA III/IV) would modify levels of oxidative stress and improve functional status and prognosis. However, supplementation with vitamin E did not result in any significant improvements .Despite the theoretical potential of adding anti-oxidants to standard heart failure medication, which might have been underestimated on the basis of these rather small trials, it has to be mentioned that standard heart failure medication already includes drugs with anti-oxidant properties, especially ACE inhibitors and angiotensin receptor blockers .On the other hand, the addition of a fixed dose of isosorbide dinitrate plus hydralazine to standard therapy for heart failure improved survival among black patients with advanced heart failure in the AHeFT trial , 136. ThAutologous blood exposed ex vivo to heat and oxidative stress induces anti-inflammatory effects in animal models . The preConsiderable epidemiological data found an association of omega-3 polyunsaturated fatty acid (ω-3 PUFA) intake, mainly from fish oils, and cardiovascular endpoints (reviewed in ). ReductDietary PUFA supplementation was studied by the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico-Heart Failure (GISSI-HF) study, a large-scale clinical trial in a chronic heart failure cohort. This trial showed a moderate, albeit significant benefit of ω-3 PUFA in addition to standard heart failure medication . The mecBased on these trials the clinical relevance of PUFAs in chronic heart failure patients is still not clear. Thus, PUFA supplementation received a class IIb, level of evidence B recommendation in the most current European heart failure guideline .There is a considerable proportion of patients with chronic heart failure, especially DCM, which have auto-antibodies . The higSeveral related studies have demonstrated that immuno-adsorption induces improvement of left ventricular function in patients with DCM , 126. ItA wide array of potential immuno-modulating effects has been attributed to intravenous immunoglobulins which demonstrated therapeutic efficacy in a variety of diseases with unrelated pathophysiology. However, clinical study results for treating heart failure are ambiguous. A small randomized trial in patients with symptomatic heart failure demonstrated a change in the balance between inflammatory and anti-inflammatory cytokines by immunoglobulin treatment. This effect was significantly correlated with an improvement in LVEF . AnotherThe underlying biological mechanism for the potential beneficial effects of immunoglobulins in DCM remains to be determined. A comprehensive overview of potential mechanism was recently provided by Gelfand . NeutralGiven the efficacy of immunoglobulins in attenuating virus replication in myocardium, it might thus be the case that the high prevalence of latent virus infection in the myocardium of DCM patients contributes to the positive effects of immunoglobulins in heterogeneous populations of DCM patients included in the above-mentioned trials.The field of therapeutic stem cell therapy has made great advance in the last years due to a number of clinical studies which are underway or just have been completed . Most anMost clinical trials were designed to improve repair/regeneration of the injured heart in the setting of myocardial infarction, while few studies yet addressed chronic heart failure patients.The TOPCARE-CHD trial tested intracoronary treatment with either bone marrow cells or peripheral progenitor cells in a crossover design in patients with chronic ischemic heart failure. Transplantation of bone marrow cells was associated with moderate but significant improvement in the left ventricular ejection fraction after 3 months  and evenAnother group reported similar promising results of intramyocardial delivery of bone marrow cells in chronic myocardial infarction and advanced chronic heart failure patients .It was speculated that progenitor cell therapy might be more promising in non-ischemic heart failure patients, as the functional capacity of endogenous progenitor cells is more severely impaired in patients with ischemic cardiomyopathy than in DCM patients . In the + stem cells in patients with DCM were reported. During the follow-up period, cell therapy was associated with a moderate but significant improvement in cardiac function (mean increase of EF 5.7 %), coming along with an improved exercise capacity and even decreased mortality [Recently, 5-year follow-up data of a well-designed, albeit not placebo-controlled and not double-blinded, randomized study investigating the long-term effects of intracoronary administration of G-CSF-mobilized CD34ortality . InteresHowever, none of the trials in chronic heart failure patients deeply assessed parameters of immune activation. Therefore, the contribution of putative immuno-modulatory effects of transferred cell preparations cannot be determined yet.There is a great variety of potential therapeutic targets in the field of pro-inflammatory signaling in heart failure. Perhaps, most experimental evidence has accumulated in experimental models of post-myocardial infarction heart failure concerning toll-like receptor and intracellular NFκB signaling .Besides numerous signaling molecules like cytokines, chemokines, and their related receptors which have meanwhile been identified and targeted in animal studies two novel targets should be highlighted here.Complement is activated during acute and chronic cardiac injury in different animal models. Michael Carroll’s group demonstrated complement activation by ischemia/reperfusion in different organs by a single IgM clone that recognizes a neo-epitope on hypoxic cells , 159. ThGlycoprotein-130 (gp130) is the common receptor of IL-6, which is elevated in patients with chronic heart failure . CardiomMBL and IgMs are independent trigger mechanisms for initiating the complement cascade. Both pathways converge on the activation of complement component C3 into cleaved C3a. Mice deficient for either C3 or the membrane receptor for C3a (C3aR) is protected against chronic left ventricular remodeling after myocardial infarction (own unpublished results).Accordingly, a prospective study in a chronic heart failure cohort comprising 182 patients demonstrated that complement activation is strongly linked to the outcome in chronic heart failure. Especially, high levels of activated C3, C3a, predicted cardiovascular events such as hospitalization and mortality . HoweverTherefore, there is a considerable experimental evidence that at least after ischemic myocardial injury, there is an ongoing complement activity in myocardium. Interventions inhibiting complement activation, especially the central component C3/C3a may represent a novel target for prevention and treatment of chronic heart failure after large myocardial infarction.+ regulatory T-cells came especially into focus.Most immuno-mechanisms studied in chronic heart failure till date address innate immunity, whereas adaptive immune mechanisms have gained much less interest so far. Recently, CD4+ T-cells comprise a subset of thymus-derived T-cells which are characterized by intracellular expression of the transcription factor Foxp3 [+CD25+CD127lowCD3+ T-cells. Regulatory T-cells are well known for preventing autoreactive immune responses which are associated with chronic myocardial diseases, such as auto-antibody formation [Regulatory CD4or Foxp3 . By meanormation . As disc+ T-cells might mediate part of the clinical effect of immuno-modulating therapeutic principles, which was already tested in clinical studies: intravenous immunoglobulins beneficially modulate several unrelated auto-immune diseases. As discussed above, clinical trials indicate that they might also have beneficial effect in inflammatory cardiomyopathies. It was speculated that sustained peripheral expansion of antigen-specific CD4+CD25+Foxp3+ regulatory T-cells might contribute to their therapeutic effect [Regulatory CD4c effect .+CD25+CD127low), and a decrease of activated T cells (CD4+CD69+ and CD8+CD69+ cells and CD28+ T cells) [Besides possible effects by neutralizing autoreactive antibodies, a recent study indicated that immuno-adsorption also modifies T cell-mediated immune reactions. Immuno-adsorption in patients with DCM was associated with a significant increase of regulatory T cells (CD4T cells) . The freT cells) .+CD25+ Foxp3+CD127low cells were found in peripheral blood of patients with chronic heart failure [+CD25+Foxp3+ T-cells in heart draining lymph nodes [+CD25+ T-cells was further reported to attenuate myocardial remodeling after experimental myocardial infarction in mice [+CD25+ cells ameliorates cardiac hypertrophy and fibrosis in response to angiotensin II [Furthermore, reduced numbers and impaired suppressive function of CD4 failure . There aph nodes . Our dat in mice , 132. Anensin II . Of noteTherefore, therapeutic expansion and activation of regulatory T-cells might tackle several immunological mechanisms contributing to the heart failure syndrome: mitigation of the expression of pro-inflammatory cytokines, suppression of auto-antibody production, and modification of recruitment, and differentiation of monocytic heart infiltrating cells contributing to both tissue repair and remodeling (own unpublished data).Besides transfer of autologous, ex vivo enriched regulatory T-cells, therapeutic expansion/activation in vivo might be more promising , 114. Mureg production by promoting acetylation of the Foxp3 transcription factor, which is a key regulator of Treg differentiation [Histone deacetylases (HDACs) catalyze the removal of acetyl groups from a variety of proteins. HDACs have been studied mainly in the context of chromatin, where they contribute to epigenetic regulation of gene expression by deacetylating nucleosomal histones. HDAC inhibitors stimulate Tntiation . On the ntiation . The mecMost completed trials on immuno-modulatory treatments were rather small including less than 100 patients/group. Data from randomized, placebo-controlled studies including more than 1,000 patients exist for TNF-α antagonists , 96. TheWhy did so many clinical trials, which were all launched on the basis of promising experimental data, show no additional benefit to current standard heart failure therapy?+Foxp3+ T-cells which have the ability to dampen pathological inflammation and auto-immune by a variety of mechanisms [As one hypothetical explanation, it must be mentioned here that standard drugs addressing neuro-humoral activation, which are obligatory co-medication in current heart failure trials also might have relevant effects on inflammatory activation. Especially ACE inhibitors have immuno-modulatory properties, as components of the renin–angiotensin–aldosterone system are expressed on leukocytes. Especially, the angiotensin–aldosterone system in T-cells came recently into the focus. T-cells contain an endogenous renin–angiotensin system that modulates pro-inflammatory T-cell function . Blockinchanisms . Accordichanisms . It remaOne might ask whether there is still a justifiable rationale for conduction of future clinical trials targeting inflammation as adjunct to our current pharmacological heart failure therapy. In our opinion, there is good experimental evidence that inflammation causally contributes to myocardial dysfunction and, thus, still constitutes a reasonable and promising therapeutic target, but some questions have to be carefully addressed before designing a clinical trial.First, which patients are best candidates for immuno-modulating treatment modalities? The hope to find a therapeutic principle that fits in all clinical scenarios of heart failure seems to be more and more unrealistic. It might be more promising in the future to individually characterize the immunologic status of a patient to decide whether he might benefit from the targeted immuno-modulation at the present time. Especially in patients with idiopathic DCM, this will have to include endomyocardial biopsies to gain more insights to define the present status of intramyocardial inflammatory activity and, especially, to exclude ongoing viral activity. Infectious  cardiomyopathy might become accessible to a completely different treatment in the future. Immuno-adsorption in patients with auto-antibodies, which have been evaluated for their cardio-specificity and toxicity, exemplifies such a first individualized approach . ComplemMonitoring of systemic, i.e., serum parameters of inflammation, especially cytokines, does likely not reflect myocardial inflammation, but is rather a marker of the overall clinical status. Therefore, one cannot conclude from the currently available serum parameters, which are often the only available biomaterial to monitor immunity in clinical studies, on the local effect on myocardial inflammation. Thus, at the moment, the real biological effect of immuno-modulating therapies, putatively beneficially influencing myocardial inflammation, cannot readily become assessed in clinical studies.As a different approach, as opposed to CHF patients, the wound healing phase after an acute myocardial infarction, which is the most important cause for heart failure in the Western word, might constitute another attractive phase for preventive immuno-modulation . ExperimSecond, how can we better identify immuno-modulating treatment principles tested in animal models to be suited for further translational clinical evaluation? Here, pre-clinical testing of therapeutic modalities warrants more attention. Several treatment modalities went into clinical trials, mainly on the basis of promising effects in rodent models, indicating that immuno-modulation, as evidenced e.g., by cytokine expression profiling or genetic lack/gain of function studies, might be causative. Here, we have to pay more attention on the transferability of immunological phenomena from small animal models to man. This does not only relate to the estimation of the putative therapeutic impact, but also to potential side effects. In addition, before going into man for phase I/II trials, large-animal studies have to be advocated. However, even a very good understanding of the biological mechanism of an immuno-modulating therapeutic tested in different species does not prevent from unexpected effects in man which can, at best lead to lack of efficacy, but also to deleterious side effects .Also essential, but widely underestimated, factors like age , co-mediTherefore, we pledge for even greater effort spent on translational cardio-immunological research which goes beyond phenomenological description of beneficial effects on myocardial function and remodeling and its association with parameters of inflammation. Given the inherent limitation to monitor the systemic and, especially local immunological processes within the myocardium in patients, a deep understanding of the biological effects of a therapeutic principle in different experimental models is vital before starting clinical studies.In addition, in this context, as many therapeutically principles have only been tested in the chronic myocardial infarction heart failure mouse model, other models of chronic heart failure, especially models of genetic and inflammatory cardiomyopathies, should receive more interest in the future.Summing up the current evidence from both experimental and clinical studies discussed here, we would like to conclude that inflammation is a promising therapeutic target in the setting of CHF. Current state of evidence from clinical trials indicates that unspecific immuno-modulation, e.g., by steroids or immuno-adsorption, holds some promise especially in the “orphan” field of idiopathic/inflammatory, non-viral DCM (see Table


Motor weakness finally disappeared 6 months after surgery. Moyamoya syndrome is a rare complication of both SLE and hypothyroidism, and the surgical indication remains controversial. By evaluating the decreased cerebral perfusion reserve capacity and the existence of leptomeningeal anastomosis, EDAS could be an efficient method for the treatment of moyamoya syndrome associated with SLE and hypothyroidism.A 54-year-old woman presented to our hospital with progressive motor weakness of the right arm. She had a medical history of systemic lupus erythematosus (SLE) and hypothyroidism. Magnetic resonance imaging indicated a watershed infarction of the left hemisphere. Cervical echogram indicated severe stenosis of the internal carotid artery (ICA) without wall thickening. Cerebral angiography indicated left ICA occlusion, development of unilateral moyamoya vessels, and leptomeningeal anastomosis. Encephaloduroarteriosynangiosis (EDAS) was performed after cerebral  Moyamoya-like angiopathy  is a rare intracranial complication of systemic lupus erythematosus (SLE) and hypothyroidism. However, the treatment strategy for moyamoya syndrome complicated with such chronic systemic vascular disease remains controversial. In this paper, we present a case of moyamoya syndrome associated with SLE and hypothyroidism with onset of cerebral ischemia, which was effectively treated by encephaloduroarteriosynangiosis (EDAS). A 54-year-old woman, who had undergone treatment for SLE and hypothyroidism for 20 years by continuous steroid administration (prednisolone 5 mg/day), experienced repeating cataplexy of the right arm for 6 months and visited neurologists at our hospital. She was diagnosed with occlusion of the left internal carotid arteries (ICA) and cerebral ischemia of the left watershed area between the frontal and temporal lobe, and treatment with antiplatelets  was initiated. The symptoms transiently improved, but recurred soon after. An increased dose of antiplatelets was administered , but no improvement in her symptoms was observed. Subsequently, she was admitted to the department of Neurosurgery for intracranial revascularization surgery.99mTechnetium-ethyl-cysteinate-dimer single-photon emission computed tomography (99mTc-ECD SPECT) imaging of the cerebral blood flow (CBF) using three-dimensional stereotactic region of interest template highlighted a reduced level of CBF in the left hemisphere under acetazolamide loading, compared with the right hemisphere (Preoperative head magnetic resonance imaging indicated cerebral infarction of the frontoparietal watershed area Figures . Carotidmisphere .On the basis of these results, surgical intervention for cerebral ischemia was considered necessary, due to moyamoya syndrome and left ICA occlusion. EDAS—a method of revascularization surgery—is an indirect procedure and was chosen because this case was complicated by the systemic progressive vascular disease that was treated with steroids, and the preoperative status of the left middle cerebral artery, which was not visualized by DSA, was not known.The postoperative course was uneventful, and the cataplexy of the right upper limb had nearly disappeared. A postoperative DSA study indicated marked neovascularization from the graft Figures . Postope Intracranial moyamoya-like vessels complicated with systemic vascular disease or immunological disorders, such as in this case, are diagnosed as “moyamoya syndrome,” rather than true moyamoya disease according to the precise criteria, and the exact pathology remains unknown , 2. Nato The surgical strategy against moyamoya syndrome complicated with systemic vasculopathy is still controversial, particularly for adult cases. Kaga et al. reported a case of adult moyamoya syndrome with reduced cerebrovascular reserve capacity, which was effectively treated by an indirect method . MoreoveBy evaluating preoperative cerebrovascular reserve capacity and the development of leptomeningeal anastomosis, the case of moyamoya syndrome associated with progressive vasculopathy would be treated effectively by indirect revascularization surgery, including EDAS, despite the earlier continuous steroid treatment.


Autogenous tooth transplantation (ATT) can be considered when there is a hopeless molar tooth and suitable donor present. This report presents an unconventional case of successful ATT of a third molar replacing the adjacent fractured second molar in a 33 year old woman. This wisdom tooth had completely developed roots. Root-end filling with Calcium Enriched Mixture (CEM) cement was performed in the third molar. The second molar was extracted non-traumatically without any bone removal; the wisdom tooth was immediately transplanted into the recipient socket. No endodontic treatment was carried out either during or after the ATT. At six-month and 2-year clinical examination the patient was asymptomatic; the transplanted tooth was still functional, with no evidence of marginal periodontal pathosis. At the same follow ups, radiographic evaluation illustrated bone regeneration, normal PDL, and absence of external root resorption. Transplantation of mature third molar seems to be a promising method for replacing a lost permanent molar tooth and restoring aesthetics and function.  Dental autotransplantation or autogenous tooth transplantation (ATT) was first reported in 1951 . The reci.e. untreatable large decay, severe periodontal disease, failed root canal treatment and root fractures/perforations  named calcium enriched mixture (CEM) cement has been developed as root-end filling material . Althougstandard -15, and d humans -18 revead humans . This ded humans  and doesd humans . AdditioThe aim of this case report is to demonstrate a successful replacement of an untreatable second mandibular molar with vertical root fractured with its adjacent completely-developed third mandibular molar.A 33-year-old Caucasian woman was referred to an endodontic practice for extraction of her mandibular left second molar. The medical history was non-contributory. Clinical examination revealed good oral hygiene but the second molar had vertical fracture. Periodontal probing depths did not exceed 3 mm, however, the involved tooth was tender to percussion and palpation. The radiographic examination showed previous endodontic treatment with poor instrumentation and unsatisfactory obturation. A large extended periapical lesion was observed. The patient was informed about the fractured tooth; and extraction was recommended to her.The examinations also showed that adjacent third molar was healthy, favourably developed, completely erupted and well positioned within the dental arch, and therefore it was an appropriate candidate for ATT . The treAt the treatment session, oral disinfection was carried out with 0.2% chlorhexidine gluconate solution. Surgery was performed under local anaesthesia with a mandibular block . First, the fractured second molar and then the wisdom tooth were extracted. The third molar was then positioned into the recipient socket to assess its adaptation. As a result of the small size of single donor root, adequate adaptation was achieved with ease.Root-end resection was made by removing 3mm of the tooth root apex. Three millimetres deep class I root-end preparations were made using an ultrasonic power unit  with ultrasonic retrotips  and irrigated with sterile normal saline solution. The root-end cavity were dried with absorbent paper points and filled with CEM cement. The tooth was rinsed in sterile saline to remove all debris and was then replaced in recipient socket with favourable distance (1 mm) to the adjacent teeth and no interference with opposing teeth . The traThe patient was recalled for clinical and radiographic follow-ups after 1 day (post-operative check-up), 1 week , 1 month , 6 and 24 months . The postoperative period was uneventful. At the 2-year follow up visit the tooth had normal occlusion, with physiological mobility and masticatory function. Periodontal probing revealed no pockets or other pathological signs, and the patient was symptom-free. The periodontal ligament appeared intact; periradicular area had normal status and no evidence of root resorption or periapical lesion was observed.Teeth with vertical root fractured and poor prognosis are classified as a genuine indication for ATT . In the It is obvious that ATT requires size compatibility between the transplanted tooth and recipient socket. In the present case, the third molar was single-rooted which was smaller than recipient site. Thus, extraoral modification of the donor root was not required and the transplanted tooth was simply inserted, well positioned and easily stabilized in the new site. It was reported that close contact of the transplanted tooth with the alveolar bone of the recipient site may provide better blood supply for the PDL cells . AlthougThe critical factor for inflammatory root resorption after ATT is the infection of root-canal system. ATT of closed-apices teeth, therefore, necessitates pulp extirpation within 1 to 2 weeks to avoid pulpal infection followed by periradicular inflammation and subsequent inflammatory root resorption . This seThe favourable results obtained in this rare case of ATT may be attributed to distinct factors, such as maintaining asepsis during surgical procedure, atraumatic surgical extraction and replacement, preservation of the PDL cellular vitality, minimal extraoral time, good occlusal interrelationship, adequate fixation as well as filling and sealing the apex with a biocompatible root-end filling material.ATT of third molars with closed apices may be considered as a viable treatment option compared to the usual prosthetic and implant treatments for both restorative and financial reasons.


The emergence of HBsAg mutants presents a challenge to HBV screening programs. Hepatitis B viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. Mutations that occur within the immunodominant epitopes of hepatitis B surface antigen (HBsAg) allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus is reduced to undetectable levels. HBsAg mutants present as false-negative results in some immunoassays. An understanding of immunoassay reactivity with HBsAg mutants is key to establishing an appropriate testing algorithm for hepatitis B virus detection programs. Over the past decade, the importance of hepatitis B virus (HBV) mutants has made a transition from an academic phenomenon of unknown prevalence to a factor for consideration during disease diagnosis. HBV infection has a major effect on world health care: more than one third of the world's population has been infected at some point; ≈350 million people are currently infected . These ORFs encode the polymerase protein (Pol gene); core antigen and e antigen (C gene); large, medium, and small surface-antigen proteins (S gene); and the X protein (X gene). From a relatively small genome, these overlapping ORFs generate 7 proteins. While this gene overlap may constrain some viral variability, mutant or variant forms have been identified for all 4 genes , in active infection, 107 base-pairing errors can be generated per day over the 3,200-bp genome  and nucleoside analogs  to inhibit polymerase activity. These treatment options can suppress wild-type HBV to undetectable levels, allowing a mutant HBV strain to emerge as the predominant form. Emergence of a mutant species can be monitored by using such techniques as real-time polymerase chain reaction (PCR) assays, restriction length polymorphism assays, quantitative fragment analysis, and primer extension assays. These powerful techniques can detect trace mutant sequences in clinical samples with a preponderance of wild-type virus, while conventional DNA sequencing cannot  protein, which is composed of 226 amino acids (aa). sHBsAg is the major structural protein of the hepatitis B viral envelope. Most HBsAg in the plasma of HBV-infected persons consists of 22-nm spherical particles composed of ≈100 HBsAg monomers each . Of greater interest are the secondary compensatory changes emerging in polymerase mutants (These studies of recombinant surface antigen mutants underscore the usefulness of mapping the epitope susceptibility of various commercially available HBsAg assays. While testing of mutant panels is voluntary in some countries, certain regulatory agencies are becoming increasingly aware of HBsAg mutants. In the United States, manufacturers of new HBsAg assays must address mutant detection in their package inserts. With a firm understanding of immunoassay mutant detection, the diagnostician can select the appropriate HBsAg screening algorithm to minimize the impact of mutants in sentinel screening programs.


Therefore, computational prediction of target sites is usually restricted to these gene regions. Recent experimental studies though have suggested that microRNAs may alternatively modulate gene expression by interacting with promoters. A database of potential microRNA target sites in promoters would stimulate research in this field leading to more understanding of complex microRNA regulatory mechanism.We developed a database hosting predicted microRNA target sites located within human promoter sequences and their associated genomic features, called microPIR (microRNA-Promoter Interaction Resource). microRNA seed sequences were used to identify perfect complementary matching sequences in the human promoters and the potential target sites were predicted using the RNAhybrid program. >15 million target sites were identified which are located within 5000 bp upstream of all human genes, on both sense and antisense strands. The experimentally confirmed argonaute (AGO) binding sites and EST expression data including the sequence conservation across vertebrate species of each predicted target are presented for researchers to appraise the quality of predicted target sites. The microPIR database integrates various annotated genomic sequence databases, e.g. repetitive elements, transcription factor binding sites, CpG islands, and SNPs, offering users the facility to extensively explore relationships among target sites and other genomic features. Furthermore, functional information of target genes including gene ontologies, KEGG pathways, and OMIM associations are provided. The built-in genome browser of microPIR provides a comprehensive view of multidimensional genomic data. Finally, microPIR incorporates a PCR primer design module to facilitate experimental validation.http://www4a.biotec.or.th/micropir.The proposed microPIR database is a useful integrated resource of microRNA-promoter target interactions for experimental microRNA researchers and computational biologists to study the microRNA regulation through gene promoter. The database can be freely accessed from:  MicroRNAs (miRNAs) are small ∼22 nt regulatory RNA molecules that are involved in post-transcriptional regulation of gene expression through inhibition of translation initiation or targeting messenger RNAs for degradation Generally, the function of a miRNA is defined by its target genes and its effect on gene expression; thus, identification of potential miRNA targets is of highest importance. Since many potential miRNA regulated genes exist in a genome, accurate bioinformatic prediction is an essential tool for identifying targets. In animals, it is well known that miRNAs control gene expression through recognition of target sequences in the 3′-UTRs of mRNAs CDH1 and CSDC2 containing complementary promoter sequences. Kim et al. cis-regulatory role of miR-320 in targeting its own genomic location, which resulted in transcriptional silencing of an adjacent downstream gene, POLR3D through an AGO1-dependent mechanism. The recent report by Younger and Corey trans. They showed that miRNAs with incomplete complementarity to their targets require AGO2 instead of AGO1, suggesting the possibility of different silencing mechanisms for different miRNA/target interactions. These studies indicated that the control of gene transcription by promoter targeting may be a general mechanism of miRNA gene regulation. Through promoter targeting, miRNAs may positively regulate gene expression Recent experimental studies have suggested an alternative miRNA mechanism for modulating gene expression by targeting outside gene bodies through promoter recognition in human cells. Place et al. So far, only a few miRNA-promoter interactions have been verified, possibly because there are few good candidate targets. A public resource providing genome-wide computationally identified potential miRNA binding sites on gene promoters could assist identification of biologically interesting candidates for validation We developed the microPIR (microRNA-Promoter Interaction Resource) database to facililate comprehensive exploration of putative miRNA binding sites within gene promoters of the human genome. In contrast to most currently available miRNA target databases, the predicted target data are managed in a way that enables the interactive and flexible exploration of putative binding sites that are most suitable with users' hypotheses, especially in the global view along with other associated genomic features and functions. The experimentally verified binding sites of AGO proteins, which have been reported to be involved in the mechanism of promoter-targeting miRNAs microPIR presents miRNA target sites on promoter regions through a web interface with supporting features not only searching and retrieving data but also the visualizations of corresponding target sites. The web interface comprises three modules: 1) query, 2) genome browser, and 3) database statistics .p-value, number of unpaired nucleotides, G-U base pairs in seed region, internal loop size, and bulge size). The target output (s) can also be filtered based on the existence of supporting experimental information, AGO binding sites and/or EST, on predicted targets. The presence/absence of other annotated genomic sequences  in association with a target site can also be used as extra screening criteria as well.Two search options, basic and advanced, are provided for users to access target site information associated with the specified target gene or miRNA. The basic search supports inputs with the following types of identifiers: HUGO gene name, NCBI gene ID, miRNA accession and miRNA ID. Users can locate target sites within a certain gene by supplying a gene ID/symbol to the search form. Alternatively, a miRNA accession number/ID can be entered in order to view the list of target genes of this miRNA. This search option displays all target sites that pass the default parameter settings (see on the basic search page of web interface). The target site parameters can be customized to be more stringent with the advanced search option, which is designed to support diverse query types according to user's specific needs . The adjhttp://www.ncbi.nlm.nih.gov/omim), and miRNA information from miRBase The results obtained from the query page include the listing of target sites matched with the search criteria used . This reEach target site can be visualized on a local genome browser see  implemenp-value. The number of target sites found to be associated with each type of annotated sequence is also shown here. This information could guide researchers to make their decisions on searching parameters. This module will be automatically updated with the new release of microPIR.The summary information and initial analysis of data deposited in the microPIR database are displayed on the statistics page. The information includes build/release of miRNA/promoter sequences, number of unique targets, number of miRNA-target interactions, distribution of binding sites in terms of chromosomal locations, positions on the upstream regions, MFE, and We constructed a novel database which facilitates a comprehensive exploration of miRNA-promoter interactions. The genome-wide promoter target sites of miRNAs were predicted, organized, and integrated with a number of publicly available annotated sequence databases including the database of CLIP-generated AGO binding sites, gene and EST databases, database of vertebrate sequence conservation scores, genetic variation  databases, and gene regulation  databases. By incorporating these databases together with functional annotation, microPIR offers the most comprehensive and integrated view of these novel target sites.p-value≤0.005), a huge number of potential target sites can be narrowed down to 402 unique target candidates in 297 genes (211 genes are found in OMIM database), when considering only non-repeat derived, highly conserved sites (average conservation score ≥0.8) accompanied with experimental support, containing AGO1 or AGO2 binding sites, and are located in the expressed genomic region, as shown by EST data.Currently, the microPIR database contains a total of about 15 million unique target sites, with an approximately similar number of sense and antisense targets associated with 22,357 annotated genes. These numbers represent all possible sites on promoter sequences which pass the default parameter settings  before considering any other parameter cutoff values. All experimentally verified cases of miRNA promoter targets cis-regulatory role of promoter targeting miRNAs can look for target sites with their associated miRNA present in the same promoter region, as shown in microPIR database was developed with the main purpose to provide a new data resource for miRNA researchers. Unlike other miRNA target databases, microPIR caters for a broad range of adjustable search options, giving a better control over various kinds of interactions interested by a user. For example, highly confident sites can be selected with the perfectly complementary binding sites by setting maximum number of unpaired nucleotide to be zero. Since it is generally accepted that endogenous miRNAs do not require complete complementarity to achieve target recognition, the stringency can also be adjusted to produce the corresponding targets. These flexible search options also allow the generation of hypotheses about possible miRNA/target relationships with other genomic elements, e.g. sequence polymorphisms, repetitive elements, TFBSs, CpG islands. Moreover, the genome browser view supports users' specific interest, e.g. researchers who are interested in the Currently, microPIR provides information of miRNA-promoter target interactions for human sequences for which experimentally determined data of AGO binding sites and other genomic data are available. In the future, we plan to include data from more organisms in order to permit the comparative features across different species. Since the knowledge on miRNA-promoter interaction is in its early stage, the computational procedures for target site prediction will soon be updated. Additional computational prediction algorithms are planned to be used in the future. The scoring system of predicted target sites will be implemented and tested once enough cases of experimentally confirmed miRNA-promoter interactions are reported. To enhance the complete picture of the miRNA regulatory network, previously determined target sites within the body of genes will be included for further analysis.microPIR is the most comprehensive database specifically developed to provide an open access repository of information for miRNA target sites within human promoter sequences. Integration of considerable resources of genomic data and functional annotation allows researchers to investigate predicted miRNA-promoter interactions and generate new testable hypotheses and relavant biological insights of promoter-targeting miRNAs. The microPIR database is equipped with flexible search features including user-friendly GBrowse graphical interface and hypertext link-outs to various databases and a primer design service. We believe that microPIR will be a useful resource for the miRNA community to facilitate the discovery of new miRNA regulatory mechanism through promoter recognition.All known human miRNAs were downloaded from miRBase release 13.0 Crosslinking and immunoprecipitation (CLIP) coupled with deep sequencing is a high-throughput experimental technique which has been applied to AGO proteins for identification of miRNA-target interactions Sequence conservation information of each predicted target site was also included into the database. Position-specific conservation scores, which were derived from multiple whole genome sequence alignments between the human and 16 other vertebrate genomes http://www.biotec.or.th/thaisnp) release 2. The locations of CpG islands were taken from the UCSC Table Browser The GenBank human EST sequences were downloaded from the UCSC Table Browser http://www.webwareforpython.org). The web interface offers three main functionalities. First, the search feature allows users to locate miRNA target sites of interest. Data can be queried through the webform, which formulates the corresponding SQL queries to MySQL using python scripting language. We used Python MySQLdb module to connect (sending and receiving SQL queries/outputs) to the MySQL database backend. Second, the viewing feature enables users to graphically view the location of miRNA target sites within a specific locus via a genome browser interface. Third, the statistics module summarizes the information contained within this database for the genomic region of interest. The link-out module is provided for users to cross-check with other related databases, e.g. gene ontology and original data sources. Furthermore, for the sake of convenience, a primer design section is included to assist in validating potential target genes. The whole microPIR framework is running on our 12-core database server (2 AMD 6-core (2.8 GHz) processors with 64 Gigabytes of RAM and 2 Terabytes of hard disk space).The overall system design of microPIR is illustrated in


Since the onset of antiviral therapy, viral resistance has compromised the clinical value of small-molecule drugs targeting pathogen components. As intracellular parasites, viruses complete their life cycle by hijacking a multitude of host-factors. Aiming at the latter rather than the pathogen directly, host-directed antiviral therapy has emerged as a concept to counteract evolution of viral resistance and develop broad-spectrum drug classes. This approach is propelled by bioinformatics analysis of genome-wide screens that greatly enhance insights into the complex network of host-pathogen interactions and generate a shortlist of potential gene targets from a multitude of candidates, thus setting the stage for a new era of rational identification of drug targets for host-directed antiviral therapies. With particular emphasis on human immunodeficiency virus and influenza virus, two major human pathogens, we review screens employed to elucidate host-pathogen interactions and discuss the state of database ontology approaches applicable to defining a therapeutic endpoint. The value of this strategy for drug discovery is evaluated, and perspectives for bioinformatics-driven hit identification are outlined. With few exceptions, therapeutic approaches to combat infectious diseases have focused in the past decades on targeting unique components or enzymes of viral, bacterial and parasitic origin. It has now become perfectly evident that this traditional, pathogen-directed strategy, while highly successful in numerous cases , is inhePrompted largely by the onset of the global HIV epidemic, the development of combination therapies based on drugs with distinct individual resistance profiles has considerably heightened the barrier against the development of pathogen resistance and frequently boosted the effectiveness of pathogen-directed therapeutics through synergistic effects . DespiteOf the strategies entertained towards this goal, targeting host factors that are essential for the pathogen life cycle, rather than pathogen components directly, has recently received increasing attention –10. WhilIndeed, the currently available data for the experimental use of host cyclin-dependent kinase (CDK) inhibitors to block HSV-1 and HIV-1 replication, for instance, has revealed a remarkably reduced frequency of viral escape from inhibition in tissue culture settings ,12. In cFrom a therapeutic perspective, the intricate and complex network of virus-host interactions yields a multitude of potential cellular targets for host-directed antivirals. In addition to the aforementioned role of CDK host protein kinases in HSV-1 and HIV-1 replication ,12, someClearly, the most desirable host target is essential for completion of the pathogen life cycle under investigation but at least temporarily dispensable for host cell survival, thus supporting the prospect that successful inhibition will combine a potent antiviral effect with manageable toxicity. Nevertheless, targeting host factors carries an inherently higher potential for undesirable drug-induced side effects than pathogen-directed antiviral therapies, particularly when the latter is highly selective. While host-directed therapies are being explored for the treatment of some major chronic viral infections such as HSV-1 and HIV-1 ,12, theyHit candidates for host-directed drug development programs have resulted from a diverse set of experimental approaches. These can be grouped largely into knowledge-driven direct identification of individual targets, automated screening of chemical diversity sets with protocols specifically designed for the discovery of host-directed hits, and systems-wide screens for host factors essential for pathogen replication.The evolving understanding of critical host-pathogen interactions through molecular virology research of individual viral families has made possible the direct selection of candidate host targets. With an arsenal of approved and experimental therapeutics for inhibition of many cellular pathways at hand, identified candidate targets may in many cases be immediately testable through repurposing of known drugs or commercially available experimental compounds with known bioactivity. The demonstration that Gleevec (Imatinib mesylate), an Abl tyrosine kinase inhibitor licensed for the treatment of several cancer forms , is a po50/EC50), the latter should result in efficient inhibition not only of the screening agent but also of pathogens of related viral families when assessed in counter-screening assays. When we explored the general feasibility of this approach conceptually using a ~140,000-entry diversity set, several chemical compound classes were identified that efficiently blocked replication of a panel of distinct members of the myxovirus families [The availability of large robotic capacities in both corporate and academic settings combined with the rapid design and production of small-molecule compound libraries in the past two decades has accelerated the pace of discovery of novel drug candidates via high-throughput screening exercises. Applied to the identification of antiviral hits with a host-directed activity profile, for instance, it should be feasible to derive suitable screening protocols based on the hypothesis that host-directed candidates will likely show some cellular interference and return a broadened pathogen target range. While the former will translate into a lower primary screening score represented by the selectivity index , and/or proteomics  studies is conceivable. Target identification not only sets the stage for possible knowledge-based scaffold optimization through rationale design in conjunction with hit-to-lead chemistry or repurposing of known inhibitors with identical target profile, but also contributes to further elucidating critical pathogen host interactions and, thus, basic insight into pathogen biology.To determine the molecular target of host-directed compounds identified through screening of chemical libraries, a combination of traditional mechanism of action studies, genomics  of protein-protein contacts  and yeasSurprisingly, however, independent large-scale screens directed at influenza virus, for instance, returned very little redundancy for essential host factors identified. This suggests that the screening efforts still lack saturation, and that cross-study bioinformatics efforts for data mining would benefit significantly by commencing on a host cell pathway rather than at the individual protein level. When a set of identified pathways essential for virus replication emerges through bioinformatics selection, individual components can be subjected to secondary screens using known drugs or experimental inhibitors to short-list desirable individual targets. A proposed workflow for a bioinformatics lead-identification process is displayed in As with the output of chemical genomic screens, genome-wide association studies (GWAS) require bioinformatics support to analyze high density data points and highlight the important gene or gene sets responsible for the phenotype under investigation. False positives need attention beyond the high throughput experiment when choosing targets for further study. These data points often arise when certain genes have been incorrectly identified due to off-target effects associated with siRNA inhibition. In one case, a single siRNA reportedly perturbed the expression of over 300 genes . The potCurrently, several curated databases in the public domain detail known gene product associations  –84. InveWhile the previously cited resources offer investigators utmost convenience in immediately accessing lists of available small molecule modulators related to a pathway of interest, other databases connect small molecule modulators with known protein targets. These require a separate pathway analysis to choose a particular set of gene products of interest. Suitable resources include DrugBank ,86, PDB/de novo drug discovery can be sustained through 3D virtual screening [Following a bioinformatics selection of target candidates, individual targets must be selected for medicinal chemistry, for instance, based on the previous discovery of small-molecule blockers or the availability of crystal structures in the Protein Data Bank (PDB). ,89. Thencreening  and struEach of the systematic studies examined in the following sections employed a unique bioinformatics approach to pathway analysis. Similar to a chemoinformatics clustering analysis of a high-throughput screen to short-list a set of chemical leads for optimization, a goal of the RNAi screening studies is to identify, by means of gene pathway or functional analysis, potential host factor targets that are essential for viral replication. A key question is whether the resulting bioinformatics short list of host factors contains suitable candidates for drug development.et al. [et al. [et al. [et al. in 2009 [For HIV, three independent siRNA studies were published in 2008 by Brass et al. , Konig e [et al.  and Zhou [et al. . All thr [et al. . Figure  in 2009 .et al. performed an overlap analysis/random distribution comparison based on these data and found associations that were statistically significant . While one may safely assume that the hit genes are enriched with respect to independently identified and confirmed host factors required for HIV-1 replication, pairwise overlaps between the studies are low, ranging only from 3 to 6%. While these were still judged statistically significant  [Bushman l pairs) , the oveet al. performed the screen in duplicate. As a case in point, the experimental data showed large variances between the replicates: 24% of hit siRNAs (141) exhibit standard deviations greater than 25% of their median values. Furthermore, Bushman et al. demonstrated that adjusting the filtering thresholds in this study strongly influences the nature of the identified genes  [Variation in individual host factors could be accounted for by a number of factors, including: (a) high experimental variance of siRNA transfection efficiencies ; (b) har et al.) . Other pbona fide host factors required for HIV replication or false positives that may have arisen from experimental variability. Equally important for hit confirmation is the organization of the data sets into groups by gene function and cellular pathways to illuminate distinct parts of the intricate host-pathogen interaction network. Using terms from the Gene Ontology (GO) database Brass et al. noted that 103 of their hit genes were assigned with 136 statistically significant  biological processes [p-value < 0.05).Independent of redundancy between studies, the question remains of whether the gene hits represent rocesses ,63,98,99p-value < 0.06 based on a geometric mean for all the terms in a group), redundancy, biological relevance and specificity returned 24 functional groups   were ideThe meta-analysis was extended by building an HIV-host factor interaction network of 1657 cellular proteins using an array of protein-protein interaction databases  . With MCLack of saturation in host genes identified through current siRNA screens, varying consistency and high overlap of genes in specific areas emerge as future challenges for application to system-wide drug discovery efforts.et al. [et al. [et al. [et al. [et al. [et al. [et al. [In addition to application of system-wide siRNA screens to the HIV system, the technology was applied to the influenza virus. Major siRNA studies were reported by Hao et al. , Brass e [et al. , Shapira [et al. , Konig e [et al.  and Karl [et al. . Other t [et al. , which i [et al. , which p [et al. ).et al. summarized five of the six systematic studies reported above and performed bioinformatics analysis on the 1,449 identified genes required for influenza replication [et al. analysis of HIV host factors [et al. and Karlas et al. studies, possibly due to the overlaps in host cell type (A549 cells) and influenza virus strain (A/WSN (H1N1)). This lack of overlap is also illustrated in the pairwise analysis given in i.e., different harvest times, detection thresholds and host cell lines, coupled with the additional complication of variability introduced through the use of different viral strains.Watanabe lication . Much li factors , 128 genAs described in the HIV siRNA analyses, each study examining influenza virus infections performed individual bioinformatics analyses on siRNA screening results. A summary of these bioinformatics data along with the methodology is reviewed by Min . In uniq50 values ranging from 0.5 to 30 μM target FRAP1, HSP90AA1, TUBB, FGFR4, GSK3B, or ANPEP. Of these, FRAP1, TUBB, FGFR4 and GSK3B home to the same GO Term cluster, protein kinase activity, recommending it as a potentially rich source for influenza virus inhibitors. The cytosolic chaperone Hsp90AAP1 was identified in a separate GO Term cluster; interestingly, previous reports have already established a link to influenza virus [Konig reported six compounds with ECza virus  and HCV za virus  replicat50 values ranging from 5.8 to 30 μM. In-house analysis of the 20 genes used to identify these compounds revealed that they are significantly enriched in metabolic processes. Similar to previous studies, the Josset report does not explicitly identify a host pathway essential for viral replication based on the small molecule inhibition studies. To appreciate the full potential of this approach for antiviral drug development, it may be informative to collect all known inhibitors of a particular host pathway and determine the complete extent of virus inhibition.While the above studies identified potential host factor targets through GO term enrichment and then followed up with small molecules available for viral testing, the Josset project searched Connectivity Map with 20 of the most perturbed genes from the 300 initially identified. Of the eight compounds available through commercial vendors, six attenuated influenza virus replication with ECet al. performed a meta-analysis of the siRNA results using the set of 128 genes found in two or more studies [Watanabe  studies . The majSuccessful outcomes for bioinformatics searches predominantly depend on the accuracy of tabulated database interactions. As detailed below, use of different databases may alter the profile of pathways that are enriched from the same gene list. In such cases, users are obligated to formulate a realistic biological interpretation of the relational data to ensure identification of meaningful candidate compounds for an antiviral drug program.As outlined above, it is a primary function of gene databases to extract biological meaning as well as potential therapeutic host factors from a high throughput RNAi screen by means of descriptive annotations of genes common to a particular biological pathway or gene function. In the realm of antiviral drug discovery, this approach aims at identifying host cell components critical for virus replication.et al. have demonstrated that inconsistencies emerge when gene association data are compared across different pathways databases [Crucial for the success of this strategy is the quality of the pathway database used, which is determined by the curation method of published experimental data of gene associations and the expertise of the curators involved. Soh atabases . This caThe Wnt signaling pathway provides a tangible example illustrating the current challenges. This pathway has been implicated in therapeutic interference with cancer and viral entry. Two-way analyses revealed approximately 80% gene similarity when based on the KEGG and Wikipathways databases (Table 4 in ). Howeveet al., gene overlap similarity has a mean value of 66.5% when comparing the KEGG database and Wikipathways [Looking on a broader scale across 26 cellular pathways described in Soh pathways . By contet al. also analyzed the comprehensiveness of the databases, which was a measure for the total number of genes from all three databases [Soh atabases . This waConcentrating in particular on viral host factors, we performed an in-house analysis that compares host proteins involved in the influenza virus life cycle across various databases. Databases used in this example included Reactome, Ingenuity IPA and PANTHER. The Reactome database records six host factor genes for influenza in each the categories associated with NS1-mediated effects and virus-induced apoptosis. Databases such as Ingenuity IPA and PANTHER lack pathway categories dedicated to influenza virus. Keyword searches for influenza in the PANTHER database identified no host factor associated with influenza virus infection . Converset al. [Applying this approach to the previously described influenza RNAi screens, we sought to address the question of how does target identification change when different pathway databases are applied to the same dataset? Databases used in this comparison were PANTHER, Reactome and STRING, and the data set analyzed was the commonly identified 128 gene list generated by Watanabe et al. . Resultset al. using GeneGO/MCODE. In all other cases, the different databases returned remarkably different top pathways when the same gene expression set was analyzed. Closer examination reveals that that other top ranking pathways  rank lower on the Reactome enrichment analysis scale. Pathways associated with B-Cell metabolism are also identified by the Ingenuity IPA and Reactome enrichments, although slightly different naming schemes are used. Since discrete databases identify certain similar pathways at different rankings, a consensus scoring function applicable to available databases appears warranted. This would afford greater confidence in the identification of individual targets for follow-up through small molecule searching.It becomes immediately obvious when examining the most enriched pathways that only the STRING database seems to reproduce the results generated by Watanabe et al. and Watanabe et al., gene lists and enriched pathways vary widely despite the pursuit of similar biological goals. Indeed, the likelihood to successfully identify novel host-directed antivirals would increase significantly if the reproducibility of individual RNAi screens were to be increased [i.e., Konig et al., Karlas et al., and Josset et al.) demonstrate that RNAi screening coupled with bioinformatics-driven triaging is a viable method to identify small molecule inhibitors of virus replication.Previous GWAS experiments have attempted to capture the most relevant cellular host pathways utilized by pathogens such as HIV and influenza virus for virus replication ,123. As ncreased . Furtherin vitro for various endpoints offer a rich source for hit candidates with optimization potential. As more databases are used to analyze potential host targets, validation methods employing siRNA are improved and small molecule knowledge is added to the genetic web, more drug discovery initiatives are likely to incorporate this approach in their portfolio of standard operations for the identification of antiviral therapeutics.Current databases that infuse chemical knowledge into schemes such as Ingenuity IPA and the Connectivity Map are limited to a small number of compounds, mostly FDA-approved drugs. This narrow focus limits their application to current translational medicine. The STITCH database makes an interesting leap by crosslinking its gene network with multiple chemical-genomic high throughput screening results archived in PubChem. These experimental chemicals along with compounds currently tested


Since alendronate became available in generic form in the Unites States in 2008, its price has been decreasing. The objective of this study was to investigate the impact of alendronate cost on the cost-effectiveness of osteoporosis screening and treatment in postmenopausal women.Microsimulation cost-effectiveness model of osteoporosis screening and treatment for U.S. women age 65 and older. We assumed screening initiation at age 65 with central dual-energy x-ray absorptiometry (DXA), and alendronate treatment for individuals with osteoporosis; with a comparator of “no screening” and treatment only after fracture occurrence. We evaluated annual alendronate costs of $20 through $800; outcome measures included fractures; nursing home admission; medication adverse events; death; costs; quality-adjusted life-years (QALYs); and incremental cost-effectiveness ratios (ICERs) in 2010 U.S. dollars per QALY gained. A lifetime time horizon was used, and direct costs were included. Base-case and sensitivity analyses were performed.Base-case analysis results showed that at annual alendronate costs of $200 or less, osteoporosis screening followed by treatment was cost-saving, resulting in lower total costs than no screening as well as more QALYs . When assuming alendronate costs of $400 through $800, screening and treatment resulted in greater lifetime costs than no screening but was highly cost-effective, with ICERs ranging from $714 per QALY gained through $13,902 per QALY gained. Probabilistic sensitivity analyses revealed that the cost-effectiveness of osteoporosis screening followed by alendronate treatment was robust to joint input parameter estimate variation at a willingness-to-pay threshold of $50,000/QALY at all alendronate costs evaluated.Osteoporosis screening followed by alendronate treatment is effective and highly cost-effective for postmenopausal women across a range of alendronate costs, and may be cost-saving at annual alendronate costs of $200 or less. Osteoporosis affects approximately 10 million individuals in the United States, most of whom are postmenopausal women Medical treatment of osteoporosis reduces fracture risk, and multiple studies have demonstrated the cost-effectiveness of osteoporosis treatment The aim of this study was to evaluate the effect of various alendronate costs on the cost-effectiveness of osteoporosis screening and treatment.We constructed a Monte Carlo microsimulation model of osteoporosis screening followed by alendronate treatment compared to no screening with treatment only if fracture occurs for US women age 65 and older. The model estimates direct costs in 2010 US dollars, quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratios (ICERs) in units of cost per QALY gained for osteoporosis screening followed by alendronate treatment. A lifetime time horizon was used. We followed guidelines of the Panel on Cost-Effectiveness in Health and Medicine We modeled initiation of screening at age 65 with DXA, with repeat screening every 5 years for individuals who test negative. With repeat screening, individuals who did not have osteoporosis at age 65 but who subsequently developed osteoporosis as their BMD declined with age would be offered treatment at the older age at which they are diagnosed. We used 65 as the screening initiation age for women in the absence of additional risk factors in accordance with current guidelines from the U.S. Preventive Services Task Force We assumed that women with positive DXA results (T-scores≤−2.5) or who experienced a fracture of the hip, vertebra , or wrist were offered treatment with 70 mg of alendronate once weekly. We assumed 5 years of treatment Fracture rates for women not on alendronate treatment were based on Study of Osteoporotic Fractures (SOF) data, with future fracture probability predicted as a function of age, femoral neck or lumbar spine BMD, and history of fracture We assumed a constant, linear decline in fracture risk reduction benefit over 5 years after completion of alendronate treatment We used US national vital statistics data for baseline mortality rates Nursing home admission rates, length of stay, and mortality data were obtained from several published sources We included direct costs of DXA screening ($97.71) We used data from a sample of older women in the U.S. for baseline health state utilites We modeled medication adverse events of esophagitis and esophageal ulceration with rates obtained from clinical trials data We performed base-case and sensitivity analyses separately for each alendronate cost evaluated. Key parameter values used for base-case analyses and the range of values used for sensitivity analyses are shown in We compared the model's fracture and life expectancy predictions with published U.S. outcomes data.Our model predicted a mean life expectancy of 19.3 years for 65-year-old women who were not screened for osteoporosis. This is similar to the U.S. National Vital Statistics figure of 19.8 years reported for 65-year-old women in 2006 Osteoporosis screening initiated at age 65 followed by alendronate treatment was more effective than no screening with treatment only if fracture occurs, resulting in 10.6 additional quality-adjusted life-days. When assuming alendronate costs of $200 or less, osteoporosis screening and treatment was cost-saving, resulting in lower total costs than no screening as well as more QALYs . LifetimProbabilistic sensitivity analyses revealed that the cost-effectiveness of osteoporosis screening followed by alendronate treatment was relatively robust to variations in input parameter estimates at a willingness-to-pay threshold of $50,000/QALY for all alendronate costs evaluated . When asOur analyses demonstrated that osteoporosis screening followed by alendronate treatment is effective and highly cost-effective for women aged 65 and older across a wide range of alendronate costs; and potentially cost-saving at annual alendronate costs of $200 or less, depending on assumptions about fracture risk, nursing home admission probability after hip fracture, and health care costs. Sensitivity analyses showed that the value (cost-effectiveness) of alendronate treatment for all alendronate costs evaluated was relatively robust to key model parameter uncertainty; but the price at which alendronate becomes cost-saving is sensitive to the parameters specified above. These results indicate that osteoporosis screening followed by alendronate treatment is an advantageous use of healthcare resources for women age 65 and older, that can be expected to improve health outcomes and may result in potential cost savings when alendronate annual costs are $200 or less, as is currently the case at discount pharmacies. As the cost of alendronate continues to fall, the value and potential for cost savings for the U.S. healthcare system resulting from osteoporosis screening of older women followed by alendronate treatment can be expected to increase, assuming appropriate selection of candidates for treatment. This is a significant finding, given how few preventive services can result in cost savings Our model has several limitations. First, our analyses did not incorporate the costs of added life days from osteoporosis screening. However, the costs of added life days would likely be small as the age of death was very similar in the screening followed by treatment and no screening model arms. Second, our analysis assumed that only women with DXA T-scores in the osteoporotic range would be offered treatment, in accordance with evidence that treatment of women with osteopenia (low bone mass) is not cost-effective In conclusion, our analyses indicate that osteoporosis screening followed by alendronate treatment is highly cost-effective for women aged 65 and older when assuming annual alendronate costs of $400 through $800, and potentially cost-saving when assuming annual alendronate costs of $200 or less, depending on key parameter assumptions. Thus, osteoporosis screening followed by alendronate treatment in appropriately selected patients represents an excellent healthcare value, and this important preventive health service should be promoted. Although our analyses were limited to alendronate, other osteoporosis treatments with similar effectiveness and costs may be expected to be similarly cost-effective. For example, other available oral bisphosphonates (e.g. risedronate) may be similarly cost-effective if costs of the medication were to decrease. However intravenous bisphosphonates, which have additional costs for the infusions, different adverse event profiles, as well as different fracture reduction outcomes would not be expected to have similar cost-effectiveness to alendronate. This would apply to other osteoporosis medications that have different costs, adverse event profiles, adherence patterns, and routes of administration.Future research should evaluate the cost-effectiveness of “real-world” osteoporosis screening and treatment practices, in which some patients will be inappropriately selected for treatment. Furthermore the effects of assuming treatment duration longer than 5 years or a drug holiday should be examined. However, assuming appropriate selection of individuals for treatment, osteoporosis screening followed by alendronate treatment in women aged 65 and old represents a superb healthcare value across the variety of alendronate costs evaluated.Table S1Key Model Parameter Assumptions.(DOC)Click here for additional data file.