G-CSF mobilization might be beneficial to ICM, but the relationship between effect/safety and the dosage of G-CSF remains unclear. In this study, 24 pigs were used to build ICM models and were randomized into four groups. Four weeks later, different dosages of G-CSF were given daily by subcutaneous injection for 5 days. Another 4 weeks later, all the animals were sacrificed. Electrocardiography, coronary arteriography, left ventriculography, transthoracic echocardiography, cardiac MRI, and SPECT, histopathologic analysis, and immunohistochemistry techniques were used to evaluate left ventricular function and myocardial infarct size. Four weeks after G-CSF treatment, pigs in middle-dose G-CSF group exhibited obvious improvements of left ventricular remodeling and function. Moderate G-CSF mobilization ameliorated the regional contractility of ICM, preserved myocardial viability, and reduced myocardial infarct size. More neovascularization and fewer apoptotic myocardial cells were observed in the ischemic region of the heart in middle-dose group. Expression of vWF, VEGF and MCP-1 were up-regulated, and Akt1 was activated in high- and middle-dose groups. Moreover, CRP, TNF-α and S-100 were elevated after high-dose G-CSF mobilization. Middle-dose G-CSF mobilization therapy is an effective and safe treatment for ICM, and probably acts via a mechanism involving promoting neovascularization, inhibiting cardiac fibrosis and anti-apoptosis. Recently, advances in both medical and invasive therapies, such as coronary artery bypass grafting and percutaneous coronary intervention, have improved the outcomes in many cases, but the overall prognosis of IHD patients who are admitted to hospital with heart dysfunction remains poor, with a 5-year survival of ∼50% and a 10-year survival of ∼10%For patients with ischemic cardiomyopathy (ICM), especially for those with multivessel lesions, severe lesions or diffuse stenosis in coronary arteries, there are no therapies available to restore dead myocardium nor to improve cardiac function. On the other hand, although heart transplantation can be regarded as a radical cure for end-stage ICM, it is not appropriate or practical for widespread clinical application in the short term.12. After myocardial infarction, bone marrow stem cells can spontaneously migrate to ischemic regions and then differentiate into myocardial cells, vascular endothelial cells, or smooth muscle cells according to the different microenvironments in which they localize, which may play vital roles in the regeneration of myocardium and the improvement of cardiac function14. However, this self-healing mechanism is too weak to completely repair the traumatized myocardium, since the number of migrated bone marrow stem cells is often too limited. From this point of view, the mobilization of hematopoietic stem cells and endothelial progenitor cells (EPC) from the bone marrow into peripheral blood circulation appears to be a key. Although the effect and safety of this method remain controversial, as a minimally-invasive approach to stem cell transplantation, granulocyte colony-stimulating factor (G-CSF) mobilization has been applied for the treatment of acute myocardial infarction (AMI)22. In face of several prior experiments and clinical trials27, whether this cytokine therapy can really benefit the patients with chronic IHD, especially those with ICM, as well as its probable biological mechanisms remain more evidence to be explored. Moreover, the relationship between G-CSF dosage and its corresponding effects are still unclear.During the past few decades, several studies have suggested that stem cell therapy might contribute to the regeneration of infarcted myocardium and enhance neovascularization of ischemic myocardiumThe aim of this study was to investigate the potential effect and safety of different dosages of G-CSF-mobilized bone marrow stem cells on myocardial regeneration and cardiac function in an animal model of ICM.P > 0.05). No complications, such as cardiac death, AMI, fatal arrhythmia, infection, or heart failure, occurred during G-CSF mobilization.Twenty-two of the 24 pigs survived after placement of ameroid constrictors. The 2 deaths were due to AMI and infectious pericarditis after surgery. One death occurred in control group, the other was in high-dose group. However, survival rates were equal among the groups . Moreover, numbers of lymphocytes, hemoglobin, and platelets remained unchanged after mobilization (P > 0.05). CD34 and CD133 expression was determined by three-color flow cytometry with CD45/PE gating  were also up-regulated with increasing dose of C-GSF .When routine blood examinations were conducted, leukocytes counts, especially those of neutrophils and monocytes, were found to peak between the 5Four weeks after placement of ameroid constrictors, coronary arteriography (CAG) revealed complete occlusions in four pigs. Distal segments of the LCX in all other pigs reached a stenosis diameter of ≥95% Table . Four weWe also compared the proximal segments of the LCX between each group. Similarly, middle-dose group displayed the mildest stenosis after G-CSF mobilization. While both low-dose and high-dose group showed improving trends without statistical significance.When analyzing collateral vessels, we found all the pigs were lack of collateral circulation around the obstructive vessel segments 4 weeks post-modeling. After G-CSF mobilization, we observed LCX distal enhancement in each of the pigs from low-dose to high-dose group during CAG.Except for LCX, the other coronary arteries in each pig had no obvious stenosis during the study.As shown in Table P < 0.001, Fig. P > 0.05). Parameters of left ventricular diastolic function, such as mitral E peak and A peak, were also detected by TTE (Table P < 0.05). However, the middle-dose group showed the greatest improvement.Baseline left ventricular ejection fraction (LVEF) detected by each measurement was balanced among the groups. Since ameroid constrictors were fitted, LVEF dropped equally in each group. Eight weeks after placement, we reexamined the LVEF of every pig and found a significant improvement of approximately 10% in middle-dose group (TE Table . Left veP < 0.05).The values of left ventricular end-diastolic diameter, left ventricular end-diastolic volume, left ventricular end-systolic diameter and left ventricular end-systolic volume, measured via both TTE and CMR, also changed with ischemic myocardium modeling and G-CSF mobilization Fig. , Table 2P < 0.05). When using CMR and SPECT to precisely evaluate the ischemic regions, we found that all three dosages of G-CSF could prevent ischemia and infarct development, but middle-dose (5 μg/kg/d) mobilization could shrink the size of the infarcted or ischemic myocardium best  staining to complete the histopathological examination. Pigs in the middle-dose group obviously presented the mildest artery intimal hyperplasia surrounding ameroid segments Fig. , and theAs shown in Fig. Western blotting, RT-PCR, and immunohistochemical techniques were used to examine the expression of vascular endothelial growth factor (VEGF) and its mRNA in the infarct zone. As shown in Fig. Serum concentrations of high-sensitivity C-reactive protein (hs-CRP) and TNF-α were measured by ELISA, to evaluate the relationship between G-CSF mobilization and inflammation. As shown in Table 10.ICM is a common type of IHD, which remains an important cause of cardiovascular death worldwide. During the pathogenesis of chronic disease, decreasing myocardial perfusion leads to ischemia, hypoxia, apoptosis, and fibrosis, gradually resulting in cardiac dysfunction and ventricular remodeling. Since conventional treatments, such as coronary artery bypass grafting and percutaneous coronary intervention, cannot directly salvage tissue from ultimate death, stem cell therapy has been attracting more attention. According to the initial hypothesis of cardiogenesis, ICM is thought to be an attractive potential indication for adult stem cell therapy, which may replace the significant cardiomyocyte loss that occurs23, which suggests some practical basis of G-CSF treatment for ICM17. However, although previous studies have partly explored the effect and safety of G-CSF mobilization regarding ICM, the outcomes remain controversial31. Several experimental studies have suggested that G-CSF can improve myocardial ischemia and prevent myocardial remodeling via different pathway, which may contribute to the pro-revascularization, anti-apoptosis, and anti-fibrosis mechanisms in ischemic myocardium35. But evidence from either humans or animals remains limited. To our knowledge, this is the first study to analyze the dose-effect relationship of G-CSF mobilization in an animal model of ICM, and to suggest that appropriate G-CSF mobilization can improve cardiac function and prevent ventricular remodeling.In recent years, animal experiments and clinical trials have indicated that stem cell therapy may improve cardiac function and prognosis in the patients with acute myocardial infarctionIn current study, after creating ICM pig models by placement of ameroid constrictors, we treated the animals with different dosages (2.5–10 μg/kg/d) of subcutaneous G-CSF daily for 5 days. Four weeks later, we used CAG, LVG, TTE, CMR, and SPECT to compare their outcomes, observing that the pigs in middle-dose (5 μg/kg/d) group had better cardiac function, smaller myocardial infarct size, richer blood perfusion, more surviving myocardium, and milder ventricular remodeling not only than the controls, but also than those treated with other dosages of G-CSF, either higher or lower (2.5 or 10 μg/kg/d). Particularly, no serious complications occurred after G-CSF mobilization during our observation period.34. In our study, after administration of G-CSF, the number of CD34+/CD45+ and CD133+/CD45+ cells increased in the infarct border zone. At the same time, we found neovascularization increasing but apoptosis of cardiomyocyte decreasing significantly. We inferred that promoting angiogenesis and anti-apoptosis along with anti-fibrosis should be the keys of moderate G-CSF mobilization treating ischemic cardiomyopathy.G-CSF can mobilize bone marrow stem cells, including EPCs, to peripheral circulation. These stem cells may play vital roles in the process of ischemia neovascularization− kinase, Ras-Raf-mitogen-activated protein (MAP) kinase, Janus kinase (Jak)-signal transducer and activator of transcription (STAT), Src family kinase pathways, and so on38. Among them, serine/threonine kinase Akt was identified as a downstream target of PI3-kinase. Activation of Akt1 is crucial to the survival of endothelium and myocardium40. When ischemia occurs, the activity of Akt1 can be abolished. In current study, after subcutaneous injection of G-CSF, the reduction of Akt1 expression turned to be weakened, while simultaneously the Bcl-2/Bax pathway is enhanced. Thus, we speculate that G-CSF can drive cellular activities, as well as inhibit myocardial apoptosis.In fact, several potential molecular mechanisms have been proposed in some former experiments concerning to acute myocardial infarction, but the exact biologic mechanisms are not fully understood. G-CSF can bind to a cell surface receptor that is a member of cytokine receptor superfamily, which can activate a variety of intracellular signaling cascades such as phosphatidylinositol (PI) 341. Hence, G-CSF may preserve myocardial viability, rebuild the micro-circulation, and protect endothelial function by regulating these cytokines.Moreover, the expression of VEGF, MCP-1, and TNF-α was up-regulated in the same region after G-CSF mobilization. VEGF has been proven to be important in the protection of endothelial stability and function, especially for those atherosclerotic vessels+ cells transplantation, G-CSF mobilization via subcutaneous injection are both more convenient, available and safety during clinical practice, which presents to be a specific advantage.These findings not only support the theories mentioned above, but also provide further practical clues to G-CSF therapy in ICM. We calculate that G-CSF can motivate stem cell homing to ischemic myocardium, ameliorating neovascularization, and recovering ventricular function. Moreover, compared with CD3443. While, these two cells are linked with atherosclerosis tightly. Since EPCs, DCs, and mesenchymal stem cells have the same precursor cells with the surface marker of CD34+, G-CSF therapy can not only mobilize hematopoietic stem cells and EPCs into peripheral blood, but also increase the number of cells and cytokines associated with inflammation44, which can promote cell over-proliferation and cardiac fibrosis46. Meanwhile, Harada et al. previously reported the existence of some G-CSF receptors on the surface of fibroblasts38. Once upon ligand binding, the expression of collagen decreases. From this aspect, excessive mobilization of G-CSF may contribute to myocardial fibrosis. In addition, over-expression of VEGF can result in over-proliferation of endothelial and vascular smooth muscle cells, which may contribute to the development of atherosclerosis directly48. In high-dose group, we have found severe endothelial over-proliferation in the distal segments of ameroid constrictors, which also supports this point of view. Hence, we hypothesize the results of high-dose G-CSF group in our study partly owing to the reasons above.Interestingly, we found that high-dose G-CSF seemed to have less of a cardiac benefit than middle dose. The levels of inflammatory markers, such as hs-CRP and TNF-α, presented to have a trend of positive correlation to the dosage of G-CSF. As a result, the expressions of these factors were extremely elevated in high-dose group, indicating that high-dose G-CSF mobilization could stimulate an over-activation of inflammation. On the other side, as a marker of dendritic cell (DC), the expression of S-100 was up-regulated with the dosage of G-CSF higher up, showing that high-dose G-CSF mobilization could induce DC filling under endothelial. Prior studies used to confirm DCs and EPCs aggregating under the atherosclerotic plaque33. Studies have found that some inflammatory factors, including TNF-α, interleukin-6, and interleukin-1, are associated with ischemic myocardial proliferation46, which may also refer to ventricular remodeling, enlargement, and dysfunction. In current study, the expression of TNF-α in infarction border zone have demonstrated to be dose-dependent on the dosage of G-CSF. Thus, we speculate that middle-dose of G-CSF may stimulate TNF-α expressing moderately, which can mobilize bone marrow stem cells homing in ischemic region and can play vital roles in anti-apoptosis and neovascularization. However, high-dose G-CSF may induce TNF-α over-expression, resulting in inflammation and fibrosis excessively.Another important potential influencing factor of our results is about the expression of TNF-α. Once cardiac infarction, inflammatory response can be activated immediately in the region of ischemic myocardium. As a result, multiple inflammatory factors and adhesion molecules lead to myocardial regeneration and fibrosis33. So that, more related signaling pathways need to be detected seriously. Consequently, whether our findings can be directly transferred to clinical practice should be considered cautiously.Although we found some beneficial effects of G-CSF mobilization therapy in pigs with ICM, several issues remain to be addressed. First, since the sample size of our study was limited, a larger-scale animal experiment will be necessary to confirm our findings. Secondly, if possible, the duration of observation should be prolonged. Thirdly, although we confirmed 5 μg/kg/d as the optimal dose of G-CSF to treat the ICM pigs, the exact therapeutic dosage and its safety in real patients requires further investigation. Besides, since we failed to found neonatal cardiomyocyte in the infarct zone while conducting the experiments, mobilized cells seem not to be the primary effectors responsible for the G-CSF effects on the ischemic myocardiumOur study has firstly confirmed the dose-dependent effectiveness and safety of G-CSF mobilization therapy in a pig model of ICM. A middle dosage (5 μg/kg/d) of G-CSF mobilization can improve cardiac function, reduce myocardial infarct size, enhance blood perfusion, protect surviving myocardium, and delay ventricular remodeling safely. These findings remind us of balancing G-CSF’s protective effect from angiogenic, anti-fibrosis, and anti-apoptosis and its side-effect such as promoting atherosclerosis and inflammation. Our study may offer new clues to stem cell therapy for the treatment of ischemic heart disease. In future, more details of the biological mechanisms of G-CSF mobilization therapy and clinical application require more evidence and further investigation to clarify.50. Penicillin  was given intramuscularly for 3 days, and oral aspirin (150 mg per day) was maintained until the animals were sacrificed. The pigs were allowed to recover for 4 weeks after surgery. Electrocardiography (ECG), CAG, LVG, TTE, CMR, and SPECT of dipyridamole 99mTc-MIBI myocardial perfusion imaging (99mTc-MIBI-SPECT) were conducted to evaluate the function of the left ventricle, the myocardial infarct and the perfusion size of every animal. A successful model was defined as one satisfying at least two of the following conditions: (1) newly-presented ST segment depression and (or) T waves changed in leads I, avL and (or) II, III, avF and (or) V4–V6 in ECG; (2) total or sub-total occlusion (a stenosis diameter ≥95%) of the LCX confirmed by CAG; (3) perfusion defects and (or) regional motion abnormalities of the left ventricular lateral walls detected using CMR; (4) perfusion defects and (or) metabolic/perfusion mismatches of the left ventricular lateral walls demonstrated by SPECT. The protocol was approved by the ethics committees of Zhongshan Hospital, Fudan University. All the experiments were performed in accordance with relevant guidelines and regulations.Twenty-four Yorkshire pigs of either sex weighing 25 to 30 kg (2–3 months old) were anesthetized with intravenous phenobarbital (30 mg/kg) after intramuscular injection with diazepam (1 mg/kg) and ketamine (1.5 mg/kg). Under mechanical ventilation, a left thoracotomy was performed through the fourth intercostal space, using sterile technique. The pericardium was opened, and an ameroid constrictor  of 1.5–2.5 mm internal diameter was placed around the LCX. After observing for a few minutes, pericardium and chest were closed in turnFour weeks after constrictor placement, the surviving successful model pigs were randomly divided into the following four treatment groups: (1) Control group: normal saline, 2 mL/d, subcutaneous injection, 5 days; (2) Low-dose G-CSF therapy group: G-CSF, 2.5 μg/kg/d, subcutaneous injection, 5 days; (3) Middle-dose G-CSF therapy group: G-CSF, 5 μg/kg/d, subcutaneous injection, 5 days; and (4) High-dose G-CSF therapy group: G-CSF, 10 μg/kg/d, subcutaneous injection, 5 days. Another 4 weeks later (8 weeks after constrictor placement), all the animals were sacrificed. with a lethal dose of intravenous potassium chloride.g) and the serum was stored at −70 °C until the final analysis. Then ELISA were conducted to measure the serum concentrations of TNF-α, hs-CRP, cTnT, BNP, and G-CSF. While, methods of western blot and reverse transcription-polymerase chain reaction (RT-PCR) were conducted to evaluate the transcriptions and expression of TNF-α, vWF, VEGF, Bcl-2, Bax, MCP-1, and PPAR-α.Venous blood samples were collected before and after modeling, on day 1, 2, 5 and 7 after the start of G-CSF mobilization, and before sacrifice. From each whole sample, 1 mL was used for analysis of blood cell count by flow cytometry (FCM), then the remainder of each sample was centrifuged , in the left decubitus or supine position. All the measurements were performed according to the recommendations of the American Society of Echocardiography and as described in previous studies52. 99mTc-MIBI-SPECT imaging was performed using a tri-head SPECT gamma camera . Pigs were scanned in the supine position with intravenous injection of a mean dose of 370 mBq.All the pigs also underwent CMR and SPECT at similar time-points to the TTE. For CMR examination, pigs were positioned in the body coil of a Siemens MAGNETOM Avanto 1.5 T  as described previouslyAfter fixation for at least 1 week, tissues from the left ventricles and vessels were sectioned into 6–8 transverse slices. Each slice was weighed, its apical surface was traced and the infarct region was determined visually based on scar tissue. Anterior and posterior wall thickness were determined in multiple transverse sections by measuring endocardial to epicardial distances. Care was taken to determine myocardial wall thickness in the same plane and location in all specimens. Then, samples from each animal were analyzed by hematoxylin–eosin (HE) staining, Mallory trichrome staining and TUNEL staining.P values are two-tailed, and a P value < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS software, version 20.0 . Continuous variables are presented as mean ± standard deviation (SD) and were compared using ANOVA, whereas categorical variables are presented as percentages and were compared using the Chi-square test or Fisher’s exact test. All reported Supplementary Figures


Determine what clinical role, if any, GM-CSF may have in the clinical treatment of sepsis in the adult patient.Advancements in the management of sepsis have led to significant decreases in early mortality; however, sepsis remains a significant source of long-term mortality and disability which places strain on healthcare resources with a substantial growing economic impact. Historically, early multiple organ failure (MOF) and death in patients with severe sepsis was thought to result from an exaggerated proinflammatory response called the systemic inflammatory response syndrome (SIRS).Numerous prospective randomized controlled trials (PRCTs) tested therapies aimed at decreasing the organ injury associated with an exaggerated inflammatory response. With few exceptions, the results from these PRCTs have been disappointing, and currently no specific therapeutic agent is approved to counteract the early SIRS response in patients with severe sepsis. It has long been recognized that there is a delayed immunosuppressive state that contributes to long-term morbidity. However, recent findings now support a concurrent proinflammatory and anti-inflammatory response present throughout sepsis. Multiple immunomodulating agents have been studied to combat the immunosuppressive phase of sepsis with the goal of decreasing secondary infection, reducing organ dysfunction, decreasing ICU stays, and improving survival. Granulocyte-macrophage colony stimulating factor (GM-CSF), a myelopoietic growth factor currently used in patients with neutropenia secondary to chemotherapy-induced myelosuppression, has been studied as a potential immune-activating agent.The applicability of GM-CSF as a standard therapy for generalized sepsis is still largely understudied; however, small-scale studies available have demonstrated some improved recovery from infection, decreased hospital length of stay, decreased days requiring mechanical ventilation, and decreased medical costs. However, this has led to the increased appearance of a new predominant phenotype of chronic critical illness (CCI) which is plagued by increased susceptibility to secondary infections, prolonged ICU stays, long-term cognitive and functional impairment, increased disposition to long-term acute care (LTAC) facilities, and a surprisingly high ongoing post-hospital discharge mortality.37 This disconnect between improved clinical outcomes and lack of short-term survival benefits is not surprising given the emerging phenotype of PICS which is characterized by long-term disability and indolent death. The effects of GM-CSF therapy on long-term outcomes have yet to be evaluated.GM-CSF has been studied across institutions and age groups with varying indications and dosage. The current body of published research has shown some benefit when examining clinical benchmarks; however, there is a consistent lack of 28-day survival benefit in the adult population.38 the clinical model of the immunologic state during sepsis has been evolving over the last few decades. The once established model of unbridled hyperinflammation, termed the “systemic inflammatory response syndrome” (SIRS) causing early death39 spurred the investigation of anti-inflammatory mediators in an attempt to decrease early mortality.40 As early survival improved due to advancements made in earlier sepsis recognition and improved critical care management, the emergence of a later immunosuppressive state that leaves the septic patient at risk of secondary infection became apparent.41 The SIRS model was amended to include a later immunosuppressive state42 or “compensatory anti-inflammatory response syndrome” (CARS)43 which again became the target for immune modulating therapies; however, the focus proinflammatory mediators , and epigenetic modifications secondary to the cell microenvironment.56 Despite a chronic inflammatory state, innate immune functions are also affected, including impaired phagocytosis, ex vivo production of inflammatory cytokines, cell surface expression of check-point regulators and immunosuppressive molecules, and the appearance of immature myeloid cells with inflammatory and immunosuppressive phenotypes.58As research into the immune state of sepsis continues, our understanding of the course of sepsis has evolved and the biphasic SIRS/CARS model has become less valuable. Surprisingly, this late period of immunosuppression, previously identified as CARS, occurs throughout the course of septic illness and is, surprisingly, associated with low grade, chronic inflammation, and an increase in inflammatory mediators, such as C-reactive protein (CRP), IL-6, IL-1ra, and sTNFR.60 These poor outcomes range from secondary bacterial infection, consisting of ventilator-associated pneumonia (VAP)61 and avirulent opportunistic infections,63 to reactivation of latent herpes virus such as CMV and HSV,64 and an increased risk of multiple organ dysfunction syndrome (MODS).66 These patients have increased hospital length of stays, increased disposition to long-term care facilities, and increased long-term mortalities.67The magnitude and duration of this immune suppression has been shown to be associated with dramatic declines in clinical outcomes. Patients with immunosuppression, as documented by either reduced mHLA-DR expression, reduced absolute lymphocyte counts (ALC), or ex vivo TNFα production in response to endotoxin stimulation, are associated with increased nosocomial infections.32 Although some studies show improved eradication of primary infection, prevention of secondary infection, and decrease latent virus activation, only thymosan α1 has been shown to decrease 28-day mortality.32 However, the importance of 28-day mortality is overshadowed by the more predominant state of chronic disability and indolent death. GM-CSF has been an ongoing area of study over the last couple of decades in populations ranging from the neonate to elderly. While it has been shown to improve clinical markers, it has failed to show a consistent short-term survival benefit.37There have been several proposed methods to combat this immunosuppressed state, from immunostimulating adjuvant therapies to extracorporeal removal of immunodepressants. Immunostimulating treatments that have been studied are interferon γ (IFNγ), IL-7, thymosan α1, and GM-CSF.+ blood monocytes has been used as a diagnostic marker of the onset of an immunosuppressive state in several studies.68 However, the recognition that immunosuppressive processes are present at the onset of sepsis should challenge the timing of GM-CSF implementation in addition to the end-points evaluated.The advancements made in the early management of the critically ill have increased short-term survival only to unmask the emergence of a new predominant phenotype of chronic illness, PICS. PICS criteria Table  utilize 69 Later in vitro models demonstrated the effects of GM-CSF on mature myeloid cell populations, and it became clear that its role was more complex than that of simply a hematopoietic-cell growth factor. Its beta-subunit in humans is shared with IL-3 and IL-5, and activation of GM-CSF stimulates at least 3 pathways .70 The effects of GM-CSF on monocytes, macrophages, neutrophils, eosinophils, and basophils are polyfunctional and range from increased cell survival to enhanced proliferation, differentiation, and activation  when these cells are stimulated with endotoxin.73 This mobilizing effect has been used clinically in patients with myelosuppression secondary to chemotherapy; it has been shown to shorten duration of granulocytopenia and promote immune cell proliferation and function.74 There is additional evidence that endogenous GM-CSF plays a role in emergency myelopoiesis in response to infection in addition to its effects on innate and adaptive immunity.70Since its discovery, GM-CSF has been extensively studied and its effects are wide-ranging. The role that it plays as a baseline hematopoietic factor is unclear. In healthy adult populations circulating levels of GM-CSF are inconsistent and often found at very low levels; endogenous production of GM-CSF usually requires inflammatory stimulation.68 These dendritic-like cells and antigen-presenting cells are both increased in number and primed for activation by a second stimulus like endotoxin which then results in secretion of significant levels of inflammatory cytokines .73 GM-CSF increases neutrophil and monocyte survival, proliferation, differentiation, phagocytosis, and bacterial killing while also increasing cytotoxicity of macrophages.75 Specific to neutrophils, GM-GSF has been shown to increase bone marrow production and function of myeloid populations including degranulation, reactive oxygen species (ROS), phagocytosis, and adhesion, while decreasing neutrophil migration from tissues effectively immobilizing them at the site of inflammation.62 Clinically, this would be expected to decrease rates of nosocomial infection and opportunistic secondary infections as well as the resulting morbidity and mortality.While it clearly has important systemic effects on bone marrow proliferation and differentiation, it also has profound functional effects on existing myeloid cell populations. Further suggesting a proinflammatory role, GM-CSF increases mHLA-DR antigen receptor molecule expression which promotes antigen presenting cells and serves to boost the adaptive immune system.76 Later studies again using GM-CSF KO models confirmed impairment of pulmonary macrophages;77 in vitro, there was impaired phagocytosis and secretion of inflammatory mediators which translated to enhanced susceptibility to pneumonia.77 Intraperitoneal injection in a murine model and introduction of GM-CSF in human peritoneal dialysate increased the number of peritoneal macrophages.78 A relative deficiency of GM-CSF during sepsis could explain the propensity toward secondary infection that is improved with endogenous GM-CSF administration. Additionally, these GM-CSF KO mice also have impaired invariant natural killer T (iNKT) cell differentiation during thymic ontogeny as well as some reduced T-cell responses to antigen.80GM-CSF knock-out (KO) mice or GM-CSF overexpressing mice have further served to elucidate the wide-ranging functions of GM-CSF. Murine KO models of GM-CSF demonstrate little evidence of impaired myeloid cell development or defense against diverse pathogens; however, these mice do show impaired pulmonary macrophage clearance of surfactant resulting in a pulmonary alveolar proteinosis.18 however, the functional capacity of these cells and the possible clinical implications has not been elucidated. When examining a GM-CSF overexpression murine model that is absent of other disease states, macrophage functions, including inflammatory cytokine production, are upregulated and a lethal myeloproliferative state is induced consisting of macrophage accumulation, tissue damage, progressive weight loss, and premature death.82 These murine models serve to highlight the potential roles of GM-CSF; however, their applicability to the human diseased state which is a complex interplay of cytokines and cells with a varying amount of GM-CSF is limited.Human studies of GM-CSF therapy in sepsis have also documented increased T-cell lymphocytes;84 there are several studies that implicate the role of GM-CSF as a predominantly local effector. GM-CSF has been extensively studied in autoimmune disease processes and has been implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and other inflammatory conditions such as asthma. Increased concentrations of GM-CSF are found at sites of inflammation in autoimmune diseases and GM-CSF depletion has been consistently shown to be beneficial in suppressing several autoimmune disease states.34 In RA, GM-CSF in synovial fluid acts as a biomarker for disease severity and responsiveness to therapy.85 Paradoxically, GM-CSF administration, rather than suppression, has also been proven to be effective in certain instances. While many of these studies have focused on clinical endpoints, there has also been a great deal of immunologic study.Although GM-CSF appears to have a systemic role and is systemically elevated in states of inflammation and infection,86 GM-CSF action in vitro appears to be limited in the absence of additional stimulus , and both TNFα and IL-1 increase GM-CSF production by several cell types.89 Interestingly, GM-CSF can be produced circulating T-cells suggesting a humoral role, or by resident tissue cell types such as keratinocytes, smooth muscle cells, endothelial cells, epithelial cells, and neurons, suggesting a more paracrine effect.88 The increased production of GM-CSF by various cell types in response to IL-1 and TNFα and the positive feedback have led some to suggest the presence of a “CSF network” that potentiates a chronic inflammatory state.90 The ability of GM-CSF to increase host defenses could result in decreased microorganism load and explain the benefits of administration in sepsis.Further supporting GM-CSF as a proinflammatory agent, there is a link between GM-CSF and proinflammatory cytokines TNFα and IL-1, as well as the IL-23-IL-17 pathway.91 prevent atherosclerosis progression,92 and have dual function, as both inflammatory and anti-inflammatory, in pulmonary fibrosis.94 The explanation for these findings is unknown but could be attributed to dose, specific disease states, or other as yet undiscovered immune complex interplay. These far-ranging proinflammatory effects spurred a debate regarding timing of administration using the previous model of SIRS/CARS with an attempt to target the later CARS immunosuppressive state.In stark contrast to these proinflammatory findings, GM-CSF also has the capacity to function as an anti-inflammatory agent. It has been shown to enhance viral replication and promote viral infection,18 there is no standardized method of administration and timing of administration varies in the published literature. However, the emergence of PICS creates an entirely new ideology from which to approach the timing of GM-CSF administration.Since GM-CSF promotes both the number and function of inflammatory cell populations, there has been concern that early administration, during the hyperinflammatory phase, could exacerbate tissue injury by causing a detrimental proinflammatory cascade of cytokines via macrophage stimulation. Therefore, the timing of GM-CSF administration, specifically after the onset CARS, was thought to be crucial to its efficacy. While one study utilized mHLA-DR as a marker for immunosuppression and initiated therapy at a chosen threshold,The PubMed database was queried using keywords GM-CSF, sepsis, and sargramostim . This yielded articles that included pediatric populations, adult populations, and murine models. Pediatric findings were excluded. Journal articles as well as meta-analyses written in English and dealing specifically with GM-CSF as it pertains to clinical outcomes were included. Institutional review board (IRB) approval was not necessary as this is a review paper.GM-CSF has been extensively studied in both murine and human models and across age groups. The primary endpoint of interest in these clinical studies has been 28-day survival. Although these studies have universally failed to show a reduction in short-term mortality, several clinical secondary endpoints have been shown to be significantly improved; however, these endpoints may not be generalizable to the heterogeneous population of septic patients. The disconnect between improved clinical status and lack of 28-day survival benefit has led some to conclude that GM-CSF may be of limited benefit; however, the recognition of PICS begs for the evaluation of long-term end-points.The murine model of sepsis has well known benefits and drawbacks, especially when attempting to translate and reproduce findings in humans. Specific to GM-CSF therapy is again the issue of timing of GM-CSF administration. The murine model allows administration that ranges not only from early to late, but also as prophylactic administration.52 The implications and feasibility of implementing this in a human model have not been well studied.Implementation of GM-CSF therapy in a prophylactic manor in the human sepsis population presents many clinical challenges. However, prophylaxis in a murine model of sepsis has consistently shown increased host resistance to bacteria as well as a survival benefit.95 An ex vivo study has similarly found that GM-CSF inhibits neutrophil migration across IL-1-activated endothelium; interestingly, they also found that GM-CSF enhances neutrophil migration across unstimulated endothelium.17 This could potentially explain the findings from murine models of sepsis where early administration is associated with adverse outcomes. The finding of hepatic damage in GM-CSF treated animals has been observed in other GM-CSF studies in murine models. These findings suggest that systemic GM-CSF administration may be detrimental in those clinical conditions associated with an early exaggerated inflammatory response. This study also raises the question of whether GM-CSF should be administered systemically or whether it is meant to act locally at the site of the primary infection.95Early studies, concerned with the safety of GM-CSF given its potential to exacerbate the exaggerated early inflammatory state of sepsis, studied the effects of GM-CSF administration in a septic model on vital organ function. One study administered a single dose of GM-CSF 3 hours after the induction of peritoneal sepsis by a cecal ligation and puncture model (CLP), and found increased centrilobular degeneration and necrosis in the liver, decreased leukosequestration in the peritoneal cavity suggesting impaired migration, and earlier death.51 Survival benefits were also demonstrated in a murine model of trauma.30 These studies are not as generalizable to a sepsis model as they include other confounding clinical factors.A later study found improved survival with GM-CSF therapy in a more complex murine model that included CLP, burn, and transfusion.Importantly, no human studies to date have shown significant adverse effects from GM-CSF administration. Due to the wide-ranging effects of GM-CSF, it may produce dramatically different results depending on when it is administered in the time course of illness. The difficulty when comparing clinical trials is the lack of homogeneity regarding dosage, route of administration, pharmacologic GM-CSF subtypes used, patient characteristics, and outcomes measured. The primary outcome most frequently studied has been 28-day all-cause mortality. Most studies were performed with implementation of treatment after the onset of sepsis and with the goal of implementing GM-CSF therapy after the acute inflammatory phase had ended, which is based off of the SIRS/CARS model. Both the unpredictable nature of sepsis in an inpatient population and the frequent presence of sepsis on admission make prophylactic administration of GM-CSF in adults challenging, and the efficacy of this prophylactic administration is lacking in the published literature.96 as well as another therapy (n = 8 RCTs).97 There was no difference in 14-day, 28-day, or in-hospital mortality; however, earlier resolution of infection was significantly increased and there were no significant adverse events. The drawback of this meta-analysis is that GM-CSF was not the sole therapy studied in several of the studies, and was the focus of study in a minority of papers. A major critique of many early studies was the implementation of GM-CSF therapy without documentation that the patient was in a state of immunosuppression. To address this, a multi-institutional trial has utilized mHLA-DR expression on CD14+ blood cells as a biomarker for severity of immunosuppression, and subsequently monitored immune recovery with GM-CSF therapy.18 This importance of the timing of administration since the recognition of PICS is less clear; due to the concurrent nature of inflammatory and anti-inflammatory processes, one might argue for the implementation of GM-CSF at the onset of disease. While there was again no difference in 28-day mortality, there were significant improvements in alternative clinical end-points as detailed below.The number of study participants in published GM-CSF papers is often very low, a meta-analysis is helpful in evaluating the potential benefits for GM-CSF therapy. Four studies, discussed in greater detail below, were included in a meta-analysis that consisted of 12 placebo-controlled randomized controlled trials (n = 2380) that included GM-CSF (n = 4 RCTs)68 Lower mHLA-DR expression has been shown to be indicative of poor immune cell function,14 increased rates of nosocomial infection,13 and has been associated with reduced survival;99 however, some controversy remains as some authors did not find this association.100 One study was not powered to evaluate mortality, which was not significantly different. Their goal was to evaluate mHLA-DR expression as a possible biomarker of sepsis, and to validate previous ex vivo studies showing increased mHLA-DR expression in response to GM-CSF while tracking clinical end-points to 28-days. This was a prospective, randomized, double-blind, placebo-controlled multi-institutional study (n = 38) that evaluated patients in a state of severe sepsis or septic shock with low mHLA-DR expression. These patients were treated for 8 days with GM-CSF therapy and their outcomes were followed for 28 days.In an attempt to quantify immune suppression, mHLA-DR expression was studied as a potential biomarker to both diagnose immune suppression and monitor response to therapy.18 Although this study noted quantitative differences, they did not study the qualitative differences; however, a prior study did examine the effects of GM-CSF on leukocyte function.This study found that GM-CSF therapy increased mHLA-DR expression; however, the study unfortunately did not fully assess monocyte function. Increases in neutrophils, monocytes, and T-lymphocytes were documented but again, the function of these cells was not fully assessed. A change in cytokine profile toward inflammatory cytokines  with a concurrent decrease in anti-inflammatory cytokines (IL-10) in the GM-CSF-treated patient population was observed. Clinically, GM-CSF therapy significantly decreased length of mechanical ventilation, and there was a trend toward decreased ICU and hospital days as well as disease severity score. Of note, the control group had a trend toward more severe disease. No adverse events directly attributed to GM-CSF therapy were reported.96 Of note, one-third of the study population was undergoing organ transplant, severe sepsis and septic shock were excluded, and there was no standardization of antibiotic regiment. The GM-CSF group was found to have a significantly higher increase in total leukocyte counts which corresponded to increased rates of infection clearance and clinical improvement. However, there was again no observable difference in mortality. Ex vivo assays demonstrated an increase in inflammatory activation in GM-CSF-treated patients. Specifically, there was an increase in adhesion molecules that aid in transendothelial migration of neutrophils and monocytes. This study additionally confirmed an increase in mHLA-DR expression in the GM-CSF-treated patients.96 While the clinical efficacy of GM-CSF appeared limited to the rate of infection clearance, this is a largely underpowered study and more homogenous septic populations have resulted in additional significant improvements in clinical markers.A randomized, un-blinded, placebo-controlled prospective study (n = 40) evaluated GM-CSF efficacy on clinical markers to 28-days, and on leukocyte function in septic patients.22 Only clinical markers were measured, leukocyte number and function was not reported. There were multiple significant improvements in clinical end-points. GM-CSF significantly decreased hospital length of stay, median duration of antibiotic therapy, number of infectious complications, and direct medical costs.22 While this study has limited applicability to the larger heterogeneous population of septic patients, it further reinforces with the idea that GM-CSF therapy may be beneficial especially in subsets of patients.To control for the heterogeneity of the septic population, 1 trial limited patient selection to abdominal sepsis. A randomized, double-blinded, placebo-controlled clinical trial (n = 58) studied GM-CSF effects in patients with nontraumatic abdominal sepsis that underwent surgical intervention.38 There was also increased function of circulating neutrophils and pulmonary phagocytes. Again, no survival benefit was found and no adverse events attributable to the drug were documented.An additional study again chose to focus on a specific patient population consisting of patients with severe sepsis and respiratory dysfunction. This study was a randomized, double-blind, placebo-controlled study (n = 18) that showed improved gas exchange which corresponded to decreased alveolar neutrophils.There are, unfortunately, many limitations to the conclusions that can be drawn from the current body of research since most of the studies are small and have been underpowered for clinical outcomes, especially survival. However, multiple studies have demonstrated the benefits of GM-CSF therapy on clinically significant patient end-points, and 1 study on medical costs. Further research is needed to identify those patient populations most likely to benefit from GM-CSF treatment and larger trials in these patient subsets may provide more insight into possible survival benefits. Although no adult trials have documented survival benefit from GM-CSF therapy, there has been a significant survival benefit in neutropenic septic neonates. In the context of PICS immune dysfunction the role of GM-CSF in preventing chronic disease states progressing to indolent death demands to be studied. GM-CSF appears to combat some of the immune processes that are attributed to PICS as detailed earlier and the potential of this agent, possibly in combination with other agents, may be the as-yet undiscovered therapy to prevent and combat PICS.102 Due to differences in their baseline immune function, it would follow that they may have differing responses to GM-CSF therapy when compared with the adult population. Although there have not been any studies focusing specifically on the elderly, the clinical impact of GM-CSF in the neonatal population requires a separate review because of the extensive body of published research. This body of research is growing, and like the adult studies there have been promising results in several clinical end-points. Interestingly, there also appears to be a significant impact on survival in a small subset of neonatal neutropenic patients. A Cochrane review published in 2003 evaluated 7 studies that examined administration of GM-CSF after the onset of sepsis and 3 studies that employed GM-CSF as a prophylactic therapy.103 While they found no decrease in all-cause mortality, there was significant reduction in mortality in neonates with systemic infection and neutropenia found in a prior meta-analysis.104 However, these studies had small numbers and require further validation. Like the adult population, research into the applicability of GM-CSF therapy in subpopulations of septic patients is ongoing.The elderly and the very young have been shown to have unique immunologic deficits that predispose them to increased rates of sepsis and increased resultant morbidity and mortality.34 Studies that either focused on G-CSF or failed to differentiate between the 2 are therefore difficult to interpret. The failure to recognize GM-CSF and G-CSF as distinct compounds is a shortcoming of several available meta-analysis publications.Although GM-CSF and granulocyte-colony stimulating factor (G-CSF) have many overlapping downstream effects, they also have functions that are unique. G-CSF has been shown to more heavily impact antimicrobial defense, while GM-CSF has been shown to restimulate antigen-presenting cell function and bolster adaptive immunity.Although GM-CSF is FDA-approved for chemotherapy-induced neutropenia, its off-label use for sepsis has resulted in highly variable dosing regimens. This has led to varying routes of administration and dosage among publications; some studies gave continuous infusion while others implemented several days of varying dosages and dosage intervals. The potential differences of implementing GM-CSF systemically versus locally have also not been evaluated; systemic administration appears to effect a mobilization from the bone marrow while local administration likely affects the proinflammatory effects demonstrated in vitro. In contrast to murine models that prophylactically implemented GM-CSF, essentially all of the human adult studies implemented GM-CSF therapy after the onset of disease. The timing for starting GM-CSF therapy varied; some studies started administration at the time of diagnosis, others chose a specific day status-post onset of sepsis , while others measured biomarkers thought to be indicative of an immunosuppressed state and only administered GM-CSF once the patient was proven to be in an immunosuppressed state. A critique of many of the early papers is the failure to stratify patients according to their immunological states by utilizing biomarkers or direct measures of immune function; however, in the context of PICS this concept needs to be re-examined. Another variable to consider is the lack of standard manufacturing of GM-CSF resulting in pharmacologic subtypes, mostly varying in their glycosylation patterns. Additionally, the variability in patient characteristics alone can be difficult to control for and the inclusion and exclusion criteria varied between studies; some studies specifically evaluated severe sepsis and septic shock while other studies excluded these populations. Patient selection is further obfuscated by comorbid conditions, age, and genetic differences that are in some cases impossible to take into account. These trials often enter patients who will have good outcomes regardless of the intervention. The complexity of these variables could explain some of the observed differences across publications and the lack of cohesive study findings.105 Onset of disease can be secondary to infection from a number of organ systems. Infectious agents can vary from bacterial, to fungal or viral. The severity of the disease burden can vary extensively and can be difficult to quantify. Classification of sepsis into subcategories can be difficult. The feasibility of studying only 1 subtype of sepsis can limit enrolment numbers and present a challenge of power to the study. The analysis of certain subtypes of sepsis also limits the applicability of study findings to a broader patient population. The heterogeneity inherent when studying sepsis has likely been a major factor over the last several decades of pharmaceutical research; there are still no Food and Drug Administration approved drugs in the treatment of sepsis.106Sepsis is a complex disease state with variable etiologies.Sepsis continues to be a significant source of morbidity and mortality despite advancements made over the last few decades. Many of these advancements have been in the recognition and management of the early acute hyperinflammatory sepsis or SIRS; however, these successes have led to the realization of a later stage of sepsis anti-inflammatory CARS. On further study, this biphasic model is an oversimplification of a more complex immune phenomenon of concurrent inflammation and immune suppression that can lead to a state of chronic inflammation with concurrent immunosuppression, termed PICS. This state leaves a patient not only vulnerable to secondary infection but in a state of protein catabolism that eventually leads to indolent death. The advent of CARS resulted in a growing body of research focusing on immunostimulatory agents to bolster the immune system and to improve outcomes without inducing exaggerated inflammatory activities. GM-CSF, a growth factor, with wide-reaching effects on the immune system has been studied in murine, ex vivo, and human models with the hope of improving survival to microbial infections and sepsis. The current body of research has many short-comings, 1 of which is studies that are underpowered to detect survival advantage and that examine only short-term survival. There has been no evidence of short-term survival benefit from GM-CSF therapy; however, select patient populations have demonstrated significantly improved clinical outcomes without deleterious side effects. These clinical improvements include more rapid recovery from infection, decreased hospital length of stay, decreased days requiring mechanical ventilation, and decreased medical costs. The widespread efficacy across all septic patient populations appears to be limited; however, for patients with abdominal sepsis and pneumonia, implementation of GM-CSF may improve short-term clinical outcomes and decrease direct medical costs.These findings, specific to GM-CSF, bring up several important questions when approaching immune-modulating treatment for sepsis. While GM-CSF does not appear to have a significant impact in all septic patient populations, there does seem to be a subset of patients that may benefit from therapy. For example, Presneill et al suggested that GM-CSF may have a homeostatic role in sepsis-related pulmonary dysfunction. It may be that the heterogeneity of septic foci and severity play a dominant confounding role and that select patient populations need to be defined and studied as largely separate entities. In addition, the heterogeneity of immune dysfunction present in septic patients means that targeting a sole cytokine or implementing a single agent therapy may not be able to significantly impact morbidity and mortality. Defining patient-specific immune deficits, finding diagnostic techniques that can be implemented in clinical practice, and then targeting these defects with a cocktail of therapies may be the only way to significantly impact mortality.The studies reviewed here were influenced by the SIRS/CARS model and they were implementing GM-CSF therapy in response to a late CARS state. However, PICS with a concurrent state of pro- and anti-inflammatory processes demands a re-evaluation of the timing of administration and pertinent end-points to be evaluated. The predominance of early death or death in the hospital setting from sepsis has been decreasing due to advances in critical care management and is giving way to a chronic disease state that leads to a late indolent death. It is this profound disability and late death that needs to be closely evaluated and targeted. The far-reaching immunologic impact of GM-CSF therapy should be able to combat many of the immunologic derangements that characterize PICS; the potential of GM-CSF to prevent and combat a PICS disease state is promising. A large prospective multi-institutional study with standardized administration of GM-CSF to all sepsis patients should be performed. The lack of significant GM-CSF deleterious effects argues the safety of broad application in early trials. The goals should be to stratify patient and disease states to identify subpopulations most likely to benefit from therapy. The end-points should be clinically relevant to pertinent in-hospital endpoints as well as long-term disability, hospital readmission, return to functional life, and late death.


To prevent disease transmission, 0.05% chlorine solution is commonly recommended for handwashing in Ebola Treatment Units. In the 2014 West Africa outbreak this recommendation was widely extended to community settings, although many organizations recommend soap and hand sanitizer over chlorine. To evaluate skin irritation caused by frequent handwashing that may increase transmission risk in Ebola-affected communities, we conducted a randomized trial with 91 subjects who washed their hands 10 times a day for 28 days. Subjects used soap and water, sanitizer, or one of four chlorine solutions used by Ebola responders , sodium dichloroisocyanurate (NaDCC), and generated or pH-stabilized sodium hypochlorite (NaOCl)). Outcomes were self-reported hand feel, irritation as measured by the Hand Eczema Score Index (HECSI) (range 0–360), signs of transmission risk , and dermatitis diagnosis. All groups experienced statistically significant increases in HECSI score. Subjects using sanitizer had the smallest increases, followed by higher pH chlorine solutions (HTH and stabilized NaOCl), and soap and water. The greatest increases were among neutral pH chlorine solutions (NaDCC and generated NaOCl). Signs of irritation related to higher transmission risk were observed most frequently in subjects using soap and least frequently by those using sanitizer or HTH. Despite these irritation increases, all methods represented minor changes in HECSI score. Average HECSI score was only 9.10 at endline (range 1–33) and 4% (4/91) of subjects were diagnosed with dermatitis, one each in four groups. Each handwashing method has benefits and drawbacks: soap is widely available and inexpensive, but requires water and does not inactivate the virus; sanitizer is easy-to use and effective but expensive and unacceptable to many communities, and chlorine is easy-to-use but difficult to produce properly and distribute. Overall, we recommend Ebola responders and communities use whichever handwashing method(s) are most acceptable, available, and sustainable for community handwashing.Trial Registration: International Standard Randomized Controlled Trial Registry ISRCTN89815514 First characterized following an outbreak in Zaire (now the Democratic Republic of Congo) in 1977 . The disPrior to the 2014 outbreak, Ebola emerged in remote, low population density settings, and outbreaks were contained primarily by medical responders. The West African outbreak began in Guinea with the death of a 2-year-old child in a remote forested area. It then spread to urban areas via healthcare workers, and then grew exponentially after the death of a businessman in the capital of Conakry . This waThe Ebola virus is transmitted through contact with an infected person or animal, their bodily fluids, or contaminated surfaces ,10. ContThere is a lack of clear evidence on the safety of these three handwashing methods that has led to inconsistent and even contradictory international guidelines. Doctors Without Borders (MSF) recommends that buckets of 0.05% chlorine solution are prepared and placed around ETUs for regular hand washing use by staff and caregivers . By contIn an effort to contain the sustained transmission of Ebola in urban centers, these handwashing recommendations initially meant for ETU settings were extended to the community level in the West African outbreak. In particular, handwashing with 0.05% chlorine solution has been commonly adopted by government, health, and commercial facilities in West Africa . RespondAll of the recommended handwashing methods can lead to dermatitis, a condition of skin inflammation and skin barrier disruption that can cause transepidermal water loss, itching, redness, swelling, and an increase in disease transmission risk . There aBecause ICD does not require specific sensitization and can occur in a large percentage of the population, it presents a worrisome risk for groups required to perform numerous handwashings with potentially irritating solutions and therefore put individuals at risk of Ebola infection. ICD has been demonstrated in users of soap and water ,22, ABHSDifferences in recommendations cause us to question the best methods to prevent disease transmission when ETU recommendations are extended to the community setting. We investigated the impact of six recommended handwashing methods on skin irritation and dermatitis to better understand the risk that community level handwashing may pose during an Ebola outbreak.We conducted a randomized study during which subjects washed their hands 10 times per day for 28 days, and other potentially irritating personal-care products and activities were controlled. The six handwashing methods were soap and water, ABHS, 0.05% HTH solution, 0.05% NaDCC solution, 0.05% NaOCl solution produced with an electrochlorinator, and stabilized 0.05% NaOCl solution produced from laboratory grade NaOCl stock.The study took place on the Tufts University campus in Medford, MA beginning with recruitment on September 11, 2015 and concluding with endline data collection on November 18, 2015. The clinical outcomes were: 1) patch test results, 2) self-rated hand evaluation, (secondary outcomes) 3) Hand Eczema Score Index (HECSI) evaluation, and 4) diagnosis of dermatitis (primary outcomes). We completed the following activities during which these factors were assessed: 1) recruitment and enrollment, 2) baseline assessment and patch testing, 3) daily handwashing intervention and evaluation, and 4) endline patch testing. The study was approved by the Institutional Review Board at Tufts Medical Center and Tufts University Health Sciences Campus (#11818); Harvard University ceded review to the Tufts Institutional Review Board. Here we describe first the key study outcomes, and then the study activities during which outcomes were assessed. This study was not registered as a clinical trial prior to the start of subject enrollment because the Institutional Review Board did not consider the study to be a clinical trial. The authors confirm that all ongoing and related trials for these interventions are registered.Patch testing is the gold standard for assessing whether a substance induces an allergic reaction on the skin, and testing was performed prior to the intervention and after the completion of the study period according to a typical patch testing protocol used by dermatologists . ResearcSubjects used a scale from 0–10 developed by the research team to self-rate the level of discomfort that they were currently experiencing on their hands along with nine different symptoms, including itchiness, pain, redness, flaking, cracks in the skin, skin thickening, swelling, bumps, and blisters.The HECSI system is a sensitive and standardized clinical grading approach for hand eczema and dermatitis, and allows comparison of minor changes in skin irritation . The sysSubjects would receive a consultation with the dermatologist if they received a HECSI score >68. To ensure protection of subjects, two photos, one of the backs of hand and one of the palms were also taken daily, uploaded to a secure study server nightly, and reviewed within 24 hours by the board-certified dermatologist to screen for issues of clinical concern. Subjects would be removed from the study if they received a HECSI score >270, or if concern was noted by the dermatologist.Dermatitis was diagnosed by a board-certified dermatologist who examined subjects in person on campus at Tufts University on the last day of the study and classified cases as mild, moderate, or severe. The dermatologist was blinded to the intervention arm of each subject examined.A total of 108 subjects were recruited and signed written informed consent forms . SubjectOne week before the intervention, a baseline assessment was used to assess usual hygiene behaviors and confirm that subjects met study criteria. Researchers administered a short survey, including questions about demographics; history of atopic  disposition; handwashing behavior; and current skin, household, and hygiene product use. Additionally, enumerators asked subjects to respond to the self-rated hand score assessment. Subjects’ hands were examined and scored on the HECSI scale by a trained researcher. Lastly, subjects were patch tested.Subjects who completed the baseline assessment and met all study criteria were randomly assigned to one of six handwashing treatment arms . Block randomization with a block size of 18 was performed by a researcher not involved in day-to-day study activities after all subjects were enrolled using a random number generator to assign 18 subjects per arm. Subjects were informed of whether they were part of the soap and water, hand sanitizer, or chlorine arms and given a drawstring bag with materials and instructions for handwashing. Subjects were asked to wash their hands ten times each day with their assigned method according to the instructions provided, and returned to the study office each day for 28 days for hand examination, supply refill, and to answer a short questionnaire.Soap and water and ABHS handwashing protocols were based on WHO’s hand hygiene technique guidelines , and chl™ Cleansing Bar  for use when showering or for handwashing beyond 10 times a day, and commercially available heavy-duty vinyl gloves  for use during household activities such as dishwashing. Subjects were also provided with commercially available Vaseline petroleum jelly  as a moisturizer. These products were chosen because they are hypoallergenic and fragrance free. In particular, control soap was chosen to be less irritating than intervention soap to prevent interference with intervention effects.Subjects were asked to refrain from using their usual shampoo, conditioner, soap, and moisturizer and to avoid contact with cleaning substances to control for other potential sources of irritation. Subjects were provided with donated Free and Clear Shampoo and Conditioner and a VanicreamChlorine solutions were produced daily in the Environmental Sustainability Laboratory at Tufts University and their concentrations confirmed to be within 10% of a target 0.05% solution daily using Hach Iodometric Titration Method 8209 . NaDCC solution was produced using donated Klorsept granules (previously Aquatabs) with 50% active chlorine . HTH was produced from commercially available granular calcium hypochlorite with 65% available chlorine , and the solution allowed to settle for 24 hours and decanted to avoid precipitate. Two types of NaOCl were produced by: 1) using table salt without added iodine and an Aquachlor on-site sodium hypochlorite generator , and 2) diluting a 5.25% lab-grade pH stabilized bleach stock solution . Milli-Q deionized water was used for mixing all solutions, and pH was measured daily using Hanna Instruments HI 9811–5 portable pH/EC/TDS/°C meter, calibrated with non-expired solutions . Expected pH values were 6–7 for NaDCC, 10.4–10.8 for HTH, 10.8–11.4 for stabilized NaOCl, and 9 for generated NaOCl.Each day subjects visited the study office for a survey, hand evaluation, and supply refill. Subjects first filled out a self-administered survey asking them about their handwashing behavior and use of provided control products and products outside the protocol. Additionally, subjects were asked about household cleaning activities during the previous day. Subjects then had their hands examined and scored according to the HECSI system by a trained researcher. Lastly, subjects were required to bring their handwashing materials with them each day and were provided with refills as needed. Researchers observed whether or not the materials appeared to be used, recording the amount of chlorine and ABHS remaining in the bottle, and observing if the soap was wet.On day 28 of the intervention, subjects performed normal daily evaluation activities, and also: 1) reported their level of compliance over the course of the study anonymously on paper forms, identifying themselves only by group assignment, 2) were examined by a board-certified dermatologist for dermatitis, and 3) had a second round of patch testing applied.Data were collected electronically using Qualtrics software , and analysis was completed using Stata 14 . A chi-square test or Fisher’s exact test was used to assess differences in baseline group characteristics. Student’s paired t-test was used to evaluate differences in self-rated hand score and HECSI score from baseline to endline. Linear regression was used to evaluate the association between the HECSI score, intervention arm, and other potential effect modifiers including gender, atopic disposition, weather, and moisturizer use. We constructed two models: 1) using soap as a comparison because it is the most common recommendation, and 2) using the handwashing method with the lowest HECSI score as a comparison. Subjects who reported a history of asthma, seasonal allergies, hayfever, or childhood eczema were categorized as having an atopic disposition. We also divided the six components of the HECSI score into higher and lower transmission risk categories. Higher transmission components included vesicles, scaling, and fissuring (i.e those manifestations with greatest disruption to the skin barrier), and erythema, papulation, and edema were considered lower transmission risk. Logistic regression was used to evaluate the association between the presence/absence of high transmission risk components with the same explanatory variables as the linear regression. There were no adjustments made for multiple comparisons and nominal p-values are reported.Overall, 108 people registered for the study, 99 completed the baseline assessment, and 91 completed all study activities. Subjects who left the study prior to the baseline assessment were found not to meet study criteria or removed themselves due to inability to complete all study evaluations. All 8 subjects who left the study after baseline described their reason as an inability to complete daily evaluations with the study team. Thus, all data is presented with an n = 91.Subjects were 58% female, 57% self-described their race as “white,” and 49% of subjects were classified as having an atopic disposition . Broken Subjects self-reported handwashing an average of 8.8 to 9.2 times by study group with their handwashing study method and there was no significant difference in compliance by group found by linear regression . All chlAt the baseline assessment, subjects reported an average self-reported hand score of 0.88 out of a possible 90 (range 0–9). On the final intervention day, subjects reported an average score of 1.18 (range 0–11). This change in discomfort over the course of the intervention was not statistically significant (p = 0.262). At baseline and endline the most prevalent self-reported sign of irritation was redness, present in 16% at baseline and 31% at endline.At baseline, the average HECSI score was 1.76 out of a possible 360 (range 0–10) . At endlAcross all arms, days of handwashing (p<0.001) and male gender (p<0.001) were associated with a higher HECSI score, while atopic disposition (p<0.001) and higher average humidity (p<0.001) were associated with a lower HECSI score .In comparison to the soap treatment, ABHS resulted in a significantly lower HECSI score (p = 0.019), while NaDCC resulted in a significantly higher HECSI score (p<0.001) . We wereThe group with the lowest HECSI was ABHS, which was therefore used as a reference group for a second model. In comparison to the ABHS treatment, soap (p = 0.019), NaDCC (p<0.001), and generated NaOCl (p<0.013) were all associated with a higher HECSI score when corrected for potentially confounding factors and effect modifiers . We wereErythema (redness) was the greatest contributor to the HECSI score . Over thIn logistic regression analysis, days of intervention (p = 0.002) and male gender (p<0.001) were significantly associated with higher odds of transmission risk . HistoryIn comparison to soap, ABHS (p = 0.006), HTH (p = 0.001), and stabilized NaOCl (p = 0.020) treatments were all associated with a significantly lower odds of transmission risk, and NaDCC (p = 0.306) and generated NaOCl (p = 0.477) did not have significantly different odds .In comparison to ABHS, soap (p = 0.006) and generated NaOCl (p = 0.050) had significantly higher odds of transmission risk, but there was no significant difference with HTH (p = 0.500), NaDCC (0.102), or stabilized NaOCl (p = 0.726) .At endline, 4% (4/91) subjects presented with clinical dermatitis, one in each of four of the six total groups: soap, NaDCC, generated NaOCl and stabilized NaOCl. There was no significant difference in dermatitis among groups (p = 0.824), and all cases were classified as “mild.”We completed a randomized study including 91 subjects who washed their hands with six different commonly used methods for 28 days with a high level of compliance. Subjects demonstrated low levels of irritation at baseline, and across all groups there were significant increases in skin irritation. ABHS was associated with the least irritation and fewest signs of potential Ebola transmission risk related to skin barrier disruption followed by high-pH chlorine solutions (HTH and stabilized NaOCl), and then soap and water. Neutral-pH chlorine solutions (NaDCC and generated NaOCl) resulted in the most irritation. Handwashing with soap and water was associated with the greatest odds of signs of potential transmission risk, while ABHS and HTH were associated with lowest odds. These results suggest that irritation varies with handwashing method and chlorine chemistry, particularly pH. Only 4% of subjects developed mild dermatitis by the end of the study period, despite the observed increases in irritation. Thus we found that no handwashing method presented a significant transmission risk by compromising skin integrity.Our study showed some surprising results, such as the lower odds of transmission risk associated with history of atopic disposition. In many studies atopic diathesis, and specifically atopic dermatitis, has been associated with more severe hand dermatitis ,37. BecaThe most surprising result of the study was that higher pH dilute chlorine solutions were associated with lower HECSI scores. Both scientific literature and recommendations amongst producers and users of hygiene products suggest that pH-neutral or slightly acidic skin preparations should to help preserve the “acid mantle” of the outer layers of the skin by matching skin pH ,38,39. WOther results of our study are consistent with findings in the literature on skin irritation and dermatitis, including the impact of humidity and use of moisturizer as effect modifiers reducing irritation . Our resMaintaining skin integrity is essential to prevent Ebola transmission. We documented a slight increase in skin irritation related to handwashing; however, people often have compromised skin integrity for other reasons. Our subject population of primarily university students had small wounds on their hands such as burns, paper cuts, or blisters from recreational activities. In a population that does more work with their hands, we would expect these percentages to be higher. Thus, community handwashing is unlikely to represent the biggest assault on skin integrity.We also note that significant variation exists within all three recommended handwashing methods tested. Soap may refer to a range of products, from moisturizing formulas intended for frequent use in hospitals to harsher detergents and bar soaps. Alcohol-based hand rubs produced as gels with elevated levels of glycerine are more hydrating . We expeSimilarly, chlorine solutions may be made from the range of compounds we tested in this study. Guidelines for chlorine use do not take into account the variation in chlorine properties or specify a recommended type, and these differences have not been well investigated until now. It has been suggested that NaDCC may be preferable because it has a pH similar to that of human skin . HoweverAside from skin irritation, there are benefits and drawbacks to each of the methods. Soap is widely available, familiar, and acceptable, but it requires water, can be easily stolen from handwashing stations, and does not inactivate the Ebola virus. ABHS is the easiest and fastest method, requires no water, and inactivates viruses similar to Ebola. However it is very expensive because it must be imported or produced locally using ingredients which must be imported , and it Our study focused on simulating a level of handwashing typical at the community level during an Ebola outbreak. Healthcare workers wash their hands more frequently than the ten times daily that was required of our subjects—around 2–15 times per hour or 5–42 times per day/shift . More frOur study is limited by differences in context between the Tufts campus in Medford, MA and the centers of the Ebola outbreak in West Africa, including weather and subject characteristics, and also by sample size. The weather during our investigation was cooler and drier than most Ebola affected areas, and we did not collect information on time spent outdoors. The majority of our subjects were both young and full time students. Skin loses collagen and elasticity as people age ,49, and Our results suggest that the recommendation that chlorine should only be used for handwashing if there are no other options available is overly general and may not represent the best approach to limit increased disease transmission risk from development of dermatitis. We found that no handwashing method should be considered a significant transmission risk at frequencies of handwashing typical to communities. Further research should be done to explore the impact of these handwashing formulations in a context more similar in demographic makeup and environment to the outbreak area in West Africa, and should take into consideration the interaction of these methods with PPE and other potential irritants. Lastly, handwashing behavior change and compliance is a well-documented challenge . Thus, wS1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.


Introduction: Exercise performance is reproducible in experienced athletes; however, less trained participants exhibit greater variability in performance and pacing. To reduce variability, it is common practice to complete a familiarization prior to experimental testing. However, there are no clear guidelines for familiarizing novice participants to a cycling time-trial (TT), and research findings from novice populations may still be influenced by learning effects. Accordingly, the aims of this study were to establish the variability between TTs after administering differing familiarization protocols (duration or type) and to establish the number of familiarization trials required to limit variability over multiple trials.Methods: Thirty recreationally active participants, with no prior experience of a TT, performed a 20-km cycling TT on five separate occasions, after completing either a full , a half  or an equipment familiarization .Results: Variability of TT duration across five TTs was the lowest after completing FF  compared to HF  and EF . In the FF group after TT2, the effect size for changes in TT duration was small (d < 0.49). There were large differences between later TTs in HF  and EF . The variability in mean power output profiles between trials was lowest within FF, with a similar pacing profile reproduced between TT3-TT5.Discussion: Familiarization of the exercise protocol influenced reproducibility of pacing and performance over multiple, maximal TTs, with best results obtained after a full experience of the exercise compared to HF and EF. The difference of TT1 to later TTs indicates that one familiarization is not adequate in reducing the variability of performance for novice participants. After the FF and an additional TT, performance changes between TTs were small, however, a reproducible pacing profile was not developed until after the FF and two additional TTs. These findings indicate that a minimum of three full familiarizations are necessary for novice participants to limit systematic error before experimental testing. During exercise testing, particular care is needed to ensure reliability from testing procedures, equipment, and the “internal” ability of participants to achieve the goals of the task Hopkins, . As suchPrevious studies have shown that cycling TT duration and mean power are relatively reproducible in trained cyclists  participants who were recreationally active, whilst relatively inexperienced at cycling, volunteered for this study (Table full (FF), half (HF), or equipment (EF) familiarizations. In FF, participants performed a 20-km TT; in HF, they performed a 10-km TT, whilst in EF they performed 5 min of constant pace (75 W) cycling, which enabled participants to learn the mechanics of the bike, without experiencing a self-paced TT . Prior to each testing session, a factory calibration was performed using the Accuwatt “run down” verification program (RacerMate Inc.) accompanying the ergometer software. Within the familiarization, participants set the ergometer to their own specifications, with values recorded and replicated for subsequent sessions. All TT protocols were controlled via Velotron Coaching software  with all courses being flat with no wind effect. TTs were conducted in the same laboratory, with regulated environmental conditions . No fan was provided to participants in all TTs, although they were permitted to drink water Preceding the TT, a warm-up (5-min cycling at 75 Watts) was conducted. In all TTs, participants were instructed to finish the required distance “as quickly as possible” by being free to change gear and cadence throughout the trial to what felt appropriate at the time. Changing of gear utilized the ergometer electronic gearing system with all TTs started in the same gear. To overcome flywheel inertia, participants were instructed to obtain a self-selected comfortable cadence immediately prior to beginning the trial, with the TT commencing with a verbal 3-s countdown from the researcher. Throughout all TTs, participants were blinded from all performance information, except for distance covered, and received no encouragement from investigators. Participants did not receive any information on how they performed until all TTs were completed  and peak power output (PPO). The maximal incremental test involved incremental stages of 30 W/min commencing after a 3-min baseline period, cycling at a 30 W , or 60 W  resistance which is like the protocols used for those unaccustomed to cycling tasks  and CD-3A (CO2), AEI Technologies Inc., Pittsburgh, PA). VO2peak was calculated as the highest 30-s mean VO2. Peak power was extrapolated by using the formula [Peak power last completed stage (Watts) + time in the last stage (s)/60 × 30 (Watts)] . When normality assumptions were violated an equivalent non-parametric test was performed. Tests for homogeneity of variances were performed to ensure normality of the cohort for dependent variables. When homogeneity of variances was violated Welch F-ratio is reported. The level of significance for all tests was set at P < 0.05. In the instance of a significant main or interaction effect, post-hoc Sidak comparisons and t-tests were conducted to examine differences and its magnitude (effect size). Effect sizes for one-way and repeated-measures ANOVAs are reported as partial eta squared (ηp2) with a small effect at 0.01–0.059, medium effect 0.06–0.139 and a large effect > 0.14. Effect sizes for t-tests are reported as Cohen's d with a small effect being 0.2–0.49, medium 0.5–0.79 and large > 0.8  with data reported as mean ± 8 Cohen, .For data analysis, given the inter-participant differences in TT power output, power has been reported as a percentage of the individual's PPO obtained from the maximal incremental . To examine differences between participants in the three different groups , one-way ANOVAs were conducted on anthropometric variables.n = 3), with follow-up t-tests conducted to determine the effect size between familiarization protocols for each TT. To examine differences between trials within-groups, one-way repeated-measures ANOVAs  were conducted, with follow-up t-tests conducted to determine the effect size between TTs for each familiarization protocol. In addition, as the FF group familiarization was the same protocol as the TTs, t-tests were conducted to determine the difference between the familiarization trial and all other TTs in the FF group.To explore whether the three different familiarization protocols had an influence on performance in terms of TT duration and TT mean power, we conducted a three  by five (TT) repeated-measure ANOVA. Follow-up one-way ANOVAs were conducted to investigate differences between familiarization protocols , we calculated the coefficient of variation (CV) using the formula R2) for each mean power output profile. To compare the within-groups between trials variance, we considered the magnitude of change in R2 between trials as an indicator of variability in pacing profiles, with smaller changes in R2 considered as lower variability between trials.To prepare for pacing profile analysis, mean power output profiles were established by normalizing each TT to 2,000 data points  and mean power  with large effect sizes was found for TT duration  was found to be large (Table d) was smaller in the FF group compared to HF and EF. For FF group, there were small effect sizes between the familiarization and TT1, whilst between TT1 and TT2, the effect sizes were moderate. For HF and EF there were large effect sizes between TT1 and the other trials  for differences in TT1 duration and a moderate effect size for TT1 mean power. Between HF and EF groups, there was a large effect size (d) for TT1 duration and mean power. Between HF and FF groups, there was a large effect size (d) for TT1 duration, whilst mean power effect size (d) was moderate. The comparisons between FF and EF had a small effect size (d), whilst moderate effect sizes (d) were observed in TT2, TT3, TT5 duration between HF and EF groups  for all trials was established with the application of a cubic regression model. Change in R2 between the five trials was lowest in FF group, ranging from 0.43 to 0.58  for changes across all five TTs for performance measures (Table d) between TT1 and all other TTs in FF group  for performance changes between later TTs was small  was lowest within FF group (Figure Previous experience allows for an understanding of the physiological demands of the exercise so that an appropriate intensity can be initially set that requires less refinement throughout the task (Mauger et al., p Figure . Althougp Figure . Most imp Figure . This obp Figure  presumabp Figure . HoweverIn all groups, there was an apparent difference in the pacing profile of TT1 Figure , with EFAnother indication of pacing variability is that the end spurt of TTs became larger as TTs were repeated in both HF and EF groups. This likely indicates the adoption of a different pacing strategy in the early part of TTs. Specifically, with more experience, participants have a greater understanding of exercise demands and presumably adopt a more manageable approach during the TT that allows a higher intensity toward the end of the bout (Micklewright et al., In conjunction with the influence of prior experience, the knowledge of the actual performance of a task is also crucial in setting a successful pacing strategy (Micklewright et al., This investigation refines guidelines for familiarizing novice participants to a cycling TT. Application of a full exercise familiarization produced the greatest reproducibility in pacing and performance over multiple trials, compared to a half exercise familiarization and no experience. Within the FF group, there were small differences in TT duration between TT2 and other TTs, indicating only two familiarizations may be required. Yet, a stable pacing profile was not developed until TT3 Figure . Taken tPrior experience is an important moderator of self-paced performance. Therefore, it is important for participants to gain experience in an exercise before conducting experimental testing to establish a reproducible baseline performance. This study demonstrates that three familiarization trials of the exact experimental protocol should be administered to reduce variability across multiple trials in novice participants. This finding should be considered when interpreting the results of interventions that utilize self-paced tasks and unfamiliar participants. In conclusion, it is recommended that future investigations administer three familiarization trials to reduce systematic error before experimental testing.Conceived and designed the experiments: AH, FB, MV, and RP. Collected and analyzed data: AH. Interpreted results of research: AH, FB, and RP. Drafted manuscript and prepared table/figures: AH. Edited, critically revised manuscript and approved the final version of manuscript: AH, FB, MV, and RP.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Testicular sonography has contributed greatly to the preoperative diagnosis of testicular torsion in the pediatric patient and is the mainstay for evaluation of acute scrotal pain. Despite its high sensitivity and specificity, both false-negative and false-positive findings occur. Presence of documented Doppler flow within the testis might be a dissuading factor for surgical exploration with resultant testicular loss in the false-negative cases. Our goal is to illustrate key sonographic features in the spectrum of testicular torsion with preserved testicular flow, and to describe how to differentiate testicular torsion from epididymitis in order to avoid the under-diagnosis of testicular torsion. We simplify the anatomy of the bell clapper testis. We also describe our sonographic protocol for testicular torsion and share valuable tips from our approach to challenging cases.The online version of this article (10.1007/s00247-018-4093-0) contains supplementary material, which is available to authorized users. Recent investigators have emphasized the concept of intermittent and partial testicular torsion, which can be difficult to diagnose with sonography because these cases have either subtle decreased flow or flow that appears to be symmetrical with the contralateral testis, and symptoms can wax and wane . Such caComplete torsion occurs when the testis twists 360° or greater, usually leading to absence of intratesticular flow on color Doppler exam; however sometimes the flow is preserved or decreased. Intermittent torsion is defined as sudden onset of unilateral testicular pain of short duration with spontaneous resolution. In partial or incomplete torsion, the degree of spermatic cord twist is less than 360°, allowing for some residual perfusion to the testis. However there is no spontaneous resolution of pain .Testicular torsion has been reported to have a bimodal distribution, an initial peak in the first year of age where the torsion is of extravaginal type, and a second surge in adolescence where intravaginal torsion is common. Cases of intravaginal torsion with preserved flow and their critical sonographic findings are described in this review. Preserved flow indicates these cases did not have completely absent flow  on color Doppler exam. Instead, the affected testis had either decreased or symmetrical preserved flow compared to the contralateral unaffected side, sometimes even increased flow . In manyThe bell clapper anomaly has been defined as an abnormally high attachment of the tunica vaginalis parietal lamina to the spermatic cord such that the tunica vaginalis completely encircles the epididymis, the distal unattached spermatic cord and the testis rather than attaching to the posterolateral aspect of the testis Fig.  3].Fig..Fig.3].FNormally, the epididymis extends along the full length of the testis posterolaterally so that the upper and lower poles of the testis are covered and the tunica vaginalis parietal lamina is anchored to the epididymis. In a bell clapper anomaly, not only is there a lack of posterior attachment of the tunica vaginalis parietal lamina to the epididymis, but the epididymis–testis connection is also incomplete and the epididymis might be detached from the testis lower pole . DependiThe “whirlpool sign” is defined as an abrupt change in the course of the spermatic cord with a spiral twist at the external inguinal ring or in the scrotal sac  . AsymmetThe clinical features considered predictive for testicular torsion include pain <24 h, nausea/vomiting, abnormal cremasteric reflex, and high position of the testis . The besEpididymitis is inflammation of the epididymis Video 2(AVI 4892 kb)Video 3(AVI 9569 kb)Video 4(AVI 4047 kb)Video 5(AVI 5098 kb)Video 6(AVI 5497 kb)Video 7(AVI 1216 kb)Video 8(AVI 2159 kb)Video 9(AVI 3195 kb)Video 10(AVI 4450 kb)Video 11(AVI 1455 kb)


The postfunctionalization of the main chain alkyne moieties of carbazole containing poly(arylenebutadiynylene)s was attempted by using a high yielding addition reaction between electron rich alkynes and a strong acceptor molecule, tetracyanoethylene (TCNE). After successful postfunctionalization, the polymer band gap decreased due to the intramolecular donor-acceptor interactions. The resulting donor-acceptor alternating polymer showed a very broad charge-transfer band in the visible region as well as redox activities in both anodic and cathodic directions. The optical band gap showed good agreement with the electrochemical band gap. Furthermore, the thermal stability was enhanced after postfunctionalization. These features of the donor-acceptor alternating polymer are expected to be useful for high performance activities in organic solar cell applications. Since the discovery of conducting polyacetylene films, organic conjugated polymers have become an important class of materials for applications in the next generation of organic electronic devices . IntroduRecent trends of conjugated polymer development have been directed toward the design of more complex monomer repeat units. For example, donor-acceptor type conjugated polymers are composed of donor-acceptor repeat units and are now recognized as promising p-type semiconducting polymers for applications in bulk heterojunction organic solar cells . These pA conventional synthetic method for achieving donor-acceptor type conjugated polymers is based on the metal catalyzed polycondensation between a bifunctional electron donating monomer and a bifunctional electron accepting comonomer . This teWe recently started a new project on the synthesis of donor-acceptor type polymers by a novel postfunctionalization method based on a high yielding addition reaction between electron rich alkynes and strong acceptor molecules, such as tetracyanoethylene (TCNE) . The reaIn this article, we apply the high yielding TCNE addition reaction to aromatic polyamines containing main chain alkynes and show for the first time the postfunctional synthesis of purely organic donor-acceptor alternating conjugated polymers .N-hexadecyl-3,6-carbazolylene)butadiynylene] P1 and poly[butadiynylene] P3, were prepared by the acetylenic oxidative coupling of diethynyl-N-hexadecylcarbazole derivatives under the Hay conditions  -phenyl-C61-butyric acid methyl ester (PCBM) as an n-type semiconductor , 3.68 , 6.86–6.89 , 7.65 , 8.15 ppm . IR (KBr): ν 2921.6, 2851.2, 2223.5, 2156.0, 1730.8, 1623.8, 1592.9, 1520.6, 1481.1, 1462.7, 1384.6, 1349.9, 1286.3, 1238.1, 1133.0, 1019.2, 882.3, 804.2, 720.3, 590.1, 512.0 cm−1. Elemental analysis calcd for [(C32H39N)0.4(C38H39N5)0.6]n: C 83.52, H 7.78, N 8.71; found: C 83.18, H 8.27, N 8.55%.In a glass vessel, 15.01 mg  derivative was successfully converted into the donor-acceptor type polymer, while the 2,7-carbazole-based counter polymer did not react with TCNE due to insufficient activation of the alkyne moieties. The resulting donor-acceptor type polymer was characterized by