Dependent on the tissue, HDL is either resecreted (retro-endocytosis) or degraded after endocytosis. Finally, findings on HDL transcytosis across the endothelial barrier will be summarized. We suggest that HDL endocytosis and resecretion is a rather redundant pathway under physiologic conditions. In case of disturbed lipid metabolism, however, HDL retro-endocytosis represents an alternative pathway that enables tissues to maintain cellular cholesterol homeostasis.HDL removes excess cholesterol from peripheral tissues and delivers it to the liver and steroidogenic tissues via selective lipid uptake without catabolism of the HDL particle itself. In addition, endocytosis of HDL holo-particles has been debated for nearly 40 years. However, neither the connection between HDL endocytosis and selective lipid uptake, nor the physiological relevance of HDL uptake has been delineated clearly. This review will focus on HDL endocytosis and resecretion and its relation to cholesterol transfer. We will discuss the role of HDL endocytosis in maintaining cholesterol homeostasis in tissues and cell types involved in atherosclerosis, focusing on liver, macrophages and endothelium. We will critically summarize the current knowledge on the receptors mediating HDL endocytosis including SR-BI, F •HDL endocytosis and resecretion is accompanied by cholesterol exchange.•HDL endocytosis and resecretion might be an alternative pathway for lipid exchange.•HDL is catabolized by the liver and in tissues with high cholesterol demand.•Several redundant receptors were shown to mediate HDL endocytosis.•HDL endocytosis might be relevant in hepatocytes, endothelial cells and macrophages. Importantly, HDL removes excess cholesterol from peripheral tissues and cells. These cells include macrophage foam cells, which are directly involved in atherosclerotic plaque formation. HDL then transports this cholesterol to the liver for disposal into the bile. This process of peripheral cholesterol efflux to HDL and delivery to the liver is termed reverse cholesterol transport (RCT) and is important for the anti-atherogenic properties of HDL. Thus, high plasma HDL cholesterol levels are generally associated with reduced risk for cardiovascular diseases Recent studies suggest that HDL is not a mere transport vehicle for lipids, but is also a carrier of non-lipid cargo such as microRNAs In this review, we will summarize receptors and mechanisms for HDL endocytosis and resecretion focusing on liver, endothelial cells and macrophages as central tissues in atherosclerosis development.2Even though HDL endocytosis was described in the mid-seventies of the last century It was not until 1996 that scavenger receptor class B, type I (SR-BI) was cloned and characterized as an HDL receptor by the group of Krieger The transport of lipids from HDL to SR-BI is generally thought to occur via selective cholesteryl ester uptake, where only the lipid load of HDL is transferred without concomitant catabolism of the HDL particle itself Endocytosis and resecretion is not limited to HDL, but instead occurs with almost all lipoprotein classes: Similar to HDL endocytosis and resecretion, uptake and resecretion was described for LDL as well as Lp(a). It is estimated that approximately 20% of endocytosed LDL escapes lysosomal degradation By now, HDL endocytosis has been shown to occur in cell lines originating from various tissues (summarized in 3While LDL endocytosis is known to occur via clathrin-coated pits Studies in our lab have focused on extensive characterization of HDL endocytosis using different methodologies: Using ultrasensitive fluorescence microcopy in SR-BI over-expressing cells we found that HDL is endocytosed in clusters of approximately 10 particles. HDL clusters are rapidly transported towards the Golgi-apparatus (v = 0.52 μm/s), a process requiring functional microtubules Although the intracellular trafficking during HDL endocytosis has been characterized in many aspects, it is still unclear, if endocytosis is definitely necessary for selective lipid uptake. Several lines of evidence suggest that HDL endocytosis is not required for selective uptake: Liposomes containing purified SR-BI are sufficient for selective CE uptake It is thus unclear if HDL endocytosis is essentially required for selective uptake. However, it is clear that cholesterol exchange occurs during endocytosis, as several reports suggest modification of the HDL particle after endocytosis: In SR-BI overexpressing cells HDL size is more heterogeneous after resecretion, indicating HDL particle modification during retro-endocytosis 4While the majority of LDL is catabolized and degraded in lysosomes, degradation of HDL is generally hard to quantify. In human fibroblasts, degradation of HDL less than 5% compared to LDL 1-ATPase; see below), is a possible candidate mechanism facilitating HDL degradation. The independence of HDL degradation of SR-BI makes inhibition of HDL degradation a putative therapeutic strategy for raising HDL levels without concomitant inhibition of cholesterol clearance from plasma by selective uptake.The liver as a central organ in lipoprotein metabolism does not only mediate selective CE uptake from HDL, but also HDL endocytosis and degradation. HDL degradation in hepatocytes seems to be regulated by leptin and is impaired in ob/ob mice In contrast to the liver, the kidney is generally not regarded as a central organ in lipoprotein metabolism, because lipoproteins are too large to pass the glomerular filtration barrier. However, an important role is attributed to the kidney in apoA-I catabolism. The role of the kidney in lipid metabolism as well as the function of the HDL receptors cubilin and megalin are excellently reviewed elsewhere Taken together, HDL degradation occurs in metabolically highly active tissues and in cases, where extracellular cholesterol is limited. It would be interesting to study HDL degradation in malignant tumors, as they might rely on high amounts of exogenous cholesterol for proliferation. Indeed, SR-BI expression correlates with the degree of malignancy in melanoma cells , suggesting an increased demand for cholesterol in malignant metastatic tumors.55.1The liver is a complex organ composed of various cell types of parenchymal (hepatocytes) and non-parenchymal  origin. Besides nascent HDL formation, the liver plays a major role in the clearance of HDL derived cholesterol. After injection of radiolabeled HDL, 22% of liver associated HDL was associated with Kupffer and endothelial cells, suggesting the importance of these cell types for HDL metabolism 1-ATPase), which is normally localized in the inner mitochondrial membrane 1-ATPase induces the hydrolysis of ATP to ADP which in turn activates HDL endocytosis by a low affinity receptor that remains to be identified .In contrast to these in-vitro studies in SR-BI over-expressing cells, SR-BI knockout mice display unchanged holo-HDL uptake into the liver, whereas selective lipid uptake is considerably diminished entified . This enAnother receptor for hepatic holo-HDL particle uptake is scavenger receptor cluster of differentiation 36 (CD36), as recently reported by Rinninger's group 5.2Macrophages play a crucial role in the pathogenesis of atherosclerosis. Endothelial inflammation leads to recruitment and invasion of monocytes/macrophages which excessively internalize modified lipoproteins. Excess cholesterol uptake triggers macrophage foam cell formation and pronounced inflammatory responses. Subsequent accumulation of cells, lipid and matrix leads to necrotic breakdown of some lesions and thrombosis 2 showed a higher uptake than HDL32-like particles and hypothesized that HDL resecretion facilitates removal of excess cholesterol from cells HDL uptake and resecretion in cholesterol-laden mouse peritoneal macrophages was the topic of one of the first reports of HDL retro-endocytosis 5.3Interactions of lipoproteins with the vascular endothelium play a critical role in lipid homeostasis and atherosclerosis: First, endothelial cells mediate lipolysis of triglyceride-rich lipoproteins. Second, vascular tone and endothelial integrity can be regulated by lipoproteins . ThGlycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) was initially described as an HDL receptor and is highly expressed in endothelial cells in-vivo While vascular endothelial cells are studied extensively in terms of HDL transcytosis, HDL transport through lymphatic endothelial cells has been largely neglected previously. A recent report showed that lymphatic endothelial cells express ABCA1 and SR-BI, but not ABCG1 5.41-ATPase and its expression increases during adipocyte differentiation 1-ATPase mediates endocytosis and resecretion of free apoA-I. However, this apoA-I recycling is not accompanied by cholesterol efflux and its physiological function is unknown yet 1-ATPase is also involved in the regulation of HDL endocytosis remains to be investigated.Adipose tissue is the major storage site for triglycerides and thus plays a central role in energy metabolism. In addition, it is the largest reservoir for free cholesterol and a direct modulator of cholesterol metabolism: Adipocyte ABCA1 mediates cholesterol efflux in-vitro 5.5Platelets are directly involved in atherosclerotic plaque formation and thrombosis. As HDL cholesterol levels are inversely related to thrombosis risk, the interaction of HDL with platelets is under extensive investigation 6Despite the fact that HDL endocytosis has been known for more than 40 years, its physiological relevance is still under debate. HDL endocytosis does not seem to be a major contributor to maintain cellular cholesterol homeostasis. It is generally accepted that selective lipid uptake exceeds HDL protein uptake and is highly efficient In SR-BI knockout mice, HDL endocytosis in adrenals is enhanced, suggesting a compensation for reduced selective lipid uptake In case of low cholesterol availability and high cholesterol demand, cells seem to adapt to HDL endocytosis and degradation to supply themselves with cholesterol Another hypothesis for an essential function of holo-particle uptake is cholesterol delivery to distinct cellular pools. In hepatocytes, for instance, there is evidence for distinct functional cholesterol pools, one for bile-acid synthesis and one for direct cholesterol secretion into the bile Taken together, the role of HDL uptake and resecretion in cholesterol homeostasis is still insufficiently defined. However, the function of HDL as a transport vehicle is not restricted to cholesterol. Instead, HDL cargo comprises a multitude of lipid species, hormones, proteins and nucleic acids


Heterotopic ossification is a rare complication of musculoskeletal injuries, characterized by bone growth in soft tissues. Percutaneous antegrade intramedullary nailing represents the ‘gold standard’ for the treatment of femur shaft fractures. Minor bone growth is frequently seen around the proximal end of reamed femoral nails , which are asymptomatic and lack a therapeutic implication. The occurrence of excessive, symptomatic heterotopic ossification around the entry site of an antegrade femoral nail is rarely described in the literature.We present the case of a 28-year-old Caucasian woman who developed extensive heterotopic ossification around the reaming seeds of a reamed femoral nail. She developed severe pain and significantly impaired range of motion of the hip joint, requiring revision surgery for heterotopic ossification resection and adjunctive local irradiation. She recovered full function of the hip and remained asymptomatic at her two-year follow-up appointment.Severe heterotopic ossification represents a rare but potentially detrimental complication after percutaneous femoral nailing of femur shaft fractures. Diligent care during the reaming procedure, including placement of a trocar to protect from osteogenic seeding of the soft tissues, may help decrease the risk of developing heterotopic ossification after reamed antegrade femoral nailing. Heterotopic ossification (HO) represents a debilitating condition characterized by bone formation in soft tissue that normally does not calcify -3. HO oc2). The procedure was performed uneventfully and without intraoperative complications by a fellowship-trained orthopaedic trauma surgeon. The piriformis fossa starting point was easily identified intraoperatively in a standard fashion under fluoroscopic guidance, without excessive attempts for identifying the ‘perfect’ starting point. The length of the percutaneous incision was 2cm, as previously described [A 28-year-old Caucasian woman was an unrestrained passenger in a high-speed motor vehicle collision. She sustained a blunt chest trauma, a right closed transverse mid-shaft femur fracture Figure A, and a escribed . The IMNAfter surgery, the patient was mobilized with full weight-bearing on the right side (femur fracture), and with touch-down weight-bearing on the left side (nonoperative acetabular fracture). She was placed on low-molecular-weight heparin  for venous thromboembolism prophylaxis and discharged home after clearance by physical therapy on hospital day five. The patient followed up in the outpatient clinic for a wound check and staple removal at two weeks, and for radiographic assessment of fracture healing at two months Figure . At thatHeterotopic ossification (HO) after antegrade reamed femoral nailing is a common postoperative problem that, however, frequently lacks a therapeutic implication ,7,12. Bret al. demonstrated a significant decrease in HO incidence with nonreamed femoral nailing compared to traditionally reamed IMN (9.4 percent versus 35.7 percent) [Ongoing debate as far back as the 1980s centered on the notion that small incisions are not necessarily associated with a decreased extent of soft tissue trauma , a problpercent) ,17.Intuitively, one would argue that copious soft tissue irrigation prior to wound closure would decrease the risk of HO by washing out the osteogenic reaming debris. However, the study by Brumback and colleagues failed to support this notion, since a standardized wound irrigation with 3L of saline by pulsatile lavage did not decrease the incidence of HO . IschemiSymptomatic HO after reamed IMN of femur shaft fractures represents a common postoperative complication, albeit without clinical relevance in most cases. As outlined in the present case report, patients may rarely develop significant HO characterized by subjective pain and compromised hip function, up to full joint stiffness (ankylosis). Osteogenic reaming debris and operative soft-tissue injury appear to represent the main known root causes of HO after femoral nailing. Despite the absence of strong evidence, the diligent intraoperative care of the soft tissues in conjunction with the use of a trocar during the reaming process to avoid local seeding of reaming debris, and copious fluid irrigation, appear to represent intuitive parameters that can be influenced by the surgeon. Further research is required to fully understand the pathogenesis of HO and to determine risk factors, root causes, and preventability of this potentially detrimental complication.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.Drs Hak, Hammerberg, and Stahel have received occasional speaker’s honoraria from Synthes  in the past five years. The authors declare no other financial competing interests related to this case report.SB drafted the first version of this manuscript. PFS performed the patient’s revision surgery, designed the case report, and revised the initial draft of the paper. CM, EMH, and DJH wrote the discussion and significantly contributed to the critical revisions of the final manuscript. All authors read and approved the final version prior to submission.


We examine the physiological and lifestyle adaptations which facilitated the emergence of ostracods as the numerically dominant Phanerozoic bivalve arthropod micro-benthos.2 of modern normoxic seawater is 21 kPa (air-equilibrated water), a level that would cause cellular damage if found in the tissues of ostracods and much other marine fauna. The PO2 of most aquatic breathers at the cellular level is much lower, between 1 and 3 kPa. Ostracods avoid oxygen toxicity by migrating to waters which are hypoxic, or by developing metabolisms which generate high consumption of O2. Interrogation of the Cambrian record of bivalve arthropod micro-benthos suggests a strong control on ecosystem evolution exerted by changing seawater O2 levels. The PO2 of air-equilibrated Cambrian-seawater is predicted to have varied between 10 and 30 kPa. Three groups of marine shelf-dwelling bivalve arthropods adopted different responses to Cambrian seawater O2. Bradoriida evolved cardiovascular systems that favoured colonization of oxygenated marine waters. Their biodiversity declined during intervals associated with black shale deposition and marine shelf anoxia and their diversity may also have been curtailed by elevated late Cambrian (Furongian) oxygen-levels that increased the PO2 gradient between seawater and bradoriid tissues. Phosphatocopida responded to Cambrian anoxia differently, reaching their peak during widespread seabed dysoxia of the SPICE event. They lacked a cardiovascular system and appear to have been adapted to seawater hypoxia. As latest Cambrian marine shelf waters became well oxygenated, phosphatocopids went extinct. Changing seawater oxygen-levels and the demise of much of the seabed bradoriid micro-benthos favoured a third group of arthropod micro-benthos, the ostracods. These animals adopted lifestyles that made them tolerant of changes in seawater O2. Ostracods became the numerically dominant arthropod micro-benthos of the Phanerozoic.The POOur work has implications from an evolutionary context for understanding how oxygen-level in marine ecosystems drives behaviour. This simple respiratory mode that requires no specialized organs may also have been used by larger sheet-like Ediacaran organisms Molecular, geochemical and fossil evidence suggests that the first animals (eumetazoans) appeared during the late Precambrian s; see 2 . In mammWaptia fieldensis, The transportation of oxygen to the tissues via a circulating fluid (hemolymph) and a cardiovascular system  aquatic crustaceans with a dorsally articulated bivalve carapace. The most numerically abundant arthropods in the fossil record, through their half billion-year history they have adapted to environments ranging from the abyssal depths of the oceans, to freshwater lakes and damp leaf litter. Earlier studies of the fossil record have documented the radiation of ostracods from the benthos into the marine zooplankton sensuUsing uniformitarian principles applied to carapace morphology the oldest benthic ostracods have been identified from early Ordovician strata , experimental studies 2 gradient of the sediment in order to reach sediment layers where the PO2 of the water is much lower (ca 3–5 kPa). This low-oxygenation (hypoxic) level that occurs within a few millimetres below the water-sediment interface seems to be the preferential environment of numerous podocopid ostracods.Vannier and Abe th limb of myodocopid ostracods. Importantly, the ventilatory movements are similar to what is observed in living green crabs Carcinus maenas or crayfish Astacus leptodactylusLeptocythere, Cyprideis, Loxoconcha, Cytheropteron and Argilloecia) indicate that podocopids lack regulatory mechanisms for adapting their ventilation to changes in water oxygenation in contrast to decapod crustaceans which can control their ventilation. Corbari et al. Conchoecia, Vargula, Cylindroleberis mariae, which possesses a system of gas transfer , also demonstrated the inability of myodocopid ostracods to regulate their ventilatory and circulatory activities Cylindroberis mariae build the nests in which they are buried during daytime, re-breathing together in a confined environment. They become active during the night and swim into the water-column in normoxic water.All ostracods possess a pair of ‘ventilatory appendages’ that beat rhythmically, creating currents between the inner side of the valves and the animal body over the main gas exchange tissue surfaces. Ventilation is produced by fan-like exopodial features with primary radiating bristles and closely packed secondary setae that offer resistance to water, typically on the 52 was low in the water column at the beginning of the Palaeozoic Era  that live permanently within the water column, being active swimmers, possibly avoid high concentrations of O2 in their tissues not by hiding in low-oxygen niches but by “burning” O2 via a relatively high metabolism.The oxygenation status at tissue and/or cellular level is not only dependant on the ambient POCytherella, a platycopid that seems to have developed various adaptations to resist hypoxia. Cytherella is quite different from other ostracods in that it has very thick valves and the ability to close them hermetically (using powerful muscles). Moreover, its activity level and ventilatory frequency is only half that of ostracods studied previously. When subjected to a decrease in oxygenation, it demonstrates the beginnings of ventilatory adaptation which is unknown in the other ostracod groups , a strategy that may have enabled it to withstand intervals of seabed anoxia.In marine and non-marine benthic aquatic environments most podocopid ostracods are found at and above the sediment-water interface and living interstitially within the sediment as meiofauna e.g. . One spePreserved respiratory organs in fossil ostracods are rare, but examples from fossil Lagerstätten in the Silurian Cyclotron) approaches 6 mm in length Cyclotron, These animals possessed a dorsally articulated bivalve shield which covered the entire body, the largest of which  to 17 mm length (e.g. in Petrianna) for adults and this probably covered much of the body , and at the Drumian-Guzhangian boundary  being stratigraphically coincident with the beginning and peak of the SPICE event Cyclotron, Vestrogothia, Falites, Veldotron, Trapezilites and WaldoriaPhosphatocopids also appear during the ‘CARE’ , but havIn this section we examine the feedback mechanisms that may have driven changes in bradoriid and phosphatocopid diversity. We begin by assessing the level of Cambrian seawater oxygen. We then examine how changes in marine oxygen may have led to the demise of bradoriids and phosphatocopids, but favoured a third group of arthropod micro-benthos, the Ostracoda.13Ccarb) and sulphur (δ34SCAS – carbonate associated sulphur) isotope excursions associated with this event would have been associated with net O2 production 2 (taken from the Berner model), Salzmann et al. 2 at about 30 kPa  both possess biramous appendages. The endopods of these limbs had an obvious crawling function suggesting that the animals lived in contact with the seabed. The exopods are leaf-like and although no microstructures are preserved (e.g. hemolymph sinuses) the exopods may have functioned both for respiration and swimming. Bradoriids are likely to have been highly mobile arthropods the contrary of slow moving animals, moving rapidly at the water sediment-interface or just above it.Bradoriids originating during the ‘CARE’ mostly possessed large shields that exhibit clear evidence of hemolymph networks and a cardiovascular system There is abundant evidence of gills in early Cambrian arthropods  and they also lacked the ability to tightly close their shields, thus negating their ability to control oxygen levels in the waters immediately adjacent to their respiratory organs.Benthic bradoriids remained rare in the late Cambrian, as did all small bivalved arthropod micro-benthos Olenus Biozone strata peltura Biozone, ca 490 Ma) may be linked to the elevated oxygen-levels of late Cambrian shelf seas that kept deep water anoxia at bay Even in those successions most intensively studied for bivalved arthropod micro-benthos, the occurrence of phosphatocopids is sporadic Altajanella and Vojbokalina that possess shields which appear to conform to ostracod ‘body plan 2’ of Vannier and Abe peltura trilobite Biozone  may have been selectively favoured. Ostracods tolerate normoxic sea conditions and even during intervals of elevated oxygen can retreat to the interstices of sediment where hypoxic conditions prevail Vojbokalina, Septadella and Altajanella are therefore plausible early ostracod micro-benthos.Post-DICE event  shelf-maIn tandem with changes in marine oxygenation, there were also significant changes in marine substrate evolution that may have benefited a burrowing (ostracod) micro-benthos. During the Cambrian, seabed sediments became increasingly bioturbated, for example by worms 2 levels of the late Cambrian that may have restricted the environmental distribution of ostracods, despite their ability to retreat from normoxic waters. For those organisms with more limited mechanisms to control the oxygen levels of their body tissues this may have been catastrophic.The arthropod micro-benthos remained impoverished throughout the post-SPICE interval  even whe2-levels (between 10 and 15 kPa). Their biodiversity declined during bouts of Cambrian marine shelf anoxia and may also have been curtailed by elevated late Cambrian oxygen-levels that dramatically increased the PO2 gradient between seawater and bradoriid tissues. Phosphatocopids responded to Cambrian anoxia differently, reaching their peak in environments characterised by seabed dysoxia. As late Cambrian marine shelf waters became well oxygenated, phosphatocopids went extinct. Changing seawater oxygen-levels and the demise of much of the seabed bradoriid micro-benthos may have favoured a third group of Early Palaeozoic arthropod micro-benthos, the ostracods. These animals have lifestyles and strategies which make them tolerant of changes in seawater oxygen-level, able to close their valves tightly and able to burrow into sediment both to utilise food sources there, escape predation and also occupy zones of lower (hypoxic) oxygen conditions. It was the ostracods which went on to become the numerically dominant bivalve arthropod micro-benthos of the Phanerozoic, radiating into a vast range of aquatic environments from the Ordovician onwards.The fossil record of Cambrian bivalve arthropod micro-benthos suggests strong feedbacks between marine oxygen level and evolution. Benthic bradoriids evolved well-developed cardiovascular systems that favoured their colonization of oxygenated marine shelf waters but possibly in a fairly narrow range of O


Coexistent Brugada syndrome and Wolff-Parkinson-White (WPW) syndrome is rare, and as such poses management challenges. The overlap of symptoms attributable to each condition, the timing of ventricular stimulation after accessory pathway ablation and the predictive value of programmed stimulation in Brugada syndrome are controversial. We describe a case of coexistent Brugada syndrome and WPW syndrome in a symptomatic young adult. We discuss our treatment approach and the existing literature along with the challenges in management of such cases. Both cardiac channelopathies and ventricular pre-excitation are important causes of sudden cardiac death in young adults. The coexistence of these conditions appears to be rare, and as such poses management challenges. Advances in translational medicine between molecular genetics, experimental, and clinical electrophysiology have begun to shed light on the genetic background of primary electrical disorders. However, the relationship between genetic mutations and clinical conditions are both complex and only beginning to be understood. We describe a case of coexistent Brugada syndrome and Wolff-Parkinson-White (WPW) syndrome in a symptomatic young adult.A 23-year-old otherwise healthy Hispanic male presented to the emergency department with one month of paroxysmal palpitations associated with lightheadedness and presyncope. The patient denied any prior true syncopal episode or any history of sudden cardiac death in his family. An electrocardiogram (ECG) showed normal sinus rhythm with evidence of right septal pre-excitation, incomplete right bundle branch block, and ST segment elevation in V1-V2 consistent with type 1 Brugada pattern . PhysicaBased on the ECG findings and the recurrent symptoms the patient underwent electrophysiological study. Endocardial electrograms were bandpass filtered at 30-300 Hz and recorded using a computerized multichannel EP workmate 4.1 . Baseline conduction intervals were notable for a short HV interval of 16 milliseconds (msec). The AH interval was 60 msec, QRS duration 118 msec, and RR interval 1067 msec. Diagnostic catheters were placed via femoral access in the high right atrium , His region , and RV apex , as well as the coronary sinus . During catheter manipulation, pre-excited atrial fibrillation was induced with the shortest RR interval of 210 milliseconds . The patPost ablation, the type 1 Brugada pattern persisted on ECG . ProgramThe following day an ECG showed a type 2 Brugada pattern and no pre-excitation. An implantable cardioverter defibrillator (ICD) was implanted after discussion with the patient. However, patient refused genetic testing. The patient had no siblings or children.Brugada Syndrome is described as a clinical condition associated with characteristic ECG findings and an increased risk of sudden death due to ventricular arrhythmias . CharactThe Brugada syndrome together with the WPW syndrome has been reported before in the medical literature ,6. HowevThe optimal approach for sudden death prevention in these patients is unclear, and our case raises a number of interesting questions. First, our patient's symptoms might have been attributable to a tachyarrhythmia arising from either WPW or Brugada syndrome. Did our patient have just a Type 1 Brugada pattern ECG, or true Brugada syndrome? He had not had true syncope or observed nocturnal apnea, but his presyncopal symptoms were profound. Secondly, how should this patient be treated and risk stratified? EP study and ablation of a rapidly conducting accessory pathway is curative of WPW syndrome and the possibility of associated sudden death. Programmed ventricular stimulation in a Type 1 Brugada patient is reasonable, even without symptoms, but nonetheless remains controversial. Finally, it is possible that the altered repolarization of the ventricular myocardium in the period after loss of AP conduction might have affected the inducibility of ventricular fibrillation. Memory T-waves are common and may persist for weeks after ablation of manifest accessory pathways, reflecting this altered repolarization phenomenon . In our


Infectious diseases after solid organ transplantation (SOT) are one of the major complications in transplantation medicine. Vaccination-based prevention is desirable, but data on the response to active vaccination after SOT are conflicting.Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitides, tick-borne encephalitis, rabies, varicella, mumps, measles, and rubella.In this systematic review, we identify the serologic response rate of SOT recipients to post-transplantation vaccination against tetanus, diphtheria, polio, hepatitis A and B, influenza, Of the 2478 papers initially identified, 72 were included in the final review. The most important findings are that (1) most clinical trials conducted and published over more than 30 years have all been small and highly heterogeneous regarding trial design, patient cohorts selected, patient inclusion criteria, dosing and vaccination schemes, follow up periods and outcomes assessed, (2) the individual vaccines investigated have been studied predominately only in one group of SOT recipients, i.e. tetanus, diphtheria and polio in RTX recipients, hepatitis A exclusively in adult LTX recipients and mumps, measles and rubella in paediatric LTX recipients, (3) SOT recipients mount an immune response which is for most vaccines lower than in healthy controls. The degree to which this response is impaired varies with the type of vaccine, age and organ transplanted and (4) for some vaccines antibodies decline rapidly.Vaccine-based prevention of infectious diseases is far from satisfactory in SOT recipients. Despite the large number of vaccination studies preformed over the past decades, knowledge on vaccination response is still limited. Even though the protection, which can be achieved in SOT recipients through vaccination, appears encouraging on the basis of available data, current vaccination guidelines and recommendations for post-SOT recipients remain poorly supported by evidence. There is an urgent need to conduct appropriately powered vaccination trials in well-defined SOT recipient cohorts. The numbers of solid organ transplant (SOT) recipients have substantially increased in recent decades. Transplant rejection rates decreased with improved immunosuppression and quality of life increased with better post-transplant care. Donor- and recipient- derived, opportunistic, nosocomial or community acquired infections including newly emerging infectious diseases and malignancies remain a problem, however Vaccination is the most efficient and cost effective intervention to prevent infectious diseases in healthy persons Despite current recommendations emphasizing the importance of vaccinating SOT candidates prior to transplantation, reality tells a different story: vaccine coverage studies have demonstrated that this goal is far from being achieved. In the pre-transplantation phase practical problems of timely vaccination are difficult to overcome and concerns by both patients and doctors about vaccine-induced side effects are the main reason for the reluctant vaccination uptake in the post-transplantation period Guidelines and recommendations for vaccination of SOT recipients are poorly supported by evidence and are largely extrapolated from what is known for healthy persons. For new immunosuppressive regimens and recently approved vaccines, recommendations are solely based on theoretical considerations.We systematically review published data on the vaccination response of SOT recipients to summarize current knowledge and to identify areas where research is needed.We systematically reviewed published work in accordance with the PRISMA guidelines We included original research papers published in peer-reviewed scientific journals reporting results on adult or paediatric SOT recipients that were vaccinated with currently licensed vaccines see . We inclSerologic immune response to vaccination had to be assessed at least once after vaccination. All types of vaccines that are currently in use independent of the vaccination schedule, combination and dosing were accepted. No restriction regarding study design was applied.We excluded studies on vaccination of transplant candidates or organ donors, experimental vaccines or adjuvants, comparative trials on routes of application, trials on vaccines that are out of use and trials on pandemic vaccines. We excluded articles where humoral (serologic) response) was not assessed, key data were missing or not extractable , when no primary data were given , case and conference reports. Further, we excluded trials on vaccination for prevention of hepatitis B recurrence due to HBV-related liver disease, transplant recipients with a history of HBV infection or anti-HBc positive grafts. If the same group of SOT recipients was described in more than one article, only the report with the more detailed data presentation was included.All articles obtained in the search were reviewed on the basis of the title in a first step, if the title was unclear, the abstract was additionally reviewed (exclusion step one). After subtraction of duplicates, articles were checked for inclusion criteria on the basis of the abstract or the full text (exclusion step two). Articles not fulfilling the inclusion criteria were excluded (exclusion step three). Hard copies of the articles fulfilling all inclusion criteria were obtained and the data extracted with a data extraction sheet. Additionally, the reference list of relevant articles and reviews were searched , type of vaccine, vaccination schedule (number of doses and timing), number of SOT recipients and controls vaccinated, proportion of subjects  with vaccine response, time of follow up , cut-offs used for definition of vaccine response and immunosuppressive regimen.As outcome of interest we choose the serologic response to vaccination and report the overall proportion of SOT recipients with vaccine response at two time points after vaccination. For our analysis the vaccination response defined by the authors of the individual studies was accepted as valid. Short-term response was defined as the humoral  vaccine response measured as rate of seroconversion where this information is available, or as serological response defined as “protective” by the authors two weeks to three months after vaccination. Long-term response was defined as humoral  vaccine response measured after 12 months or longer. If a shorter time interval was exceptionally accepted as long-term response, this is indicated .Streptococcus pneumonia, Haemophilus influenzae, and Neisseria meningitides). Category E includes all live virus vaccines  and category F includes vaccination against pathogens indicated in certain geographical areas  . Category C includes vaccination against seasonal influenza; category D includes vaccination against bacterial pathogens (s virus) .We calculated the percentage of seropositive individuals at follow-up by dividing the number of patients with positive response by the total number of patients with exact binomial 95% confidence intervals (CIs) separately for each vaccination group in each study and displayed these in forest plots. Studies which only reported calculated percentage numbers were not included. For influenza forest plots, only studies which assessed response to every one of the three single antigens  were included in the forest plot. We used meta-regression to compare response proportions, for example between adult and paediatric transplant recipients. The standard error for the response proportion in a study was derived from the lower and upper confidence limit of the response proportion. Serological responses in transplant patients were compared with those of comparison groups when available in each study with the use of the risk difference (RDs) and 95% CIs either assuming a common-effect by using the Mantel-Haenszel method or by using the DerSimonian and Laird random-effects meta-analysis as implemented in the “metan” command in Stata In total, we obtained 2478 articles in the search . Based o64 articles obtained through the initial search fulfilled all inclusion criteria. By searching the reference lists of relevant articles, reviews and books, 8 papers were additionally identified. In total 72 papers were included into the review.Of the trials that fulfilled the inclusion criteria , publicaStreptococcus pneumoniae (9 articles), hepatitis B (7 articles), tetanus (6 articles), varicella (6 articles), diphtheria (4 articles), mumps, measles, rubella (4 articles), hepatitis A (3 articles), vaccination against Haemophilus influenzae (2 articles), rabies vaccine (2 article), polio (1 article), Neisseria meningitides (1 article) and tick-borne encephalitis vaccine (1 article). Some studies investigated more than one vaccine in the same cohort. No studies were found on Bordetella pertussis, rotavirus, human papilloma virus vaccines or vaccines recommended for certain geographic areas  apart from tick-borne encephalitis vaccine and rabies.Numbers of SOT recipients vaccinated ranged from 1 to 165 and healthy control numbers ranged from 7 to 109. There were 47 trials on vaccination of adult SOT and 25 of paediatric SOT recipients. Most trials included renal transplant recipients , followed by liver transplant , heart transplant , lung transplant  or mixed cohorts of organ transplant recipients (9 articles). Of the vaccines studied, inactivated influenza vaccination was the most common (36 articles), followed by vaccination against Most trials only assessed the short-term response within two weeks to three months after vaccination. Only a few also measured long-term responses at a second time point. Data on long-term response were not always extractable for all SOT recipients with a positive response at first assessment after vaccination. Most trials, despite small patient numbers, addressed vaccine safety issues. None of the trials neither on inactivated or on live vaccines, however, reported severe adverse events (SAEs) or transplant-related complication such as organ rejection after vaccination during the time period of the trial.Category A: Vaccination against tetanus, diphtheria, polio.We retrieved six trials investigating the response to tetanus toxoid vaccination in SOT recipients with publication dates ranging from 1995 to 2010    while immunosuppression with anti-CDFour trials were identified with SOT recipients vaccinated against diphtheria with publication dates ranging from 1997 to 1999  Only one trial with an inactivated polio vaccine (IPV) in SOT recipients was identified published in 1997 , group ACategory B: Vaccination against viral hepatitis A and B.Three trials on hepatitis A (HAV) vaccination in SOT recipients were included with publication dates ranging from 1999 to 2001, two were prospective uncontrolled and one prospective controlled  , 4, 5. WCategory D: Vaccination against bacterial pathogens.Nine studies were identified which investigated pneumococcal vaccine in SOT recipients with publication dates ranging from 1998 to 2011 , heart , and three trials on mixed cohorts.In four trials responses to a randomized or mixed vaccination scheme with both types of vaccines were investigated. Overall response ranged from 32% to 100% with comparable responses in the control group, if included We retrieved two prospective trials on Hib vaccination in SOT recipients published in 1998 and 1999, one prospective controlled in RTX and one prospective uncontrolled in HTX recipients  with substantial heterogeneity (I-squared = 76%) . Among other reasons (such as studies conducted over more than 30 years) the varying seasonal composition of influenza vaccines may play a role and makes comparisons between trials difficult. When comparing response rates of SOT recipients with controls, several studies showed a clear trend to a less pronounced response in SOT recipients with overall a 10% to 16% lower response rate in SOT recipients. The difference in response to control patients was in renal and lung transplant recipients somewhat less pronounced than in the other SOT groups. In some studies, however, the response rate in SOT recipients was even higher than in controls, which might reflect the play of chance or the fact that in some of these studies different vaccination schemes were used in the two groups (i.e. double dose for SOT recipients and single dose for controls) or highly differing pre-transplant antibody levels (higher in SOT recipients than in controls). Almost all trials, however, observed a measurable vaccine response at least in a subset of SOT recipients after single dose vaccination with a trend to lower response rates in SOT recipients compared to healthy controls in most of the trials, which is encouraging and is in accordance with findings from other authors 20 monoclonal antibody showed a decreased response rate compared to conventional immunosuppressive medication (p = 0.043 in meta-regression). All studies with diphtheria vaccination in the post-transplant phase show comparable high response rates (I-squared = 0%). From the data reviewed serological response to tetanus appears to be longer lasting than to diphtheria vaccination. Long-term response for tetanus showed persistence of antibodies without relevant decrease while the response for diphtheria had a 17% (95% CI: 7%–27%) decline over time in SOT recipients.Good evidence and encouraging results were found for vaccinating SOT recipients against tetanus, diphtheria and polio. Vaccination with tetanus toxoid in the post-transplant period elicits a high rate of responders in SOT recipients with conventional immunosuppression with no significant difference to healthy controls (p = 0.96 on only two studies) while immunosuppression with anti-CDResponse to polio vaccination also seems to be elicitable in SOT recipients, even though only one trial has investigated this vaccine. However, as polio eradication suffers from backlashes and international travel of SOT recipients is increasing, polio vaccination in SOT recipients is important and deserves further exploration.Vaccination trials for viral hepatitis A and B have largely been conducted in LTX recipients and results can only be extrapolated to other SOT types with great care. Results for HAV vaccination show a high degree of heterogeneity (I-squared = 97.7%), while one study shows high response rates for both RTX and LTX, others did not confirm this finding. Response rate to HBV after transplantation in adult SOT recipients is poor while in paediatric SOT recipients a 58% (95 CI: 37%–80%) higher response rate was seen (p = 0.001 from meta-regression). The results of primary vaccination after SOT are disappointing as early response rates were low in almost all trials and long-term responses have not been assessed on a large scale. In one study, however, long-term response was assessed and antibodies for HAV were declining rapidly. The response for hepatitis A had a 41% (95% CI: 26%–57%) decline over time in SOT recipients.In the light of this finding, the importance of hepatitis A and B vaccination prior to transplantation should be markedly stressed in pre-transplant assessment and counselling. The immunogenicity of both vaccines needs to be extended beyond LTX and should be evaluated in recipients of organs other than liver.Streptococcus pneumoniae studies was above 50% with a summary estimate of 83% (95% CI: 83%–93%) with substantial heterogeneity (I-squared = 81%), for Haemophilus influenzae vaccines (Hib) a response rate above 50% in SOT recipients in both studies was observed as well as for Neisseria meningitides (100%).Vaccination against bacterial pathogens is of great importance for immunosuppressed recipients. Response rate in all Assessment of the response to pneumococcal vaccines is difficult due to the large numbers of serotypes included in the vaccines (conjugate vaccine with 7 serotypes and polysaccharide vaccine with 23 serotypes) and the unclear impact of the seroresponse measured on protection. The response rate assessed here might be overestimated as we accepted the serological response to a single antigen as positive response. However, even in healthy children and adults, vaccine-, serotype-, and population-specific differences in immune response is not readily understood Vaccines for protection of travel-related infections in SOT recipients have, with very few exceptions, not been studied so far. Due to increasing quality of life, SOT recipient are willing to travel and a thorough assessment of their vaccination status is therefore necessary Vaccination of SOT recipients with live-attenuated viral vaccines remains controversial and is clinical studies are currently limited to paediatric SOT recipients Vaccination aims for long-term protection after initial immunization. To which extent this can be achieved in SOT recipients is unclear. Only few studies assessed a long-term response after ≥12 months. Long-term response for tetanus showed persistence of antibodies without relevant decrease while response for diphtheria had a 17% (95% CI: 7%–27%) and the one for hepatitis A 41% (95% CI: 26%–57%) decline over time in SOT recipients. Based on this limited number of studies that assessed both short- and long-term response to vaccinations no recommendation on adaptation of general recommendations for booster intervals for SOT recipients can be given.Limitations of our review are that, despite the use of systematic search strategies, some trials may have been missed, particularly since the search was limited to trials published in English. We did not assess the quality of the trials. All trials included in this review were, however, published in peer-reviewed journals. Only publications in which essential key data were missing were excluded. Since we did not restrict our search to a specific publication period, older trials using out-dated immunosuppressive regimens, which are no longer relevant, may have been included. We are aware that the type of immunosuppressive treatment impacts on the vaccine response. Stratification by type of immunosuppressive treatment would have, however, further fragmented the presentation of available data with the result of even smaller numbers per stratum. Equally, we did not exclude trials on the basis of the time period between SOT and vaccination despite the fact that the first 6 months after transplantation is regarded as the period during which immunosuppression is highest.Additional potential sources of heterogeneity in positive response to vaccination were substantial loss to follow-up and often unknown and possibly varying levels of pre-transplant immunity to the infections covered in the respective vaccines. An unknown vaccination status of a SOT recipient represents, however, a situation frequently seen in clinical practice and requires a pragmatic approach.With these caveats in mind, we believe that this review comprehensively presents the current knowledge on vaccination response in SOT recipients.Vaccine-based prevention of infectious diseases is far from satisfactorily in solid organ transplant recipients. Despite the large number of vaccination trials preformed over the past decades, knowledge on vaccination response after SOT is still limited. Even though the protection, which can be achieved in SOT recipients through vaccination, appears encouraging on the basis of available data, current vaccination guidelines and recommendations for post-SOT recipients remain poorly supported by evidence. There is an urgent need to conduct appropriately powered vaccination trials with relevant endpoints in well-defined SOT recipient cohorts, as well as to increase the awareness of clinicians for timely pre-transplant vaccination.


Accumulating evidence supports the notion that thyroid cancer is initiated by tumor-initiating cells (TICs) (commonly known as cancer stem cells), which are thought to play a crucial role in malignant progression, therapeutic resistance and recurrence. Thyroid TICs have been isolated and identified using specific biomarkers (such as CD133), the side population, sphere formation and aldehyde dehydrogenase activity assays. Although their characteristics remain largely unknown, TICs provide an attractive cellular mechanism to explain therapeutic refractoriness. Efforts are currently being directed toward the identification of therapeutic strategies that could target these cells. The present review discusses the cellular origins of TICs and the main approaches used to isolate and identify thyroid TICs, with a focus on the remaining challenges and opportunities for anticancer therapy. Thyroid cancer is the most common endocrine malignancy, and its incidence continues to rise worldwide ,2. ThyroRecent progress in thyroid cancer biology has revealed that the histological appearance and biological behaviors of thyroid carcinoma often exhibit heterogeneity, with cells exhibiting distinct proliferative and differential capacities –5. Emerget al   –24. Theson (EMT) . The Ameon (EMT) . Furtheron (EMT) . Based oAlthough the method is debated, putative TICs derived from thyroid carcinoma cell lines and tumor biopsy specimens were recently isolated using current prevailing cancer stem cell methods –30.The approaches used to identify and isolate these putative TICs were based on the notion that TICs should have conserved stem and progenitor cell functions and phenotypes. TICs have been frequently isolated using specific markers from normal stem cells of the same organ ,15. Accoet al . Friedman et al  and FRO (6.32%), but not in well-differentiated PTC cell lines (NPA and WRO). CD133+ cells harbor stem cell features characterized by high expression of stem cell gene Oct-4 and rapid long-term tumorigenesis. Ke et al (+ cells were present not only in the ATC cell line ARO (61.3%), but also in the PTC cell line CG3 (5%) and FTC cell line WRO (1.5%). A further study demonstrated that these CD133+ cells showed higher radioresistance and an undifferentiated status. Additionally, Zhu et al (+ subpopulation from MTC cell lines (MZ-CRC-1 and TT) exhibited the TIC features of self-renewal and multiple lineage differentiation.Surface markers have been used to identify and isolate adult stem/progenitor cells in human thyroid glands . Recent et al  found than et al  detectedKe et al  recentlyhu et al  suggeste+ fraction ranging from 7.02 to 64%  assay is based on the differential potential of cells to efflux Hoechst dye  via the ATP-binding cassette (ABC) family of transporter proteins expressed within the cell membrane . The abiow cells . SP cellow cells . Hoshi es  isoforms and the IGF receptor (IGF-IR) and that treatment with IGF increases the stemness features of thyrospheres. Li et al  did not give rise to tumors after more than 10 months following fat-pad implantation, despite generating spheres in culture. Therefore, the sphere-formation ability is not restricted to TICs and self-renewal cannot solely be defined in the context of a sphere assay.However, there is little definitive information regarding which cancer cells are propagated under these non-adherent and serum-free conditions. Visvader and Lindeman observed +-dependent enzyme involved in the detoxification of intracellular aldehydes to weak carboxylic acids and is significantly more highly expressed in stem/progenitor cells  possess the ability to self-renew and re-initiate serial transplantable tumors that recapitulate the phenotype and metastatic behavior of the parental tumors promoted by the activation of Met and Akt. In another study, Klonisch et al  is an NAD(P)or cells ,48,49. Ior cells . The basactivity . Todaro y  and vice versa. The strategies for identification of thyroid TICs are illustrated in in vivo strategy, serial transplantation is the golden standard recommended by the AACR workshop to determine stem cell properties  pathway has been shown to be required for self-renewal of TICs in several types of cancers, including hepatoma and glioblastoma ,56. Micrectively . Sox-2 iectively . Accumuly  and enhanced radioiodine uptake. The effects of cucurbitacin I on normal thyroid stem/progenitor cells remain unclear. Of note, differentiation therapy should involve selective targeting of TICs while having no effect on normal stem cells.TICs facilitate tumor growth through enhanced self-renewal and limited differentiation capacity. Therefore, an alternative approach to the eradication of TICs may be achievable by overcoming the blockage of differentiation by TICs. In the 1970s, this approach led to the identification and clinical use of allleukemia  and metaleukemia . Tseng ea  may be the two main reasons for TIC insensitivity to 131I therapy. Undifferentiated thyroid carcinomas have an absent or decreased ability for iodide uptake due to decreased or undetected sodium-iodide symporter (NIS) expression (+ cell differentiation by upregulation of NIS expression and thus enhances radioiodine uptake (131I uptake by TICs. A phase II study used retinoids as redifferentiation agents to increase the iodine uptake for metastatic thyroid cancer (et al (et al (+ thyroid cancer cells exhibit reduced IR-induced cell death, particularly apoptosis. The critical mechanisms determining the resistance of thyroid TICs to radiation remain elusive and warrant further investigation. TICs can be effectively eradicated by combined methods that overcome IR resistance and restore radioiodine uptake with 131I therapy.ng et al  observedpression . As mente uptake . Therefod cancer . Furtherd cancer ,73. Tsenr (et al  and Ke el (et al  reportedAlthough accumulating evidence supports the existence of TICs in thyroid cancer, and potential therapeutic strategies for targeting thyroid TICs are currently underway, some unanswered questions remain. First, the most basic cellular origins of thyroid TICs are transplantations from other malignancies. Thus, the origin of thyroid TICs should be explored in further studies to prevent tumor initiation. Second, the TIC fractions in thyroid cancer are highly impure, and the reported frequencies in the same tumor types have varied enormously among different research groups. This reflects the fact that TICs themselves are heterogeneous. This highlights the importance to identify additional specific markers or to use combinations of markers. Finally, the existence of TICs may depend on the specific niche. Accumulating evidence suggests that the tumor microenvironment is necessary for sustaining the stem-like properties of TICs ,75. Thus


Tyrosine kinase inhibitors are directly targeting different kinases and are known to regulate effector NK cells and expression of NKG2D ligands (NKG2DLs) on tumor cells. Demethylating agents, histone deacetylases, and proteasome inhibitors interfere with the epigenetic regulation and protein degradation of malignant cells. There are first hints that these drugs also sensitize tumor cells to chemotherapy, radiation, and NK cell-mediated cytotoxicity by enhanced expression of TRAIL and NKG2DLs. However, these pharmaceuticals may also impair NK cell function in a dose- and time-dependent manner. In summary, this review provides an update on the effects of different novel molecules on the immune system focusing NK cells.There is a wide range of important pharmaceuticals used in treatment of cancer. Besides their known effects on tumor cells, there is growing evidence for modulation of the immune system. Immunomodulatory drugs (IMiDs In addition to classical chemotherapeutic treatments, the novel classes of IMiDs® have been developed for the treatment of multiple myeloma (MM), myelodysplastic syndrome (MDS), chronic lymphatic leukemia (CLL), and B cell lymphoma (Immunomodulatory drugs (IMiDslymphoma .Tyrosine kinase inhibitors (TKIs) are administered in patients with tumors overexpressing kinases like BCR/ABL, stem cell factor receptor (c-KIT), platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR). TKIs are used in first-line for chronic and acute myeloid leukemia (CML and AML) with BCR/ABL overexpression as well as for gastrointestinal stromal tumors (GIST) with c-KIT mutations . FurtherDemethylating agents such as azacytidine and decitabine have been approved for treatment of AML, MDS, and other hematopoietic disorders .Histone deacetylases inhibitors (HDACis) have been designed as direct antitumor agents with major anti-proliferative effects. Some HDACis have been approved for the treatment of cutaneous T cell lymphoma (CTCL) and recently also for peripheral T cell lymphoma. HDACis are further tested in clinical phase II and III trials for multiple entities such as cervical, breast, and ovarian cancer, sarcoma, lymphoma, myeloma, and leukemia .In addition, the proteasome inhibitor bortezomib has been approved for treatment of MM and mantle cell lymphoma .Current studies addressing the use of immune modulating pharmaceutics in different other malignant diseases are in progress. Interestingly, these new pharmaceuticals do not only show direct antitumor effects, but also impact cellular immunity. In this review, we sum up the recent knowledge of the influence each of these drugs has on natural killer (NK) cells.Natural killer cells are a heterogeneous lymphocyte population with cytotoxic antitumor capacity and multiple immunoregulatory properties. They express a wide range of activating receptors , as well as inhibitory receptors  that regulate NK cell activation and tolerance . In the ® have been developed  and pomalidomide  but further thalidomide analogs are under clinical trials  from healthy donors that was mediated by the phosphoinositide (PI)-3 kinase signaling pathway causing enhanced IL-2 transcription and secretion in T cells  in NK, T, and NKT cells  mice with human lymphoma xenografts. The combination of IMiDs® together with rituximab prolonged the median survival and was abrogated by NK cell depletion  and their secreted cytokines  and nilotinib (Tasigna®) that specifically inhibit BCR/ABL, PDGFR, and c-KIT, as well as dasatinib (Sprycel®) that additionally targets the SRC kinase (®) and sorafenib (Nexavar®) have been developed inhibiting various tyrosine kinases  line with sorafenib did not affect HLA class I expression but increased membrane-bound MICA and decreased soluble MICA resulting in enhanced NK cell-mediated cytotoxicity. Sorafenib led to a decline of the metalloprotease ADAM9 that is usually upregulated in human HCC resulting in MICA shedding . Also, ipatients .+ NK cells and fewer NKp30-dependent lytic potential, both were at least partially restored during imatinib therapy. On the other hand, NKG2D showed a normal expression on NK cells in GIST patients, but nevertheless imatinib increased NKG2D-dependent cytotoxicity. Additionally, after 2 months of therapy, imatinib led to increased IFN-γ production of patient-derived NK cells after restimulation with IL-2 or DCs in vitro  and decitabine  are cytidine analogs irreversibly inhibiting the DNA methyltransferase and in addition induce genome-wide damages in a dose-dependent manner. Demethylating agents have already revealed their direct effects on malignant cells  on various tumor cell lines . Interacin vitro setting did neither alter the KIR profile of NK cells nor the NKG2DL expression of target cells that has been described previously. Altogether, the inhibitory effect of azacytidine seemed to be caused by impaired mRNA synthesis, whereas decitabine increased the responsiveness to activation stimuli resulting in enhanced gene transcription , romidepsin (Istodax®), and chidamide (CS055/HBI-8000).Histone deacetylases (HDACs) play an important role in the epigenetic modulation of gene expression affecting cell-cycle arrest, differentiation and apoptosis in all nucleated cells. Mutations in genes encoding HDACs and alterations in HDAC expression have been found in many types of cancer. Therefore, inhibition of HDACs seems to be a good point of attack in tumor therapy . Mentionin vitro in treated leukemic blasts from AML patients that enhanced MICA/B and ULBP1 expression, thereby increasing cytolysis by KIR–HLA class I-mismatched cells , cytotoxic enzymes (granzyme and perforin), and molecules important for apoptosis (FASLG) was observed , an important proteasome inhibitor, acts, may differ dependent on tumor targets and still have to be further examined .Table 1).In summary, the heterogeneous impact on NK cell function reported for the different immune modulating substances, referred in this review, is mediated by either indirect or direct effects on NK and tumor cells and is further depending on the tumor entity, timing, and dosing of the different pharmaceutics (® predominantly mediates immune stimulation positively influencing NK cell-mediated antitumor capacity. TKIs, HDACis, and proteasome inhibitors exhibit various effects. They display mostly negative impacts on NK cell function although treatment of tumor cells may result in contradictory results by sensitization to apoptosis-induction. Exceptions were the new HDACi chidamide and the TKI imatinib that showed a positive effect on NK cell activation. Both of the demethylating agents described exhibit opposite effects on NK cell function.The majority of IMiDsFor better understanding of the underlying mechanisms, unambiguous data on NK cell function from preclinical and clinical studies are still needed. In addition to the complex balance of both tumor and NK cells, there are multiple effects on the other players of the immune system such as antigen presenting cells and cytolytic T cells that regulate the outcoming immune effects.Finally, an ideal therapeutic concept for cancer patients might combine inhibitors of immunosuppression with tumor sensitizers and cellular therapy protocols such as adoptive NK cell transfer. This review underlines the urgent need of further immunomonitoring studies that should address functional aspects of immune cells on autologous patient-derived tumor cells under therapy with immune modulating agents. Growing knowledge from such studies will allow the right choice of the ideal immune modulators at the optimal dosing and timing to obtain the best possible outcome for the patients.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.


PLOS Pearls article.To discriminate self from non-self, the innate immune system evolved a large repertoire of germline-encoded receptors that detect molecular patterns associated with infections macromolecular, non-self structures in order to mark them as targets for innate immune responses. Once bacteria or PVs are marked as aberrant or non-self structures, they can be delivered to microbicidal lysosomes, a process that frequently involves the autophagic machinery Intracellular bacterial pathogens reside and replicate either within the cytosol or inside membrane-bound, pathogen-containing vacuoles (PVs) Whereas the importance of autophagy in cell-autonomous host defense is well established, we are only beginning to understand the mechanisms by which the host can specifically recognize invading pathogens and PVs as non-self structures. In the following I give three examples describing how the innate immune system can locate intracellular pathogens. Each example highlights one of the three basic types of pattern associated with intracellular pathogens and recognized by the innate immune system, namely the non-self, the aberrant-self, and the missing-self.Salmonella enterica serovar TyphimuriumS. Typhimurium that had exited from the protective surroundings of Salmonella-containing vacuoles (SCVs).One marker that labels cytosolic bacteria and PVs as substrates for defense pathways is the small protein ubiquitin in vitro through its LRR domain How is LRSAM1 able to detect cytosolic bacteria? The LRSAM1 protein contains a leucine-rich repeat (LRR) domain, a common feature of PRRs that is often instrumental in the direct binding of microbe-derived molecules by PRRs LRSAM1 represents the first member of what will most likely emerge as a larger group of cytosolic PRRs that bind directly to non-self ligands decorating microbial cell surfaces. As effective as these LRSAM1-like PRRs may turn out to be in fighting cytosolic pathogens, they cannot provide protection against pathogens residing within the confines of PVs. Therefore, in order to locate pathogens hidden inside vacuoles, the innate immune system must be able to detect additional patterns that discriminate non-self PVs from self vacuoles.Intracellular bacterial pathogens have developed complex mechanisms to create and maintain PVs as their intracellular residence Shigella flexneri and Listeria monocytogenesSalmonella. One galectin specifically, Galectin-8, has been shown to translocate to disrupted SCVs and to recruit the autophagy adaptor protein NDP52 to SCVs, thereby delivering S. Typhimurium for degradation inside autolysosomes Galectins constitute a family of β-galactoside–binding proteins, which exist as transmembrane, secreted, and cytosolic forms Legionella pneumophila and S. Typhimurium. Both of these pathogens secrete specific bacterial effector proteins into the host cytosol to prevent PV disruption Salmonella effector protein SifA is required to maintain SCV membranes sifA mutants colocalize with galectins at significantly higher rates than coisogenic wild-type bacteria Because loss of membrane integrity is an aberrant-self pattern that can be recognized by the innate immune system, vacuolar pathogens must have evolved mechanisms to either inhibit detection or prevent the occurrence of the pattern itself. The latter strategy is indeed employed by at least two bacterial pathogens, namely by Chlamydia trachomatis forms rigid cages made out of actin and intermediate filaments to stabilize its vacuole Pathogens invest considerable resources into preserving the stability of their surrounding vacuoles. For example, the obligate intracellular pathogen C. trachomatis, cytosolic GKS proteins translocate to C. trachomatis inclusions IRGM proteins belong to a family of immunity-related GTPases (IRGs) C. trachomatis inclusions on the other hand are missing substantial amounts of IRGM proteins and are therefore unable to block GKS protein activation and membrane binding C. trachomatis inclusions remains mysterious, our studies show that the lack of endogenous IRGM proteins serves as a missing-self pattern associated with a subset of PVs In order to bind to membranes, GKS proteins must transition into the “active” state by exchanging GDP for GTP To direct cell-autonomous defenses specifically to cytosolic bacteria and PVs, the host cell must be able to discriminate intracellular self membranes from non-self membranes. Only very recently have we begun to identify membrane-sensing PRRs that can survey the vesicular landscape of a mammalian cell: this class of PRRs includes LRSAM1, galectins, and IFN-inducible GTPases. How these PRRs interact with the network of immune surveillance and how they orchestrate innate as well as adaptive immune responses are important, unanswered questions. Finding answers to these questions will vehemently advance our understanding of innate immunity and infectious disease.


Decisions under risk and with outcomes that are delayed in time are ubiquitous in real life and can have a significant impact on the health and wealth of the decision-maker. Despite its potential relevance for real-world choices, the degree of aberrant risky and intertemporal decision-making in patients suffering from major depressive disorder (MDD) and posttraumatic stress disorder (PTSD) has received little attention to date.We used a case-control design to compare decision-making in healthy control subjects (N=16) versus untreated depressed subjects in a current major depressive episode (N=20). In order to examine how major depressive disorder (MDD) may impact decision-making, subjects made decisions over (1) risky outcomes and (2) delayed outcomes in the domain of gains and losses using choice paradigms from neuroeconomics. In a pre-planned analysis, depressed subjects were subdivided into those with primary PTSD along with comorbid MDD (MDD+PTSD) versus those with primary MDD without PTSD (MDD-only). Choice behavior was modeled via a standard econometric model of intertemporal choice, a quasi-hyperbolic temporal discounting function, which was estimated for each subject group separately.Under conditions of potential gain, depressed subjects demonstrated greater discounting for gains across all time frames compared to controls. In the realm of losses, both subgroups of depressed subjects discounted more steeply than controls for short time frames. However, for delayed losses ranging from >1-10 years, MDD+PTSD subjects showed shallower discounting rates relative to MDD-only subjects, who continued to discount future losses steeply. Risk attitudes did not contribute to differences in intertemporal choice.Depressed patients make choices that minimize current pain and maximize current reward, despite severe later consequences or lost opportunities. Anxiety associated with PTSD may serve as a partially protective factor in decision-making about long-term potential losses compared to MDD patients without PTSD. Major depressive disorder (MDD) is a chronic and debilitating disease with a lifetime prevalence of 13-17% . In addiPosttraumatic stress disorder (PTSD) affects 2-7% of the population ,2. PTSD An important but understudied clinical issue for patients suffering from depression is decision-making behavior. Depressed patients often make choices that are not in their best interest, such as neglect of social relationships, reacting passively to potential defeats, and failing to pursue new opportunities [9.10]. Such choices can serve to continue or intensify the external circumstances that act to reinforce the negative mood state. Furthermore, depressed patients may look to use short-term external environmental factors to improve their mood, choosing to pursue immediate rewards to the neglect of long-term benefits or costs. This decision-making style is consistent with observations that depressed patients may overeat, abuse substances, or shop excessively as efforts at mood repair ,12, despNeuroimaging studies have identified alterations in the brains of patients with MDD and PTSD. Structurally, both MDD and PTSD patients demonstrate reduced volumes in anterior cingulate cortex, hippocampus and thalamus relative to healthy controls . FindingSeveral of these brain regions affected in MDD and PTSD also serve as important processing areas within networks associated with valuation and decision-making . InterteIn psychiatry, greater discounting of future rewards has been most consistently demonstrated for patients with addictive disorders ,33. WhetIn the current study, we compared intertemporal decision-making in healthy controls with subjects currently experiencing a major depressive episode. Furthermore, we assessed whether decision-making among MDD patients differed between subjects for whom MDD was the primary illness (MDD-only) versus those for whom MDD was secondary to a primary diagnosis of PTSD (MDD+PTSD). Specifically, we explored: 1) whether there are differences in risk-taking and temporal discounting between depressed subjects and psychiatrically healthy subjects; and 2) whether MDD-only subjects differ in their choice behavior from MDD+PTSD subjects in the context of choice scenarios probing risk and intertemporal economic preferences. Because differences in risk attitudes may affect intertemporal choice behavior ,38, thesWe used a case-control design to compare subjects with depression and healthy, non-depressed control subjects on factors related to decision-making. The Emory Institutional Review Board reviewed and approved the study protocol. The study was conducted in accord with the Declaration of Helsinki and its amendments.Participants were recruited from ongoing studies of MDD and PTSD in the Mood and Anxiety Disorders Program of Emory University and from flyers posted on the Emory University campus. All subjects were English speakers between the ages of 18-65 years and signed a written informed consent form to participate.To be eligible, depressed subjects had to meet criteria for MDD and be in an active major depressive episode diagnosed using the Structured Clinical Interview for DSM-IV (SCID) , and scoExclusionary criteria for all subjects included a lifetime diagnosis of bipolar disorder or a psychotic disorder, acute clinically significant suicide risk, substance abuse or dependence in the previous three months, or active central nervous system-related illness that would affect ability to complete the test procedures.Anxiety was assessed with the Hamilton Anxiety Rating Scale (HAM-A) . Self-reAfter completion of the clinician interviews and self-report questionnaires, subjects completed a series of standard computerized decision-making tasks, using either points or hypothetical monetary payouts as outcomes.In the Risky Gains task , subjectIn the Delay Discounting Task , subjectInitial analyses compared responses of HC and the depressed groups to assess questionnaire and behavioral differences between the two groups. In a pre-planned analysis, we repeated all comparisons using the three groups  to examine the moderating effects of primary PTSD within the depressed group. For the risky gains task, the percentage of risky choices overall and after punishment was compared across subject groups using nonparametric tests. Specifically, for comparisons of two groups, the two-sample Wilcoxon test was employed, while for comparisons of three groups, the Kruskal-Wallis test was used for initial group comparisons and, if significant differences were identified, follow-up analyses were conducted using the two-sample Wilcoxon test. Repeating all non-parametric analyses using the equivalent parametric approach (one-way between-subjects ANOVA and independent samples t-test) identified the same trends.For the Delay Discounting task, subjective values reflective of the indifference point between the choice options offering immediate and delayed rewards were entered as response variables into a standard econometric model ,48. SpecNon-linear least squares regression implemented in R (http://cran.us.r-project.org) was employed to assess differences in temporal discounting slopes for gains and losses as a function of group. The use of a quasi-hyperbolic model allowed us to estimate group-specific parameters reflective of discounting slopes for early  and late  choice options as well as for gains and losses. Our model extracted parameters for representative subjects that were clustered based on their diagnosis. Using aggregate data to estimate behavioral parameters based on choices in economic games and choice tasks is a standard econometric approach in behavioral economics as well as neuroeconomics -52. We cThirty-six participants consented to participate in the study: 20 depressed subjects  and 16 HC. Data from one HC could not be extracted and was excluded from the Delay Discounting Task analysis. Demographic data for the sample are presented in 2  = 2.2973, p = 0.3171; or safe choice after punishment: X2  = 1.731, p = 0.421]. These results indicate that the presence of depression (with or without PTSD) did not differentially impact choice frequencies over options involving risks over hypothetical outcomes.As shown in Specifically, the groups demonstrate different early discounting slopes as indicated by the significant interaction term β * g, such that MDD-only and MDD+PTSD subjects show steeper slopes than HC reflective of greater temporal discounting for both gain and loss choice options in the immediate future . For intertemporal choices involving potential gains over delays of >1-10 years, MDD-only and MDD+PTSD subjects continue to show significantly steeper slopes than HC as indicated by the significant interaction term . These results indicate that both depressed groups value long-term future rewards significantly less relative to immediate outcomes than HC. In the domain of losses, MDD-only subjects do not adjust their discounting functions like the other two groups , indicating that, compared to MDD+PTSD subjects and HC, MDD-only subjects have greater preference for larger later losses over smaller immediate losses. Importantly, choice patterns of MDD+PTSD subjects differ significantly from the other two groups in that they show a significantly greater change from short- to long-term outcomes as indicated by the significant interaction term (δ* g * l). Specifically, while choice patterns of MDD+PTSD subjects are similar to MDD-only subjects for outcomes after short-delays and gains after >1-10 year delays, their choice patterns look more like HC for losses over the >1-10 year delay period. These results indicate that the effect of loss framing was greatest on choice patterns of MDD+PTSD subjects relative to the MDD-only and HC .On the FDMQ, depressed subjects scored higher than HC on Hypervigilance, Procrastination and Buck-Passing factors. HC scored significantly higher than depressed subjects on Vigilance. Post hoc testing of Vigilance scores did not find differences between MDD-only and HC, but MDD+PTSD subjects were significantly lower than HC.In this study of subjects in a current major depressive episode and HC subjects, we found substantial differences in temporal discounting between healthy and depressed subjects. Furthermore, the effect of PTSD modified the discounting for losses observed among subjects in a current major depressive episode. These differences in discounting did not derive from different risk attitudes, as we observed no differences between the groups on the level of risk taking, nor in the sensitivity to punishment on the Risky Gains Task.The most intriguing result of this study was the dissociation between depressed subjects with and without PTSD in the area of long-term (>1 year) discounting of losses. Both groups showed the expected steep discounting for gains in both short-term and long-term time frames, indicating a greater preference than controls to take smaller immediate outcomes rather than await a later, larger gain. In the domain of losses, however, significant differences between the depressed groups were observed. Although both groups of depressed subjects showed steeper discounting than healthy subjects for losses delayed up to a year, PTSD subjects demonstrate a stark and significant recovery from steep discounting for loss outcomes beyond one year. This finding was contrary to our hypothesis that the PTSD symptom of a foreshortened future would drive steeper discounting over both losses and gains.The shallower discounting of future losses in the healthy controls and MDD+PTSD versus the MDD-only group suggests the MDD-only group did not experience disutility in dreading a known long-term loss, which others have observed in healthy subjects . The MDDThis willingness of MDD+PTSD subjects to accept an immediate loss to avoid a larger later one may reflect an anxious attitude about negative future events. For example, victims of domestic violence who have PTSD often continue to live with their abusers for years before moving out to a shelter or some other source of refuge. Although many practical aspects may affect such decision-making, one conceptualization consistent with this study’s findings is that accepting the pain of physical abuse now is more acceptable to depressed PTSD patients than larger perceived long-term losses incurred from being dispossessed of one’s home and belongings. It is also possible that the disutility associated with dreading a long-term high cost is greater for anxiously depressed PTSD patients than MDD patients without PTSD. Interestingly, the negative experience of dread of future pain is processed partly through the anterior cingulate cortex , a regioThe finding of reduced discounting of losses over the >1-10 year frame suggests that the presence of anxiety associated with PTSD in the setting of a major depressive episode may serve protective functions against overly pessimistic-based choices in MDD-only subjects. Although the presence of anxiety in patients suffering from MDD predicts poor response to antidepressant treatments in terms of symptom reduction , real-woTogether, these results inform future research investigating the underlying affective and cognitive processes, as well as related neural mechanisms, of the observed choice distortions in patients with MDD. Research in neuroeconomics has made considerable progress in identifying the neural mechanisms involved in intertemporal decision-making. From a number of recent studies it has become apparent that intertemporal choice is a complex behavior that engages multiple affective and cognitive processes that are mediated by distinct but interacting neural networks ,47,56-61The choice distortions observed in patients with MDD in the current investigation are likely due to changes in multiple interacting brain systems. Primary among those are systems known to be altered by depression and anxiety, including (1) valuation, which is expected to be particularly distorted in the presence of anhedonia, a common sympton of MDD; (2) choice conflict, which is expected to be biased in the presence of anxiety about aversive events in the future, a common symptom in PTSD; and (3) the ability to simulate future outcomes, which our results suggest to be attenuated in patients with MDD only. Interestingly, the functional connectivity between ACC and hippocampus has been associated with reductions in impulsive intertemporal decision-making , both ofSome have argued that intertemporal choice behavior may be confounded by risk attitude because delays entail an inherent risk that the future event may never occur ,63. FurtOur results both agree and conflict with the only prior study of intertemporal choice in depressed patients. Consistent with our results, Takahashi and colleagues  reportedA potential limitation of this study is the use of hypothetical rewards. Specifically, artificial reward “points” formed the basis of motivation in both the Risky Gains and the Temporal Discounting tasks. Although real payoffs may increase salience and more strongly engage anticipatory emotions in decision-making, previous work suggests the use of hypothetical rewards does not lead to meaningfully different choices from those involving real outcomes ,70. StreThe results of the present study suggest that there are significant differences between healthy subjects and depressed patients in the way they make intertemporal decisions. Intertemporal decisions are ubiquitous in real life and have been shown to significantly impact important facets of life, such as relationships, physical health and wealth . The obsFinally, the differences we identified in choice behavior between depressed subjects with a primary diagnosis of PTSD versus those MDD subjects without PTSD suggest changes in the underlying neurobiological mechanisms involved in intertemporal choice . Key netEmploying (neuro-)economic decision-making paradigms to investigate choice distortions in patients with psychiatric disorders can convey important insights into clinical features of mood and anxiety disorders. Anxiety or dread may lead depressed patients with a primary diagnosis of PTSD to make more conservative decisions about long-term losses that provide partial protection from negative consequences derived from the short-term outlook that drives decision-making among other patients with MDD.File S1Text S1, Text S2, Figure S1, Tables S1-S6.Text S1. Description of Additional Questionnaires. Text S2. Model Selection. Figure S1. Frequencies of selecting the safe choice option during the risk task in all trials (A) and after punishment only (B) across three groups. The risk task was analyzed using both two (results reported in main paper) and three groups, in order to account for the moderating effects of comorbid PTSD. Results for the three-group analysis investigating the moderating effect of comorbid PTSD replicated findings for the two-group analysis. No difference between the groups were found for choice frequencies of safe option overall (A) and after punishment (B). Specifically, as illustrated in the box plots risky choice behavior did not differ between the HC, MDD-only and MDD+PTSD groups . Table S1. Model comparison results across three commonly used discounting functions for the combined MDD analysis (see Text S2). The winning model is highlighted in bold. Table S2. Model comparison results across three commonly used discounting functions when stratifying the MDD group by comorbid PTSD diagnosis (see Text S2). The winning model is highlighted in bold. Table S3. Model comparison results across three commonly used discounting functions for control subjects (see Text S2). The winning model is highlighted in bold. Table S4. Model comparison results across three commonly used discounting functions for all MDD subjects (see Text S2). The winning model is highlighted in bold. Table S5. Model comparison results across three commonly used discounting functions for MDD only subjects (see Text S2). The winning model is highlighted in bold. Table S6. Model comparison results across three commonly used discounting functions for MDD+PTSD subjects (see Text S2). The winning model is highlighted in bold.(PDF)Click here for additional data file.


Drosophila melanogaster, there are three BMP-encoding genes: decapentaplegic (dpp), glass bottom boat (gbb) and screw (scw). dpp and gbb are found in all groups of insects. In contrast, the origin of scw via duplication of an ancestral gbb homologue is more recent, with new evidence placing it within the Diptera. Recent studies show that scw appeared basal to the Schizophora, since scw orthologues exist in aschizan cyclorrhaphan flies. In order to further localise the origin of scw, we have utilised new genomic resources for the nematoceran moth midge Clogmia albipunctata (Psychodidae). We identified the BMP subclass members dpp and gbb from an early embryonic transcriptome and show that their expression patterns in the blastoderm differ considerably from those seen in cyclorrhaphan flies. Further searches of the genome of C. albipunctata were unable to identify a scw-like gbb duplicate, but confirm the presence of dpp and gbb. Our phylogenetic analysis shows these to be clear orthologues of dpp and gbb from other non-cyclorrhaphan insects, with C. albipunctata gbb branching ancestrally to the cyclorrhaphan gbb/scw split. Furthermore, our analysis suggests that scw is absent from all Nematocera, including the Bibionomorpha. We conclude that the gbb/scw duplication occurred between the separation of the lineage leading to Brachycera and the origin of cyclorrhaphan flies 200–150 Ma ago.Bone morphogenetic proteins (BMPs) play key roles in development. In The online version of this article (doi:10.1007/s00427-013-0445-9) contains supplementary material, which is available to authorized users. Drosophila melanogaster, there are two known families of BMP-encoding genes: decapentaplegic (dpp)—a member of the vertebrate BMP2/4 family—as well as glass bottom boat (gbb) and screw (scw)—members of the BMP5/6/7/8 family  patterning during early embryogenesis  group perform key roles in morphological and physiological processes in all metazoan phyla. They have been variously referred to as a superfamily or family depending on the author  is found in all groups studied so far, there are differences in the number and types of gbb-like genes  also have two closely related gbb genes, but these duplications appear to have occurred independently in the mosquito lineage  with its two copies of gbb (gbb1 and gbb2), which show distinct expression patterns, but are more closely related to each other than to any other gbb  also exhibits two gbb-related genes, again more closely related to each other than to any other gbb duplicates. Arthropod species outside the holometabolan insects—such as the water flea Daphnia pulex (Crustacea), the human louse Pediculus humanus (Phthiraptera) and the pea aphid Acyrthosiphon pisum (Hemiptera)—have only one copy of gbb  with high sequence conservation across lineages, and a second gbb-like gene in some groups (gbb2 or scw), which arose by independent duplication events. It would be interesting to investigate whether the origin of the scw duplication can be located more precisely within the dipteran lineage.Several studies have focused on the evolution of BMP-encoding genes in dipterans and other insects , and the scuttle fly Megaselia abdita, have orthologs of dpp, gbb and scw, which show expression patterns similar to the ones in D. melanogaster . Despite some recent controversy over the placement of Psychodidae  by BLAST using dpp, gbb and scw sequences from D. melanogaster, M. abdita and E. balteatus (retrieved from GenBank). In addition, we searched a preliminary assembly of the C. albipunctata genome (our unpublished data) and the genome of the Hessian fly Mayetiola destructor  with these same sequences. PCR primers for C. albipunctata dpp and gbb were designed from transcriptome sequences . Amplified sequences for C. albipunctata dpp and gbb have been deposited in GenBank (accession numbers KC810051 and KC810052). Fragments were cloned into the PCRII-TOPO vector (Invitrogen) and used to make DIG-labelled riboprobes for in situ hybridisation.We searched the early embryonic transcriptome of C. albipunctata embryos were collected at blastoderm and post-gastrulation stages as described in García-Solache et al. . The sequences were initially aligned using T-Coffee , a dipteran species whose lineage branches basally to the Brachycera, and thus Cyclorrhapha . Next, we searched a preliminary genome assembly for C. albipunctata . We identified the same single copies of the putative dpp and gbb/scw orthologues. No additional gbb duplicate is present in the genome. At this point, we cannot rule out the possibility that C. albipunctata has lost a scw-like duplicate secondarily.We obtained putative BMP members from the transcriptome and genome of C. albipunctata dpp and gbb/scw sequences using maximum likelihood methods and Bayesian inference  either. Therefore, our phylogenetic analysis suggests that the gbb/scw duplication occurred within the Brachycera. Further research in non-cyclorrhaphan Brachycera  (PDF 27 kb)Online Resource 2Bayesian phylogenetic analysis of BMP sequences Below is the link to the electronic supplementary material.