The present results suggest that a relatively short-term application of an optimal concentration of TNF-α may protect neurons against ischemic injury by elevating the expression of EAAT2 in astrocytes.The aim of the present study was to determine whether tumor necrosis factor (TNF)-α regulates the expression levels of excitatory amino-acid transporters (EAATs) in primary astrocytes and its roles in brain ischemia. Exogenous TNF-α was administered to pure cultured astrocytes and the expression levels of EAAT1, EAAT2 and glial fibrillary acidic protein (GFAP) were evaluated. The results showed that TNF-α at 10 ng/ml enhanced the expression of EAAT2 in a time-dependent manner, while the expression levels of EAAT1 and GFAP did not change. To determine whether the elevation in the levels of the EAAT2 protein induced by TNF-α had a beneficial effect on ischemic insult, TNF-α was applied to  Acute ischemic stroke is one of the leading causes of adult disability . The excet al  is an important inflammatory factor and its expression levels markedly increase in the brain following ischemia . For a let al  demonstrl . The experimental procedures were approved by the Animal Ethics Committee of Zhejiang University and were carried out in accordance with institutional guidelines. Recombinant rat TNF-α was obtained from BioVision .6) were plated on coverslips coated with poly-D-lysine  or on coverslips in Neurobasal® medium supplemented with 2% B27  and 0.2 mM L-glutamine  was added to the cultures two days after plating to block the proliferation of non-neuronal cells. One-half of the culture medium was changed every four days. After culturing for 12 days, neurons were then co-cultured with astrocytes.Rat hippocampal neurons were prepared as previously described, with certain modifications . BrieflyAstrocytes in primary culture were prepared from the cerebral cortices of newborn  rat pups as previously described . In brieCo-cultures of astrocytes and neurons were prepared as previously described, with certain modifications . Briefly2 and 5% CO2 at 37°C for 2 h. Exposure was terminated by removing the glucose-free DMEM and adding Neurobasal medium. After 22 h of recovery in a normoxic incubator, the coverslips were carefully transferred, face-up, to a new 24-well plate containing Neurobasal medium and the cell viability was assessed as described below. In each experiment, cultures exposed to OGD were compared with normoxic controls supplied with DMEM containing glucose and maintained in standard incubation conditions.OGD was performed as previously described . BrieflyCell viability was measured using the MTT (Sigma-Aldrich) assay. Cells were gently washed with fresh culture medium, and DMEM and the serum-free medium containing MTT (5 mg/ml) was added and incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 150 μl dimethyl sulfoxide. Absorbance was measured at 490 nm.A total of 6 male rats were anesthetized by an intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg) and mounted in a stereotaxic frame on a heating blanket. TNF-α was administered according to previously described techniques , with ceCerebral cortical tissues or astrocytes were lysed on ice in radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Sigma-Aldrich) containing a mixture of several protease inhibitors, including AEBSF at 104 mM, Aprotinin at 80 μM, Bestatin at 4 mM, E-64 at 1.4 mM, Leupeptin at 2 mM and Pepstatin A at 1.5 mM. Protein content was estimated using the bicinchoninic acid assay method. Immunoblotting was performed as described previously . FollowiData are expressed as the mean ± standard deviation. Comparisons between groups were analyzed using the Student’s t-test or one-way analysis of variance, followed by Dunnett’s post hoc test. P<0.05 was considered to indicate a statistically significant difference.in vivo, recombinant rat TNF-α was administered by i.c.v. injection and the expression levels of the EAATs in the cerebral cortical tissue were assessed by western blot analysis. Compared with the EAAT2 levels in the saline control group, EAAT2 expression markedly increased (P<0.01) after TNF-α treatment for 24 h. However, no changes were observed in the expression level of EAAT1  on primary cortical astrocytes was analyzed . The optTo further explore the association between incubation time and the expression levels of EAATs in astrocytes, the astrocytes were treated with the optimal concentration of TNF-α (10 ng/ml) for different incubation periods . The EAAT2 protein expression showed a time-dependent increase, followed by a time-dependent decrease, with a maximum level (P<0.01) after 12 h . Howeverin vitro, a co-culture system of astrocytes and neurons was established and cell viability was assessed following OGD injury. After different periods of incubation (0–24 h) with 10 ng/ml TNF-α, OGD was applied for 2 h, followed by 22 h of reoxygenation .Clinical studies have demonstrated that a transient ischemic attack (TIA) within a narrow time-window may enhance the tolerance of the brain against a more severe ischemic insult –22. Notaet al (in vivo and in vitro. This was consistent with the findings of Davis and Patel (et al (Numerous studies have been performed to investigate the mechanisms of ischemic tolerance caused by TNF-α exposure. Glazner and Mattson found that TNF-α pretreatment reduced calcium influx and upregulated the gene transcription of neuroprotective factors . In addiet al  revealednd Patel , who deml (et al , in whicet al (et al (Consistent with the present results, Tilleux et al  reportedl (et al  revealedin vitro, the viability following OGD injury in a co-culture system of astrocytes and neurons was assessed. Corresponding with the in vivo results, it was revealed that TNF-α increased the cell viability following OGD injury, suggesting that it has a neuroprotective effect by promoting EAAT2 expression in astrocytes.To further determine whether the elevated levels of EAAT2 protein induced by TNF-α had a beneficial outcome The present study had several limitations. Firstly, based on previous findings, TNF-α may have dual effects on the expression and activity of EAATs. Thus, detection of the activity of EAATs under these experimental conditions is required. Secondly, it is necessary to demonstrate whether the protection is eradicated following the blockade of glutamate uptake. Finally, an advanced mechanistic study is also required. Therefore, these issues should be addressed in future studies.In conclusion, the present study revealed that an optimal concentration and time-course of TNF-α elevates EAAT2 expression in astrocytes and has a beneficial effect on subsequent ischemic insult.


In recent years, the partial differential equations, both fractional and integer orders, have been recognized as a powerful modeling methodology. They are inspired by problems which arise in diverse fields such as biology, fluid dynamics, physics, differential geometry, control theory, materials science, and engineering.The purpose of this special issue is to report and review some recent developments in methods and applications of partial differential equations. The majority of the papers contained in this special issue are based on areas of research ranging from functional analytic techniques and singularity methods as well as numerical methods that are applied to both partial and ordinary differential equations. There are papers which deal with fractional partial differential equations and in addition papers analyzing equations that arise in engineering as well as classical and fluid mechanics.This special issue contains the papers addressing the recent theoretical advances and experimental results on the topics such as operational calculus and inverse differential operators, global existence and energy decay rates for a Kirchhoff-type wave equation, nonsymmetric system of Keyfitz-Kranzer type, different methods for numerical solution, recent progress on nonlinear Schrödinger systems with quadratic interactions, upper semicontinuity of pullback attractors, numerical modeling, mean-variance portfolio selection, a novel iterative scheme, and the iterative methods of linearization.The guest editors very much hope that the papers published in this special issue will be useful to a large community of researchers and will arouse further research in the topics presented as well as in the connected fields.


Alterations in TNF-α expression have been associated with cerebral aneurysms, but a direct role in formation, progression, and rupture has not been established.Cerebral aneurysms were induced through hypertension and a single stereotactic injection of elastase into the basal cistern in mice. To test the role of TNF-α in aneurysm formation, aneurysms were induced in TNF-α knockout mice and mice pretreated with the synthesized TNF-α inhibitor 3,6′dithiothalidomide (DTH). To assess the role of TNF-α in aneurysm progression and rupture, DTH was started 6 days after aneurysm induction. TNF-α expression was assessed through real-time PCR and immunofluorescence staining.TNF-α knockout mice and those pre-treated with DTH had significantly decreased incidence of aneurysm formation and rupture as compared to sham mice. As compared with sham mice, TNF-α protein and mRNA expression was not significantly different in TNF-α knockout mice or those pre-treated with DTH, but was elevated in unruptured and furthermore in ruptured aneurysms. Subarachnoid hemorrhage (SAH) occurred between 7 and 21 days following aneurysm induction. To ensure aneurysm formation preceded rupture, additional mice underwent induction and sacrifice after 7 days. Seventy-five percent had aneurysm formation without evidence of SAH. Initiation of DTH treatment 6 days after aneurysm induction did not alter the incidence of aneurysm formation, but resulted in aneurysmal stabilization and a significant decrease in rupture.These data suggest a critical role of TNF-α in the formation and rupture of aneurysms in a model of cerebral aneurysm formation. Inhibitors of TNF-α could be beneficial in preventing aneurysmal progression and rupture. Cerebral aneurysm rupture is associated with significant morbidity and mortality ,2. A sigInflammation has been implicated in the pathogenesis of intracranial aneurysm formation and rupture . AlteratThe goals of the present study were to: 1) assess the direct role TNF-α in a model of cerebral aneurysm formation; 2) determine if there are alterations in TNF-α expression in cerebral aneurysm formation and rupture; 3) evaluate if TNF-α inhibition decreases the incidence of aneurysm formation; and 4) test whether TNF-α inhibition after cerebral aneurysm formation may lead to aneurysm stabilization and inhibition of rupture.Guide for the Care and Use of Laboratory Animals of the National Research Council [This study was carried out in accordance with the recommendations in the  Council . The pro Council ,12,13 wiTo induce hypertension, mice underwent nephrectomy followed by implantation of deoxycorticosterone acetate pellet  1 week later . On the Blinded daily neurological examination was carried out using a previously described method ,16-18. NSystolic blood pressure was measured by the tail-cuff method with the BP-2000 Blood Pressure Analysis System  after 3 days of training to allow for acclimation and then before aneurysm induction surgery (day 0) and every week until day 28 after surgery .To test whether TNF-α inhibition decreased the incidence of cerebral aneurysm formation, progression, and rupture, the TNF-α inhibitor 3,6′dithiothalidomide (DTH) was synthesized as previously described ,21 and wQuantitative real-time PCR and immunohistochemistry were carried out as previously described ,22. DetaPrimary outcomes were the incidence of aneurysm formation (both ruptured and unruptured) and the incidence of ruptured aneurysms. Further description of the methods of statistical analysis can be found in Additional file To test the role of TNF-α in the formation of intracranial aneurysms, the TNF-α inhibitor DTH was synthesized as previously described ,21 to grP = 0.002) and 4 of 12 mice  treated with DTH  and those receiving DTH  and those treated with DTH were 4.1 times  less likely to have aneurysm rupture as compared to mice receiving vehicle.Cerebral aneurysm formation occurred in 18 of 22 (81.8%) animals receiving only vehicle as compared to 3 of 12 TNF-α knockout mice  and those treated with vehicle (18 of 22, 83%, Cerebral aneurysm rupture leads to disability or death in the majority of patients ,2. Treatin vivo can increase the expression of TNF-α [TNF-α has also been found to be elevated in humans with ruptured cerebral aneurysms ,6, but mof TNF-α . We haveof TNF-α ,24, and of TNF-α . AdditioEnvironmental factors may activate TNF-α in both macrophages and smooth muscle cells. In this study we have found that TNF-α co-localized to both macrophages and smooth muscle cells. We have previously found that hypertension, hemodynamic stress, and cigarette smoke may activate TNF-α and induce phenotypic modulation in smooth muscle cells . AlthougThe role of TNF-α in aneurysm progression and rupture is unknown. Increased expression of TNF-α in human ,6 and moin vivo and in humans to assess the role of TNF-α in cerebral aneurysm progression and rupture. Further in vivo experiments following upregulation of TNF-α both in wild type and TNF-α knockout mice would be beneficial to further clarify the molecular mechanisms downstream of TNF-α upregulation in aneurysm formation and rupture. Limitations of this study include detection of aneurysm rupture through assessment of alterations in neurological examination, which may fail to detect subclinical or asymptomatic hemorrhages. Despite this drawback, examination of brains in asymptomatic mice did not reveal signs of significant SAH. Additionally, although DTH has been found to be a specific inhibitor of TNF-α synthesis [Additional experiments are indicated both ynthesis ,21, it min vivo model of cerebral aneurysm formation. TNF-α expression was decreased in TNF-α knockout mice, those pre-treated with the synthesized TNF-α inhibitor DTH, and controls versus unruptured and ruptured aneurysms. Additionally, the incidence of aneurysm formation and rupture was significantly decreased in TNF-α knockout mice and those pre-treated with DTH. Finally, treatment with DTH after aneurysm formation resulted in aneurysm stabilization and markedly decreased the incidence of rupture.In summary, we have demonstrated that TNF-α is increased in both unruptured and ruptured cerebral aneurysms in an BAPN: Beta-aminoproprionitrile; DAPI: 4′,6-Diamidino-2-phenylindole; DTH: 3,6′Dithiothalidomide; H & E: Hematoxylin and eosin; IL-1β: Interleukin 1-beta; MCP-1: Monocyte chemoattractant protein-1; MMP: Matrix metalloproteinase; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; SAH: Subarachnoid hemorrhage; SM-22α: Smooth muscle cell 22 alpha; TNF-α: Tumor necrosis factor alpha; VCAM-1: Vascular cell adhesion molecule-1.The authors declare that they have no competing interests.RS and AD made substantial contributions to conception and design, carrying out experiments, acquisition of data, and analysis and interpretation of data. NC, KW, and KS made substantial contributions to carrying out experiments, acquisition of data, and analysis and interpretation of data. PJ, ST, LFG, RR, DH, and GO made substantial contributions to conception and design, and analysis and interpretation of data. NG made substantial contributions to conception and design, and carrying out experiments. All authors were involved in drafting the manuscript and revising it critically for important intellectual content; have given final approval of the version to be published; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Quantitative real-time PCR, immunohistochemistry, and statistical analysis. Figure S1. TNF-α expression in TNF-α knockout mice and those pretreated with DTH. Figure S2. Beta-actin mRNA expression.Click here for file


Tumor necrosis factor (TNF) is an ancient and widespread cytokine required in small amounts for much physiological function. Higher concentrations are central to innate immunity, but if unchecked this cytokine orchestrates much chronic and acute disease, both infectious and noninfectious. While being a major proinflammatory cytokine, it also controls homeostasis and plasticity in physiological circumstances. For the last decade or so these principles have been shown to apply to the central nervous system as well as the rest of the body. Nevertheless, whereas this approach has been a major success in treating noncerebral disease, its investigation and potential widespread adoption in chronic neurological conditions has inexplicably stalled since the first open trial almost a decade ago. While neuroscience is closely involved with this approach, clinical neurology appears to be reticent in engaging with what it offers patients. Unfortunately, the basic biology of TNF and its relevance to disease is largely outside the traditions of neurology. The purpose of this review is to facilitate lowering communication barriers between the traditional anatomically based medical specialties through recognition of shared disease mechanisms and thus advance the prospects of a large group of patients with neurodegenerative conditions for whom at present little can be done. In 1975 the name TNF was given to a novel function of a semipurified peptide detected in the serum of mice receiving parenteral bacterial lipopolysaccharide (LPS) several weeks after they had been infected with Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis. On transfer to mice bearing transplanted sarcomas a purified form of this peptide caused necrosis of these tumors equally well as did LPS, but it was devoid of detectable LPS [Nearly forty years ago the term TNF appeared in the literature. First, to set a common source of confusion to rest, we note that the synonym TNFable LPS . Though  in vitro biologically generated specific anti-TNF agents) has become an enormous success story in a number of chronic inflammatory diseases outside the brain but curiously has not yet been taken seriously enough by the relevant medical specialists for its possible widespread adoption to be objectively examined for the chronic neuroinflammatory disease states. A 2010 editorial marking the 40th anniversary of the journal Stroke laments the dearth of advances leading to new targets for poststroke therapy, treatment ideas for which have stagnated [TNF neutralization by biological therapy , it generated side effects that mimicked the onset of severe sepsis . Yet whiNevertheless certain acute iatrogenic states that mimic sepsis can be treated preemptively. As reviewed , a numbe in vitro anti-TNF antibody reduced IL-1 as well as TNF [The challenge of disease states in which inflammation does not resolve is complex , but chrl as TNF , 60. Thil as TNF . The impl as TNF  led to al as TNF . With exl as TNF  and led l as TNF . Subsequl as TNF  and Crohl as TNF . Hence 1As we have reviewed , the duaAs noted earlier, neuroscience knows the cytokine TNF through copious literature on its central roles in much normal brain physiology and, when in excess, chronic brain disease states in which function and behavior are altered , 71. As http://www.pharmatimes.com/article/12-06-26/Ablynx_plans_to_partner_RA_drug_lifted_by_immunogenicity_data.aspx), a “Trojan Horse” trans-BBB transport method [In large part, an explanation for this delay may lie in cerebral diseases being technically more difficult to treat because the specific anti-TNF agents currently in use, such as infliximab, etanercept, and adalimumab, are too large to pass through the blood-brain barrier in any meaningful amount after subcutaneous or intravenous administration except in massive experimental doses , 84. Appt method , 87, andt method , followet method –91. Perit method , 93. As t method , the patt method –94 and ct method  on head-The nanobody and Trojan Horse approaches have not yet generated human data, but the perispinal approach has done so, albeit only in open trial  and obseThe principle of encouraging rational therapy by grouping diseases under their pathophysiology rather than immediate cause or anatomical location was thoughtfully expanded in 2010, again from the perspective of the wide relevance of specific anti-TNF agents as rational therapy . These aUnfortunately, the current division of medicine into anatomically focused clinical specialties does not always help the above logic gaining a firm foothold. Arguably, this arises because some disease specialists schooled and experienced within a particular narrow, albeit deep, knowledge base rarely rub shoulders with those with expertise in cytokine biology, a key to understanding much disease pathogenesis across the board. The cytokine-disease link has now become a formidable body of information compared to its earlier size, when, for example, rheumatologist needed to assimilate this literature, so the challenge is now correspondingly larger, though not impossibly so, for new entrants such as neurologists. In contrast, cytokine scientists working on disease mechanisms need to cross anatomical boundaries in order to remain competitive and for decades have necessarily ignored conventional specialty barriers in their reading. Hence they are, in general, better equipped to accept the rationale of specific anti-TNF therapy for neuroinflammatory disease more readily and, having observed its effects here, advocate its further development –6.Perhaps this relative lack of exposure to the broad cytokine literature is a large part of why this treatment concept has to date been dismissed by the clinical neurology community, despite not having observed its short- or long-term effects, as essentially impossible or dangerous , 106. Thhttp://www.sciencebasedmedicine.org/enbrel-for-stroke-and-alzheimers/) written by a clinical neurologist, recently being used to further the argument, in reference 33 of an American Academy of Neurology publication [http://www.sciencebasedmedicine.org/enbrel-for-stroke-and-alzheimers/), since each has a different mechanism and so requires a different treatment. Current broad knowledge base of cytokine biology in disease would have allowed the neurologist author to appreciate that anti-TNF agents, already accepted as a logical treatment for RA, psoriasis, Crohn's disease, Jarisch-Herxheimer reaction, adverse reactions to OKT3 therapy, and acute graft-versus-host disease , ankyloences in , sarcoidences in ), are alences in , after iences in , after cences in , and in ences in  and RA [ences in . This brences in , are imphttp://www.amgen.com/media/rapid_cognitive_improvement.html). This posting, still current at the time of writing, also ignores a number of confirmatory reports of a rapid response [Moreover, a serious student of cytokine biology would never regard specific anti-TNF agents simply as immunosuppressives (see above website), as if they were ibuprofen or aspirin. TNF in excess can be a proinflammatory cytokine but, as summarized earlier, it has many other activities that are as much a part of normal physiology as those of insulin and like it can become pathophysiological through loss of homeostasis . In the response , 98, 129response . Stroke editorial [ alert to new models of disease that may vertically integrate basic, clinical, and epidemiological disciplines. For example, could advances in the understanding of infectious diseases or inflammation dramatically change our thinking about stroke pathogenesis?Other criticisms on the quoted (reference 33 of ) websiteClearly, understanding advances in infectious diseases and inflammation would take neurologists directly into the world of basic TNF biology and thence how the corrupting effects of its excessive poststroke production in the brain would lead to neurodegenerative disease. This points them to a treatment based on a logical, testable hypothesis, one amply demonstrated for some years in open trials and ripe for formal testing with etanercept. As summarized in this review, its prospects are far better than any other treatment, extant or proposed.


Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection.B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes.First of all, B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.This study demonstrated that PAFR has a compelling involvement in The online version of this article (doi:10.1186/s12866-016-0685-8) contains supplementary material, which is available to authorized users. Brucella spp. are intracellular pathogens responsible for a chronic mammalian zoonotic disease, which has emerged as an ongoing public health problem worldwide [Brucella spp. to invade and persist are assumed to aid the organism's ability to avoid the killing mechanisms within cells [Brucella may bind to distinct phagocytic receptors, such as Fc gamma receptors and scavenger receptors, but may also bind to unknown receptors resulting in subsequent engulfment by zipper-like phagocytosis [Brucella invades phagocytes through lipid raft microdomains [orldwide . These oorldwide . The virin cells , but theocytosis . It has odomains , 6.Platelet-activating factor receptor (PAFR) is a member of the G protein-coupled receptor superfamily (GPCR) that is expressed on various cell types, including neutrophils, macrophages, monocytes, and epithelial cells . This reJanus kinase (JAK) 2 is generally known as a key linker of cytokine-mediated signal transduction and modulation of immune responses . It has B. abortus interacts with PAFR and what phagocytic mechanisms of B. abortus are associated with PAFR-linked signaling have not been defined. Here, we address a novel phagocytic mechanism and reveal that PAFR facilitates the phagocytosis of B. abortus, and this event is associated with intracellular JAK2 pathways leading to amplified rearrangement of the actin cytoskeleton. In correlation with the interaction of B. abortus with PAFR-linked JAK2 signaling, there is evidence that PAF production is notably increased by B. abortus infection, which triggers trafficking of PAFR from the membrane to the endosomes. Taken together, this study suggests that the PAFR-linked JAK2 signaling contributes to the entry of B. abortus into macrophages. These findings highlight an important receptor-mediated signaling pathway involved in the B. abortus phagocytic mechanism.It has been demonstrated that PAFR stimulation activates divergent signaling pathways. Whether B. abortus and PAFR or its ligand, PAF, has not yet been investigated. We first determined whether B. abortus infection induces PAF production through activation of PAFR. The PAF content increased at a high level at 5 min post-infection with B. abortus (2.12-fold increase) compared to resting cells -treated and AG490 (JAK2 inhibitor)-treated cells during B. abortus uptake. As a result, the levels of PAF secretion during B. abortus infection were significantly attenuated in both CV3988- and AG490-treated cells, demonstrating approximately 1.40-fold reductions. Collectively, PAF production is enhanced during B. abortus infection through a JAK2-mediated pathway. Additionally, the PAF content was not significantly increased in cells infected with heat-killed B. abortus compared to resting cells or live B. abortus, while in the case of E. coli O157:H7 infection, it was remarkably increased (2.18-fold increase) as much as those of live B. abortus.Previous studies elucidated the action of PAFR on bacterial infections , 17, howlls Fig. . PAF has abortus . ConsequB. abortus could augment the activation of LPCAT2 as a key step for PAF production, hence we investigated the phosphorylation of immunoprecipitated LPCAT2 in B. abortus-infected cells. As a result, B. abortus infection and PAF stimulation resulted in notable increase in the values of LPCAT2 activation by 53.63 % at 5 min post-infection compared to resting cells  as much as that of live B. abortus , an enzyme involved in the synthesis of PAF from lyso-PAF, enhances PAF production in endotoxin-stimulated macrophages . We hypolls Fig. . In conttus Fig. . In corrB. abortus-induced PAF production is involved in PAFR-mediated signaling pathway, and accumulating studies have shown that PAFR has important roles for bacterial infections [B. abortus. Macrophage cells were stimulated with various concentrations of PAF, which effectively acts through PAFR, in an artificial manner for a range of incubation times followed by infection with B. abortus. The uptake of B. abortus was significantly increased by the stimulation of PAF for 5 min in a dose-dependent pattern compared with untreated cells (P < 0.001)  , the numbers of internalized bacteria were significantly increased at MOI 100 than MOI 10 in control cells but also in PAF-treated cells  during B. abortus uptake , in contrast to the reduced uptake in JAK2-inactivated cells, was slightly altered by PAF stimulation /well in unconjugated bacteria vs 5.12 ± 0.542 CFU (×105)/well in Alexa flour 405-conjugated bacteria). As expected, the stimulation of PAFR by PAF augmented F-actin polymerization upon uptake of B. abortus and showed high levels of F-actin rearrangement as much as that of B. abortus-infected control cells  and a rightward intensity shift compared with non-stimulated or non-infected cells  to transduce extracellular signals , 24. FolB. abortus infection. The levels of colocalization of PAFR with endosomes at 30 min post-infection in B. abortus-infected and PAF-treated cells increased to 24.12 % and 24.07 %, respectively, compared to resting cells . Because a direct contact between PAFR and B. abortus was not detected, we thus assessed the effects of a PAFR ligand, PAF, on signal transduction during an entry step in macrophage infection by B. abortus.The invasion of pathogenic organisms into the host elicits a series of immune responses through interactions between a variety of pathogen virulence factors and the immune surveillance mechanisms of the host. Host-pathogen interactions are commonly initiated via host recognition of conserved molecular compositions known as pathogen-associated molecular patterns (PAMPs) , which aB. abortus infection led to a remarkable production of PAF during invasion. Furthermore, we postulated that PAF produced upon B. abortus infection affects PAFR-mediated events for the advanced uptake of B. abortus, hence uptake of B. abortus into macrophage was investigated by extrinsic treatment with PAF. The number of internalized B. abortus was significantly increased in PAF-stimulated cells, indicating that the stimulated PAFR enhances B. abortus uptake into macrophages. This phenotype is dependent on PAF stimulation time as our results show that treatment with PAF for 5 min more significantly increases internalization than treatment for 30 min. In contrast, the inhibition of PAFR function notably reduced the uptake of B. abortus. Possibly, the same mechanism occurs in PAFR through B. abortus-induced PAF on phagocytosis of B. abortus into host cell. Collectively, these findings suggest that the stimulation of PAFR actively influenced B. abortus phagocytosis and confirmed that PAFR has the potential to serve as a trigger for the uptake of B. abortus.PAF has diverse immunomodulatory actions for the host defense against bacterial infections, including stimulation and degranulation of granulocytes, monocytes, and macrophages , 33. In B. abortus infection, and the B. abortus-induced JAK2 activation was augmented through PAFR stimulation by PAF. In contrast, upon inhibition of PAFR, the elevated JAK2 activation by B. abortus infection was obstructed at the internalization stage. Indeed, these findings suggest that PAFR-involved JAK2 activation could be a virulence strategy critical for B. abortus invasion into host cells.The PAFR-associated intracellular signaling pathway was found to involve the intracellular tyrosine kinase JAK2, which resulted from the findings that JAK2 fails to become phosphorylated in a PAFR mutant , 18. In B. abortus infection of macrophages engaged in JAK2 activation, we observed the reorganization of the actin cytoskeleton and cytosolic activated JAK2 during B. abortus uptake by PAFR-stimulated or -suppressed macrophages. As expected, the microscopy results revealed that the F-actin polymerization and the redistribution of activated JAK2 were augmented by stimulation of PAFR. Furthermore, FACS analysis showed increased F-actin fluorescence intensity upon PAFR stimulation, which correlates with our microscopy results. Therefore, the B. abortus-induced effect on actin polymerization enhanced by PAFR-associated JAK2 activation could be essential for macrophage infection by this pathogen. In addition, regarding the infection dose, we verified that the number of internalized bacteria and intensity of F-actin were enhanced in higher infection dose (MOI 100) than in lower one (MOI 10), indicating the effects and levels of PAFR activation on B. abortus phagocytosis into macrophage have a tendency to be proportional to the infection dose.It has been known that PAFR activation induces the recruitment of intracellular kinases leading to distinct actin rearrangement at the plasma membrane , 23. To B. abortus infection induced intracellular lyso-phosphocholine acetyltransferase (LPCAT) activation, which subsequently participated in PAF synthesis. Advanced studies have shown the complex pathway connecting the stimulation of PAFR through enhanced PAF production by extracellular stimuli which initiates receptor internalization [Brucella infection in comparison with killed bacteria and E. coli O157:H7 as a non-Brucella control revealed that the positive effect on LPCAT2 activation-related PAF production is practically related with live Brucella infection but not killed bacteria. But the same positive effects of E. coli O157:H7 infection as much as those of live B. abortus corresponded with previous study that described a significant increase in PAF production by human colonic tissue for 4 h after infection by enterohemorrhagic E. coli (EHEC) [Brucella infection also induced PAF production through activation of LPCAT2 similar to other bacteria but we particularly verified that this enhancement is linked to the JAK2-mediated pathway. Moreover, the internalization and degradation of PAFR were induced by stimulation of PAF, which can be maintained by proteasome and lysosomal pathways [B. abortus could induce the production of PAF, which may be advantageous for invasion. Subsequently, the PAFR stimulation by B. abortus-induced PAF secretion causes mobilization and trafficking in membrane-engaged PAFR compared with the resting condition. This finding is supported by the evidence showing the increased trafficking of PAFR to endosomes, which is in accordance with the internalization of PAFR from the membrane upon B. abortus infection. Considering the relevant evidence that Brucella cause a weak response in innate immune system, of which mechanism is likely that Brucella do not rely on single distinct virulence factor [B. abortus infection could indirectly regulate PAFR-mediated signaling activation in an integrated aspect of host-pathogen interaction.lization , 24. On i (EHEC) . Thus, opathways . In linee factor , the eveB. abortus utilizes a supporting mechanism (non-binding membrane receptor PAFR), which modulates the activation of PAFR-mediated JAK2 signaling pathway during the entry step of infection.Thus, this sequential processing and correlation implies that B. abortus promotes PAF production, which induces PAFR-linked JAK2 signaling activation to successfully invade macrophages. It seems likely that B. abortus may enable us to further elaborate mechanisms of intracellular signaling cascades connected to membrane receptor activation. Thus, we establish a correlation between receptor-mediated cellular signaling and the pathogenic strategy of B. abortus.In summary, this study concludes that 2 atmosphere in RPMI 1640 medium  containing 10 % heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin . RAW 264.7 cells were seeded (1 × 105 cells/well) in cell culture plates and incubated for 24 h before infection for all assays.The RAW 264.7 murine macrophage cell line was obtained from the American Type Culture Collection  and was grown at 37 °C in a 5 % COB. abortus strain used in this study was derived from 544 (ATCC 23448), which is a smooth, virulent B. abortus biovar 1 strain. The B. abortus organisms were maintained as frozen glycerol stocks (glycerol 80 % v/v) at -70 °C. In all experiments, the contents of freshly thawed vials were cultured in Brucella broth  or Brucella agar without antibiotics for 3 days at 37 °C with aeration; whereas, enterohemorrhagic E. coli (EHEC) O157:H7 (ATCC 43894) cultures were grown in Luria-Bertani (LB) broth. Bacteria were grown at 37 °C with vigorous shaking until they reached stationary phase, and the bacteria were suspended in PBS and then the viable counts were measured by plating serial dilutions on Brucella agar. For generating the killed bacteria, B. abortus organisms grown to stationary phase were washed five times in sterile PBS, heat killed at 80 °C for 20 min. Total absence of B. abortus viability after heat killing was confirmed by the absence of bacterial growth in Brucella agar.The B. abortus as described previously [g at 22 °C for 10 min, and were then incubated at 37 °C in 5 % CO2 for 0, 15, and 30 min. The cells were washed once with medium and were then incubated with medium containing gentamicin (30 μg/ml) for 30 min to kill the remaining extracellular bacteria. For evaluation of viable bacteria at different time points, the infected cells were washed three times with PBS and were then lysed with distilled water. The number of viable bacteria was determined by CFU counts from serial dilutions of cell lysates on Brucella agar plates. All of the assays were conducted in triplicates and repeated at least three times on different days.To detect internalization efficacy of the bacteria, RAW 264.7 cells were infected with eviously . In brieB. abortus-infected cells in 6-well plates (5 × 105 cells/well) were lysed using ice-cold lysis buffer for 30 min at 4 °C. Total cell lysates were collected by centrifugation at 18,000 × g at 4 °C for 15 min. For the isolation of plasma membrane fractions, the infected cells were suspended in 50 mM Tris-HCl buffer , and then were sonicated on ice 2 times . The sonicated samples were ultracentrifuged at 265,000 × g at 4 °C for 2 h, and the resultant pellet (membrane fraction) was resuspended in 50 mM Tris-HCl buffer. After purification of all proteins, protein concentration was determined by Bradford protein assay . For the immunoprecipitation studies to analyze LPCAT2 activation, the cell lysates were incubated with goat anti-LPCAT2 antibody  overnight at 4 °C. The cell lysates were then mixed with protein G Sepharose beads (Santa Cruz Biotechnology). The final immunoprecipitated product, the initial total cell lysates and plasma membrane proteins were separated by SDS-PAGE and were transferred to a PVDF membrane . The blots were blocked for 1 h with 5 % (w/v) bovine serum albumin in TBS-T  and were probed by a mouse anti-phosphoserine antibody  for LPCAT2, rabbit anti-JAK2  and rabbit anti-p38α , and rabbit anti-PAFR  phosphospecific antibodies. Pan antibodies and rabbit anti-β-actin antibody  were used to probe stripped blots to verify that equivalent amounts of proteins were loaded per lane. Mouse anti-Sodium potassium ATPase (Na/K ATPase) antibody  was applied for quality control of plasma membrane. The binding of primary antibody was visualized using HRP-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies  followed by detection with enhanced ECL . The immunoblot ECL signals were quantified using NIH Image J software.B. abortus for 5 min. The PAF concentrations in cell culture supernatants were measured by mouse PAF ELISA  according to the manufacturer’s instructions. All assays were conducted in triplicates and repeated at least three times on different days.Cells cultured in 96-well plates were infected with B. abortus was labeled with Alexa Fluor 405  as follows; Ten mg of bacteria (approximately 1010 CFU) was dissolved in 1 ml of 0.1 M sodium bicarbonate buffer (pH 8.3), and then slowly added with 50 μl of Alexa Fluor 405. The reaction mixture was incubated for 1 h at room temperature with continuous stirring. After conjugation, viable bacteria were counted by culture on Brucella agar, it was ascertained that the conjugation of bacteria had no negative effect on viability. Macrophages were cultured in 12-well plates with 18 mm-diameter glass coverslips (105 cells/well) for 24 h before the infection. Cells were infected with unconjugated or Alexa Fluor 405 -conjugated B. abortus for 5 min. The samples were fixed with 4 % (w/v) paraformaldehyde (Sigma) and were permeabilized with 0.1 % Triton X-100 for 10 min at 22 °C. After 30 min incubation with a blocking buffer (2 % goat serum in PBS), the samples were stained with specific antibodies in blocking buffer for 1 h at 37 °C. For F-actin staining, the cells were incubated with 0.1 μM rhodamin-phalloidin  for 30 min at 22 °C. For the detection of intracellular JAK2 localization, the cells were incubated with phospho-JAK2 and then FITC-conjugated goat anti-rabbit IgG (Sigma). For detection of colocalization of PAFR and endosomes, B. abortus in medium containing 1 mg/ml of TRDx were deposited onto cells by centrifugation at 150 × g at 22 °C for 10 min, and were incubated at 37 °C for 5 and 30 min. Immunostaining was conducted with Alexa Fluor 350-labeled PAFR. Finally, the preparations were washed and mounted with fluorescent mounting medium . Fluorescence images were collected with an Olympus FV1000 laser scanning confocal microscope. The images were processed with Adobe Photoshop and NIH ImageJ software [For using fluorescent-conjugated bacteria, software .B. abortus , we performed a FACS assay for F-actin as previously described [6 cells/ml) were harvested and fixed with 4 % (w/v) paraformaldehyde at room temperature for 30 min. Next, the samples were permeabilized and stained with 20 μg/ml lysophosphatidylcholine (Sigma) containing 1 μM TRITC-phalloidin (Sigma). After centrifugation at 500 × g at 4 °C for 5 min, the cells were washed with PBS, and the F-actin content was quantified by FACS analysis using a FACSCalibur flow cytometer . For gating in FACS, two kinds of gating strategy were used; first, the viable cells were gated based on forward-scatter (FSC) and side-scatter (SSC) and F-actin fluorescence was measured by the second plot gated on the viable cells. Second, the population of B. abortus-infected cells was drawn to tightly encompass the majority of the viable cells based on FSC and SSC, and F-actin fluorescence was determined by the third plot gated on the infected cells, which was drawn to encompass an expanded area of positive cells. The data were collected as log-scaled fluorescence histograms from 10,000 cells, and the average F-actin content of a population was expressed as the mean of the fluorescence intensity. Experiments were performed in duplicates and repeated at least three times.To determine the relative content of F-actin in cells infected with Alexa Fluor 405-unconjugated or conjugated escribed . In briet-test or one-way ANOVA followed by the Newman-Keuls test were used to make a statistical comparison between the groups. The results with P < 0.05 were considered significantly different. *, **, *** denote P < 0.05, P < 0.01, P < 0.001, respectively.The data are expressed as the mean ± standard deviation (SD) for the replicate experiments. Statistical analysis was carried out using Graphpad-Prism software, version 4.00 . The Student’s Not applicable.Not applicable.The dataset supporting the conclusions of this article is included within the article and its additional files.


The overexpression of tumor necrosis factor (TNF)-α leads to systemic as well as local loss of bone and cartilage and is also an important regulator during fracture healing. In this study, we investigate how TNF-α inhibition using a targeted monoclonal antibody affects fracture healing in a TNF-α driven animal model of human rheumatoid arthritis (RA) and elucidate the question whether enduring the anti TNF-α therapy after trauma is beneficial or not.A standardized femur fracture was applied to wild type and human TNF-α transgenic mice (hTNFtg mice), which develop an RA-like chronic polyarthritis. hTNFtg animals were treated with anti-TNF antibody (Infliximab) during the fracture repair. Untreated animals served as controls. Fracture healing was evaluated after 14 and 28 days of treatment by clinical assessment, biomechanical testing and histomorphometry.High levels of TNF-α influence fracture healing negatively, lead to reduced cartilage and more soft tissue in the callus as well as decreased biomechanical bone stability. Blocking TNF-α in hTNFtg mice lead to similar biomechanical and histomorphometrical properties as in wild type.High levels of TNF-α during chronic inflammation have a negative impact on fracture healing. Our data suggest that TNF-α inhibition by an anti-TNF antibody does not interfere with fracture healing. Inflammatory diseases such as rheumatoid arthritis (RA), do not only increase the risk of fractures ,2 but maTNF-α is also an important regulator of fracture healing . Aside fTherefore, TNF-α is an important mediator during different phases of fracture healing. However, the influence of TNF-α blockade, as in treatment of RA patients under chronic inflammatory conditions, is still unknown. A retrospective study of rheumatoid patients treated with TNF- α antagonists showed no decreased risk of fractures . Since TGeneration of heterozygous human tumor necrosis factor transgenic (hTNFtg) mice strain Tg197) were described previously . Homozyg97 were dFmax = 250 N, XLC, Lloyd Instruments, Meerbusch, Germany) that detected the torsional force. The data were recorded using Catman32 software  and analyzed by Microsoft Excel . The angle of failure and the maximum load were determined as material characteristics and the torsional stiffness (N/°) was calculated as a biomechanical parameter. Each parameter was compared with the values of the non-fractured contralateral femur (ratio of fractured/non-fractured femur (100%)). Data shown are medians with a range of up to 8 measurements for each group.For biomechanical analyses, mice  were euthanized 28 days after surgery. Both femurs were dissected and prepared for biomechanical testing as described previously ,18. BrieFor histomorphological analyses fractured femurs  were embedded undecalcified in methylmethacrylate . Sagittal serial sections (5 μm) were stained with alcian blue. Image Pro Plus software  was used to determine and calculate the following histological parameters within the area of interest  was carried out using GraphPad Prism 5.02 . p-values >0.05 were regarded as significant, p-values between 0.05 and 0.1 indicate tendency.As described previously, the hTNFtg mice are smaller in size and weight compared to their wild type littermates ,19. A trTwo different material characteristics were measured, that are influenced by the tissue composition during fracture healing. Firstly, the force that is needed to break the bone, characterized by maximum torque, as well the angle of failure that is used as a parameter of the flexibility of the bone, were measured. Secondly, the torsional stiffness was calculated from these values as a measure for the biomechanical stability of the bone. After 28 days of healing there was no significant difference in the maximum torque between the wild type group and both hTNFtg groups, although the standard deviation was high in the hTNFtg group without treatment Figure . The fraFurther investigation of the callus tissue composition provided an indication for the altered biomechanical properties of the three different groups. Sagittal sections of femurs were stained for cartilage using alcian blue staining and for determination of the whole callus area Figure A. The arThe hTNFtg mouse is considered to be a valuable model of human RA ,20. In tThe importance of TNF-α signaling during fracture healing is commonly accepted today. On the one hand, it is known that TNF-α expression increases soon after injury ,21. DuriTNF-α-inhibitors like Infliximab are used in treatment of rheumatic patients to bind TNF-α with high affinity to reduce inflammatory symptoms ,24. The Elevated TNF-α level have a negative impact on the physiological development of the callus and thereby impair fracture healing in this murine model of chronic inflammation. We have found that the treatment of this chronic inflammation during fracture healing is beneficial and restores the biomechanical and morphological condition as found in the wild type. Further studies on fracture healing under chronic inflammatory conditions in animal models and rheumatoid arthritis patients will be necessary to further elucidate the mechanisms of this positive effect of anti-TNFα-treatment.TNF-α: Tumor necrosis factor alpha; RA: Rheumatoid arthritis; hTNFtg: Mice with a human TNF alpha transgenic background; IL: Interleukin; TNFi: AntiTNFalpha antibody treatment; MSC: Mesenchymal stem cell; AOI: Area of interest.The authors declare that they have no competing interests.MT, WB, HH, BW and RS collected the data. RS, TP and JZ designed the study. TP, MR and GS contributed to the data analysis and interpretation. MT, RS and JZ wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/184/prepub


Clear evidence based guidelines on the best and safest method of achieving and maintaining normal body weight during inpatient treatment of Anorexia Nervosa (AN) are currently not available. Oral feeding with food alone, high-energy liquid supplements, nasogastric feeding and parenteral nutrition all have the potential to achieve weight gain in the treatment of AN but the advantages and disadvantages of each method have not been comprehensively evaluated.A literature search was undertaken to identify papers describing feeding methods used during inpatient treatment of AN. The selection criteria searched for papers that described the feeding method; and reported weight change variables such as admission and discharge weight in kilograms, or Body Mass Index; or weight change over the course of inpatient treatment.Twenty-six papers were identified, describing a total of 37 samples with a mean sample size of 58.9 participants, and a range from 6 to 318. The majority (84.6%) of papers were observational cohorts and retrospective chart reviews. The most common feeding method described was nasogastric feeding and food, then high-energy liquid supplements and food.There is limited evidence on the efficacy of feeding methods used in the refeeding and nutritional rehabilitation of AN, therefore no conclusion can be made about the most effective method of achieving weight gain during inpatient treatment. While there are a number of papers exploring this issue there is no consistency in the way the information is reported to enable comparisons between the different methods. There is an urgent need for research in this area to guide decision-making in the inpatient management, refeeding and nutritional rehabilitation of AN. Under-nutrition and its sequelae are a major consequence of Anorexia Nervosa (AN) with refeeding and restoration of nutrition status a primary aim of the initial treatment. Guidelines on the treatment of AN -6 highliThere are several feeding methods used as part of the refeeding process in AN, such as with food alone, using high-energy liquid supplements, nasogastric  feeding, and parenteral nutrition -10. ThisLiquid supplements, nasogastric feeding and parenteral nutrition have the potential to provide one hundred percent of an individual’s recommended daily intake of nutrients or may be used to provide additional energy to any food that is eaten, in order to meet nutritional requirements and assist with weight gain. Nasogastric feeding and parenteral nutrition have an established use in acutely ill hospitalised patients who are unable to eat secondary to their illness or injury ,16. All This paper aims to review the published peer-reviewed literature on the feeding methods used in the inpatient treatment of AN, and to report any advantages and disadvantages of each method. Additionally, the authors aimed to determine if there is evidence of one feeding method being superior to another in terms of weight gain during inpatient treatment.A keyword search of the databases Medline and PsycINFO was undertaken between June and December 2012, to identify papers describing feeding methods used during inpatient treatment of AN. Five searches were undertaken using the key word “anorexia nervosa” combined with a second key word. The second key words used for the literature search, when combined with “anorexia nervosa”, were “nasogastric”, “enteral”, “feeding”, “parenteral” and “refeeding”. Additionally, a hand search was undertaken of key eating disorder journals as was snowballing of the published literature to identify any additional papers. The selection criteria for this review were papers which: described inpatients with a diagnosis of AN; provided a description of the feeding method used; and included weight change variables such as admission and discharge weight in kilograms, or Body Mass Index (BMI), or weight change over the course of inpatient treatment. Articles meeting the criteria (N = 26) were reviewed for study design, sample size, age of participants, length of stay, and prescribed energy intake. Papers that did not meet the inclusion criteria but described benefits or adverse effects of a feeding method were identified and pooled..Papers were excluded if: they were case studies with less than 10 participants; there was no description of the feeding method used; it was not clear which feeding method was being used; the subjects were not inpatients or were combined cohorts of inpatients and outpatients and unable to be separated; or it did not report weight change, or admission and discharge BMI. Only articles in English were reviewed. The results were tabulated in PASW Statistics GradPack 18.0In this literature search of feeding methods and inpatient treatment of AN, 26 papers were identified that met the inclusion criteria Table . As 10 pSearch one of “nasogastric” and AN identified 23 papers, of which 10 were reviewed, and seven were kept. Four of these seven papers included a comparator group. Reviewed papers were excluded because they were case series with less than 10 participants (n = 2), and did not describe any weight data (n = 1). Search two of “enteral” and AN identified 104 papers; 17 were reviewed and three were kept. Two of these three papers included a comparator group. Reviewed papers (n = 14) were excluded because they were case studies with less than 10 participants (n = 8), had insufficient weight data (n = 3), were a descriptive paper (n = 1), and presented data that could not be extracted (n = 2). Search three of “feeding” and AN identified 613 papers. Thirty-two papers were reviewed and three were kept. Two of the three papers included a comparator group. Reviewed papers were excluded because they had insufficient weight data (n = 10), were case series with less than ten participants (n = 2), did not adequately describe the feeding method (n = 13); or were review studies (n = 4). Search four, which used the keywords “parenteral” and AN identified 101 papers. Thirteen were reviewed, one was kept, which did not have a comparator group. Reviewed papers were excluded because they were case series with less than 10 participants (n = 5), had insufficient detail of the feeding method (n = 1) or were descriptive papers with no data (n = 6). Search five, using the keywords “refeeding” and AN identified 279 papers. Eleven were reviewed, and two were kept. Nine reviewed papers were excluded because there was insufficient detail on weight variables (n = 3), they were case series with less than 10 participants (n = 3), had insufficient detail on the feeding method (n = 1), the feeding method was not clear (n = 1), and described pooled data from 30 separate treatment facilities (n = 1). None of these papers had a comparator group. Ten relevant papers were identified using the hand search and snowballing methods, of which one paper had a comparator sample, and one paper had two comparator samples.The majority (84.6%) of papers were observational cohorts n = 10) and retrospective chart reviews n = 12). Two papers were randomised controlled trials, both by the same author, and one study had a prospective longitudinal design. Numerical data was extracted for age, sample size, admission and discharge BMI, total weight change in kilograms, prescribed energy in kilocalories, and length of stay in days, and are tabulated for each paper  reported numerical data for all seven variables . The most frequent feeding method described was nasogastric feeding and food, then liquid supplements and food. There were only three studies meeting the inclusion criteria that described the use of parenteral nutrition. Food only was described in six studies where it was the only method of refeeding however in 11 papers, food was the comparator group for the other feeding methods, resulting in a total of 17 food only samples. Most information about the benefits and adverse effects of the feeding methods were extracted from descriptive papers, which are pooled in Table The initial aim of this paper was to undertake a meta-analysis of all papers describing the feeding methods used for the inpatient treatment of AN, to determine which feeding method was the most effective, and to document occurrence of any adverse effects in the samples examined. As the majority of studies (84.6%) were observational cohorts and retrospective chart reviews, and much of the content of these papers were based on descriptions of practice rather than being based on data obtained from a robust research method, it was not possible to complete a meta-analysis.While there is much to be learnt from clinical expertise it is impossible to determine the superiority of any feeding methods based on this alone. Instead, an attempt was made by the authors to extract data from papers that met the inclusion criteria on sample size, age, admission and discharge weight, weight gain during treatment, length of stay and energy prescribed during treatment. This task was a difficult one, due in most cases to the data being presented in a non-standardised or inconsistent way that made it impossible to compare the results between papers. For example, 17 papers reported admission weight in kilograms, 31 reported admission BMI, seven reported percentage of ideal body weight, and only seven reported height measurements. Some papers reported more than one weight variable, for example, admission weight in kilograms and admission BMI. Discharge weight variables were reported in 31 of 37 samples, however 20 reported discharge weight in kilograms, 21 reported discharge BMI, and four reported percentage of ideal body weight. For the 33 samples that reported a weight change variable over the course of treatment, weight change was reported in kilograms for 24 samples, three reported weight change in grams per day, and seven reported the change in BMI. It could be argued that these variables  be mandatory in any research describing inpatients with AN to further knowledge of the effectiveness of treatment.All feeding methods, apart from six papers that used food only, were mixed feeding methods. That is, the method of feeding was a combination of oral food intake as meals and snacks, and liquid supplements, nasogastric feeding or parenteral nutrition. The majority of papers described the use of a second feeding method, in addition to food, to increase the intake of energy to achieve weight gain. High-energy liquid supplements were used to supplement meals and snacks if the prescribed amount of food was not consumed at the meal . NasogasThe authors’ recommendation is that a randomised prospective study (with sufficient power) be undertaken exploring different feeding methods. It should collect all the necessary data to make an informed decision about the relative effectiveness of each feeding method. We note that good clinical practice may include mixed methods and as such this should be seen as a feeding method and described in detail to allow for appropriate comparisons.The largest numbers of participants were treated with food only n = 1189), and notably with less disadvantages reported when compared to nasogastric feeding (n = 560) and parenteral nutrition n = 173)  reported that two out of 51 participants died after inpatient treatment when they were followed up 25 months after discharge . An addiWhile nasogastric feeding and parenteral nutrition appear to have more adverse effects than food only and liquid supplements, it is not possible to determine whether this is a result of the feeding method or resulting from another factor. For example, it is possible that subjects treated with nasogastric feeding or parenteral nutrition were more unwell, had greater severity of illness, or were less motivated to eat food orally. In most papers, confounding factors were not discussed in consideration of the results presented. For example, in most papers there was no discussion of the role of meal supervision in ensuring that nutrients that are prescribed are actually ingested. Only two papers were identified in this literature search, which described the benefits of meal supervision, and defined the processes involved ,52. Nota2, higher energy intake and total score on the Eating Disorder Inventory. However, there were no differences between the nasogastric feeding and food only groups in regards to psychological recovery, satisfaction with treatment or medical complication frequency. This paper did demonstrate that nasogastric feeding is equally safe as food only feeding owing to the lack of reported medical complications in this study.The best that could be achieved from this review was a description of benefits versus adverse effects of the feeding methods, which are summarised in Table The study by Zuercher 2003) showed differences in mean weight gain between 226 receiving food only compared to 84 patients receiving nasogastric feeding (0.82 versus 1.0 kg per week) and delivery of more energy via nasogastric feeding . However003 showeDiamanti, et al. 2008) compared 104 food only feeding patients and 94 parenteral nutrition patients and found no significant differences with respect to weight gain, recovery and rehospitalisation rate  compared-56. MelcBased on the results of this literature review, there is limited evidence on the efficacy of feeding methods used in the refeeding and nutritional rehabilitation of AN, therefore no conclusion can be made about the most effective method of achieving weight gain during inpatient treatment. Considering the importance of re-nutrition in the overall treatment of AN, this is a concern. While there are a number of papers exploring this issue there is no consistency in the way the information is reported to enable comparisons between the different methods. There is an urgent need for research in this area to guide decision-making, as currently little research directs clinicians towards best practice. The literature does provide direction for further research which can be achieved if clinicians working in the area work collaboratively to further knowledge and understanding of refeeding and nutritional rehabilitation in AN. The summary of benefits and adverse effects in Table In order to improve our knowledge of feeding methods in AN, there should be a minimum data set reported on research describing inpatient treatment for AN that includes admission and discharge BMI (or weight and height), weight change during treatment, and length of stay. Additionally, details of the feeding regime, such as nutrient composition and energy delivered would be useful. The authors also recommend that the short term benefits of feeding methods used in the treatment of AN, such as weight gain, and long term benefits, such as recovery rate of participants, be examined using a robust study design.It is a limitation that only English articles were reviewed. Additionally, it was difficult to extract papers from the literature search using key words, as there was diverse usage and no consistent keywords used, which made identifying useful papers difficult. Some authors have published a number of papers, and it is unclear whether the papers describe the same or different cohorts.AN: Anorexia nervosa; BMI: Body mass index; kg: Kilograms; kg/m2: Kilograms per metre squared; kg/week: Kilograms per week; kCal: Kilocalories; NG: Nasogastric feeding; PN: Parenteral nutrition; SUP: High-energy liquid supplements; RCT: Randomised controlled trial.The authors declare that they have no financial or non-financial competing interests.SH – Completed literature search; analysed papers and determined which papers met inclusion and exclusion criteria; wrote and reviewed manuscript. RF - Analysed papers and determined which papers met inclusion and exclusion criteria; wrote and reviewed manuscript. SA - Wrote and reviewed manuscript. JR - Wrote and reviewed manuscript. All authors read and approved the final manuscript.


Various pathways have been implicated in the pathogenesis of heart failure (HF) with preserved ejection fraction (HFPEF). Inflammation in response to comorbid conditions, such as hypertension and diabetes, may play a proportionally larger role in HFPEF as compared to HF with reduced ejection fraction (HFREF).This study investigated inflammation mediated by the tumor necrosis factor-alpha (TNFα) axis in community-based cohorts of HFPEF patients (n = 100), HFREF patients (n = 100) and healthy controls (n = 50). Enzyme-linked immunosorbent assays were used to investigate levels of TNFα, its two receptors (TNFR1 and TNFR2), and a non-TNFα cytokine, interleukin-6 (IL-6), in plasma derived from peripheral blood samples. Plasma levels of TNFα and TNFR1 were significantly elevated in HFPEF relative to controls, while levels of TNFR2 were significantly higher in HFPEF than both controls and HFREF. TNFα, TNFR1 and TNFR2 were each significantly associated with at least two of the following: age, estimated glomerular filtration rate, hypertension, diabetes, smoking, peripheral vascular disease or history of atrial fibrillation. TNFR2 levels were also significantly associated with increasing grade of diastolic dysfunction and severity of symptoms in HFPEF.Inflammation mediated through TNFα and its receptors, TNFR1 and TNFR2, may represent an important component of a comorbidity-induced inflammatory response that partially drives the pathophysiology of HFPEF. Heart failure (HF) continues to have a significant clinical burden with a high mortality and morbidity, and has a worse one-year prognosis than cancer Biomarker studies have differentially implicated various pathways in HF, including fibrosis and extracellular matrix remodeling, oxidative and cardiomyocyte stress, and inflammation This study conforms with the conventions outlined in the Declaration of Helsinki, it received internal ethics board approval at the Universities of Alberta and Calgary for use of human subjects, and all subjects gave written informed consent Heart Failure Etiology and Analysis Research Team) project, community-based, ambulatory patients with clinical diagnoses of HF and healthy age- and gender-matched controls (n = 50) were consecutively recruited for comprehensive clinical, echocardiographic and biomarker analyses during a three-year period beginning in 2010 through the end of 2012. Blood pressure was recorded sitting or recumbent, and detailed clinical data were compiled at the time of enrollment. Transthoracic echocardiograms were performed using the Phillips IE33 ultrasound platform. Echocardiograms were interpreted by cardiologists with specialized echocardiography training who were blinded to both the clinical classification and biomarker analyses. LVEF was assessed using Simpson's biplane method of disks. HF patients were adjudicated as HFREF (n = 100) or HFPEF (n = 100) using an LVEF cutoff of 50%, according to clinical practice guidelines As part of a prospective clinical study, the Alberta HEART  read in an automated DxI 800 immunoanalyzer  at provincial heath laboratories in the province of Alberta Blood for plasma analysis was collected during a one-day baseline enrolment which included the acquisition of the echocardiograms and ECGs. Patients were rested and sitting while blood was collected into cooled lithium-heparin tubes, which were immediately placed on ice prior to plasma fractionation and deep freezing at ≤−75°C. Commercially-available enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, TNFR1, TNFR2 and IL-6  were used as previously described 2  at pH 6.5. Assay buffer was made to contain various protease inhibitors: 10 µM captopril ; 5 µM amastatin ; and Protease Inhibitor Cocktail  dissolved to achieve a concentration of 10 µM bestatin (aminopeptidase inhibitor), 1 µM E-64 , 770 nM pepstatin A (aspartyl protease inhibitor), 154 µM AEBSF , 75 nM phosphoramidon (neprilysin inhibitor), 15 nM aprotinin (serine protease inhibitor) and 75 nM leupeptin . The use of protease inhibitors in the buffer solution was meant to ensure that the fluorogenic substrate and specific ACE2 inhibitor (both described below), which are integral to the assay, were not cleaved by other proteases naturally present in human plasma. Samples were incubated with Dnp-quenched Mca-containing fluorogenic substrate  at a final concentration of 10 µM at 37°C. To find the fluorescence increase due to ACE2 activity, plasma samples were assayed both in the presence and absence of the specific linear ACE2 inhibitor, DX600 . Fluorescence was measured with excitation and emission settings at 320 nm and 405 nm, respectively, using SpectraMax M5 plate reader . The maximal fluorescence increase due to ACE2 activity was determined from the maximum fluorescence difference between inhibited and uninhibited aliquots; this was normalized to a standard curve for Mca-containing fluorescent peptide  and scaled for time of measurement and plasma volume over 24 hours, with 1 hour as the baseline. All ACE2 enzymatic activity values herein are expressed in pmol/hr/mL, which describes the amount of substrate turned over per unit time per unit volume of plasma. Inter- and intra-assay coefficients of variation were 10.2% and 7.5% (n = 10), respectively.Plasma ACE2 activity was measured using a previously established immunofluorescence protocol 2 per WHO criteria 2 for males and ≥95 g/m2 for females per previously published criteria Continuous data in tables are expressed as median with interquartile range (IQR) in parentheses. Continuous data in figures are expressed as box and whisker plots on a logarithmic scale to account for non-normal distributions, where boxes represent IQR with median as a bisecting line, while whiskers represent the minimum and maximum values. Categorical data are expressed as percentages. All categorical data were compared using Pearson Chi-square tests, while continuous variables analyzed by category were compared using Mann-Whitney U Test or Kruskal-Wallis Test with Mann-Whitney U Test for pairwise comparisons, where appropriate. Associations between categorical factors or continuous covariates and circulating inflammatory markers were tested using binary logistic or linear regression analyses, respectively, with log-transformed inflammatory marker levels to account for non-normal distributions. Associations between increasing grade of diastolic dysfunction or NYHA functional class and plasma biomarkers were tested using ordinal logistic regression analyses with log-transformed inflammatory marker levels. Forest plots are used to graphically display results from logistic regression analyses. A p-value<0.05 was considered significant for all statistical analyses. Statistical analyses were conducted using SPSS Statistics Version 20 . Obesity was defined as BMI≥30 kg/mDemographic and clinical information for the control, HFPEF and HFREF groups is displayed in Heart rates did not differ significantly between controls or the two HF groups; however, a significantly greater number of HFPEF patients were in AFib on the day of study than HFREF patients , a sheddase involved in proteolytic processing of TNFα, TNFRs, and angiotensin-converting enzyme 2 (ACE2) might represent a mechanism of increased circulating TNFα and TNFRs in HF In this comparative analysis of healthy controls, and HF patients with preserved or reduced left-ventricular ejection fraction, we found that our cohorts recapitulated previous characterizations, whereby, HFPEF subjects were more likely to be older, hypertensive and obese Improvement in exercise capacity in a cohort of HFPEF patients after an exercise training regimen was largely mediated by peripheral improvements, which suggests a systemic component to HFPEF, including skeletal muscle dysregulation We found that low eGFR, hypertension, smoking status and history of atrial fibrillation were significantly associated with elevated TNFα levels, while elevated TNFR1 levels were associated with aging, low eGFR, hypertension, diabetes and PVD, and elevated TNFR2 levels were associated with aging, low eGFR, diabetes and atrial fibrillation. The advanced age and greater prevalence of comorbidities in the HFPEF population could drive the observed elevation in circulating inflammatory markers relative to HFREF. Indeed, non-cardiac comorbidities are more prevalent in HFPEF, with a larger fraction of adverse clinical outcomes attributable to non-cardiac events compared to HFREF 2+ handling proteins involved in myocardial relaxation Our finding that circulating TNFR2 levels are significantly associated with increasing average E/e' ratio and grade of diastolic dysfunction in HFPEF, but not in HFREF, suggests a greater role for TNFα-mediated inflammation in this cohort. However, we found that TNFR1 and TNFR2 levels were significantly associated with increasing NYHA class in both HF phenotypes, which shows that part of the HFREF phenotype can also be attributed to inflammation. The association of diastolic dysfunction and symptoms with TNFR2 in HFPEF is consistent with findings in experimental models, as the TNFR1/TNFR2 axis is involved in mediating divergent effects: TNFR1 has been implicated in adverse cardiac remodeling and adipogenesis, while TNFR2 antagonizes the pathological effects of TNFR1, and also stimulates angiogenesis et al. implicate TNFR1, while our study implicates TNFR2 in HFPEF et al. explored risk of incident HF, while we examined existing HF populations. Indeed, we found TNFR1 to be associated with the greatest number of HF risk factors, which is congruent with a role for TNFR1 in precipitating incident HFPEF. Meanwhile, our data show that TNFR2 might then be a better biomarker for gauging severity of established HFPEF.The differential role of TNFRs might explain why TNFα is not associated with function or symptoms: the variable response to TNFα is mediated at the receptor level. Congruent with a recent report, we found TNFα was not strongly associated with either HF phenotype; however, Marti Circulating fragments of TNFα, TNFR1 and TNFR2 are generated from full, transmembrane proteins through the activity of TACE Taking our data together with previous reports indicates that the TACE/TNFα/TNFR1/TNFR2 inflammatory axis is a part of the pathogenesis of HFPEF et al. is a strong contribution Our study, while larger than most other studies that compared levels of TNFα and its receptors in HFPEF versus HFREF, is still relatively small in terms of clinical studies. Indeed, TNFα might also be informative with respect to differentiating HFPEF from HFREF, but our study cannot resolve whether this is the case. In addition, the results reported herein represent a single cross-section across healthy controls, and HFPEF and HFREF patients, so follow-up measurements are not included, nor are outcomes data. Further studies with a longitudinal component will be important for implicating various molecules or pathways in the pathophysiological development of HF, for which work by Marti


This editorial deals with the state of the Iranian Journal of Basic Medical Sciences (IJBMS) in 2013. In the previous year, we received 613 manuscripts for publication. After peer-review by expert national and international reviewers, 15 % of submitted papers were accepted for publication. As presented in The publication frequency was doubled by switching from bimonthly to monthly, with around 12 papers per issue. Most of the published papers were original articles; review articles were around 5% of all published papers . Contributions of the international authors were increased compared to the previous years . In 2013Crocus sativus). As Saffron is one the most famous plants cultivated in Iran and has various pharmacological effects including anti-asthmatic, anti-carcinogenic, anti-mutagenic, anti-depressant, and immunomodulating and has antioxidant-like properties, it was selected for a special issue. The second issue was conducted with 20 papers on Human T-cell lymphotropic virus-I (HTLV-I), as this virus is endemic in several regions of the Middle East including Iran; various concepts about this virus and related diseases were discussed.Two special issues were published in 2013. The first one focused on Saffron  is expected for the current year.


Immunologists currently face daunting challenges, as a result of the rapid development of new methods for immunological data collection, from high-throughput phenotyping to deep sequencing . These aAt about the same time, the organizers mentioned above were approached by the Frontiers editorial staff with the idea for a “Frontiers in Immunology” research topic, which was to provide a comprehensive, online, open access snapshot of the current state of the art on immune system modeling and analysis. The research topic was launched, edited, and finalized with the kind help of co-editors Rob de Boer, Miles Davenport, Carmen Molina-Paris, Michal Or-Guil, and Veronika Zarnitsyna. It has been a success, with 35 papers accepted for publication, which attests to the timeliness of the topic.The papers included in this Research Topic reflect many of the issues that theoretical immunologists are struggling with. Some of the papers address old questions – such as the targeting of somatic hypermutation  and the My thanks go to the above-mentioned co-editors, to the responsive and efficient Frontiers editorial staff, to all the authors who contributed papers, and to the reviewers whose work has made publication of all these papers possible.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Immunotherapy for type I allergies is well established and is regarded to be the most efficient treatment option besides allergen avoidance. As of today, different forms of allergen preparations are used in this regard, as well as different routes of application. Virus-like particles (VLPs) represent a potent vaccine platform with proven immunogenicity and clinical efficacy. The addition of toll-like receptor ligands and/or depot-forming adjuvants further enhances activation of innate as well as adaptive immune responses. CpG motifs represent intensively investigated and potent direct stimulators of plasmacytoid dendritic cells and B cells, while T cell responses are enhanced indirectly through increased antigen presentation and cytokine release. This article will focus on the function of VLPs loaded with DNA rich in nonmethylated CG motifs (CpGs) and the clinical experience gained in the treatment of allergic rhinitis, demonstrating clinical efficacy also if administered without allergens. Several published studies have demonstrated a beneficial impact on allergic symptoms by treatment with CpG-loaded VLPs. Subcutaneous injection of VLPs loaded with CpGs was tested with or without the adjuvant alum in the presence or absence of an allergen. The results encourage further investigation of VLPs and CpG motifs in immunotherapy, either as a stand-alone product or as adjuvants for allergen-specific immunotherapy. Respiratory allergies today are some of the most prevalent chronic diseases . AccordiAllergen-specific immunotherapy over 3–5 years requires numerous allergen applications either subcutaneously or sublingually , 8, leadAll of the above strategies are based on the dogma that AIT requires the allergen. This dogma has been challenged, however, by recent data , 42••. OVLPs spontaneously assemble upon expression of virus or bacteriophage capsid proteins Fig. . Some ofBecause of their natural function as carriers of genetic information, virus or bacteriophage capsid-derived VLPs can readily be filled with DNA or RNA. VLPs stabilize and protect these molecules from enzymatic degradation by DNAses and RNAses. Bacteriophage-derived VLPs filled with immunostimulatory CpGs have been used to display tumor antigens and were found to efficiently induce tumor antigen-specific cytotoxic CD8 and CD4 T cells in melanoma patients –50. SimiVLPs can be subcutaneously or intramuscularly injected. Their diameter of approximately 30 nm facilitates entry into lymphatic vessels and direct drainage into local lymph nodes –54. OnceDCs can be subdivided into two main populations. While myeloid DCs (mDCs) produce mainly interleukin (IL)-12, plasmacytoid DCs (pDCs) produce mainly interferon (IFN)-α –60. CpGsThe complete lack of genetic information with regard to viral replication enhances safety. In contrast to attenuated vaccines, no reversion to the virulent form is possible.Escherichia coli or yeast, in accordance with Good Manufacturing Practice (GMP) guidelines.As VLPs assemble spontaneously, they can be produced cheaply in The size and form of VLPs enhance lymphatic drainage and uptake by antigen-presenting cells in the local lymph nodes.Immunostimulatory CpGs or RNA can be packaged into VLPs and are stabilized and protected from enzymatic digestion inside the VLPs . Many prOverall, the immune response against VLPs is characterized by immunological properties similar to those seen with live viral vaccines, but, in contrast to the latter, VLPs have the following advantages:The human immune system can be activated by pathogen-associated molecular patterns (PAMPs), which are common to nearly all pathogens. These include nonmethylated CG-rich DNA sequences, which are common in the bacterial genome but not in the genome of vertebrates, where these motifs are usually methylated. Numerous such CpGs have been characterized and can be synthesized as oligodeoxynucleotides (ODNs), using either the naturally occurring “phosphodiester” bonding or the more stable “phosphorothioate” bonding in the backbone of the molecule, hence the designation “C–p–G”. CpGs bind to TLR9, which activates (via the adaptor molecule MyD88) the nuclear factor (NF)-κB pathway, which starts the inflammatory cascade .Three different types of CpGs have been distinguished—namely, A-, B-, and C-type CpGs. Type A CpGs contain their CG motifs in a palindromic sequence, surrounded by guanosines. The backbone of type A CpGs consists of phosphodiester bonds, which are readily digested by DNAses. In contrast, the backbone of type B CpGs consists of phosphorothioate bonds, which are DNAse resistant. Type C CpGs are characterized by phosphorothioate bonds, as well as CG motifs in palindromes on a phosphodiester backbone. These three different types of CpGs differ in their immunological activities. Type A CpGs induce mainly IFN-α, whereas type B CpGs are potent activators of B cells but barely induce IFN-α. In mice, type B CpGs are potent inducers of IL-12 in mDCs. But, as mentioned above, as human mDCs do not express TLR9, the IL-12 response to this type of CpG is inefficient in humans. In addition, pDCs essentially fail to respond to type B CpGs, as they produce only low levels of IL-12 and do not respond with the production of IFN-α to stimulation with this type of CpG. Thus, human DCs respond very poorly to B-type CpGs. Finally, type C CpGs have the characteristics of both A- and B-type CpGs.While, in mice, TLR9 is expressed in both pDCs and mDCs, in humans, TLR9 is expressed only in pDCs. TLR9 is also expressed in B cells, mast cells, neutrophils, eosinophils, basophils, and keratinocytes . TLR9 isThe human immune system has evolved to efficiently recognize highly repetitive structures, because such structures are typically found on the surfaces of viruses and bacteria. To speed up the antibody response, such repetitive arrays of epitopes can crosslink such large numbers of B cell surface receptors that antibody production is induced even in the absence of T cell help. Therefore, repetitive antigens, such as those found on the surface of VLPs, are known as “Th-independent antigens” ••. In adCreticos et al. performed a clinical trial using the major ragweed allergen Amb a 1 conjugated to a type B CpG-ODN in 25 ragweed-allergic patients ••. In thE. coli-derived RNA ligand for TLR7 and 8. The vaccine was administered three times at 4-week time intervals. Twenty-four healthy volunteers participated in this study, and antibody titers were followed for 18 months [We have performed an open-label phase I clinical trial with a house dust mite (HDM) Der p 1-derived peptide of 16 amino acids in length, which was covalently coupled by a chemical linker to bacteriophage Qβ-derived VLPs filled with 8 months •. The va8 months • (Table In an additional clinical trial, we investigated the safety and tolerability, as well as the clinical efficacy, of type-A CpGs packed in Qβ-derived VLPs. The CpG-loaded VLPs were mixed with HDM allergens and used as an adjuvant in specific immunotherapy  Table 11. In ordIn the following clinical trial, we therefore used the same Qβ-derived VLPs filled with the same type A CpG, named G10. The study was a randomized, double-blind, placebo-controlled phase IIb trial in 299 patients suffering from allergic rhinoconjunctivitis due to HDM allergy •• (TableAnimal models suggest that type-A CpG-induced Th1 responses and IFN-α inhibit Th2 responses. Furthermore, symptom amelioration could also be due to direct effects of CpGs on mast cells, which also express TLR9 –74. CpGsLast ,but not least, it should not be forgotten that if clinical trials are being performed in HDM allergy, the latter allergens are nearly ubiquitous and present during the entire season. It can therefore not be excluded that the natural exposure to allergen plays a role in the “allergen-free” treatment regimens being discussed.Thus, treating allergies with CpG-loaded VLPs remains an attractive option and may allow desensitization of a large number of patients in an efficient manner in the absence of risk of anaphylactic reactions. However, there are a number of questions that still need to be resolved, including finding the optimal dose and the duration of the therapeutic effect. Further clinical trials are therefore required to elucidate whether “allergen-free” immunotherapy is a valuable long-term treatment option for allergies.