Juvenile pulmonary alveolar proteinosis (PAP) due to CSF2RA mutations is a rare disorder with only a few cases described worldwide.We identified nine children with severe diffuse interstitial lung disease due to CSF2RA mutations. Clinical course, diagnostic findings and treatment were evaluated and correlated to the genotype. Functional impairment of the intracellular JAK/pStat5 signaling pathway was assessed using flow-cytometry of peripheral mononuclear cells (PBMC) and granulocytes.We identified six individuals with homozygous missense/nonsense/frameshift mutations and three individuals homozygous for a deletion of the complete CSF2RA gene locus. Overall, four novel mutations  were found. Reduced STAT5 phosphorylation in PBMC and granulocytes was seen in all cases examined (n = 6). Pulmonary symptoms varied from respiratory distress to clinically silent. Early disease onset was associated with a more severe clinical phenotype (p = 0.0092). No association was seen between severity of phenotype at presentation and future clinical course or extent of genetic damage. The clinical course was favorable in all subjects undergoing whole lung lavage (WLL) treatment.Our cohort broadens the spectrum of knowledge about the clinical variability and genotype-phenotype correlations of juvenile PAP, and illustrates the favorable outcome of WLL treatment in severely affected patients.The online version of this article (doi:10.1186/s13023-014-0171-z) contains supplementary material, which is available to authorized users. SFTPB), surfactant protein-C (SFTPC), member A3 of the ATP-binding cassette family of transporters (ABCA3) . In In our cohort, treatment varied from regular follow-up investigations in asymptomatic patients to frequent, periodical WLL, which were conducted over several years to either maintain a stable clinical course, or to even improve the clinical outcome. It has been repeatedly shown that WLL may lead to symptomatic relief ,13. For Several lines of evidence suggest that WLL treatment may not only provide symptomatic relief, but may act in favour of a benign clinical disease course. Of note, two cases of siblings with identical GM-CSF receptor α chain mutations and proven functional receptor impairment in flow cytometry investigations have been reported where only one sibling developed PAP . It was While such individual concepts currently remain the treatment of choice, novel developments point towards advanced therapeutic options such as organotopic transplantation of marcrophage progenitors . FurtherIn summary, the current study extends the clinical body of knowledge on juvenile PAP. During the prolonged clinical follow-up, a clear symptomatic benefit of repeated WLL therapy was seen in severely affected patients. Mild cases were usually self-limiting over time. While all CSF2RA mutations led to a severe impairment of the STAT5 signaling pathway, no correlation between the severity of the clinical phenotype and the extent of genetic damage was seen. However, we observed a tendency towards earlier disease onset in patients with loss of the transmembranous domain of the GM-CSF receptor α chain. Our approach to the infant and child with pulmonary alveolar proteinosis of unclear origin is to locate the defect and administrate its most effective treatment as soon as possible. STAT5 functional analysis in peripheral blood cells and subsequent genetic testing is the method of choice to classify the pathogenetic nature of this rare condition. Long-term, web-based clinical follow-up of patients with CSF2RA mutation related juvenile alveolar proteinosis in registers will be a helpful tool to further extend the body of knowledge on this rare condition, and to create ready-to-trial populations for emerging therapeutic approaches.www.ojrd.com“.The data set supporting the results of this article is available online as Additional file


Multiciliated cells (MCCs) in the respiratory epithelium play crucial roles in the innate defense system against airborne pathogens and pollutants. Each MCC possesses 200-300 cilia on their apical surface, which beat in a synchronized manner to clear mucus with foreign particles from the respiratory tract. Ciliary dysfunction leads to serious respiratory diseases including chronic infection as seen in patients with primary ciliary dyskinesia (PCD), a ciliopathy with defective multicilia. However, the molecular mechanisms underlying the differentiation of MCCs are poorly understood.de novo via an acentriolar pathway that involves centriole nucleation from fibrogranular structures called deuterosomes. In contrast, a canonical centriolar pathway, in which centrioles replicate using the existing centriole as a template, contributes to a small portion of the total centriole population. Nascent centrioles mature by acquiring accessary structures, which include distal appendages (DAs). Upon docking to the apical membrane, centrioles and DAs transform into basal bodies and transition fibers (TFs), respectively. DAs/TFs are fibrous structures that emanate from the distal end of the B-tubules of each of the nine centriole/basal body triplet microtubules, and are important for membrane docking [In MCC precursor cells, downregulation of Notch signaling induces expression of the coiled-coil protein Multicilin (Mcidas), which in turn upregulates expression of the ciliogenesis transcription factors Myb, RFX and FoxJ1 that direct MCC differentiation. The majority of centrioles are generated  docking , althougChibby (Cby) is an evolutionarily conserved 15-kD coiled-coil protein that localizes to the base of cilia. We previously reported that Cby−/− mice suffer from chronic respiratory infection due to a lack of effective mucociliary transport . ConsistRecently, using SIM and STORM super-resolution microscopes, we found that Cby protein clusters at the base of mature cilia as a ring with a diameter of 300 nm and a height of 100 nm . IntriguSince the first morphological EM study on MCCs in rat lungs in the 1960s , the molWe are only beginning to understand the molecular details for ciliary vesicle assembly. Recently, the membrane shaping proteins EHD1 and EHD3 and the SNARE membrane fusion regulator SNAP29 have been shown to function in ciliary vesicle formation . However


Apart from its application in human diagnostics, magnetic resonance imaging (MRI) can also be used to study the internal anatomy of zoological specimens. As a non-invasive imaging technique, MRI has several advantages, such as rapid data acquisition, output of true three-dimensional imagery, and provision of digital data right from the onset of a study. Of particular importance for comparative zoological studies is the capacity of MRI to conduct high-throughput analyses of multiple specimens. In this study, MRI was applied to systematically document the internal anatomy of 98 representative species of sea urchins (Echinodermata: Echinoidea).GigaDB repository can be accessed using open source software. Potential uses of the dataset include interactive exploration of sea urchin anatomy, morphometric and volumetric analyses of internal organs observed in their natural context, as well as correlation of hard and soft tissue structures.The dataset includes raw and derived image data from 141 MRI scans. Most of the whole sea urchin specimens analyzed were obtained from museum collections. The attained scan resolutions permit differentiation of various internal organs, including the digestive tract, reproductive system, coelomic compartments, and lantern musculature. All data deposited in the The dataset covers a broad taxonomical and morphological spectrum of the Echinoidea, focusing on ‘regular’ sea urchin taxa. The deposited files significantly expand the amount of morphological data on echinoids that are electronically available. The approach chosen here can be extended to various other vertebrate and invertebrate taxa. We argue that publicly available digital anatomical and morphological data gathered during experiments involving non-invasive imaging techniques constitute one of the prerequisites for future large-scale genotype—phenotype correlations. Despite the fact that sea urchins (Echinodermata: Echinoidea) have served as model organisms in various biological disciplines for over a century and a half , the intIn recent years, morphology has fallen behind data gathering, deposition and transparency practices considered as standard in other biological disciplines, such as proteomics or genomics . Apart fThe deposited dataset comprises 141 MRI scans from 98 representative extant sea urchin species. The scanned specimens were whole sub-adult or adult individuals ranging in diameter or length from 5–43 mm. Most of the specimens were obtained from museum collections, where they are preserved in ethanol for long-term storage. Some of the specimens were collected and fixed more than 135 years ago, while others were collected a few months prior to scanning. Additional file Basic information on the protocols used for specimen preparation, contrasting, and scanning are provided in Additional file Forty-four two-dimensional (2D) and 97 three-dimensional (3D) scans were obtained using various high-field MRI scanners. Table For each scan, one image stack in tagged image file format  was created based on the standard Bruker MRI 2dseq raw image file using the software ImageJ . To faciThe suitability of a given specimen for MRI was ascertained through visual inspection of the MRI scout images and the low-resolution scans performed prior to scanning at high resolutions . The achin vivo study of sea urchins whose gonads are intended for human consumption.The methodological approach employed here allows conducting high-throughput analyses of hundreds or even thousands of zoological specimens . PotentiThe dataset presented here constitutes a representative sample of sea urchin structural diversity. No significant differences in scanning results were observed when employing freshly fixed or museum specimens , while tDue to the digital nature of data based on non-invasive imaging techniques, the rapid online dissemination of morphological and anatomical information has finally become possible. This development is bound to lead to an unprecedented level of data availability and transparency in zoomorphology and paleontology, ultimately resulting in more widespread data mining in these two scientific fields . FurtherGigaDB [The dataset supporting the results of this study is available in the GigaScience repository, GigaDB . AdditioGigaDB  for a di2D: Two-dimensional; 3D: Three-dimensional; MRI: Magnetic resonance imaging; TIFF: Tagged image file format.The authors declare that they have no competing interests.AZ designed the study, drafted the manuscript, and gathered, analyzed and curated data. CF, SM, NN, and LS designed the experiments and gathered data. All authors read and approved the final manuscript.Supplementary specimen information and tables. Table S1. Overview of the dataset comprising 141 MRI scans of 98 extant sea urchin species. Table S2. Overview of Bruker MRI file types.Click here for file


Predictive biomarkers can be used to identify most suitable treatments for patients. To maximise patient benefit valid predictive biomarkers need to be used and several trial designs have been proposed to achieve this.We aimed to review the evidence considered sufficient by the European Medicines Agency (EMA) for inclusion of a predictive biomarker in a drug indication or contraindication and this required development of a methodology to summarise key information.We reviewed the evidence supporting 49 biomarker-indication-drug combinations (37 different biomarkers and 41 drugs) within EMA licensing. The main issues considered were: biomarker evaluation design, sample size, representativeness of patients in trials, primary outcome type and strength of findings. We developed a novel visual overview of these dimensions in radial plots. We explored whether there were differences in the evidence base depending on authorisation recommendation, disease area, biomarker type and orphan designation.155 studies were identified: one biomarker strategy design, 45 enrichment studies, 37 RCT subgroup analyses and 72 other designs . A median of 2 studies (range: none to 19) supported a biomarker-indication-drug combination. Comparison of evidence depending on factors outlined above will be reported.There was a wide variation in type and quantity of evidence supporting biomarker-indication-drug combinations and the radial plots were a useful means of assessment. Study designs used were not always robust. It was not always clear why certain predictive biomarkers were included in the indication. The role of the biomarker evaluation context seemed important.


Squalius alburnoides. This complex is composed by several genomotypes connected by an intricate reproductive dynamics. We developed a model that predicts the hybrid complex can persist when females exhibit particular mate choice patterns. Our model is able to reproduce the diversity of population dynamic outcomes found in nature, namely the dominance of the triploids and the dominance of the tetraploids, depending on female mate choice patterns and frequency of the parental species. Experimental mate choice trials showed that females exhibit the preferences predicted by the model. Thus, despite the known role of assortative mating in driving speciation, our findings suggest that certain mate choice patterns can instead hinder speciation and support the persistence of hybrids over time without speciation or extinction.Previous studies have shown that assortative mating acts as a driver of speciation by countering hybridization between two populations of the same species (pre-zygotic isolation) or through mate choice among the hybrids (hybrid speciation). In both speciation types, assortative mating promotes speciation over a transient hybridization stage. We studied mate choice in a hybrid vertebrate complex, the allopolyploid fish  Many studies have shown that assortative mating acts as a driver of speciation –3, especSqualius alburnoides is an Iberian freshwater fish originated by the hybridization of females of the still sympatric Squalius pyrenaicus  with males from an extinct species related to the extant Anaecypris hispanica  [A sperm) . These iA sperm) ). This dSqualius alburnoides natural populations vary in their composition of genomotypes. Two distinct population types with utterly distinct reproductive dynamics may be defined, namely triploid-dominated and tetraploid-dominated populations (S. pyrenaicus non-hybrid species (PP) ; and c) PAAA offspring from crosses with PAA males  (reviewed in [ulations . The oveies (PP) . PAA femiewed in , 16). InS. alburnoides self-sustainable genomotype (PPAA). The PPAA genomotype has a balanced sex ratio, with males and females producing allodiploid (PA) sperm and oocytes through meiosis [Squalius non-hybrid species. This independency not only allows for a much simpler reproductive dynamics in tetraploid-dominated populations . The study was not carried out on private land and all specimens were returned alive to the collecting site after the experiments.Fish captures were carried out with the permission of Instituto da Conservação da Natureza e das Florestas (permit numbers 140/2012/CAPT and 239/2013/CAPT). Although the taxa studied are threatened, the population chosen for sampling  was not imperiled and the sample size was chosen to avoid depletion of the natural stock. Fishes were handled following recommended ethical guidelines . ElectroS. alburnoides population, using female mating preference and S. pyrenaicus frequency as model parameters.We formulated a theoretical model for the dynamics of the genomotype frequencies in a S. alburnoides is a multiple spawner, females can breed with distinct male genomotypes and produce distinct offspring in a single reproductive season. Considering that, at time t, a particular male genomotype (Mj) occurs with a frequency fMj in the population, and a particular female genomotype (Fi), occurring with fFi frequency, has φFi→Mj preference for that male genomotype, then the probability (pFi×Mj) of a cross between females i and males j is:The frequency of each male and female genomotype in a population determines the encounter probability of two particular genomotypes. This encounter probability would be directly proportional to mating success if there were no other factors, such as differential mate choice, affecting or biasing cross occurrence. In the former case, the probability of a female mating with a male of a given type would be a linear function of the frequency of those males. Adding the effect of female mate preference leads to a non-linear response and an increase of the mating probability with the favored males . Note thi and males j, assuming that: a) the encounter probability of a pair of genomotypes is only a function of their frequencies in the population, not being affected by other factors such as differential spatial segregation, search rates and conspicuity, among others; b) males are not choosy, meaning they breed indifferently with any female genomotype; c) individual females have similar reproductive successes per reproductive season, regardless of genomotype; d) male genomotypes have similar reproductive capabilities, being equally able to fertilize oocytes ; and e) the viability and survival of the offspring produced is similar for all cross types. Note that our model aims at assessing how mate choice shapes population dynamics and does not address whether the simulated preferences are adaptive.This probability also represents the proportion of the particular offspring genomotype arising from crosses between females φFi→Mj) for each female genomotype from the reproductive dynamics of each population type, that is, triploid- and tetraploid-dominated populations. Thus, we assumed assortative mating to occur only among the self-sustainable PPAA genomotype and allowed PAA females to have a more flexible mate choice pattern, due to their obligatory reproductive interdependency. Although there might be up to four male genomotypes in triploid-dominated populations , which father PA offspring with PAA females; and c) type III males, the PAA ones, which produce PAA sperm and father PAAA offspring with PAA females. In triploid-dominated populations, PAA is the most frequent genomotype, followed by the PA and, lastly, by the PAAA one (absent in most populations). These relative frequencies suggest that the male genomotypes which father PAA offspring  may have a higher reproductive success than the ones fathering PA offspring  and a much higher than the ones fathering PAAA offspring , being thus denominated here as type I, type II and type III males, respectively. These differential male reproductive successes may be due to a higher preference of PAA females towards type I males than towards the type II ones (0<φFPAA→MII< φFPAA→MI<1). Nonetheless, we simulated the entire range of preferences towards these two male types (0.002 steps), allowing either type I or type II males to be favored by PAA females . Offspring sex ratios (Rg) applied in the model for each genomotype were based on joint data from well-studied triploid- and tetraploid-dominated populations . Note that the sympatric S. pyrenaicus (PP), whose males also participate in S. alburnoides reproductive dynamics  and independent population dynamics. For this reason, its frequency among the overall fish population  was kept fixed over all generations of each simulation. The entire range of possible PP frequencies  was tested in the model.We assessed multiple initial population compositions by varying the relative proportions of each genomotype, namely by increasing or decreasing the frequency of PAA and PPAA genomotypes  and, thudynamics , does nobg is a binary variable assuming a value of 1 when the cross between females i and males j originates offspring of genomotype g and a value of 0 otherwise. All simulations were performed in R software v2.15.2 [Thus, the overall dynamics of our theoretical model is v2.15.2 .N = 41) of S. alburnoides  and S. pyrenaicus  was collected from Ocreza River during the reproductive season. The capture was performed randomly, trying to cover all available habitats in order to guarantee that the genomotype composition of the sample would be representative of the one found in the studied population. Note that S. alburnoides and S. pyrenaicus are threatened fishes, classified as Vulnerable and Endangered [β-actin gene [We assessed mate preferences of PAA females through mate choice experiments. Trial females were allowed to choose among the male genomotypes present in a triploid-dominated population (Tagus drainage). This population was also used as one of the main references for the model, namely regarding sex-ratios. A random sample , a single individual of each male genomotype  was inserted randomly in each of the male compartments and a single PAA female in the central neutral area of the experimental tank. Due to the rarity of some male genomotypes, some stimulus males were used more than once in the affiliation trials . Note that adult genomotypes have distinct characteristic lengths [N = 11) were allowed to swim freely across the tank and visit each of the males during a period of 1h 30min. The trials were recorded using a digital camera for ulterior video analysis in which the proportion of time spent by females near each male was measured. The first half hour of each trial was considered habituation period to the experimental tank and, thus, discarded from the analyses. The proportion of time spent by females near each male was used as a measure of preference [S. pyrenaicus (fPP) and the preference of PAA females towards type I males (φFPAA→MI), were used to run the model. All statistical procedures were performed in StatSoft Statistica v12 [The experimental trials started after a two-week habituation period to captivity, also ensuring that the small portion of tissue collected from the terminal edge of the fins was fully regrown. Individual recognition was performed using scale patterns . Each ex lengths , 29, maktica v12 .fPP<1, 0.002 steps) and 500 distinct mate preferences of PAA females towards type I males , whereas the ones favoring the dominance of the PAA genomotype also allowed the maintenance of the PA one  showed differential mate preferences towards the available male genomotypes  . Post-hoc tests using the Bonferroni correction revealed that PAA females had a significant higher preference for PPAA males than for PA (p = 0.041) and PAA males (p = 0.035). Males from the sympatric S. pyrenaicus non-hybrid species (PP) were in an intermediate position, not differing significantly from any of the S. alburnoides male genomotypes. Note that, as previously stated, adult genomotypes have distinct typical lengths [In order to experimentally evaluate the role of female preferences (one of the main parameters of our model) in n trials . Repeate lengths , 29, whiF2,20 = 6.597, p = 0.006). Post-hoc tests using the Bonferroni correction revealed that PAA females’ preference towards type I males were significantly higher than towards type III males (p = 0.007) and nearly significantly higher than towards type II males (p = 0.052). However, their affiliation tendency was similar between type II and type III males.In order to compare the experimental results with the outcome our model, the set of preference levels obtained from the mate choice trials was reorganized by male type, namely type I (PA and PPAA), type II (PP) and type III males (PAA). Thus, PAA females’ preference levels towards PA and PPAA male genomotypes  were summed for each tested female, reorganizing the data according to the offspring genomotype females would produce with each male group . All statistical analyses were repeated for this new male structure . RepeateS. pyrenaicus species observed in the studied natural population (fPP = 0.39) and the average joint preference of PAA females towards type I males (PA and PPAA) obtained from the mate choice trials (φFPAA→MI = 0.58) were used to run the theoretical model. Simulated S. alburnoides genomotype frequencies  were close to the ones observed in the referred natural population .Lastly, the frequency of the sympatric S. alburnoides genomotype composition over the final equilibrium reached in each simulation reveals that even a low frequency of the PPAA genomotype in a triploid-dominated population may tetraploidize it and even a low frequency of the PAA genomotype in a tetraploid-dominated population may triploidize it. Thus, the evolutionary route a given population will follow is independent of its current genomotype composition, but, according to our findings, seems highly influenced by female preferences and frequency of the sympatric Squalius species. A closer look at the role of both these forces in shaping S. alburnoides genomotype composition over time reveals that their relevance relies on the production of PA males. This intermediary genomotype seems to play a central role in tetraploidizing populations, but it is also indispensable for the persistence of triploid-dominated populations. This happens because both PAA and PPAA females restock their own genomotype by crossing with PA males. On the one hand, when the frequency of the sympatric S. pyrenaicus (PP) is high and/or when PAA females show a higher preference towards these males  is less frequent and/or the PAA females’ preference towards type I males (PA and PPAA) is higher than it is towards the type II ones (PP)  and if the observed values of both these variables were random among natural populations, the overall outcome of our model indicates that 55.8% of S. alburnoides natural populations would be tetraploid-dominated (against 38.8% of triploid-dominated ones), because the set of conditions favoring tetraploidization is wider than the one leading to triploidization. However, triploid-dominated populations abound across S. alburnoides geographic range and only two tetraploid-dominated populations were found so far, suggesting that the values composing the range of the studied factors do not seem to be equally probable to occur among natural populations. Both known tetraploid-dominated populations [Squalius species, S. carolitertii (CC genome), has a Least Concern status, being more common and abundant than the sympatric species of southern regions, S. pyrenaicus and S. aradensis, classified respectively as Endangered and Critically Endangered [Squalius species might have helped the tetraploidization of those tetraploid-dominated populations , most populations of the northern region are also triploid-dominated (CAA genomotype). In these other populations, the preference of triploid females towards type I males is probably high enough to promote triploidization and counter the effect of the assortative mating occurring among the tetraploid genomotype , crosses that produce PAA offspring, thus, contributing to triploidize populations. However, female preference differed significantly between the two type I male genomotypes, with PPAA being favored over PA. This difference may be related to the fact that PPAA males undergo meiosis as reproductive mode and, therefore, contribute to a higher genetic variability of the offspring, a factor already proposed as relevant when choosing a mate –34. The Type III males (PAA) were the least preferred choice of triploid females, a predictable result considering the extreme rarity of the PAAA genomotype  in natural populations. However, this result is still particularly interesting because PAA females seem to avoid mating with males of their own genomotype (disassortative mate choice) in order to produce offspring of their own genomotype (assortative mate choice). To our knowledge, this assortative-disassortative mate choice pattern was never reported before.Squalius species in S. alburnoides reproductive dynamics seems to be equally relevant in routing populations towards tetraploidization. Hybrid speciation seems only possible if the sympatric parental species is frequent in the population, suggesting that sympatry is mandatory for speciation in this hybrid complex, contrary to what is commonly argued [Because hybrid organisms with nonsexual reproductive modes have altered gametogenesis and lack regular sexual mechanisms , they were for long considered evolutionary dead-ends . Howevery argued –40. Indey argued , 41.Our findings add an important and almost neglected piece to the puzzling persistence of some hybrid animal populations without speciation or extinction. Among vertebrates, namely in amphibians and fishes , 42–46, S1 TableN = 11) spent interacting with each male genomotype.Preference values refer to the proportion of time tested females Click here for additional data file.


Immunotherapies for cancer have progressed enormously over the past few decades, and hold great promise for the future. The successes of these therapies, with some patients showing durable and complete remission, demonstrate the power of harnessing the immune system to eradicate tumors. However, the effectiveness of current immunotherapies is limited by hurdles ranging from immunosuppressive strategies employed by tumors, to inadequate specificity of existing therapies, to heterogeneity of disease. Further, the vast majority of approved immunotherapies employ systemic delivery of immunomodulators or cells that make addressing some of these challenges more difficult. Natural and synthetic biomaterials–such as biocompatible polymers, self-assembled lipid particles, and implantable biodegradable devices–offer unique potential to address these hurdles by harnessing the benefits of therapeutic targeting, tissue engineering, co-delivery, controlled release, and sensing. However, despite the enormous investment in new materials and nanotechnology, translation of these ideas to the clinic is still an uncommon outcome. Here we review the major challenges facing immunotherapies and discuss how the newest biomaterials and nanotechnologies could help overcome these challenges to create new clinical options for patients. Over the past 50 years cancer diagnosis, prognosis, and treatment have progressed immensely, improving the outcomes and quality of life for millions of patients each year. Many persistent challenges, however, continue to limit our ability to stop cancer upon detection and to prevent relapse following successful treatment. The barriers undermining these broad goals are diverse, spanning drug toxicity and poor selectivity , the immex vivo or in vivo . Ea. Eas.c. ®). Sipuleucel-T is a vaccine consisting of autologous DCs pulsed with a fusion protein of human GM-CSF linked to a prostate cancer antigen (PAP) expressed in more than 90% of prostate tumors [The success of several new immunotherapy strategies in the clinic have also revitalized the potential of cancer vaccination. One important example is targeting of the immune checkpoints, CTLA-4 and PD-1, which have been highly effective in recent trials , 50. Ipie tumors . Phase Ie tumors . In phase tumors , 73. Thee tumors . With thin vivo, and may not persist long enough to strongly activate DCs-effects that could limit efficacy, or even promote tolerance against TAAs if DCs present the antigens after adjuvants are cleared [One of the fundamental challenges facing cancer vaccines in the clinic is poor immunogenicity of TAAs. Biomaterials can help combat this challenge by improving the likelihood that vaccine components reach DCs or other immune cells or tissues together. TLRas, for example, are being used to enhance TAA-specific immune response; however, systemic co-administration of soluble TLRas with TAAs does not ensure that both components reach DCs together. Additionally, soluble vaccine adjuvants are rapidly cleared  cleared . Biomate cleared , tumor l cleared –80, prot cleared , 81–83,  cleared –89, DNA  cleared  and mRNA cleared .In addition to increasing immunogenicity of TAAs by providing co-delivery with immunostimulants, biomaterials have also been utilized to facilitate cross-presentation of antigens for the generation of CTL responses . Antigenet al. recently demonstrated that NP cancer vaccines can be enhanced by targeting DCs using PLGA NPs conjugated to CD40 antibody [Another key advantage of biomaterial vaccine delivery systems, in particular for MPs or NPs, is the ability to preferentially target DCs, which specialize in phagocytosing particulate matter such as cells, antigens, or vaccine particles. The targeting of NPs to DCs can actively be enhanced through the conjugation of antibodies or other specific ligands. Rosalia antibody . These pantibody .et al. have also revealed that the intrinsic immunogenicity of degradable polymeric carriers can change during degradation [Beyond the use of biomaterials as passive vaccine delivery vehicles, a developing area in the biomaterials and vaccine fields is the intrinsic and self-adjuvanting properties of polymers and other materials . Some ofradation . Understex vivo expansion of anti-tumor T cells, for example, have also recently been investigated as vaccines to prime effective anti-tumor CTLs in vivo [et al., in which albumin shuttling was exploited in mouse models of melanoma and cervical cancer [Several of the NP-based strategies discussed in previous sections are also relevant to improved cancer vaccination. The aAPCs discussed earlier for  in vivo , 106. Ad in vivo . This prl cancer .in vitro and are then administered to patients. However these strategies have resulted in limited efficacy, likely due to cell death. This hurdle leads to poor persistence and co-localization of the signals and tumor antigens from the engineered cancer cells. To address these limitations, injectable or in situ forming scaffolds have been widely studied as next generation cancer vaccines [in vivo. Scaffolds delivering a variety of immune cues have been investigated, including chemokines (CCL20) or cytokines  for DC recruitment, TLRas  for activation of DCs, and antigens  for the generation of strong and specific anti-tumor response [Whole cancer cell based vaccines have been widely investigated in the clinic and have demonstrated the potential of localized co-delivery of tumor antigens and immunomodulators. These strategies aim to overcome the generally poor immunogenicity of cancer cells by modifying the cells with a variety of immune signals to amplify the response against cancer antigens . A commovaccines , 90, 109response , 90, 109et al. revealed these scaffolds function to enhance anti-tumor immunity by recruiting and priming phenotypically distinct populations of DCs towards TAAs [H1 polarizing IL-12, and incorporation of CpG in the scaffold results in infiltration by plasmacytoid DCs (pDCs) that secrete high levels of type-1 IFNs. The recruitment of heterogeneous populations of DCs corresponded with high amounts of CTLs specific for Trp2–a melanoma antigen–infiltrating tumors, and increased ratios of CD8+ effector T cells to TREGs in tumors. These effects provided therapeutic anti-tumor immunity in the B16-F10 mouse model, where implantation of vaccine scaffolds 9 days after the establishment of tumors resulted in survival of 47% of mice at the end of the 100 day study, while all mice vaccinated with GM-CSF-secreting irradiated B16-F10 cells succumbed by 36. Bencherif et al. recently demonstrated this strategy can be expanded to scaffolds consisting of implantable cryogels–porous hydrogels formulated at subzero temperatures to mimic tissue properties–for co-delivering irradiated whole B16-F10 cells, CpG and GM-CSF. These cryogel-based scaffolds can be injected with a syringe [The Mooney Lab is one of the pioneers in this area, developing PLGA or other polymer-based scaffolds to serve as a platform for local juxtaposition of multiple immune signals to promote effective anti-tumor response –80, 109. syringe , demonst syringe .The clinical success of immunotherapies targeting immune checkpoints has sparked interest in combining vaccination with these ground breaking therapies. Through this combination, pathways that restrain pro-immune function might be released to create opportunities for vaccines to generate efficacious responses. For example, GVAX, a whole cell pancreatic cancer vaccine utilizing GM-CSF-expressing tumor cells showed initial promise but was ultimately halted in phase III clinical trials due to lack of efficacy . GVAX isREG, myeloid derived suppressor cells (MDSC)). These immunosuppressive signals can be targeted using small molecule inhibitors or siRNA as a strategy to overcome tumor immunsuppression. However, the effectiveness of systemic delivery of these cargos is limited by poor targeting to tumors and rapid degradation.The idea that GVAX vaccine may be enhanced by the addition of checkpoint blockade therapies highlights one important area related to this idea of combination therapy–how to overcome the immunosuppressive microenvironment of tumors. Although vaccines may generate anti-tumor immune responses, these cells are often inactivated after infiltrating tumors. In addition to the expression of checkpoint blockade ligands by tumor cells, the anti-tumor efficacy of infiltrating immune cells may also be inhibited by suppressive cytokines such as TGF-β  or enzymet al. recently demonstrated the potential to combine biomaterial based immunotherapies and vaccines to alter the tumor microenvironment for enhanced immunity in the B16-F10 mouse melanoma model [in vivo cytotoxicity assay which tested the ability of vaccinated mice to lyse splenocytes from naïve mice, after the splenocytes were pulsed with Trp2 peptide and adoptively transferred. However, the late stage vaccine was able to match the anti-tumor efficacy of early vaccination only with the combination of LCP NP-mediated TGF-β knock down. Importantly, mechanistic investigations revealed TGF-β knockdown depleted TREGs in tumors and caused higher levels of CD8+ T cells infiltrating tumors in late-stage vaccination. These effects were minimal following immunization with the monotherapy vaccine. Together, the ideas and examples discussed in this section demonstrate the ability of biomaterials to enhance pre-clinical cancer vaccination, but these combination approaches still remain uncommon and are largely untapped in the clinic, highlighting an important investment area that could “rescue” or improve previously ineffective strategies such as cancer vaccination.Biomaterials have recently been used to improve the delivery of immunomodulators targeting a variety of immunosuppressive pathways to actively enhance the cancer vaccination. Xu ma model . In thisma model . LHA NPsWith recent developments in immunotherapies, new attention is warranted to strategies combining immunotherapy with not just checkpoint blockade, but also with chemotherapy, radiation, ablation, or resection. This year the FDA granted accelerated approval for the first combination of cancer immunotherapies–checkpoint inhibitors nivolumab and ipilimumab–following phase II studies in patients with unresectable stage III or IV BRAF V600 wild-type melanoma . HoweverOne promising area already being explored as a combination therapy using biomaterials is the stimulation of multiple TLRs through co-delivery of multiple TLR agonists (TLRas) –121. Sigs.c. with Incomplete Freund's Adjuvant (IFA), ii) s.c. with IFA and a TLR-7 agonist (Imiquimod) applied topically at the injection site, iii) i.d. with topical Imiquimod application, and iv) intra-lymph node injection using ultrasound guidance [s.c. immunization revealed swelling in draining lymph nodes, as well as local inflammation at vaccine sites, confirming drainage to lymphatics still plays a role in this vaccine's efficacy , macrophages (CD14+), and CD4+ and CD8+ T cells while driving a 40% reduction in tumor burden relative to paclitaxel delivered alone [Although in the clinic chemotherapeutics are regularly combined with immunotherapy, application of biomaterials to this area has been relatively sparse , 131. Thin situ vaccine by delivering an adjuvant to sites where ablation kills tumor cells and exposes additional tumor antigens. Thus far, however, the platform has been investigated in vitro. For these studies, 4T1 tumor cells were ablated with the NPs and exposed to DCs. The DCs were activated by the TLRa-loaded NPs and phagocytosed the remnants of the ablated tumor cells [in situ vaccines by combining them with treatments like radiation, ablation and surgeries that expose new tumor antigens. Moreover, as we learn more about the unique connections between each arm of combination therapies, this synergy may also be able to help reduce relapse, for example, by promoting memory T cell populations specific for neoantigens.Radiation and ablation also provide unique opportunities for combination with immunotherapy because recruitment of immune cells to the local tissues in the treatment area can allow identification of neoantigens by immune cells infiltrating these sites. This mechanism has been hypothesized as a potential explanation for tumor regression observed in areas of the body not directly treated via radiation or ablation –134. In or cells . This stDespite the clinical success of some immunotherapies and vaccines in generating tumor specific CTLs, the anti-tumor efficacy of these treatments is limited by poor migration to tumors, as well as inactivation of cells that do reach the tumor microenvironment . Biomateet al. demonstrated 30nm poly(propylene sulfide) NPs administered i.d. accumulate at high levels in the spleen and draining LNs, and are efficiently taken up by cellular populations of the myeloid lineage. In particular, NPs are efficiently internalized by myeloid derived suppressor cells (MDSC) in tumors, tumor-draining LNs and spleens. This represents a potential clinical strategy to deliver immunomodulatory cues to suppressive cell subsets [In addition to targeting tumors at the tissue level, NP size can be tuned to passively target specific immune cells within tumors. Kourtis  subsets . Another subsets .et al. demonstrated a strategy to deliver NPs encapsulating a chemotherapeutic (SN-38) to lymphoma tumors through the conjugation of NPs to polyclonal T cells from C57BL/6J mice [ex vivo in the presence of rapamycin, which polarized T cells toward phenotypes that retain high levels of lymphoid homing receptors  after expansion. NP-conjugated T cells effectively trafficked to lymphoid tumors after adoptive transfer, causing increased concentrations of SN-38 in these lymph nodes compared to administration of unconjugated NPs. This active targeting of tumors resulted in increased survival of tumor bearing mice at greatly reduced doses.NPs may also be actively targeted to lymphatic tumors for the delivery of therapeutics by taking advantage of the ability of lymphocytes to home to lymphoid organs. Huang /6J mice . T cellsi.t. injection of non-resectable tumors offers a simple route to directly deliver immunostimulants that locally reactivate exhausted immune cells in the tumor microenvironment. By causing these reactivated APCs to be primed against TAAs within tumors, localized i.t therapy may thus offer exciting potential to generate systemic anti-tumor immunity to combat distal tumors, or memory that helps address relapse [et al. demonstrated that CpG can be retained locally in tumors by conjugating CpG to lipophilic moieties, which readily insert into tumor cell membranes after i.t. injection [et al. involves activation of the immune system to clear tumor cells after resection to prevent relapse. In this study, PLGA sutures closing the incision from resection were used to encapsulate and sustain the release of CpG locally at this site [The above examples demonstrate the benefits of passively targeting tumors after administration at a distant site from the tumor. In some cases,  relapse . Using bnjection . A strathis site . Using tet al. has enhanced IL-2 based immunotherapy in mice using nanoparticles co-loaded with IL-2 and a small molecule inhibitor of TGF-β1 (SB505124) [In addition to targeting the tumor microenvironment with immunostimulants, strategies aimed at neutralizing immunosuppressive cytokines may also allow anti-tumor immunity to overcome the suppressive mechanisms of the tumor microenvironment. IL-2 therapy, for example, shows potential by augmenting the activation and expansion of anti-tumor CTLs. Clinical trials testing IL-2 during metastatic melanoma demonstrate 4-6% of patients achieve complete remission , 142. OnB505124) . In thisA major barrier to cancer immunotherapy is the highly heterogeneous nature of tumors within individual patients, in different types of cancer, and across patient populations , 145. Tude novo.The effectiveness of a cancer vaccine depend in part on the individual MHC alleles expressed by the patient and the level of expression of TAAs targeted by the vaccine . FrequenResected tumors theoretically contain all antigens present in an individual's tumor, including neoantigens resulting from mutations. Thus these tissues may be used as effective antigen sources in personalized cancer vaccines. Using irradiated tumor cells or tumor lysates as antigen sources in vaccines may likewise offer the potential to better combat tumor immunoselection . BiomateAdvances in sequencing technologies have enabled efficient exosome mapping of tumor cells . In thiset al. recently demonstrated a biomaterial strategy that enables efficient capture of CTCs using implantable scaffolds, which function to preferentially redirect metastatic tumor cells from other solid organs [hiCD11b+ MDSCs accumulated in scaffolds to a high degree, consisting of 66% of total leukocytes in the scaffold, compared to 89% of total leukocytes in the lungs 28 days after tumor inoculation [hiCD11b+ cell infiltration in the scaffold was increased significantly by day 14 after implantation, compared to scaffolds delivering a control vector (Figure hiCD11b+ cells to enhance tumor cell capture in the scaffold. This study also demonstrated a potential strategy to address heterogeneity of disease with respect to metastatic potential, where early metastases can be detected by label-free in situ imaging of scaffolds based on optical properties of cancer cells. This capability may allow for patients to be treated based on expected aggressiveness of their disease. Additionally, metastatic cancer cells may be removed from scaffolds or other “sensor” devices for analysis of biomarkers or antigen expression. An especially exciting aspect is the multi-functionality that these types of devices could play as both a diagnostic tool to address heterogeneity, and as a strategy to delay or inhibit metastases to allow time for parallel treatments to work.Another new strategy aimed at CTCs is based on altering or exploiting the homing of these cells. Azarin d organs . In thisd organs . In contculation . By utilr Figure . AdditioIt is clear that immunotherapies for cancer treatment have progressed greatly in recent years, revealing new possibilities through which the immune system can be used to treat cancer. The successes and failures of immunotherapies in the clinic have given researchers newfound understanding of the significant challenges presented by cancers that limit current immunotherapies. Nanotechnologies have been investigated extensively for cancer immunotherapies and vaccination pre-clinically, but have had comparatively little impact for patients thus far. One common occurrence is clinicians and engineers coupling cutting-edge ideas from their respective field with basic–or sometimes outdated–ideas from the other field. For example, much nanoparticle research is being performed on small molecules drugs delivered as a monotherapy that are never used as a primary therapy in patients. Similarly, most clinical therapies involve systemic delivery, while many pre-clinical engineering strategies exist for targeting specific cells or tissues. Further, as can be observed by range of papers reviewed here, a great deal of pre-clinical work involving biomaterials is conducted in fairly basic models–for example, B16-F10. Although these are valuable starting points, utilization of more sophisticated animal models that recapitulate cardinal features–such as pathology and metastatic tendency–could improve the translatability of new material strategies. This gap highlights the potential for impact that materials can have as the focus on cross-disciplinary understanding and collaboration grows; there is outstanding synergy at the interface of nanotechnology, bioengineering, and immunotherapy. This nexus creates enormous opportunities for teams of researchers that understand both the clinical and technological needs of new cancer therapies.


R2 > 0.98) and robust parameter estimates for 42 datasets collected for 21 plant species under the same initial values. It suggests that the DE algorithm can efficiently resolve the issue of hyper initial-value sensitivity when using local optimization methods. Therefore, the DE method can be applied to fit the light-response curves of various species without considering the initial values.Selecting appropriate initial values is critical for parameter estimation in nonlinear photosynthetic light response models. Failed convergence often occurs due to wrongly selected initial values when using currently available methods, especially the kind of local optimization. There are no reliable methods that can resolve the conundrum of selecting appropriate initial values. After comparing the performance of the Levenberg–Marquardt algorithm and other three algorithms for global optimization, we develop a general method for parameter estimation in four photosynthetic light response models, based on the use of Differential Evolution (DE). The new method was shown to successfully provide good fits ( For biologically meaningful parameters, using the estimates directly from experiments would be a first try for assigning the initial values. However, for model specific parameters that cannot be measured in experiments, choosing appropriate initial values when fitting the model could become problematic. For example, two of the parameters in Eq. (4) are constants without direct biological meanings. Even for those parameters with direct biological meanings, assigning their initial values in the model fitting could still be time consuming. For instance, all the three parameters in the exponential model  have souThere are many statistical software packages currently available for fitting nonlinear models; to list a few, SAS, R, GenStat, MatLab, Sigmaplot, and SPSS. The built-in functionality to fit nonlinear models in these software packages normally implement algorithms of local optimization for regression and parameter estimation, such as the Gauss-Newton, Levenberg-Marquardt and Nelder-Mead algorithms. As local optimization methods are highly sensitive to the setting of initial values, failed convergence often occurs due to wrongly assigned initial values. However, there are no reliable methods available for selecting initial valuesTo develop effective methods for resolving the problem of assigning appropriate initial values when using these nonlinear models, we need to go beyond local optimization. Genetic algorithms and simulated annealing algorithms have been widely applied to search global optimal solutions in multi-dimensional space12131415161820The objectives of this study are to: (1) test the performance of the Levenberg-Marquardt (LM) algorithm, a local optimization method, and three global optimization algorithms: Genetic Algorithm (GA), Generalized Simulated Annealing (GSA) and the Differential Evolution (DE) algorithm; (2) develop a reliable method for fitting photosynthetic light-response models using the global optimization algorithm and simultaneously solve the issue of hyper initial-value sensitivity in local optimization algorithms.θ = 0, it becomes the rectangular model5Brassica rapa var. chinensis (L.) Kitam., Camellia sinensis (L.) Kuntze and Impatiens balsamina L. were all close to zero, the other three parameters of the nonrectangular model were also equal to those estimated from the rectangular model  for the rectangular and modified rectangular models; however, the values of R2 almost remained the same for the exponential and nonrectangular models . After 500 iterations, the estimates from the three optimization methods were used to evaluate their performance. To avoid biasing the results, we used the same lower and upper bounds for parameter estimation when using the three global optimization .


Early childhood is a sensitive period in which infant-caregiver experiences have profound effects on brain development and behavior. Clinical studies have demonstrated that infants who experience stress and adversity in the context of caregiving are at an increased risk for the development of psychiatric disorders. Animal models have helped to elucidate some molecular substrates of these risk factors, but a complete picture of the biological basis remains unknown. Studies continue to indicate that environmentally-driven epigenetic modifications may be an important mediator between adverse caregiving environments and psychopathology. Epigenetic modifications such as DNA methylation, which normally represses gene transcription, and microRNA processing, which interferes with both transcription and translation, show long-term changes throughout the brain and body following adverse caregiving. Recent evidence has also shown that telomeres (TTAGGG nucleotide repeats that cap the ends of DNA) exhibit long-term changes in the brain and in the periphery following exposure to adverse caregiving environments. Interestingly, telomeric enzymes and subtelomeric regions are subject to epigenetic modifications—a factor which may play an important role in regulating telomere length and contribute to future mental health. This review will focus on clinical and animal studies that highlight the long-term epigenetic and telomeric changes produced by adverse caregiving in early-life. The time immediately after birth is a sensitive period during development that is characterized by rapid, continuing neural maturation. During this time, the brain is especially susceptible to environmental insult, and humans, non-human primates, and rodents are extremely dependent on adequate caregiving that will ensure survival and proper development  which can alter gene expression. Specifically, epigenetic modifications include DNA methylation and demethylation, histone modifications, and non-coding RNAs. Recent evidence has also suggested that telomeres, the end sequences that protect DNA, are also subject to the very same epigenetic modifications. This review will focus primarily on: (1) the long-term impact of DNA methylation and miRNA processing; (2) how they change in response to adverse caregiving and early-life stress; and (3) how telomeric changes offer an important avenue for future epigenetic research.DNA methylation is an epigenetic modification that has been widely studied as a potent regulator of gene expression. This occurs through the addition of a methyl group to a cytosine  via the enzyme DNA methyltransferase 3a  in the hippocampus than high-LG offspring , an HDAC inhibitor. A subsequent study showed that GR methylation was mainly present in gene bodies, not promoters, which have different effects on transcriptional regulation   and TET proteins  axis. For example, Murgatroyd et al. separated pups from the dam thirty minutes per day for the first ten days of life and measured methylation of hypothalamic corticotropin-releasing hormone (CRH) and increased CRH mRNA in adulthood  gene in the prefrontal cortex (PFC) as a whole  24 h after the last caregiver manipulation and persisting through adulthood. Moreover, maltreated-female rats showed aversive caregiving behaviors toward their own offspring, and their own offspring in turn showed similar PFC bdnf methylation alterations.Our lab has characterized some of the epigenetic effects produced by early-life stress in an animal model of caregiver maltreatment. By restricting a lactating dam’s bedding material in a novel arena, we can elicit adverse pup-directed caregiving behaviors, including stepping on, dragging, dropping, and actively avoiding pups  can be reversed in the adult rat PFC by zebularine treatment, an inhibitor of DNA methylation  promoter in blood in adults 18–59 years of age  diagnosed with major depression and bipolar disorder  carrying a risk allele for the FKBP5 gene and that experienced childhood abuse exhibit altered FKBP5 methylation . This gene is a major player in serotonergic neurotransmission, and low levels of 5HTT mRNA and protein have been associated with psychopathology , hypermethylation of these gene may be one mechanism by which childhood maltreatment confers risk for psychopathology . In a study using rhesus macaques, maternally deprived infants showed increased methylation of 5HTT (and decreased 5HTT mRNA) in peripheral blood mononuclear cells (PBMCs) compared to maternally reared infants  patients approximately 30–50 years of age  with depressive symptomology that were separated from their parents during childhood had greater genome-wide DNA methylation in whole blood compared to both non-stressed and healthy males . Unlike DNA methylation, miRNAs contribute to gene silencing  Ambros, . Primary Ambros, . Certain Ambros, . AdditiomiR132, miR124, miR9, and miR29a in the mPFC of adult rats that experienced maternal deprivation  and D2 (DRD2) mRNA in the nucleus accumbens. Stressed rats with increased miR-504 expression also exhibited depressive-like behaviors, suggesting that the gene-suppressing effect of miR-504 on dopamine receptor expression could be mediating the effect of early-life stress on adult behavior  and miRNA Let-7a (correlated with lower serotonin receptor-4 expression) in the hippocampus  is conserved across mammalian species, telomere length can substantially vary across different cell-types and between mammals, including those commonly employed for investigating the impact of early-life stress ), mouse strains (~30–150 kb), and rat strains (~20–100 kb)  and 69.7 (SD = 10.14) year old adults, respectively, who reported (via the childhood trauma questionnaire) physical and emotional neglect during childhood exhibited shorter telomeres in peripheral blood mononuclear cells  years of age—suggesting that telomeres shortening following early-adversity may be associated with mental-health related disorders in adulthood  and lower maternal care in females (23.5 years of age SD = 1.9) predicted shorter leukocyte telomere length in adults  was associated with shorter leukocyte telomere length  that may moderate the long-term impact of early-life stress on telomere length in the periphery. Research using rodent models has extended our understanding of whether early-life stress induces long-term telomeric changes in the brain. Botha et al.  found thde novo addition of TTAGGG nucleotides a single base at a time onto the 3’ telomeric overhang , without a longitudinal study design a better term may be “long-term”, as it is possible the alterations were not present early in development but emerged much later in life (as we have seen with some of our rodent work). Several groups however have documented such alterations present in infants and children exposed to adversity (for example Oberlander et al., 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.


A pathological hallmark of asthma is chronic injury and repair, producing dysfunction of the epithelial barrier function. In this setting, increased oxidative stress, growth factor- and cytokine stimulation, together with extracellular matrix contact produces transcriptional reprogramming of the epithelial cell. This process results in epithelial-mesenchymal transition (EMT), a cellular state associated with loss of epithelial polarity, expression of mesenchymal markers, enhanced mobility and extracellular matrix remodeling. As a result, the cellular biology of the EMT state produces characteristic changes seen in severe, refractory asthma: myofibroblast expansion, epithelial trans-differentiation and subepithelial fibrosis. EMT also induces profound changes in epithelial responsiveness that affects innate immune signaling that may have impact on the adaptive immune response and effectiveness of glucocorticoid therapy in severe asthma. We discuss how this complex phenotype is beginning to be understood using systems biology-level approaches through perturbations coupled with high throughput profiling and computational modeling. Understanding the distinct changes induced by EMT at the systems level may provide translational strategies to reverse the altered signaling and physiology of refractory asthma. Currently it is estimated that over 300 million adults and children suffer from asthma, representing a major public health problem worldwide. Over the past two decades, there has been a rise in asthma prevalence, such that about 8% of the population, more than 25 million Americans, were affected in 2010 .a priori assumptions about the mechanisms underlying the response, allowing for the identification of new and less expected findings [Recent exciting work has yielded insight into the dynamic and central role of the epithelium in asthmatic inflammation and remodeling. In this review, we discuss the cellular biology of an epithelial transcriptional reprogramming event known as epithelial-mesenchymal transition (EMT). Specifically, we examine the current knowledge of how growth factors and inflammation interface to produce EMT, of the role of innate immune cells in this process, and phenotypic modulation by the NF-κB signaling pathway. Due to the complexity of interacting signals and consequent global transcriptional reprogramming, systems-level studies are needed to fully understand critical pathways affected by EMT. The “systems” concept relies on global, experimental measurements of the genome, transcriptome, proteome, metabolome, and epigenome in response to external perturbation. One advantage of this approach is that it makes no findings . Systemsfindings , and envfindings . Here, wThe airway mucosal barrier is produced by a relatively impermeable epithelial sheet connected by tight junctions that restrict fluid loss and limit inhaled particulate access to the internal milieu. The airway mucosa is a regionally diverse spectrum of highly differentiated epithelial cell types, each playing a specialized role in normal pulmonary function and host defense. For example, flattened simple squamous type I pneumocytes promote gas exchange, provide a barrier to minimize water loss, and prevent pathogens and toxins from access to the internal architecture; secretory goblet cells produce and secrete protective mucins into the airway lining fluid; ciliated epithelial cells produce protective epithelial lining fluid and mucociliary escalator for particulate clearance; and type II pneumocytes secrete surfactants responsible for maintaining alveolar patency . In pseuMaintenance of epithelial integrity is critical to normal cellular signaling, pulmonary homeostasis, and response to toxicants and allergen exposures. Moreover, this dynamic and plastic cell type plays a key role in initiating innate signaling programs in response to physical, chemical and biological challenge through coordinating cytokine and defensin release, and secreting alarmins and Th2-differentiating cytokines . DespiteAsthma is disease driven, in part, by epithelial injury and repair. Representing the principal cell type between the environment and internal milieu, the epithelial cell not only plays a critical role in the activation and coordination of the innate immune response, but also in tolerance and control of airway hyper-reactivity. It is well established that increased epithelial cell fragility with attendant denudation/shedding of epithelial cells and the consequent disruption of its barrier function enhances allergic sensitization . CC chemUnder normal conditions, the airway epithelial cells signal to an attenuated sheath of subepithelial mesenchymal cells, forming an epithelial-mesenchymal unit (EMU). Integrity of the EMU depends on growth factors secreted from epithelial cells; growth factor secretion is rapidly increased in response to epithelial injury to promote regeneration of this critical mucosal surface . Injury,Because TGFβ is the most potent and most well described inducer of EMT, we will discuss its role in modifying the airway epithelia in detail. In the airway mucosa, TGFβ induced type II EMT leads to disruption of mucosal barrier function by inducing the loss of apical polarity, reduced epithelial cadherin (ECad) and disruption of epithelial adherens junctions 1/2, zebra (ZEB)1/2, and Twist 1/2 families . In the Several studies suggest that the ability of TGFβ to induce EMT is modulated by innate cytokine stimulation, morphogen signaling, allergen exposure and through ECM interactions. Studies of transformed alveolar basal epithelial cells show that TGFβ-induced EMT is accelerated by the presence of members of the proinflammatory TNF/ IL-1 superfamily of cytokines -45. TNF/The actions of TGFβ on EMT are further controlled by the Notch pathway, a highly conserved morphen control pathway involved in epithelial intercellular communication and cell fate specification. In cultured rat alveolar epithelial cells, Notch activation increases the expression of α-SMA, collagen I and vimentin and decreases expression of epithelial markers. Notch promotes EMT in airway epithelia by increasing expression of TGFβ ligands and inducing myofibroblast differentiation of alveolar epithelial cells via the TGFβ -Smad3 pathway .Vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and Th2-type cytokines  produced by adaptive immune cells in the lung have also been observed to modulate EMT -58. MoreThe ECM is an interlocking mesh of collagens, elastic fibers and glycoproteins that plays a critical role in epithelial polarization, proliferation and signaling . DynamicFinally, extrinsic environmental stimuli can induce EMT. One prominent example is the house dust mite aero-allergen; exposure to house dust mite potentiates TGFβ-induced EMT of airway epithelial cells by stimulating myosin light chain phosphorylation and actin reorganization and enhanced β-catenin signaling . This isAirway remodeling, in addition to inflammation, abnormal neurogenic and contractile response, has recently been appreciated as one of the contributors to the symptoms, abnormal physiology and natural history of asthma and therefore is likely to generate manifestation of the disorder as phenotype. This novel concept led to a series of studies delineating cellular and molecular mechanisms of airway remodeling and clinical trials aiming to identify subpopulations of asthmatics with similar disease mechanism (endotype) ,65. AirwHowever, there are several observations suggesting that airway remodeling in asthma may occur without significant involvement of eosinophils  or even The correlation of airway remodeling with features of EMT, expression of TGFβ1, severity of disease, resistance to glucocorticoid therapy and airway eosinophilia or neutrophilia can be used to define distinct molecular phenotypes of asthma and to some degree asthma endotype. The asthma “phenotype” is the result of a complex constellation of pathophysiological processes, whereas an “endotype” represents a subtype of disease defined functionally and pathologically by a singular molecular mechanism translating into a treatment response ,87. AlthFuture research exploring the role of EMT in airway remodeling may delineate that EMT may play a role common to several endotypes. For example, EMT may contribute to steroid insensitivity as suggested by decreased induction of anti-inflammatory genes by glucocorticoids in TGFβ1-EMT transformed cells A549 cells . In neutIn conclusion, future studies aiming at defining asthma endotypes should consider identification of mechanisms and respective biomarkers of TGFβ1 mediated EMT. Search for specific endotype biomarkers must focus on homogenous group of patients according to underlying disease mechanisms described by histological features , cytokine signatures  and responsiveness to anti-cytokine and glucocorticoid therapy. Systemic studies need to be directed at the cellular, molecular, and genetic factors that are responsible for determining why only some asthmatics develop significant remodeling. The information gathered from these studies will impact strategies employed to combat this increasingly complex and heterogeneous disorder.In addition to affecting glucocorticoid response discussed above, changes in the phenotype of epithelial cells in asthma may determine effects on innate signaling responses . Our earFurther systems level studies will help to understand the complexity of the epigenetic reprogramming with the inducible phenotype produced by EMT. These approaches include mathematical modeling of signaling pathways to infer how EMT influences dynamic responses, systematic phosphoprotein profiling in response to cellular stimulation to identify coupling of intracellular signaling pathways and computational prediction of transcription factor binding sites at a genome-wide level. Application of this latter methodology has identified motifs that likely regulate dynamics of Polycomb-mediated histone modifications during murine stem cell differentiation . FurtherSevere asthma is associated with defects in innate immunity , includiAlthough the effect of type II EMT on innate and adaptive immunity in the airway is incompletely delineated, the understanding of type III EMT on adaptive immunity has been extensively investigated. Epigenetic modifications induced in type III EMT suppress transcription of the NK cell activating receptor, NKG2D , secretiDiscussed above, the complex events produced by transcriptional reprogramming and global epigenetic rearrangement by EMT fundamentally change the inducible phenotype of the airway epithelial cell. To better understand the various asthma phenotypes, systems level studies of EMT are needed. Systems biology is an iterative process that involves the application of high throughput measurements to cellular perturbations. These data are used to develop predictive models that drive subsequent rounds of experimentation, perturbations, and predictive model refinement Figure . ApplicaA classic example of implemented framework of systems biology was the comprehensive study of the compiled gene expression data from five publicly available mouse microarray datasets and 4,305 gene annotation sets to provide the functional knowledge base . A Modulet al. compiled the asthma biomarkers from genomics, proteomics, epigenetics and few experimentally validated datasets, and derived significant enrichment of pathways, including adipokine and ROS pathways [High-throughput proteomics and metabolomics have helped to classify asthmatic patients by many proteins found in the airways, which can be used clinically as useful biomarkers ,106. Sahpathways . Similarpathways  and micrpathways . Parallepathways , which hpathways ,112.A recent study focused on the consequences of EMT to pro-survival signaling using a correlation analysis of global measurements of protein, phosphoprotein and RNA transcript abundance . These mBy contrast, fewer systems level studies have been applied to understand type II EMT. Although there are likely to be some important commonalities between types II and -III EMT, type II EMT is qualitatively different process because EMT of primary epithelial cells is not produced in the milieu of p53 mutation, activation of Wnt/β catenin or constitutive activation of NFκB pathways, pathways that modulate EMT. To better understand the effects of type II EMT on innate signaling, we have applied systems level studies of type II EMT using innate perturbations. Time-course perturbation experiments were used to develop parameters of mathematical models of the innate pathway . ComputaFurther applications of systems approaches will provide comprehensive understanding of this complex phenotype through deterministic modeling and multidimensional genomic and proteomic profiling Figure . This kiEMT is a complex, dynamic epigenetic reprogramming event that plays a homeostatic role in airway response to injury. Left unchecked, EMT contributes to pathophysiology of severe asthma. Understanding how to reverse EMT has significant translational implications. For example, EMT affects the response to glucocorticoids and inflammatory cytokines in epithelial progenitor cells, an effect that probably plays a significant pathophysiological component in a number of asthma phenotypes. It is possible that reversal of the EMT phenotype may promote allergen tolerance, enable anti-inflammatory treatment response, reduce viral-induced exacerbations, and reverse of sub-epithelial fibrosis in patients with severe asthma. The targets for reversing EMT are only beginning to be defined, and could involve targeting modulatory TGFβ signaling pathways or epigenetic modifiers. A recent investigation into possible regulators of airway remodeling found that inhibition of aldose reductase prevents TGFβ1-induced activation of the PI3K/AKT/GSK3β signaling pathways in a Smad-independent manner . A separin vitro, animal and human studies suggest that chronic and refractory asthma is associated with type II EMT. EMT induces loss of mucosal barrier function, acquisition of mesenchymal characteristics and enhanced motility, and secretion of fibrotic proteins accounting for the remodeling phenotype of this disease. Here we review the evidence for EMT in producing defects in inducible phenotype of the epithelial cells including alterations in signal response characteristics of the innate response of the airways. Applications of systems level studies will be needed to unravel the dynamic and cross-talk signaling mechanisms characteristic of EMT. These exciting studies suggest that modulation of EMT may have therapeutic implications for reversing airway remodeling, restoring glucocorticoid response, and/or the innate immune defect observed in severe asthma.A hallmark of early asthma is the presence of epithelial injury/repair and expansion of subepithelial fibrosis. In the setting of chronic TGFβ stimulation from innate eosinophils and other leukocytes, modulated by ECM, epithelial cells undergo dramatic cellular transition known as type II EMT. Accumulating evidence from ECad: E cadherin; ECM: Extracellular matrix; ECP: Eosinophilic cationic protein; EGF: Epithelial growth factor; EMT: Epithelial mesenchymal transition; EMU: Epithelial mesenchymal unit; FGF: Fibroblast growth factor; MUC: Mucin genes; NF-κB: Nuclear factor-κB; RV: Rhinovirus; SMA: Smooth muscle actin; SNAI: Snail; TGFβ: Transforming growth factor β; Th: T helper cell type; VIM: Vimentin.The authors declare that they have no competing interests.All authors contributed to literature review, writing the manuscript and editing the figures. All authors have read and approved the final manuscript.


Pedicle screw fixation allows purchase of all three spinal columns without encroaching into the spinal canal improving fracture fixation, as well as deformity correction. Fortunately, neurologic injury associated with pedicle screw malposition is rare.A 19-year-old boy was surgically treated for severe right thoracic scoliosis associated with a Chiari Type 1 malformation and a C6 to T7 syringomyelia. Six months after the initial surgery, the patient was referred to our institution after three weeks of gait disturbances and repeated falls. Imaging showed the gross misplacement of the left T5 pedicle screw, which crossed the center of the vertebral canal. The initial surgery used a freehand technique of pedicle screw insertion, with anteroposterior and lateral postoperative X-ray control. During the surgery, no SEP modifications were noted during pedicle screw placement. However, after insertion of the second rod and scoliosis correction by posterior translation technique, SEP responses decreased considerably. Revision surgery was performed to remove the misplaced screw. During the first three months after screw removal, repeated clinical examinations showed progressive recovery of the neurological deficits. Gait and bladder functions were normal six months after screw removal, and clinical signs of spasticity disappeared. SEP explorations performed at final follow-up showed similar responses to those performed before the initial surgery for scoliosis correctionNeurologic injury associated with pedicle screw malposition is rare. In early or delayed neurological status worsening, intraoperative or postoperative imaging must be done to detect pedicle screw misplacement. In the current case, thanks to cobalt-chromium and titanium use, MRI and CT scan allowed good visualization of the spinal canal and spinal cord. Experimental studies have shown that neurophysiological monitoring of the spinal cord does not detect moderate compression. In that way, neurophysiological monitoring is an all-or-nothing technique which can misdiagnose early stage of spinal cord injuries. Major penetration of the spinal canal by pedicle screw may conduct to hardware removal.In early or delayed neurological status worsening, intraoperative or postoperative imaging must be done to detect pedicle screw misplacement. In the current case, thanks to cobalt-chromium and titanium use, MRI and CT scan allowed good visualization of the spinal canal and spinal cord. Major penetration of the spinal canal by pedicle screw may conduct to hardware removal. The patient was operated on by posterior approach on a Hall frame and with permanent sensory-evoked potential (SEP) monitoring. SEP monitoring protocol consisted in a continuous SEP monitoring with bilateral posterior tibial nerve stimulation and cortical and cervical detection. SEP amplitudes and latencies were recorded in a continuous fashion from patient positioning under general anaesthesia to the end of the surgical procedure.A 19-year-old boy was surgically treated for severe right thoracic scoliosis associated with a Chiari Type 1 malformation and a C6 to T7 syringomyelia. The Chiari Type 1 malformation was first operated on at the age of 14; the result was considered satisfactory, and the syringomyelia was considered stable between ages 14 and 19. Because Cobb angle increased to 95°  of the right psoas, the right flexor digitorum profundus, and the right triceps. There was a right Babinsky sign with increased deep tendon reflexes in both lower limbs. Romberg sign was positive. The patient also presented severe dysuria with bladder distention requiring urinary catheter. There was no recent history trauma, no fever and no remaining biological sign of infection. Axial, sagittal and coronal planes of a thoracolumbar CT scan confirmed the gross misplacement of the pedicle screw, which crossed the center of the vertebral canal (Fig. /5 of theRevision surgery was performed to remove the misplaced screw. No spinal canal exploration was performed at this time. No cerebrospinal fluid effusion was noted during screw removal, and no meningocele occurred postoperatively. During the first 3 months after screw removal, repeated clinical examinations showed progressive recovery of the neurological deficits. Gait and bladder functions were normal 6 months after screw removal, and clinical signs of spasticity disappeared. The patient reported no motor or sensory deficit at final follow-up, 18 months after screw removal. SEP explorations performed at final follow-up showed similar responses to those performed before the initial surgery for scoliosis correction Fig. , and finPedicle screw fixation allows purchase of all three spinal columns without encroaching into the spinal canal. This theoretical advantage has been translated to superior clinical results in fracture fixation, as well as in deformity correction (Dickman et al. Fortunately, neurologic injury associated with pedicle screw malposition is rare. One author evaluated 3204 screws and reported no vascular, neurologic or visceral injuries (Kim et al. Routine MEP after insertion of each pedicle screw in the thoracic spine, as well as triggered EMG stimulation of each pedicle screw to rule out breaching once all screws have been inserted, could improve safety in challenging cases (Calancie et al. As patient safety becomes more and more of a concern, the use of intraoperative CT scanning, with real CT imaging or CT fluoroscopy of pedicle screws post-insertion, may become a standard of care in the future (Flynn and Sakai Some technical tricks, such as the funnel technique Gaines , allow dThis uncommon case of completely misplaced pedicle screw crossing the spinal canal draw the reader’s attention to the risk of pedicle screw placement in severe thoracic curves. In case of early or delayed neurological status worsening, intraoperative or postoperative imaging must be done to detect pedicle screw misplacement. In the current case, thanks to cobalt-chromium and titanium use, MRI and CT scan allowed good visualization of the spinal canal and spinal cord. Major penetration of the spinal canal by pedicle screw may conduct to hardware removal.


Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location , or more severe patterns affecting more than one location , was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszky's disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under controlled conditions. Furthermore, more research including other important pathogens, such as gastro-intestinal nematodes, will be necessary to complete this picture.Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between  Co-infections  are ubiquitous in nature but most research on relevant diseases largely relies on a “one-disease-one-pathogen” perspective. From the point of view of community ecology, a host can be considered a complex ecosystem composed of parasites that directly or indirectly interact among themselves and with their own environment, the host Oryctolagus cuniculus). In this host-parasite model, respiratory infection by Bordetella bronchiseptica facilitates secondary helminth (Graphidium strigosum) infections that, at the same time, collaborate in maintaining the bacterial persistence in the upper respiratory tract B. bronchiseptica infection does not only facilitate Heligmosomoides polygyrus reproduction, another gastrointestinal nematode Trichostrongylus retortaeformis) from the small intestine Among all possible interactions, bacteria-helminth co-infections are one of the most studied models for exploring how co-infection drives disease dynamics and severity. Helminths mostly induce cytokines associated with a T-helper cell type 2 (Th2) immune response, which simultaneously tends to down-regulate T-helper cell type 1 (Th1) cytokines involved in intracellular microparasite control Mycobacterium tuberculosis), susceptibility and severity are shaped by co-infection with different types of pathogens. Since the first report describing severe pulmonary Mycobacterium spp-helminth co-infected patients in the mid 1940's Mycobacterium spp co-infection in humans is one of the best-known examples. In fact, the HIV infection is considered one of the main risk factors predisposing patients to tuberculosis as well as the progression to active disease, increasing the risk of latent tuberculosis reactivation 20-fold Shifts in disease severity are also common in a broad range of co-infected hosts. Concomitant infections often exacerbate the effects of single infections, independently of the diversity in parasite groups involved in the infection process. For example, in human tuberculosis (caused by Mycobacterium bovis is one of them and it is present in a broad range of wild hosts across different geographic regions Meles meles) in Great Britain, brush tailed possums (Trichosurus vulpecula) in New Zealand, buffalo (Syncerus cafer) in South Africa and wild boar (Sus scrofa) in the Iberian Peninsula are common reservoirs of this infectious disease Wildlife is an excellent model for exploring whether co-infection drives infectiousness of major diseases since they are almost always co-infected by several pathogens Mycoplasma hyopneumoniae, Haemophilus parasuis and Actinobacillus pleuropneumoniae) and a nematode (Metastrongylus spp) in 165 wild boar. Two hypotheses were tested. The first investigated whether contact with a selected group of pathogens in Mycobacterium spp infected wild boar occurred by chance or, on the contrary, was due to a structured community of pathogens (hypothesis i). The second was supported by links between tuberculosis severity and concomitant viral and nematode infections observed in both human ii).The aim of this work was to assess whether bTB severity in wild boar from mid-western Spain is associated with the contact with a selected group of common pathogens. Evidence of infection by means of serology and/or pathogen detection was carried out for viruses , bacteria . This study did not need to be approved by any animal ethics committee since animals were not killed for scientific purposes.Quercus ilex and Q. suber trees with understoreys dominated by Q. coccifera, Cistus ladanifer and Erica arborea. Wild boar density in the studied area ranged between 6.5 and 30 wild boar/hectare Cervus elaphus) and, in some cases, with fallow deer (Dama dama), roe deer (Capreolus capreolus) or extensive herds of cattle.This study was carried out on 20 wild boar game estates in mid-western Spain . In thisA total of 165 hunter harvested wild boar were collected between October 2011 and February 2013. The sex and age of these animals were determined on the basis of the observation of their sexual organs and the eruption of dentition pattern, respectively M. bovis as well as on the presence of microscopic granulomatous bTB lesions. Since the combination of microbiological culture and histopathology increases the sensitivity and specificity of bTB wild boar surveys Diagnosis of bTB in wild boar was carried out based on the isolation of M. bovis, microbiological cultures from intact (not handled or cut) submandibular or retropharyngeal lymph nodes and from a piece of caudal lung lobes (both with gross bTB-like lesions when possible) of each animal were carried out. For bacterial culture, tissue samples were sectioned and dissected, trimming the fat and connective tissue, using sterile scissors and forceps for each individual sample. Two grams of tissue were homogenized in 10 ml of sterile water with 0.2% albumin  for 4 minutes in a mechanic homogenizer . The homogenized material was then decontaminated by the hexadecyl pyridinium chloride method M. tuberculosis complex by PCR and “Spoligotyped” following standard methods M. bovis.To detect the presence of In addition, to assess the presence of tuberculosis granulomas, a piece of submandibular or retropharyngeal lymph node and lung of each animal sampled were fixed by immersion in neutral, buffered-formalin  and sections of 4 µm were cut and stained with hematoxylin and eosin for histopathological examination. The tissue pieces used for the histopathological analysis were chosen based on the presence of macroscopic bTB-like lesions when present. For wild boar in which bTB-like lesions were not found, a piece of submandibular lymph node and caudal lung lobe were identically processed for histopathology.To consider the extent of bTB, wild boar were classified into two groups: animals with a localized lesion pattern and animals with a generalized lesion pattern. Based on the distribution of lesions, generalized bTB implies more severe disease and a greater bacterial load than localized bTB Animals showing a localized pattern were those with bTB lesions in one location, mainly submandibular or retropharyngeal lymph nodes . On the A complete lesion assessment could not be carried out in 25 of 165 animals included in this study, since these animals were partially eviscerated upon arrival at the inspection area. This meant we could not ensure the lack of bTB-like lesions in abdominal organs and, therefore, their lesion pattern could not be completely determined.Haemophilus parasuis and Actinobacillus pleuropneumoniae, was determined in lungs of the sampled animals. DNA from a piece of cranial lobe from one of the lungs was extracted using a commercial QIAamp DNA Mini kit  following the manufacturer's recommendations. Later, specific PCRs were carried out in order to detect the presence of H. parasuisA. pleuropneumoniaeM. hyopneumoniae were detected using a commercial blocking-ELISA assay for swine  that was carried out using duplicate serum samples from each animal, following the protocol and cut-off values proposed by the manufacturers to differentiate seropositive and seronegative animals  <0.40× negative control OD).The presence of concomitant bacterial respiratory pathogens, such as Concerning viruses, a serologic survey for contacts with viral pathogens was carried out using commercial ELISA kits for swine and also following the manufacturer's recommendations to differentiate seropositive and seronegative animals. The presence of antibodies against PCV2 , ADV  >0.35), SIV  and PRRSV  was evaluated using duplicate samples of sera obtained from blood samples.Pulmonary nematodes were collected by lung dissection. During examination, the trachea and main bronchi were opened longitudinally with scissors, carefully examined and then placed under running water to collect adult worms on a sieve. In addition, the pulmonary parenchyma – and in particular the affected areas – were dissected carefully under a dissecting microscope to extract adult nematodes. Permanent preparations of adult specimens were made with lactophenol cotton blue solution and genus identification was based on previous descriptions A brief summary of the techniques used for antibodies and pathogen detection and the clinical picture produced by bacteria, viruses and parasites assessed in this work are shown in M. bovis, PCV2, ADV, SIV and Metastrongylus spp. Neither differences in pathogen prevalences, pathogen community structure nor in the effect of co-infection on bTB severity were determined at the game estate scale, in part because the sample size never exceeded 30 individuals per estate.For the analyses described below, all estates showed the presence of the most prevalent pathogens, e.g., Mycobacterium spp occurred by chance was studied using the null model analysis. Data were organised as presence/absence matrices in which each row represented a pathogen species and each column represented an individual wild boar. In a presence/absence matrix, “1” indicates that a species is present at a particular site or host, and “0” indicates that a species is absent Whether or not contact with other pathogens in wild boar infected by The C-score was used as a co-occurrence index for exploring co-occurrence patterns In addition, to compare the degree of co-occurrence across data, a standardised effect size (SES) for each matrix was calculated. The SES measures the number of standard deviations that the observed index (C-score) is above or below the mean index of the simulated communities.The association between viruses, bacteria and nematode species  and bTB severity  was assessed by a Partial Least Squares regression approach (PLSr). This statistical tool is an extension of multiple regression analysis, where associations between groups of variables are established with factors, i.e., combinations of dependent variables extracted from predictor variables that maximise the explained variance in the dependent variables. PLSr is probably the least restrictive of the multivariate techniques In the present study, bTB severity was considered as a single explanatory variable, while concomitant pathogens represented the × predictor factor. For PLSr modelling, the age of animals was considered in months. The “plspm” library version 0.3.7 M. bovis isolates were obtained from 85 animals (51.51%). Within infected animals, 28 showed M. bovis infection in both submandibular/retropharyngeal lymph nodes and lungs , whereas 57 only showed M. bovis infection in submandibular/retropharyngeal lymph nodes . On the other hand, bTB-like microscopic lesions were observed in 80 submandibular/retropharyngeal lymph nodes  and 28 lungs . Nine out of 85 animals infected by M. bovis (10.58%) did not show evidence of microscopic bTB-like lesions, while this microorganism could not be isolated from four animals that showed typical bTB granulomatous lesions in their lymph nodes (5%). Thus, since the combination of microbiological culture and histopathology increases the sensitivity of bTB diagnosis in wild boar M. bovis and showed 12 different spoligotype patterns (SB0119 (15.29%), SB0121 (25.88%), SB0134 (4.71%), SB0296 (3.53%), SB0339 (23.53%), SB1091 (8.23%), SB1142 (8.23%), SB0120 (1.18%), SB0152 (3.53%), SB0848 (3.53%), SB1142 (8.23%), SB1174 (2.35%)).The A detailed lesion severity assessment was carried out in 71 out of 89 animals positive to bTB. Generalized lesion patterns were detected in 28 bTB affected animals (40%), whereas localized lesions were observed in 43 affected animals (60%). Some of the animals with generalized patterns also displayed gross bTB-like lesions in organs such as liver, spleen or mesenteric lymph nodes; however, no bTB-like lesions were found in these organs in animals that did not show bTB-like lesions in the lungs (see ).M. hyopneumoniae (13.94%). A. pleuropneumoniae and Metastrongylus spp were detected in 4.84% and 50.51% of the animals, respectively, whereas evidence of infections with PRRSV or H. parasuis was not detected.The percentage of animals positive for selected respiratory pathogens is shown in M. bovis infected juvenile, yearling and adult wild boar are shown in M. bovis infected showed antibodies against PCV2 and ADV.The most common helminth, bacteria and viruses assemblages observed in The null model analysis showed that the observed C-scores were greater than expected by chance (O >E) indicating the existence of a competitively structured community; that is, bTB-infected wild boar have contacted with some specific, non-random pathogen combinations . This faMetastrongylus spp, sex and age provided a first PLSr X's component explaining 20.90% of the observed variability  Mycobacterium spp. later Regarding the order and timing of infections, the current study does not allow the determination of whether contact with these other pathogens was previous, simultaneous or subsequent to the precise time of infection by Diagnosis of some of the pathogens assessed in this work was based on serological techniques. Detection of antibodies against one pathogen does not necessarily mean a current infection since high titers of antibodies may remain for a long time after the clearance of the agent. However, this limitation might not influence the results obtained in this study since bTB is a chronic disease and lesions may persist in an affected wild boar for a long time Differentiation between seropositive and seronegative animals was carried out using the cut off values recommended by the ELISA's manufacturers, as has been previously done in similar surveys carried out in wild boar Metastrongylus spp and ADV on bTB severity suggests that sanitary measures focused on the control of these agents , may help to reduce the bTB prevalence in the affected areas.According to our results, certain pathogen combinations are correlated to more severe bTB lesions in wild boar. Since the presence of animals displaying severe bTB lesion patterns has been recently related to higher bTB prevalence in wild boar populations M. bovis may influence the success of measures applied to reduce bTB prevalence in wild boar populations, such as bTB vaccination Metastrongylus spp in wild boar vaccinated against bTB may reduce the effectiveness of this vaccine whereas measures such as previous deworming may be useful in the estates where vaccination will be carried out. In fact, a significant improvement in mycobacterial-specific immune responses occurs following anthelmintic therapy in vaccinated humans Importantly, the interaction between the above-mentioned pathogens and M. bovis in these populations.On the other hand, it is noteworthy that the greatest effects of co-infections in other wild hosts have been observed in seasons of food shortage To conclude, results obtained in this work provide a new point of view for bTB control based on community ecology principles. Removing specific members of the wild boar pathogen community could be considered in further bTB management plans in wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under controlled conditions. Furthermore, agents assessed in this work represent a limited group of wild boar pathogens. Therefore, the influence that other agents  have in wild boar bTB severity should be explored in future studies to obtain a more complete picture.Table S1Results obtained in characterisation, laboratory diagnosis and lesional assessment carried out in the 165 wild boar included in the study.(XLSX)Click here for additional data file.