Vaccine-elicited HIV-specific T cell responses were associated with improved control of viral replication following antiretroviral interruption (ATI from w24 to w48). We show an inverse relationship between HIV-specific responses  and the peak of viral load during ATI. Here we have performed an integrative systems vaccinology analysis including: (i) post vaccination (w16) immune responses assessed by cytometry, cytokine secretion, and Interferon-γ ELISPOT assays; (ii) whole blood and cellular gene expression measured during vaccination; and (iii) viral parameters following ATI, with the objective to disentangle the relationships between these markers and to identify vaccine signatures. During vaccination, 69 gene expression modules out of 260 varied significantly including (by order of significance) modules related to inflammation , plasma cells (M4.11) and T cells . Cellular immune responses were positively correlated to genes belonging to T cell functional modules  at w16 and negatively correlated to genes belonging to inflammation modules . More specifically, we show that prolonged increased abundance of inflammatory gene pathways related to toll-like receptor signaling  are associated with both lower vaccine immune responses and control of viral replication post ATI. Further comparison of DC vaccine gene signatures with previously reported non-HIV vaccine signatures, such as flu and pneumococcal vaccines, revealed common pathways across vaccines. Overall, these results show that too long duration and too high intensity of vaccine inflammatory responses hamper the magnitude of effector responses.The goal of HIV therapeutic vaccination is to induce HIV-specific immune response able to control HIV replication. We previously reported that vaccination with  Systems biology approaches applied to immunology and vaccinology aim at analyzing the whole data available from various high throughput technologies to better understand and predict diseases and mechanisms of interventions –3. In vaex vivo generated Dendritic Cells (DC) loaded with HIV-lipopeptides in HIV infected patients on antiretroviral therapy (ART). The primary report of this study (DALIA study) showed that the vaccination strategy was well-tolerated and immunogenic  sponsored by the Baylor Institute for Immunology Research and the Agence Nationale de Recherches sur le SIDA et les hepatites (INSERM ANRS). The study was approved by the IRB of Baylor Research Institute (BRI) (NCT 00796770). All patients gave written informed consent.The study design is shown in Participants received four vaccinations at w0, 4, 8, and 12. At w22, patients who had HIV-1 RNA <400 copies/mL were proposed to interrupt ART from w24 to w48. ART could be resumed from w24 to w48 at any time according to the following criteria: (i) if the patients or their doctors wished so; (ii) if CD4+ T cell count was <350 cells/μL and <25% of total lymphocytes at two consecutive measurements 2-weeks apart; and (iii) in the case of occurrence of an opportunistic infection or a serious non-AIDS defining event.Post vaccination (w16) immune responses assessment by cytometry, cytokine secretion, and Interferon-γ ELISPOT assays were described elsewhere .Tempus™ Spin RNA Isolation Kit (ThermoFisher scientific). PBMC, CD4- and CD8-lymphocytes RNA were purified on Qiagen RNeasy Micro Kit. RNA was quantified using a ND-8000 spectrophotometer  before being checked for integrity on a 2100 BioAnalyzer . cDNA was synthesized and biotin-labeled cRNA was generated by an in vitro transcription reaction using Ambion Illumina TotalPrep RNA Amplification Kits . Labeled cRNA were hybridized on Illumina Human HT-12V4 BeadChips. All steps were done following the manufacturers' protocols.Whole blood RNA was purified using in vitro stimulation of purified PBMC with HIV antigens has been performed as previously reported (6 of thawed PBMC resuspended in RPMI 1640 media with L-Glutamax supplemented with Penicillin/Streptomycin and 10% HS (R-10HS) were stimulated for 6 and 20 h in 48-well plates with 2 μg/ml of HIV LIPO-5 vaccine itself, a pool of 5 long peptides corresponding to LIPO-5 vaccine sequences  at 2 μM/peptide, or a pool of 36 peptides  at 2 μg/ml/peptide, in a humidified 37°C cell incubator with 5% CO2. Cells were then transferred in eppendorf tubes, pelleted, resuspended in 350 μL lysis buffer and frozen immediately at −80°C until transcriptomic analysis as described . Gene transcription data were pre-processed , 26 and atistics . An adapatistics . Unsuperatistics . CanonicTime-course gene set analysis was performed using TcGSA, an innovative approach relying on mixed regression models and likelihood ratio test to identify gene sets the expression which is significantly changing over time . It usesA transcriptomic signature was then derived through an integrative analysis of the gene expression at w16 from the significant modules identified by TcGSA together with the viro-immunological measurements at w16 in the DALIA trial, performed using sparse group Partial Least Squares (sgPLS) with leave-one-out cross-validation . This isFunctional analysis of the immune signature was performed with Ingenuity Pathway Analysis software  and CluePedia and ClueGO in Cytoscape , 33.http://www.ncbi.nlm.nih.gov/geo/, GEO Series accession number GSE46734).All microarray data is MIAME compliant and the raw and normalized data have been deposited in the MIAME compliant database Gene Expression Omnibus (All 19 included patients received the four vaccinations as planned  with no γ) interferon signaling pathways. The modules M4.13, annotated “activation and cell division of leukocytes,” M5.7 and M7.1 , changes in gene expression were assessed every week until week 28 and every 4 weeks until week 44. The abundance of the genes in the inflammatory modules tended to decrease over time up to 44 weeks . The dynIn a robustness analysis, the analysis of the dynamics of the genes during vaccination (preATI) was performed with the Blood Transcriptional Modules (BTMs) that are other gene sets generated using another approach than Chaussabel et al. . Overallex vivo DC vaccines (see section Methods) during 20 h. The stimulation by 15-mers led to a differential expression of a large number of genes compared to unstimulated cells  were correlated to HIV-specific cellular immune response at the same time point and the viral dynamics following ATI. As previously published , the vacWe found that a large part of the inflammatory modules  was negatively correlated to vaccine-elicited HIV specific cellular immune responses. The module 5.15, annotated “Neutrophils,” was also negatively correlated to the immune response . All theIn order to better characterize mechanisms of dendritic cell-based vaccine immunogenicity, we compared whole blood gene expression signatures identified in our trial with those described with other vaccines, for which protective correlates have been identified. We took the opportunity of published studies in which the same methods for measuring transcriptomic changes were used as the ones in the present study. For this, we revisited published data using our statistical approach based on gene set analyses of longitudinal data [here and detailed in ]. ObermaBy taking advantage of both blood and cellular analyses, a longitudinal study design and the comparison between a pre- and post ATI period, we were able to demonstrate an association between the mobilization of inflammatory pathways, the magnitude of vaccine elicited HIV cellular responses and the viral dynamics after ATI after experimental therapeutic DC-based vaccination in HIV-infected patients. The results reported here contribute to identify gene signatures of a therapeutic DC-based vaccine against HIV and to a better understanding of the actions of these vaccines.Longitudinal analysis of whole blood gene expression revealed changes in gene abundance from modules involved in “inflammation” and “activation” and “differentiation of T cell.” Interestingly, changes in abundance of genes involved in myeloid activation and trafficking were detectable at early vaccination time points. Although activation of inflammatory pathways is known as a physiological response to vaccinations, our results revealed also that modifications of genes related to differentiation and activation of T cells following vaccination behave in the opposite way at the same time points. Consistently, genes related to toll-like receptor signaling pathways and Interferon type 1 and 2 were mobilized in the blood of vaccinated individuals. Comparison between pre- and post-vaccination (w16) changes in gene expression induced by HIV antigen stimulation enriched these observations showing an induction of genes related to cellular immune responses to cytokines, T cell, and dendritic cell responses.A very specific feature of our study consists in the timing of whole blood sampling for gene expression that was performed every 4 weeks. This schedule is very different from previous studies that usually took blood few hours after vaccination e.g., , 11, 13], 1311, 1A too high and long-standing change in abundance of gene expression associated with toll-like receptor signaling pathways and monocyte/neutrophil activation appeared to be deleterious for the immune response to the vaccine. This may have an important impact on the use of adjuvants that are needed but may require refined adjustments  to avoidThe involvement of pathways including TLR4 and MYD88 raises the hypothesis that gut translocation may interfere with the responses to the vaccine as it does with other immunotherapy . Other mThese types of result should help improving the potency of vaccines by combining vaccines and immunomodulatory tools . For insIn conclusion, inflammatory pathways related to toll-like receptor signaling pathways that were committed in response to this DC-based therapeutic HIV vaccine were associated with a poorer immune response to vaccination and poorer viral control after ATI. Given a similar involvement and impact of these pathways in responses to other vaccines, it is important to control the kinetics of inflammatory responses induced by vaccines. In total, these results are helpful for the design of further strategies combining vaccines with adjuvants and/or immunomodulators.The study was approved by the IRB of Baylor Research Institute (BRI) (NCT 00796770). All patients gave written informed consent.YL and JB conceived the project and designed the experiments. HH and JS conducted the transcriptomics analyses. CL and MM conducted the immunological analyses. RT, BH, HB, and LR conducted the statistical analyses. RT and YL analyzed the data and wrote the manuscript. All the authors provided critical feedback on the manuscript prior to publication and have agreed to the final content.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.


Using HIV proviral DNA as a template may be suitable for initial detection of transmitted drug resistance mutations (TDRMs) as it is easy to handle and less expensive compared to RNA. However, existing literatures which are mainly focused on HIV-1B subtypes DNA extracted from PBMCs revealed controversial findings ranging from the detection of significantly lower or higher mutations in proviral DNA compared to historic viral RNA. Thus, to verify whether viral RNA or proviral DNA has improved sensitivity in detecting transmitted genotypic drug resistance mutations paired viral RNA and proviral DNA (which is directly extracted from stored whole blood) samples were tested from Ethiopian antiretroviral naive HIV-1C infected subjects.In the present comparative study the frequency of TDR mutations was assessed in paired samples of viral RNA and proviral DNA (extracted directly from stored whole blood) of HIV-1C infected treatment naïve patients and interpreted using the 2009 WHO drug resistance surveillance mutation lists, Stanford University drug resistance data base and International Antiviral Society-USA mutation lists.High agreement in rate of TDR between the two compartments was observed using the WHO mutation lists. While mutations G190A and E138A were concurrently found in both compartments, others such as G73S on PR and A62V, M184I, M230I on RT were identified in proviral DNA only. All signature mutations seen in viral RNA were not missed in proviral DNA.The concordance of major genotype drug resistance mutation between RNA and proviral DNA in treatment naïve patients suggests that proviral DNA might be an alternative approaches for an initial assessment of drug resistance prior to initiation of antiretroviral therapy using the WHO mutations lists in resource-limited countries. However, the clinical importance of TDRMs observed only in proviral DNA in terms of being a risk factor for virologic failure and whether they limit future treatment options needs additional investigation using more sensitive sequencing approaches such as Next Generation Sequencing (NGS). Since the introduction of antiretroviral therapy (ART), the relative rates of morbidity and mortality from acquired immunodeficiency syndrome have been reduced significantly . HoweverThus, to verify whether viral RNA or proviral DNA has improved sensitivity in detecting transmitted genotypic drug resistance mutations paired viral RNA and proviral DNA (which is directly extracted from stored whole blood) samples were tested from antiretroviral naive HIV-1C infected subjects from Ethiopian where HIV-1 transmitted drug resistance is increasing –21.+ T-cell count was performed on fresh whole blood by flow cytometry  following the manufacturer’s specifications. Details of patient’s recruitment and management has been described elsewhere .The characteristics of the study subjects (n = 41) are summarized in pol consensus sequences consisting of 1,261 nucleotides were analyzed to assess the similarity between the viral RNA and proviral DNA  of the patients. Thirty patients had wild type genotype in both PR and RT region of the pol genome from both viral RNA and proviral DNA; eleven patients 26.8%,  had a mu+ cell count < 100 cells/mm3 (72.7%) and in patients who were in the age group of 26–35 years . TDR to NNRTI was identified in 22.0% (9/41) of patients from both, viral RNA and proviral DNA, and in three patient’s resistance mutations to PIs. NRTIs related mutations M184I and A62V were only found in proviral DNA  had two TDR in their proviral DNA representing multi-drug resistance . The ress 81.8%, . Howeveriral DNA . HIV-1 pThe abundance of polymorphisms  at knownIn the present study, 83% (34/41) of the patient’s had concordant sequence profile in determining transmitted drug resistance from both RNA and proviral DNA and those major mutations conferring transmitted drug resistance in RNA (the gold-standard template for HIV drug resistance testing as it reflects the virus that is actively reproducing) were also concordantly detected in proviral DNA consistent with recent finding  and suggThe inconsistencies between plasma viral RNA and peripheral blood mononuclear cell proviral DNA in HIV-1 drug resistant strains have been reported from treatment naive individuals ranging from the detection of significantly lower , 8 or hiThe observed lower abundance of TDR in viral RNA than proviral DNA 14.6% vs 22%) could be conceivably a result of the reversion over time of HIV-1 quasispecies resistant to HAART  being ar% vs 22% Four out of the 41 paired samples from both compartments did not show a tight proximity relation to each other suggesting that circulating strains produced in the plasma compartment could arise from sources other than circulating infected leukocytes , 30. MosThe data has an obvious limitations including small sample size with few detected drug resistance mutation. Although there is technical improvements in the amplifcation of drug resiatnce genome using direct whole blood as a template, the technical improvment over sequencing is limitted. It would have been interesting to evaluate the use of more sensitive sequencing approaches such as Next Generation Sequencing (NGS) that might partially explain the observed differences between viral DNA and plasma RNA. Thus, the observd small mutation might be due to the low sensitivity nature of of the Sanger sequencing approach. Furthermore, as the nature the study was cross sectional, the clinical importance of the mutations observed only in proviral DNA in terms of being risk factors for virologic failure and whether limits future treatment options is not addressed. Nevertheless, with all these limitation we concluded the points below (Conclusions).In summary, as sequencing of HIV-1 proviral DNA which is extracted directly from stored whole blood detects all signature mutations observed in RNA and is less expensive and technically easier to perform compared to RNA, determining TDR using the WHO drug resistance surveillance mutation list from proviral DNA could be an alternative approaches especially in resource limited countries. However, the clinical importance of transmitted drug resistance mutations observed only in proviral DNA in terms of being risk factors for virologic failure and limits future treatment options in future needs additional investigation using more sensitive sequencing approaches such as Next Generation Sequencing (NGS).


The majority of patients develop posttraumatic osteoarthritis within 15 years of anterior cruciate ligament (ACL) injury. Inflammatory and chondrodegenerative biomarkers have been associated with both pain and the progression of osteoarthritis; however, it remains unclear if preoperative biomarkers differ for patients with inferior postoperative outcomes. The purpose of this pilot study was to compare biomarkers collected on the day of ACL reconstruction between patients with “good” or “poor” 2-year postoperative outcomes. We hypothesized that inflammatory cytokines and chondrodegenerative biomarker concentrations would be significantly greater in patients with poorer outcomes. Prospective cohort design.2) previously enrolled in a randomized trial evaluating early anti-inflammatory treatment after ACL injury. Biomarkers of chondrodegeneration and inflammation were assessed from synovial fluid (sf) samples collected on the day of ACL reconstruction. Participants completed Knee Injury and Osteoarthritis Outcome Score (KOOS) and International Knee Documentation Committee (IKDC) questionnaires two years following surgery. Patients were then categorized based on whether their KOOS Quality of Life (QOL) score surpassed the Patient Acceptable Symptom State (PASS) threshold of 62.5 points or the IKDC PASS threshold of 75.9 points. 22 patients , IL-1 receptor antagonist , and matrix metalloproteinase-9  concentrations on the day of surgery. Patients that failed to reach the IKDC PASS threshold  had significantly greater sf IL-1α (p = 0.02). Patients that failed to reach the QOL PASS threshold after surgery  had significantly greater sf interleukin-1 alpha (IL-1 These pilot data suggest that initial biochemical changes after injury may be an indicator of poor outcomes that are not mitigated by surgical stabilization alone. Biological adjuvant treatment in addition to ACL reconstruction may be beneficial; however, these data should be used for hypothesis generation and more definitive randomized clinical trials are necessary. Whether isolated or in concert with concomitant meniscal or articular cartilage injury, anterior cruciate ligament (ACL) rupture initiates a cascade of cytokine and catabolic enzyme activity –4. RegarOne method to objectively quantify a successful outcome is to determine whether a patient has achieved a Patient Acceptable Symptom State (PASS) following surgery –18. The 2, NCT01692756) . , 4. 3, 43)  and uCTX-II [p=0.08]) tended to be greater for those that failed to reach the QOL PASS threshold. Graft type, the prevalence of meniscus injury, and bone bruise volumes did not differ between QOL PASS groups  had postoperative KOOS QOL scores below the PASS threshold of 62.5 points . PatientS groups .α (p=0.02). While not statistically significant, sf IL-1ra (p=0.057), sf MMP-1 (p=0.10), and sf MMP-9 (p=0.10) tended to be greater for those that failed to reach the IKDC PASS threshold. No other biomarker differences were noted between those that did or did not surpass the IKDC or QOL PASS thresholds. Graft type, the prevalence of meniscus injury, and bone bruise volumes did not differ between IKDC PASS groups (Nine of 22 patients (41%) failed to reach the IKDC PASS threshold of 75.9 . PatientS groups .The purpose of this pilot study was to compare biomarkers assessed from samples collected on the day of ACL reconstruction between patients with postoperative patient-reported outcome scores above and below previously established PASS thresholds . We hypoα stimulating the production of matrix metalloproteinases thereby reducing proteoglycan content and altering cartilage mechanical properties. Previous studies have demonstrated that ACL injury triggers a biochemical cascade that worsens over the first 4-6 weeks after injury [ in vitro [α than IL-1β [The current pilot results support the previously described cascade of increased IL-1r injury –4. Inflar injury –31. It ir injury , 33. Synr injury . Synoviar injury . The supin vitro . Porcinean IL-1β .α and MMP-9 concentrations on the day of surgery were significantly higher for patients that failed to postoperatively achieve the KOOS QOL-based PASS compared to those with better clinical outcomes. In addition to increased IL-1a and MMP-9 concentrations, biomarkers of bone and cartilage turnover  were also increased for patients that failed to achieve KOOS QOL-based PASS. This overall state of upregulated catabolism and inflammation may lead to recurrent effusions and/or persistent synovitis which also appears to potentially have long-term implications for knee health.The current pilot results suggest that initial biochemical changes after injury may be prognostic of long-term consequences of injury of the knee that are not mitigated by surgical stabilization alone. Inflammatory cytokine and degradative enzyme concentrations on the day of ACL reconstruction have been previously shown to correlate with cartilage changes on MRI during the first three years after surgery , and theα and MMP-9 concentrations for those that failed to achieve a Patient Acceptable Symptom State in the current study.In addition to playing a contributing role in the progression of structural posttraumatic OA changes , increasα, pain, and both cartilage and bone metabolism may all contribute to KOOS QOL and IKDC outcomes at 2 years.Early anticatabolic and/or anti-inflammatory interventions after ACL injury or reconstruction may need to be further investigated as adjunctive treatment strategies to mitigate both the structural changes and symptoms of posttraumatic OA. CTX-II has been reported to be predictive of OA progression based on both radiographic and arthroscopic evidence –45. Partβ concentrations as 50% of the samples in the current study had values below the lower limits of detection.This study had several limitations. First and foremost, the results of this preliminary work are based on a small sample size and additional studies are necessary to confirm these results. Second, some patients had received corticosteroid injections prior to surgery which could have influenced biomarker concentrations on the day of surgery. As mentioned previously, KOOS QOL and IKDC scores did not differ between patients treated with preoperative intra-articular corticosteroid and the placebo group, and the surgical intervention and postoperative rehabilitation and medications did not differ between groups and no between-group differences were noted in either postoperative KOOS QOL or IKDC scores. Third, the prevalence of patients with postoperative KOOS QOL and IKDC scores below the PASS threshold was higher in the current study than those originally reported by Muller et al. . This maα) and a trend towards greater bone and cartilage collagen degradation (NTX-I and uCTX-II) at the time of surgery were associated with failure to achieve an acceptable symptom state two years after ACL reconstruction. These data suggest that initial biochemical changes after injury may be prognostic of long-term consequences of ACL injury that are not mitigated by surgical stabilization alone; however, due to the small sample size these data should be used for hypothesis generation and more definitive randomized clinical trials are necessary.In conclusion, the results of this preliminary investigation demonstrated that higher inflammatory cytokine activity (IL-1


A subpopulation of human CD56+ NK cells also exert strong cytotoxicity against renal cells and contribute to the progression of renal fibrosis.Mouse natural killer T (NKT) cells and natural killer (NK) cells are innate immune cells that are highly abundant in the liver. In addition to their already-known antitumor and antimicrobial functions, their pathophysiological roles in the kidney have recently become evident. Under normal circumstances, the proportion of activated NKT cells in the kidney increases with age. Administration of a synthetic sphingoglycolipid ligand  further activates NKT cells, resulting in injury to renal vascular endothelial cells via the perforin-mediated pathway and tubular epithelial cells via the TNF-α/Fas ligand pathway, causing acute kidney injury (AKI) with hematuria. Activation of NKT cells by common bacterial DNA (CpG-ODN) also causes AKI. In addition, NKT cells together with B cells play significant roles in experimental lupus nephritis in NZB/NZW F1 mice through their Th2 immune responses. Mouse NK cells are also assumed to be involved in various renal diseases, and there may be complementary roles shared between NKT and NK cells. Human CD56 When these cells are activated by various stimuli, including cytokines and bacterial components, they play crucial roles in the defense against tumors and bacterial infections via the IFN-γ/perforin pathway ,3, which T cells . However T cells .Vα24Jα18 and Vβ11 genes, which have an arrangement resembling that of mouse invariant NKT cells, were suggested to be NKT cells [+ T cells are considered to be a functional counterpart of mouse NKT cells, because (i) they express a surface marker of NK cells (CD56) and intermediate and oligoclonal TCRs [+ T cells also express CD161, a NK cell receptor protein 1 (NKR-P1) molecule to which the NK1.1 antigen in mice belongs [+ T cells), unless otherwise specified. However, it should be noted that whereas mouse NKT cells are almost exclusively either CD4+ or CD4− CD8− [+ T cells are regularly CD8 [In humans, T cells that express TCRs encoded by the KT cells . In factKT cells . On the nal TCRs ; (ii) thnal TCRs ; and , particularly in aged animals , the pre+ CD122+ T cells to produce IFN-γ, together with the subsequent lipopolysaccharide challenge that induces TNF-α production [Although definitive natural ligands of NKT cells have not been identified so far, some microbes have been shown to be their antigenic targets ,17. In aoduction ,20. Furtoduction . Taken tIn contrast to conventional T cells and B cells, NKT cells have been reported to be resistant to treatment with glucocorticoids as well as irradiation . We alsoThe pathogenic roles of NKT cells have also been demonstrated in renal ischemia-reperfusion injury (IRI) models, which are representative experimental models of AKI. One report showed that the absence of NKT cells markedly attenuated renal damage, including tubular necrosis, suggesting the role of NKT cells in the pathogenesis . More reSystemic lupus erythematosus (SLE) is a representative systemic autoimmune disease, which is characterized by the presence of autoantibodies and involves virtually any organ. Lupus nephritis is a serious complication involving almost half of all SLE patients, which results in an unfavorable renal prognosis ,29. The The effects of α-GalCer in the progression of SLE models have also remained contradictory . In MRL/NZB/NZW F1 (BWF1) mice, which is another representative SLE model, spontaneously produce autoantibodies and develop lupus nephritis-like renal lesions , and theIn adriamycin-induced nephropathy, a model of focal segmental glomerulosclerosis in which chronic proteinuric renal injury is shown, an agonist of NKT cells was reported to modulate immune responses and inhibit the development of the disease model . There hThe role of NKT cells in the field of organ transplantation is controversial; they are assumed to be involved in immune tolerance after liver transplantation , whereasSimilar to NKT cells, the frequency of NK cells as well as their activation in the kidney have been reported to be increased by renal IRI, suggesting their deleterious roles. The up-regulated expression of a NK cell ligand on tubular epithelial cells and its engagement through the activating receptor NKG2D is reportedly involved in the pathogenesis of renal IRI . The invAlthough some studies suggested that NK cells in the kidney are activated and that they may be responsible for promoting and maintaining inflammation in lupus nephritis ,57, dataNK cells have been reported to mediate transplanted kidney injury . However+ T cells, as well as (CD56+) NK cells, have been shown to play significant roles in generalized Shwartzman reaction-like responses in vitro [+ T cells stimulated by a combination of IL-2 and IL-12 demonstrates significantly stronger cytotoxicity against both glomerular endothelial cells and tubular epithelial cells than regular T cells [+ T cells have been shown to produce large amounts of IFN-γ, perforin, and soluble FasL [+ T cells therefore potentially damage intrinsic renal cells and can be integral to the processes that mediate AKI. In addition, there may be a common pathogenesis between the conditions in mouse NKT cells and human CD56+ T cells. Recently, it has been reported that γδT cells with NK cell–associated markers (including CD56+ T cells of the γδ type) are associated with renal fibrosis [Human CD56in vitro . Consist T cells . The cytble FasL ,62. CD56fibrosis . Our invfibrosis  and furt+ T cells do not produce very much IL-4, and hence the condition in mice may be different to that in humans. Indeed, a decreased number of human CD56+ T cells was reported to be associated with high levels of serum IgG and anti-dsDNA antibodies in patients with SLE [+ T cells may ameliorate human SLE. Whether human NKT cells are actually involved in the pathogenesis of lupus nephritis should therefore be carefully investigated.As described above, mouse NKT cells may cooperate with B cells to play significant roles in experimental lupus nephritis through their Th2 immune responses. In this regard, it should be noted that CD56with SLE , suggest+ T cells are involved in other human renal diseases has not been well investigated, and data showing their involvement are essentially limited to case reports and case series. In patients with Balkan endemic nephropathy, which is a chronic tubulointerstitial renal disease that is associated with increased incidence of upper urinary tract urothelial carcinoma, the number of CD56+ T cells in peripheral blood is reportedly increased during disease progression, suggesting that these cells are associated with disease pathogenesis [Whether CD56ogenesis . Case reogenesis  or IgA nogenesis .+ T cells participate in tubular necrosis or the rejection of transplanted kidneys [+ T cells in the peripheral blood were associated with an unfavorable outcome of renal grafts [One study reported that CD56 kidneys . Anotherl grafts .+ NK cells exert strong cytotoxicity against intrinsic renal cells [We have shown that when stimulated by a combination of IL-2 and IL-12, CD56al cells . As descal cells , these c+ CD56dim cells, CD16− CD56+ cells are the major NK cells in tissues, including the liver, and there is a discrepancy between NK cells in the peripheral blood and those in tissues [− CD56+ cells in peripheral blood are not cytotoxic under normal circumstances; however, when activated by several cytokines, some of them acquire CD16 expression and show up-regulated CD56 expression, and these cells produce large amounts of IFN-γ and exert strong antitumor cytotoxicity against not only MHC class I-negative but also MHC class I-positive  tumor cells [bright NK cells show significantly stronger cytotoxicity than CD56dim NK cells (and T cells) against human umbilical vein endothelial cells [bright NK cells in their urine [bright cells, both with and without CD16 expression, strongly injure renal tubular epithelial cells. In particular, the cytotoxicity of CD16− CD56bright NK cells was significantly higher than that of CD56dim cells and T cells  NK cells other than CD56dim cells [+ cells in peripheral blood, these CD56+ cells in the kidney may cause pathogenic effects. There are significantly more CD56+ cells among urinary MNCs than peripheral blood MNCs in patients with IgA nephropathy, which is a representative type of chronic glomerulonephritis [+ (and CD16+) NK cells have also been suggested to induce hematuria in IgA nephropathy [bright NK cells were also reportedly associated with the degree of fibrosis and loss of renal function and had increased expression of the activation marker CD69 and the activated NK cell receptor NKp46 [bright NK cells, play important roles in the disease progression of renal fibrosis [Although studies analyzing renal NK cells are limited, it has been suggested that normal kidneys as well as kidneys from patients with different forms of renal diseases contain a substantial number of CD56im cells . In contephritis . CD56+ (hropathy . In addihropathy ,76. CD56or NKp46 . These cfibrosis .bright NK cells has not yet been identified in mice, because mouse NK cells do not express CD56, making it difficult to further understand the biology of human CD56bright NK cells, such as their functions, activation mechanisms, and factors affecting their functions. However, activated NK cells in mice also acquire cytotoxic activity against various tumors, including NK-resistant tumors [A counterpart of human CD56t tumors , suggest+ T cells remained unchanged after renal transplantation [Both numbers of NK cells in peripheral blood and their antitumor cytotoxicity are reportedly decreased in renal transplant recipients treated with standard immunosuppressive therapy, although the number of CD56antation . Howeverantation . They prantation .NKG2D expression in NK cells has been reported to be decreased in patients with end-stage renal disease who are receiving dialysis therapy . This deIn recent years, it has become evident that NKT and NK cells play significant roles in various renal diseases, including acute kidney injury and immune-mediated glomerulonephritis, as well as in the field of renal transplantation. In this review, we provided an overview and discussed recent advances in our understanding of the roles of NKT and NK cells in kidney injury, both in mice and humans.+ T cells, a functional counterpart of mouse NKT cells, also cause damage to intrinsic renal cells, and there may be a common pathogenic pathway between mouse and human conditions. Repeated administration of α-GalCer slows the progression of experimental lupus nephritis in BWF1 mice by decreasing the number of NKT cells and suppressing Th2 immune responses in these cells as well as by decreasing B-cell function. However, whether human NKT cells are involved in the pathogenesis of lupus nephritis should be further investigated, because these cells may not induce Th2 immune responses. Mouse NK cells may also be involved in various renal diseases, including AKI. In addition, there may be interesting roles shared between NKT and NK cells, which function to avoid immunosuppressive states; the function of NKT cells is up-regulated in the absence of NK cells, whereas NK cells are under activation when NKT cells are in an anergic state. A subpopulation of human CD56+ NK cells, namely CD56bright cells, cause substantial damage to intrinsic renal cells, and these cells may also be involved in the pathogenesis of fibrotic kidney as well as immune-mediated glomerulonephritis. Data showing the involvement of NKT and NK cells in the injury of transplanted kidneys have been increasing, and future studies in the field of renal transplantation, which focuses on these innate immune cells are anticipated.Activated mouse NKT cells cause AKI with hematuria through damage to renal vascular endothelial cells via the perforin-mediated pathway, and to tubular epithelial cells via the TNF-α/Fas ligand pathway. Human CD56


Pulmonary vascular endothelium is the main metabolic site for Angiotensin I-Converting Enzyme (ACE)-mediated degradation of several biologically-active peptides . Primary lung cancer growth and lung cancer metastases decrease lung vascularity reflected by dramatic decreases in both lung and serum ACE activity. We performed precise ACE phenotyping in tissues from subjects with lung cancer.ACE phenotyping included: 1) ACE immunohistochemistry with specific and well-characterized monoclonal antibodies (mAbs) to ACE; 2) ACE activity measurement with two ACE substrates ; 3) calculation of ACE substrates hydrolysis ratio (ZPHL/HHL ratio); 4) the pattern of mAbs binding to 17 different ACE epitopes to detect changes in ACE conformation induced by tumor growth .ACE immunostaining was dramatically decreased in lung cancer tissues confirmed by a 3-fold decrease in ACE activity. The conformational fingerprint of ACE from tumor lung tissues differed from normal lung (6/17 mAbs) and reflected primarily higher ACE sialylation. The increase in ZPHL/HHL ratio in lung cancer tissues was consistent with greater conformational changes of ACE. Limited analysis of the conformational ACE fingerprint in normal lung tissue and lung cancer tissue form the same patient suggested a remote effect of tumor tissue on ACE conformation and/or on “field cancerization” in a morphologically-normal lung tissues.Local conformation of ACE is significantly altered in tumor lung tissues and may be detected by conformational fingerprinting of human ACE. Pulmonary vascular endothelium is the main site of metabolism of vasoactive peptides -angiotensin I and bradykinin  and likeACE and ACE inhibitors (ACEI) have received considerable attention in oncology as preclinical and clinical data suggested that ACEI may potentiate the effect of certain systemic antitumor therapies –12. The 2+ carboxydipeptidase with two catalytic centers . Conformational fingerprinting of ACE is a very sensitive tool, which can detect even single amino-acid substitution [Сonformational fingerprints of ACE from homogenates of cancer and macroscopically normal lung tissues are presented as tumor/normal tissue ratio in titution , 49–50. all cancer tissues in comparison to ACE from “normal” tissue  and 1B3, 1B8, 3F10 and 2H9 (C domain of ACE) was increased, while binding of mAb 5F1 (N domain) was decreased in There is mounting evidence for the role of abnormal glycosylation of proteins in cancerous transformation of cells and tumor progression . Onе of ACE from tumor tissues from patients #1 and #2 has increased ZPHL/HHL ratio Z/H in . MoreoveWe also compared conformational fingerprinting of ACE in macroscopically normal lung tissues from 5 patients. Expressing binding of 17 mAbs to ACE from these tissues as a percentage of average binding to ACE from unrelated lung tissues , clearlyIn conclusion, we performed ACE phenotyping of lung cancer tissues and found unique kinetic properties and ACE conformation with the conformation of ACE both tissue- and disease-specific. These findings may be clinically relevant especially with the recognition that tissue renin-angiotensin system is a target not only for therapy of cardiovascular disorders , but als


Biotechnology in its broad meaning is the use of organisms or their products for various purposes such as fast diagnosis of infectious diseases, inhibition of the antibiotic resistant bacteria, production of pharmaceuticals, biomaterials, and even energetic materials. Recent challenges in Biotechnology offer state-of-the art studies covering a wide spectrum of biotechnological applications in different organisms for different purposes that serve agriculture, environment, health, and industry. Genomes of both eukaryotes and prokaryotes are recently engineered for advances in food safety and development in all fields.The call for submission of manuscripts allowed researchers in Biotechnology fields to submit their original and novel findings to this special issue. Nineteen manuscripts were submitted. According to precise peer review processes as well as the policies and standards applied in the journal, only seven of them were accepted and they are in the context of this special issue. The full papers in this issue are categorized in four different subjects based on the scope of the Biotechnology applications. Candida Infection” showed the potential therapeutic capability of mannose binding lectin (MBL) against candidiasis as this recombinant MBL induced agglutination of both Candida albicans and C. glabrata. In their extended studies in the paper titled “Mannose-Binding Lectin Gene Polymorphism and Its Association with Susceptibility to Recurrent Vulvovaginal Candidiasis,” the authors studied the MBL gene polymorphism and its association with recurrent vulvovaginal candidiasis (RVVC) and found a close correlation between innate immunity gene mutation and polymorphism in MBL gene and the existence of RVVC. They concluded that MBL genotypic analysis can be used as a surrogate for MBL serum level in order to identify MBL-deficient women for alterative therapeutic options. In the same category, the paper titled “Microcrystalline Cellulose for Delivery of Recombinant Protein-Based Antigen against Erysipelas in Mice” is very interesting as it developed microcrystalline cellulose for delivery of recombinant protein-based antigen against erysipelas in mice. This recombinant surface protein (SPA) from the Gram positive pathogen Erysipelothrix rhusiopathiae was fused to cellulose binding domain from Trichoderma harzianum (CBD). This CBD-SPA fusion cassette was expressed in E. coli successfully.Three papers in this special issue discussed results in the immunotherapeutic field. The paper titled “Mannose-Binding Lectin: A Potential Therapeutic Candidate againstLraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity,” the results obtained found that Lactococcus raffinolactis BG TRK10-1 produces a novel LraI type II restriction endonuclease which is an isoschizomer of EcoR1, and the gene encoding its production was cloned and expressed successfully in the E. coli bacterium. In the other paper of this category titled “Cloning and Expression of the Organophosphate Pesticide-Degrading α-β Hydrolase Gene in Plasmid pMK-07 to Confer Cross-Resistance to Antibiotics,” the authors were able to clone and express the α-β-hydrolase gene in some strains of bacteria belonging to the genus Bacillus; this is a promising result since α-β hydrolase produced by bacteria degrades the organophosphate pesticide pollutants in soil.Molecular cloning and gene expression category include 2 papers. In the paper titled “ Enterococcus lactis 4CP3 Strain and Its Efficient Role on the Growth of Listeria monocytogenes in Raw Beef Meat”; the results obtained showed that the Enterococcus lactis 4CP3 strain could be used as meat protective against listerias growth at refrigeration temperatures. The intraspecific genetic analysis of the 4CP3 strain was assessed by random amplified polymorphic DNA polymerase chain reaction analysis.Food Biotechnology area of research includes one paper only titled “Genetic Analysis with Random Amplified Polymorphic DNA of the Multiple Enterocin-Producing Arnebia benthamii” which showed a novel protocol for the in vitro regeneration of the medicinal plant Arnebia benthamii by tissue culture techniques. It was found that the regenerated plants possessed high content of volatile/nonvolatile compounds that showed DNA protection potential against oxidants.Tissue culture Biotechnology includes one paper titled “Manipulation of Plant Growth Regulators on Phytochemical Constituents and DNA Protection Potential of the Medicinal PlantGamal EnanMohamed E. OsmanMahmoud E. F. Abdel-HaliemSalah E. Abdel-Ghany


Hspd1 and Hspa1a), which indicated that maternal serine deficiency did not affect basal oxidative status in weanling offspring. However, the weanling offspring were found to be vulnerable to oxidative challenges. Furthermore, our results suggested that the dysfunctional antioxidant system in response to oxidative stress in offspring of dams fed with serine-deficient diet was primarily caused by reduced availability of nicotinamide adenine dinucleotide phosphate. Furthermore, impairment of the antioxidant defense system caused by maternal serine deficiency was mediated by the Akt/AMPK/Sirt1 pathway. Our results indicated that maternal serine availability is important for maintaining antioxidant defense against oxidative challenge in weanling offspring.Serine plays an important role in the antioxidant defense system. However, the effects of maternal serine deficiency on the antioxidant ability of weanling offspring have not been reported. In the present study, we investigated the oxidative status of offspring of dams that are maintained on serine-deficient diet and subjected to diquat challenge. Individual pregnant animals were randomly divided into two dietary groups, namely, the control diet group and the serine- and glycine-deficient diet group. Samples were collected from weanling offspring at the age of 3 weeks after diquat challenge. Our results showed that maternal serine deficiency did not affect the levels of antioxidant enzymes and reactive oxygen species, as well as the expression of cellular and mitochondrial stress markers ( Maternal nutrition is strongly associated with offspring health. A suboptimal in utero environment often leads to higher rates of the development of cardiovascular and metabolic diseases in the offspring , which aα subunit (AMPKα) [Serine is part of a metabolic network that links the one-carbon cycle to glycolysis to support oxidant/antioxidant balance and cell proliferation , 4. Seri (AMPKα)  and acti (AMPKα) . Akt is  (AMPKα) . Sirt1 i (AMPKα) . HoweverRecent studies showed that serine is an important component for the synthesis of NADPH, a major cellular reducing agent –11. The C57BL/6J mice (10 weeks old) were purchased from the SLAC Laboratory Animal Central . After 1 week of acclimatization, all animals were fed with control diet and mated and the females were then checked for the presence of postcopulatory plugs. Pregnant animals were randomly divided into two dietary groups, namely, the control (CON) diet group  and the serine- and glycine-deficient (SGD) diet group . The components of the diet are presented in Supplementary Malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) contents in serum and liver were analyzed using commercial kits according to the manufacturer's instructions . Glutathione S-transferase (GST) activity was measured using a commercial kit according to the manufacturer's instructions .μm filters and then analyzed by an ion-exchange amino acid analyzer .After pretreatment according to the previous study , the livMitochondria were isolated from liver samples using a commercial kit . Afterwards, complex I (NADH ubiquinone oxidoreductase) and complex III (ubiquinol cytochrome reductase) activities were assessed spectrophotometrically using commercial kits according to the manufacturer's instructions (Solarbio).μm) were stained with 1 μM dihydroethidium  for 20 min at 37°C. Images were obtained and analyzed using Image Browser software .Reactive oxygen species (ROS) content was analyzed as previously described . Briefly+/NADPH Quantification kit .Lipid hydroperoxide (LPO) content in the liver sample was determined spectrophotometrically . Nicotinamide adenine dinucleotide phosphate (NADPH) level was measured using a NADPμL assay volumes containing 5 μL of SYBR Green mix (Takara), 0.2 μL of ROX, 3 μL of diethylpyrocarbonate-treated deionized H2O, 1 μL of cDNA template, and 0.4 μL each of the forward and reverse primers [Liver sample was ground, and total RNA was isolated using the TRIzol reagent . The PrimeScript RT reagent kit  was used for reverse transcription of total RNA. RT-qPCR was performed in 10  primers . The priμg of protein per lane was separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with primary antibodies against AMPKα, phosphorylated AMPKα, nuclear factor-like 2 (nrf2), Sirt1, Akt, phosphorylated Akt , and β-actin  overnight at 4°C. Then, membranes were incubated overnight with the secondary antibodies and subsequently exposed to EZ-ECL .Western blotting analysis was performed for the determination of relative protein levels of Akt, Sirt1, AMPK, and nrf2. Briefly, 20 P < 0.05 was considered statistically significant.Statistical analysis was performed by factorial ANOVA using a mixed procedure (PROCMIXED) in SAS software version 9.2 . The effects of diet (control or SGD diet), challenge , and their interactions were incorporated into the statistical model. Data were presented as least square means plus pooled SEM. Our results showed that maternal serine deficiency did not affect SOD, GSH, CAT, and MDA levels in both serum  and liveAs shown in As shown in As shown in Hspd1 and Hspa1a in weanling offspring. However, under diquat challenge, offspring of dams fed with the serine-deficient diet showed significantly higher LPO levels and expression of Hspd1 and Hspa1a and significantly lower NADPH/NADP+ levels when compared with the offspring of dams fed with the basal diet.As shown in As shown in Intrauterine malnutrition-induced stress during gestation is a major cause of diseases, such as diabetes, obesity, and insulin resistance in adult life . OffspriSerine is a nonessential amino acid that has been proven to be functionally essential because of its critical roles in the maintenance of cellular antioxidant status, cell survival, and cell proliferation. In the present study, our findings showed that the antioxidant abilities and oxidant indices of weanling offspring were not influenced by maternal serine deficiency, although serine-deficient diet caused significant decrease in serine and glycine content in both serum and liver in dams and their offspring. These results suggested that decreased serine and glycine content does not affect the activity and expression of antioxidant enzymes as well as GSH content in mice under normal condition. However, under diquat challenge, offspring of the dams fed with serine-deficient diet showed a significant downregulation of GSH, CAT, and SOD levels in both serum and the liver samples when compared with the offspring of dams fed with the basal diet. Further analysis confirmed the reduced antioxidant capacity in offspring of dams fed with the serine-deficient diet based on the observed downregulation of the expression of genes encoding the antioxidant enzymes. Additionally, GST activity exerts critical effects in cellular protection against oxidative damage and exogenous toxic chemicals including diquat. It detoxifies oxidized lipid and DNA by catalyzing the conjugation of GSH with electrophiles that are products of oxidative stress , 24. As Hspd1  and Hspa1a (a cellular oxidative stress marker) were observed in the livers of offspring of dams fed with serine-deficient diet. The above results further confirmed that the livers of offspring of dams fed with serine-deficient diet were subjected to high oxidative stress. The glutathione and thioredoxin antioxidant systems consume NADPH as a cofactor for neutralizing ROS. Therefore, NADPH availability is a critical factor that affects the strength of the cellular response to oxidative stress [Diquat challenge triggered the reaction between superoxide anion radicals with oxygen molecules, leading to ROS production . High ROe stress . Low NADe stress , 10, 27,The mitochondrial complexes I and III are generally considered to be the primary sites of ROS production . ParticuOxidative stress leads to inactivation of AMPK and Sirt1 pathways but results in the activation of the PI3K/Akt pathway. Oxidative challenge caused a significant increase in phosphorylated Akt levels but a significantly decrease in Sirt1 and phosphorylated AMPK levels in the offspring of dams fed with the serine-deficient diet, thereby suggesting that the impaired antioxidant defense system caused by maternal serine deficiency was mediated by a dysfunctional Akt/AMPK/Sirt1 pathway. Moreover, the downregulated expression of nuclear nrf2 further suggested that the Akt/AMPK/Sirt1 pathway plays a critical role in oxidative response by modulating its downstream target nrf2.Our results showed that maternal serine deficiency did not affect basal oxidative status in weanling offspring. However, the weanling offspring were more vulnerable to oxidative challenges. Further, our results suggested that the dysfunctional antioxidant systems in offspring of dams fed with the serine-deficient diet were caused by lower NADPH availability and the dysfunctional Akt/AMPK/Sirt1 pathway potentially mediates the effects of maternal serine deficiency on offspring.


Cancer is a heterogeneous disease with many genetic variations. Lines of evidence have shown copy number variations (CNVs) of certain genes are involved in development and progression of many cancers through the alterations of their gene expression levels on individual or several cancer types. However, it is not quite clear whether the correlation will be a general phenomenon across multiple cancer types.In this study we applied a bioinformatics approach integrating CNV and differential gene expression mathematically across 1025 cell lines and 9159 patient samples to detect their potential relationship.Our results showed there is a close correlation between CNV and differential gene expression and the copy number displayed a positive linear influence on gene expression for the majority of genes, indicating that genetic variation generated a direct effect on gene transcriptional level. Another independent dataset is utilized to revalidate the relationship between copy number and expression level. Further analysis show genes with general positive linear influence on gene expression are clustered in certain disease-related pathways, which suggests the involvement of CNV in pathophysiology of diseases.This study shows the close correlation between CNV and differential gene expression revealing the qualitative relationship between genetic variation and its downstream effect, especially for oncogenes and tumor suppressor genes. It is of a critical importance to elucidate the relationship between copy number variation and gene expression for prevention, diagnosis and treatment of cancer. Genetic structural variation in the human genome can be present in many forms, ranging from single nucleotide polymorphisms (SNPs) to large chromosome aberrance . In the It is generally accepted that somatic CNV is highly associated with the development and progression of numerous cancers by impacting gene expression level –19. SamuDespite a number of studies have explored CNV and differential gene expression of several classical oncogenes or tumor suppressor genes in different cancers , 18, 20,http://www.cbioportal.org/). The cell lines datasets of CCLE and NCI-60 contained 966 and 59 cell lines respectively and the TCGA datasets contained 31 types of cancer of 9159 samples  containing 1020 cell lines. The copy number was obtained from Affymetrix SNP6.0 array data with PICNIC [The copy number and mRNA expression data of Broad-Novartis Cancer Cell Line Encyclopedia (CCLE), NCI-60 and the Cancer Genome Atlas (TCGA) were collected from the cBioportal for Cancer Genomics . For CCLP, copy number amplification was obtained by the following criteria:  OR . While the criteria for copy number deletion was:  OR ). Gene expression levels were quantified by RSEM from RNA-Seq data and mRNA Z scores were computed using the tumors samples that are diploid for the corresponding gene. For each gene, Z score = (x-u)/o, where u, o represent the average expression and standard deviation of this gene across samples, respectively; x represents the specific expression of this gene in a specific sample. Differentially expressed genes (DEGs) were further filtered out as Z scores more than 2 (upregulated genes) or less than − 2 (downregulated genes). Thus, the four variation tendencies were defined as follows: amplified AND upregulated; amplified AND downregulated; deleted AND upregulated; deleted AND downregulated.copy number amplification and expression level upregulation ratio against copy number deletion and expression level downregulation, copy number deletion and expression level downregulation ratio against copy number amplification and expression level upregulation more than 50 % on each gene across 9159 tumor samples were applied to identify AUGs and DDGs with a higher ρ (> 0.4) and a higher number (> 146.5) of copy number amplification and expression level upregulation than the median level of 30 most popular oncogenes  was used to construct the PPI network of FYTTD1. The line thickness indicates the strength of data support from the sources of text mining and experiments with a cutoff value of medium confidence (0.4). Then the functional enrichment results of KEGG pathways and Gene Ontology (GO) biological process were applied to the PPI network with false discovery rate less than 0.05.Search Tool for the Retrieval of Interacting Genes .R (version 3.4.2) including R packages of r = 0.93, p < 0.0001; Fig. r = 0.97; Fig. It is known that CNV is related to alteration on gene expression. However, the correlations between CNV and gene expression change on a global cellular scale remain to be elucidated. To comprehensively exploit this question, we collected available genomic datasets from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE), NCI-60, and the Cancer Genome Atlas (TCGA). Integrated analysis of CNV and differential gene expression was performed across 31 cancer types and 2 cancer cell lines resources  and expression level (upregulation and downregulation), we divided variant trend into four classes: copy number amplification and expression level upregulation, copy number amplification and expression level downregulation, copy number deletion and expression level downregulation, copy number deletion and expression level upregulation (see Methods). We analyzed the frequencies of four variant trends across 24 primary sites of cell lines in CCLE database and 31 cancer types in TCGA. Intriguingly, the amount of copy number amplification and expression level upregulation, copy number deletion and expression level downregulation, representing our so-called highly concordant genes on CNV and differential gene expression, occupied 15 and 20% of the total variant copy number count on average for cell lines and tumor samples, respectively. Nevertheless, the mean proportion of copy number amplification and expression level downregulation, copy number deletion and expression level upregulation count only took up 0.7 and 0.5% for both cell lines and tumor samples -anchor biosynthesis Fig. a, which In addition, we found 9 highly concordant genes with a strong relation to patient overall survival including 5 AUGs and 1 DDG Table . For exar = 0.89, p = 9.089e-6; Additional file To validate our previous findings, the copy number and gene expression profiles were integrated across 1020 cell lines from CCLP, an independent dataset. Although CCLP utilized a different algorithm to calculate copy number variation, it still showed a positive correlation between copy number and expression level Fig. a by usinp values. Besides, not only in cell lines but in patients copy number amplification strikingly harbored a higher expressed level compared to copy number deletion. Secondly, we investigated every gene over the relationship of CNV and differential gene expression across 9139 tumor samples and 1025 cell lines respectively. Our results showed the majority of genes the copy number displayed a positive linear influence on gene expression, indicating that genetic variation generated a direct effect on gene transcriptional level. In addition, we validated 10 genes with a significant correlation between CNV and differential gene expression through literature  to revalidate the relationship between CNV and differential gene expression. Although CCLP applied a different algorithm to calculate copy number variation, it also showed a positive correlation between copy number and expression level Fig. a. Our reIn conclusion, this study demonstrated the close correlation between CNV and differential gene expression. Moreover, this trend is consistent across cell lines and patient samples. For the majority of genes, copy number shows a positive linear influence on gene expression , while copy number amplification barely causes gene expression downregulation and the copy number deletion hardly promotes gene expression upregulation. Furthermore, both AUGs and DDGs are remarkably enriched in the ubiquitin-proteasome system. In addition, we identified amplification and overexpression of FYTTD1 is highly related with poor prognosis in ESCA, which may be a potential prognostic marker in ESCA. Whereas, more in-depth studies are needed to further reveal molecular mechanisms between CNV and differential gene expression. Overall, it is of a critical importance to elucidate the relationship between copy number variation and gene expression for prevention, diagnosis and treatment of cancer.Additional file 1: Figure S1. Integrative analysis of the association between differential gene expression and CNV across multiple cancer types. Figure S2. Integrative analysis of the association between differential gene expression and CNV across multiple cancer types. Figure S3. Most genes’ expression changes significantly correlated with their CNVs. A. Genes were sorted according to ρ on Z score versus copy number across 1025 cell lines, green representing a positive ρ and red meaning a negative one, representative oncogenes and tumor suppressor genes next to the corresponding points. B. An example of gene with copy number correlated with expression level in cell lines dataset. C. Histograms present the distribution of r for each genes based on the linear fitting results of median Z score with each variable copy number in cell lines datasets. D. Scatter plot shows the distribution of genes based on r of fitting (x-axis) and ρ (y-axis) for cell lines dataset (left) and TCGA dataset (right). E. An example of gene with copy number uncorrelated with expression level in cell lines dataset. Figure S4. The amount proportion of copy number amplification and expression level downregulation, copy number deletion and expression level upregulation versus the total variant copy number count across each primary site of cell lines in CCLE (A) and each cancer types of TCGA (B). CNS: central nervous system; HLT: haematopoietic and lymphoid tissue; UAT: upper aerodigestive tract. Figure S5. The linear regression fitting of median Z scores versus corresponding copy number among 1020 cell lines of CCLP. Figure S6. Integrated analysis of CNV and differential gene expression of FYTTD1 in ESCA patients.Additional file 2: Table S1. Datasets of CCLE, NCI-60 and 31 cancers in TCGA and the number of samples used in this study. Table S2. KEGG pathways enrichment of top 1000 genes with r of fitting from the lowest to the highest for TCGA datasets. Table S3. KEGG pathways enrichment of top 1000 genes with r of fitting from the lowest to the highest for cell lines datasets combining CCLE and NCI60. Table S4. Lists of 30 most popular oncogenes with ρ and the count of copy number amplification and expression level upregulation across 9159 tumor samples. Table S5. Lists of 10 most popular tumor suppressor genes with ρ and the count of copy number deletion and expression level downregulation across 9159 tumor samples. Table S6. Identified 560 AUGs and 365 DDGs. Table S7. The distribution of AUGs and DDGs across 22 chromosomes. Table S8. High relation of 9 concordant genes with the development and progression of numerous cancers. Table S9. Correlation analysis of genes with a significant correlation between CNV and differential protein expression in literature. Table S10. Variation tendency validation of genes with a significant correlation between CNV and differential protein expression in literature. Table S11. Stable expression level over various copy number of small nucleolar RNAs in cell line dataset. Table S12. Stable expression level over various copy number of small nucleolar RNAs in T dataset.


Genome-wide association studies have identified a plethora of risk genes for both Crohn’s disease (CD) and ankylosing spondylitis (AS). A subset of genes found to be risk factors for CD have also been found to be risk factors for AS. The objective of our study was to assess whether CD risk genes were associated with non-invasive clinical markers of gut inflammation in patients with AS, indicating a potential subset of patients with clinical as well as genetic overlap.A total of 308 Caucasian patients who fulfilled the modified New York Criteria for AS, were assessed for bowel symptoms using the Dudley Inflammatory Bowel Symptom Questionnaire (DISQ). Of these patients, 157 also had faecal calprotectin measured. All AS patients and 568 healthy controls were genotyped for 10 CD risk loci using predesigned single nucleotide polymorphism (SNP) genotyping assays. Chi-square analysis was used to test for association between genotype and DISQ score and faecal calprotectin level.IL23R (rs11209026) conferred protection against AS. Only the association of 1q32 remained significant after Bonferroni correction for multiple testing. Stratification by DISQ score and faecal calprotectin did not influence the association of 1q32 with AS.The minor allele of two SNPs, one in chromosome region 1q32 SNP (rs11584383), and one in the gene coding for In patients with AS, the association of the CD 1q32 SNP was independent of non-invasive markers of bowel inflammation. Other CD related SNPs were not found have a significant association with AS. In recent years a new paradigm has developed around ankylosing spondylitis (AS) in which AS is considered part of a spectrum of spondyloarthropathies that also includes Crohn’s disease (CD) . These tThe Icelandic genealogy database has demonstrated the coinheritance of CD and AS . Not onlNon-invasive markers of bowel inflammation have been piloted in several observational studies in AS. The Dudley Inflammatory Bowel Symptom Questionnaire (DISQ)  has beenWe hypothesized that shared risk loci may influence the clinical spectrum of bowel inflammation in AS, and hence may associate with an excess of adverse bowel symptoms in patients who have AS but no evidence of overt CD. In order to investigate this hypothesis, the objective of our study was to assess whether the eight CD risk loci most strongly associated with AS were also associated with non-invasive indicators of bowel inflammation in patients with AS.A total of 308 Caucasian patients, who fulfilled the modified New York criteria for AS and who had no history of inflammatory bowel disease (IBD), were recruited from centres in New Zealand and Australia. A total of 568 New Zealand Caucasian healthy individuals aged over 17 years, with no personal or family history of spondyloarthritis or IBD, were used as controls . Informen = 157) using ELISA (Reference range: <50 mg/kg) .Bowel symptoms were evaluated quantitatively in all patients with AS using the spondyloarthritis modification of the DISQ, a validated outcome measure comprising 15 questions assessing bowel symptoms in axial spondyloarthritis . Each quJAK2 (rs10758669), CDKAL1 (rs6908425), IL12B (rs100454310), ZPBP2 (rs2872507), MUC19/LRRK2 (rs11175593), STAT3 (rs744166), IL23R . Genotyping was performed with pre-designed TaqMan® single nucleotide polymorphism (SNP) genotyping assays as per the manufacturer’s instructions using a Roche LightCycler 480 Real-time PCR system. End-point genotyping analysis software was used to assign genotypes to each study participant. A total of 10% of samples were re-genotyped to determine the accuracy of each TaqMan® assay. All assays were found to exhibit 100% agreement between original and repeat genotype calls.Guanidium isothiocyanate extraction was used to collect DNA from all study participants. Each participant was then genotyped for the confirmed CD risk loci , 1q32 , p = 0.0006, OR = 0.68, 95% CI [0.53–0.84]), and IL23R SNP rs11209026  both conferred protection against AS (p = 0.006). With stratification by DISQ score, the association of 1q32 SNP rs11584383 remained significant in both AS patients who had minimal bowel symptoms (p = 0.004) and those with clinically significant bowel symptoms (p = 0.02)  . Whilst  = 0.02) , compariJAK2 (rs10758669) and STAT3 (rs744166) are associated with mucosal immuno-regulation and more widely in immune-mediated disease  is itself functional, and is involved in the regulation of one or more genes. Data from GWAS have shown disease associated intergenic SNPs are often located in or near regulatory elements, suggesting that these variants may to disrupt or alter the function of key regulatory regions of specific genes (1). To determine whether this is the case for the rs115843383 would be challenging (1). However, a number of recently developed bioinformatic techniques, such as Chromosome Conformation Capture (2), Hi-C (3), and chiA-PET (4); which identify long-range chromatin interactions may help to determine whether 1q32 SNP (rs11584383) indeed has a role in the regulation of one or more genes.There is a wide body of evidence supporting the concept that defective gut mucosal immunity is a factor in initiating and perpetuating spondyloarthritis. The specific combination of genetic variants seems likely to determine whether an individual develops a more spondyloarthritis or IBD phenotype . In our Our study has several strengths. The study population met strict classification criteria for AS, came from a predominant Northern European genetic population and was carefully phenotyped. In particular, this is the first study to correlate new non-invasive markers of gut inflammation with genetic risk loci, enabling us to look at a much larger population than invasive endoscopic studies would allow.Potential weaknesses of our study include the small sample size which may have meant that modest associations were missed. However, the stringent phenotyping of our study participants and our decision to only look at loci which had previously been demonstrated to be strongly associated with AS in ethnically comparable populations likely reduced the impact of our small sample size. We chose not to use the DISQ as a continuous variable and this may have reduced our ability to detect a significant correlation with specific loci. We used categorical values to define clinical case definitions of significant bowel symptoms, based on the original validation of the DISQ in AS. Using DISQ scores rather than case defined grouping would be to assign undue significance to subjective self-reported questionnaire values, when compared with categorical objective genetic testing. Such case definitions based on questionnaire values are routinely used in the study of other human diseases, such as depression. Particularly when these are correlated with genetic testing .HLA-B27 alone, an allele which is not associated with IBD risk (We did not stratify for ethnicity, although our population was overwhelmingly of Northern European genetic lineage. More recent GWAS in AS have identified further shared genes with SNPs having the same direction of effect in IBD and AS . Only arIBD risk . TherefoTo our knowledge, this study is the first to stratify confirmed CD risk genotypes with presence of non-invasive makers of bowel inflammation in AS patients, in order to determine whether the association of these genotypes with AS is primarily due to presence of bowel inflammation. Using this approach, we provide preliminary evidence that the association of 1q32 SNP rs11584383 with AS is independent of bowel inflammation as assessed by non-invasive markers. To strengthen and confirm this preliminary finding, further studies which incorporate histology or colonoscopy are needed.10.7717/peerj.5088/supp-1Supplemental Information 1Click here for additional data file.


Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension. First identified in urine, hepcidin is predominantly produced by hepatocytes2 and when released into the circulation is able to interact with the membrane active cellular iron exporter ferroportin, causing it to be endocytosed, thereby preventing iron exit and encouraging cellular iron retention3. Together hepcidin and ferroportin currently represent the only known regulators of cellular iron export. Ferroportin is chiefly expressed in cells linked to iron uptake (from the diet) and homeostasis; examples include duodenal enterocytes, macrophages and hepatocytes.Hepcidin is a small (25 amino acid) peptide hormone largely responsible for regulation of body iron homeostasis4. Importantly, infection and inflammation also regulate the synthesis of hepcidin, a response most notably linked to IL-6 activation of the JAK/STAT pathway5. Resulting hypoferremia, described as the anaemia of inflammation, helps limit microbial virulence (reviewed in6). Consequences related to increased iron storage are likely to include deficient erythropoiesis and perturbation of cellular function related to excess iron accumulation8. In this regard, iron is also an essential requirement for cell proliferation; when available in excess, a proliferative state is encouraged9. Current perceptions suggest that most cell types express little or no ferroportin as iron is utilised for metabolic requirements alone and therefore there is no need to export this resource. However, new studies are emerging which indicate expression and or regulation of ferroportin and hepcidin linked to iron retention in cells of various cancer categories12 with iron retention being linked to cell survival and proliferation, thus suggesting the importance of this axis in proliferative disease.Hepcidin expression is regulated by plasma iron levels and stores; this transcriptional control is facilitated by the bone morphogenetic protein receptor (BMPR) coupled SMAD signalling pathway13 and hepcidin excess demonstrated8. It is characterised by regionalised hyperplasia of smooth muscle and endothelial cell components of resistance, pre-capillary pulmonary arterioles. Known as a rare disease, PAH is classified into idiopathic, heritable or forms resulting in association with specific conditions, such as connective tissue or congenital heart disease14. Genetic mutations, and in particular those related to BMPR II underscore most heritable cases and a significant proportion of sporadic cases of idiopathic PAH15. Inflammation may be the common link between dysfunctional BMP signalling and loss of iron; importantly plasma IL-6 is raised in patients with PAH16. Intriguingly, increased autophagy mediated by lysosomal action (where BMPR-II and ferroportin are both degraded) has been implicated in PAH17, indicating a link with altered iron handling.Pulmonary artery hypertension (PAH) is a disease process in which abnormal iron homeostasis has also been implicated18, without the classical haemolytic disease phenotype, may suggest additional avenues for iron acquisition by pulmonary vascular cells.As for the source of iron for cellular uptake, this would be most likely be provided via transferrin receptor-1 mediated mechanisms. However, the increasing recognition that free haemoglobin is associated with PAHThe present study was therefore undertaken to evaluate whether there is any role for the hepcidin/ferroportin axis in proliferative responses of pulmonary artery smooth muscle cells. The aims of the study were threefold. Firstly, to describe for the first time the presence of the iron export protein, ferroportin in these cells. Secondly, to modulate ferroportin expression/activity in these cells to evaluate any subsequent influence on proliferative responses. Thirdly, to assess any role for free haemoglobin in proliferation and potential mechanisms for cellular uptake. These studies may provide insight into the potential role of disrupted iron homeostasis in vascular remodelling, such as observed in PAH.Ferroportin mRNA and protein expression is demonstrated in human pulmonary artery smooth muscle cells (hPASMCs) Fig. . Basal f5 and, therefore, iron homeostasis. IL-6 treatment of hPASMCs clearly demonstrated that both mRNA and protein secretion of hepcidin was significantly up regulated by 2 hours and 24 hours respectively  resulted in a significant increase in the proliferation of hPASMCs  there was no measureable increase in iron in cells treated with either hepcidin or IL-6 for 24 hours  in the proliferation of these cells Fig. . Basal m21. In addition, under these circumstances hPASMC proliferate more, which suggests that iron retention encourages a proliferative state. In support of this notion, pre-treatment of hPASMCs with the therapeutic monoclonal antibody LY2928057 inhibited this hepcidin driven proliferative response and importantly also prevented cellular iron accumulation.This study has established for the first time the presence of the cellular iron exporter ferroportin in hPAMSCs and an operational role for this protein in the control of iron content in these cells; being linked to interaction with the iron regulatory peptide hepcidin. When elevated levels of hepcidin are present, interaction with ferroportin ensues resulting in internalisation of the complex and degradation, as confocal imagery and ELISA based studies indicate. Moreover this set of circumstances enhances iron retention, a finding that is further supported by the observed decline in cellular iron regulatory protein 2 (IREB2) levels; IREB2 levels are regulated by cellular iron content and decrease in the presence of iron22. These novel observations are intriguing and potentially indicate transcriptional control of, and a novel source of, hepcidin synthesis in hPASMCs which may be further linked to an autocrine aspect of cellular iron regulation/cellular proliferation in this cell type. It is likely that these IL-6 mediated responses are signalled via the canonical Jak/stat3 pathway; however, given the novelty of these observations a fresh examination of this and other potential signalling pathways seems warranted.Additional studies have demonstrated similar responses in hPASMCs to those reported above, in response to IL-6 treatments. Importantly IL-6 was also shown to increase hepcidin mRNA levels and hepcidin release from hPASMCs eliciting the possibility of an autocrine hepcidin response in these cells; although further studies will be required to confirm any functionality related to this observation. However, such a role for hepcidin has been suggested previously in cardiomyocytes23, supporting the above concept. These findings are also intriguing and potentially of relevance to pulmonary arterial hypertension not least because of the excessive and unrestricted PASMC proliferation and pulmonary vascular remodelling characteristic of this condition. Additionally, there is emerging evidence indicating disrupted iron homeostasis13, elevated blood levels of hepcidin8, IL-616 and associations with genetic abnormality (BMPRII) in heritable and sporadic PAH and regulation of hepcidin expression may be of mechanistic relevance in PAH.The hepcidin/ferroportin axis is a well-established regulator of cellular iron content and the role of IL-6 in cellular iron regulation is also well established; however, the operation of these regulatory mechanisms has not previously been reported in hPASMCs. Moreover, the relationship between cellular iron accumulation, these axes and proliferation has also not been described previously for this cell type. Interestingly, disordered hepcidin/ferroportin activity have been shown to promote growth of breast cancer cells, with elevated ferroportin expression responsible for inhibition of proliferation; importantly IL-6 was also shown to promote proliferation of these cells24 and is thought by some to be driven by nitric oxide scavenging and consequent vaso-restriction25. In other forms of PAH any association with free haemoglobin is less well established, but emerging studies in patients with iPAH and heritable PAH have demonstrated enhanced levels of zinc - protoporphyrin26 and elevated creatine27 as indicators of subclinical haemolysis in these patient groups. Moreover, in another study, plasma free haemoglobin, was independently associated with increased risk for admission to hospital in PAH groups28. Furthermore, decreased levels of haptoglobin are seen in PAH phenotypes suggesting increased haemoglobin binding and removal in these patients29 which is likely related to enhanced or on-going haemolysis. Studies presented herein suggest that free haemoglobin is also a pro-proliferative agent for hPASMCs and that the first description of the presence of CD163 on these cells suggests a means for cellular uptake of this iron containing protein complex. Whilst not directly associated with the activity of the hepcidin/ferroportin axis it is noteworthy that in addition to haemoglobin itself, IL-6 a known regulator of hepcidin, also seems to modulate the expression of CD163 in these cells, which may suggest some complementary activity linked to iron uptake and retention. To give some context to these preliminary studies, in a recently published study using an in vivo model of vascular remodelling driven by cellular iron accumulation the process was reversed with haptoglobin therapy30 suggesting a potential role proliferative role for free haemoglobin in this model and supporting our findings.A role for decompartmentalised haemoglobin in PAH is established for haemolytic diseases processes31; thirdly, iron was found to induce remodelling in cultured rat pulmonary artery endothelial cells32; and fourthly, plumbagin, a known iron chelator33 was found to limit proliferation in hPASMCs and to decrease distal PA remodelling in a rat model of PAH34. Conversely, monocrotaline treated rats fed an iron restricted diet were less prone to pulmonary vascular remodelling and right ventricular failure; interestingly, hepcidin levels decreased in these animals compared to those on a normal iron containing diet, suggesting that cellular iron retention is less likely in these circumstances35. There is evidence from clinical studies that suggest iron supplementation can prove of benefit to certain PAH patient groups37; all of which may seem to be at variance with our findings. However, considerations based on short-term improvement in patient wellbeing may need to be balanced against potential longer terms effects related to iron accumulation. Nevertheless, what is emerging from these various study counterpoints is that iron homeostatic control is important at the level of the pulmonary vasculature and that insufficient or excessive iron availability may contribute to vascular dysfunction; for a recent review see38.Taken as a whole our observations are further supported by several recent studies that have linked vascular remodelling with iron availability: firstly, administration of the iron chelator, desferrioxamine to rats inhibited chronic hypoxia induced pulmonary hypertension and remodelling; secondly, the same authors showed that the iron chelators, hinokitiol and HBED inhibited proliferation of cultured pulmonary artery smooth muscle cells39; importantly mitochondrial respiratory rate and hydrogen peroxide production and release into the cytosol are transcriptional signals for an array of processes including for cell cycle progression and proliferation, reviewed in40. In general terms a depressed mitochondrial respiratory rate favours proliferation. Iron loading of mitochondria will affect respiration directly thereby potentially promoting a pro-proliferative state. Moreover, increased levels cytosolic ferrous iron (Fe2+) will catalytically remove hydrogen peroxide thereby influencing redox signalling pathways which again may promote proliferation. In addition, iron loading of mitochondria may well favour switching to mitochondrial glycolytic pathway predominance, in common with cancer phenotypes and which has also been implicated in PAH, reviewed in41. The transcription factor Hypoxia inducible factor (hif) and its role in cell proliferation have featured in much recent PAH related research. Iron and oxygen are known repressors of Hif assembly, whereas hypoxia or iron deficits allow for assembly, migration to the nucleus and gene transcription42. Given that the current study was undertaken in normoxia and in cells with increased iron loading one could speculate that Hif was unlikely to be involved, however this may be somewhat of an oversimplification as iron has numerous effects in cells beyond inhibition of hif activity. Indeed, there are numerous reports of normoxic hif activity and iron related effects on cellular redox status including of mitochondria may well favour hif activation41, so a role for hif cannot be excluded. The iron regulatory proteins direct cellular iron homeostatic control43 with IRP-2 assuming the dominant role in these processes. Recent studies have implicated sustained IRP-2 activity in cancer cell proliferation45. For the current study measurement of IRP-2 was used as a surrogate for cellular iron. Levels did decline in response to hepcidin intervention and associated iron retention, but it is noteworthy that this experimental model was unable to abolish IRP-2 completely which may be of functional relevance given the emerging role for this protein complex in proliferation. Clearly cellular iron fluxes can affect many intracellular responses and interactions including those described above, establishing greater mechanistic insight into these processes seems appropriate.We have not investigated in detail potential mechanisms related to iron accumulation that may lead to proliferative responses in hPASMCs. However, it is plausible to suggest candidates given current understanding of cellular iron metabolism. Mitochondria are centres for iron uptake and utilisation, and are subject to functional perturbation linked to iron deficits or overloadin vivo; and fourthly, we have not examined the effects of hypoxia on these responses, hypoxia being of relevance to PAH. Finally, given the novelty of our findings no evaluation of ferroportin expression or otherwise has to our knowledge as yet been undertaken in subjects with or samples from patients with PAH nor indeed has any evaluation of CD163.Our studies may offer some explanation as to the aetiology of PASMC proliferation that may be of relevance to PAH onset and progression and, potentially a therapeutic option via intervention to target the hepcidin/ferroportin axis, or via IL-6 antagonists. However, further studies will be required to investigate these processes in greater depth. These will include the use of ferroportin over-expression or knock-down protocols to mimic the actions of LY2928057 or hepcidin and thereby strengthen the findings reported herein. Moreover there are several other limitations to our study: firstly, as mentioned above detailed mechanistic studies are required; secondly, we have conducted the experiments with hPASMCs from normal donors (not PAH patients); thirdly, we have not as yet investigated these proliferative processes In spite of these shortfalls, we would suggest that these studies offer avenues for further research into the mechanisms of PASMC proliferation in PAH, and potentially in other wider aspects of vascular dysfunction, where disrupted iron homeostasis linked to the hepcidin/ferroportin axis that is responsible for either iron accumulation or iron deficits may be of importance.Human pulmonary smooth muscle cells (hPASMCs) were either isolated from samples of pulmonary artery  from patients undergoing lung lobectomy at the Royal Brompton Hospital and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 15% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. In order to select only hPASMCs for study from cells isolated from pulmonary arteries. hPASMCs were selected in high serum DMEM over a very long period of time thereby excluding other cell types and characterised by morphological examination; for examples see Supplementary data Fig. 5 cells per well. Cells were incubated with either media alone or 1 µg/mL hepcidin or 10 ng/mL IL-6 for 2.5 h for RNA extraction or 24 h or 48 h for protein extractions. For some experiments , serum restricted media  was employed.A 90% confluent hPASMC monolayer was trypsinised using detach kit (Promocell) as per manufacturer’s instructions. Briefly, the monolayer was swiftly washed with HEPES, treated with 0.025% Trypsin/0.01 EDTA solution to detach the cells followed by quenching with TNS . For culturing and passaging, the cells were plated at 7500 per sq.cm. Only cells from passage 4–7 were used in the experiments. For experiments, the cells were seeded on 6 well plates at 1.5 × 10Total RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. RNA purity and concentration were measured by Nano-drop spectrophotometer. 0.6–1 μg of total RNA was used for reverse transcription to produce cDNA using oligo-dT primers (Invitrogen), 10 mM dNTP mix, RNasin plus (Promega) and M-MLV Reverse transcriptase enzyme (Invitrogen) according to manufacturer recommendations. Reverse transcriptase enzyme was omitted in negative controls. Real-time PCR using SYBR green was carried-out on a Rotor-Gene 6000 PCR machine using ferroportin and hepcidin (Qiagen) primers while β-actin was used as standard housekeeping gene. Both −RT and no template negative controls were employed. The change in expression was normalised to control untreated samples.2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 mM PMSF was used to lyse the cells. Cells were scraped from the culture plates and centrifuged to remove debris. Total protein in cell lysates was determined by Bradford protein assay (Bio-Rad).Cell lysis buffer (New England Biolabs) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM NaFerroportin (BlueGene Biotech) and IREB2 (Aviva) from cell lysates and hepcidin (R&D Systems) from cell culture supernatants were quantified by ELISA according to the respective detailed manufacturer’s instructions. Ferroportin and IREB2 levels were normalised to total protein in the cell lysates.46. After blocking with 5% milk, membranes were probed with rabbit anti-ferroportin antibodies (1:1000). Human β-actin (New England Biolabs) was used as a loading control (1:2000). Further horseradish peroxidase-conjugated anti-rabbit IgG (New England Biolabs) was used as a secondary antibody (1:2000).Proteins were resolved on 4–12% gradient SDS-PAGE Tris gels (BioRad) and transferred to nitrocellulose membranes as described previouslyhPASMCs were seeded on 8 well chamber slides at 10,000 cells per well with 15% DMEM. After initial adherence (20–22 h), the cells were treated with 0.2% DMEM alone or 1 µg/mL hepcidin or 10 ng/mL IL-6 (both made in 0.2% DMEM) for 48 hours followed by immuno-staining. Briefly, the cells were fixed using 100% freezing cold methanol for 10 minutes, followed by permeabilisation (0.2% Triton X-100 in PBS) for 5 minutes. The slides were then blocked with 1% BSA in PBS for an hour followed by incubation with rabbit anti-ferroportin or anti CD163 antibody (1:250) for another hour. A negative control without any primary antibody was also employed. Following four washes with 0.1% Tween-20 in PBS, the slides were incubated with 1:500 Alexa Flour 568 Goat anti-rabbit IgG for a further hour in dark. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope at a centralised facility at Imperial College London. For quantification of CD163 confocal images ImageJ was used. Cellular region of interest was determined by DAPI staining +25%. Integrated density was collected for CD163, minus background and normalised to DAPI. This was then expressed as fold change relative to control.The colorimetric BrdU cell proliferation assay kit (Roche) was used to measure cell proliferation. hPASMCs were plated on to 96 well plates (2500 cells/well). After adherence, the cells were serum starved (0.2% Serum) for 20–24 h prior to pre-treatment with either medium alone or 1 µg/mL hepcidin or 10 ng/mL IL-6 for 24 h. For treatments with 10 µM haemoglobin, the cells were replaced with fresh media after 2.5 h of Hb treatment. Following on, the cells were pre-incubated with LY2928057 (or mock-incubated) for 1.5 h prior to repeat treatments with either hepcidin or IL-6 for further 24 h. At this point, BrdU was introduced (at the manufacturer recommended concentration) for an additional 24 h before harvesting the plates. Proliferation was quantified using anti-BrdU-POD antibody, following the manufacturer’s instructions. All treatments were undertaken in triplicate.3+ was converted to Fe2+ which then reacted with Ferene S to produce a stable coloured complex with absorbance at 593 nm. Cu2+ interference was specifically blocked with the chelate chemical included in the buffer. 50 µl lysates were prepared in the iron assay buffer provided from treated hPASMCs grown on 6 well plates.The total iron content of the cells was estimated by employing the Colorimetric Iron Assay kit (Abcam) as per the manufacturer’s guidelines. Firstly all FeData is presented as ± SEM (standard error of mean) of the specified number of independent experiments. Statistical analysis was performed on GraphPad Prism. Student’s t test or ANOVA followed by Bonferroni post hoc test was employed to determine statistical significance. Level of significance achieved is indicated in individual figure legends.The use of normal lung tissue has been approved by the Royal Brompton and Harefield NHS Trust Research Ethics Committee (ethics number GQJW1). All procedures were carried out in accordance with the relevant guidelines and regulations. All patients gave written, informed consent prior to the use of their lung tissue.Supplementary information