Patients with short telomere syndromes and pulmonary fibrosis have increased complications after lung transplant. However, the more general impact of donor and recipient telomere length in lung transplant has not been well characterized.st 2012 and January 31st 2015. Relative donor lymphocyte telomere length was measured and classified into long (third tertile) and short (other tertiles). Relative recipient lung telomere length was measured and classified into short (first tertile) and long (other tertiles). Outcome data included survival, need for modification of immunosuppression, liver or kidney injury, cytomegalovirus reactivation, and acute rejection.This was an observational cohort study of patients who received lung transplant at a single center between January 1Recipient lung tissue telomere lengths were measured for 54 of the 79 patients (68.3%) who underwent transplant during the study period. Donor lymphocyte telomeres were measured for 45 (83.3%) of these recipients. Neither long donor telomere length  nor short recipient telomere length  were associated with adjusted survival following lung transplant. Recipients with short telomeres were less likely to have acute cellular rejection  but were not more likely to have other organ dysfunction.In this small cohort, neither long donor lymphocyte telomeres nor short recipient lung tissue telomeres were associated with adjusted survival after lung transplantation. Larger studies are needed to confirm these findings. Telomeres are repetitive nucleotide sequences at the ends of chromosomes that become truncated with successive cellular replication. Telomere shortening can trigger cellular senescence and apoptosis. Loss of function mutations in telomere maintenance pathways are associated with premature organ dysfunction including bone marrow failure, cirrhosis, enteropathies, and interstitial lung disease. Short tFor patients with advanced IPF associated with short telomere syndromes, lung transplantation is the only definitive treatment. These individuals, however, are at risk for worse outcomes following transplant both because they are more susceptible to bone marrow suppressive effects of anti-rejection medication and because of reduced extra-pulmonary organ reserve , 7. For Outside of patients with known short telomere syndrome, however, there are few data on telomere length in lung transplantation from either the donor or recipient standpoint. It is unknown what portions of donors and recipients have telomeres significantly below population means. It is also unknown whether shorter recipient telomeres, in general, are associated with need for modification of immunosuppression, extra-pulmonary organ dysfunction, or worse survival after lung transplant. Theoretically, these patients may have decreased extra-pulmonary reserve, even in the absence of a known telomere shortening mutation, which may manifest as similar complications following transplant.From the donor standpoint, longer donor telomere length might provide increased pulmonary reserve following transplant, improving survival and allograft function. Supporting this idea, among hematopoietic cell transplant (HCT) recipients with aplastic anemia, longer donor telomere length is associated with improved recipient survival. AlthougThe primary objective of this study was to characterize donor and recipient telomere length in lung transplant patients. Our secondary objective was to perform a preliminary assessment of the association of donor telomere length with survival following transplant and recipient telomere length with survival, acute rejection, and extra-pulmonary organ dysfunction. Our hypotheses were that longer donor telomere length would be correlated with improved survival and that shorter recipient telomere length would be associated with decreased survival and increased extra-pulmonary organ dysfunction.The Partners Human Research Committee Brigham and Women’s Hospital Institutional Review Board approved this study. All patients who underwent lung transplantation at Brigham and Women’s Hospital from January 1st 2012 to January 31st 2015 were eligible to participate at the time of transplant. Written informed consent was obtained from patients who participated. Patients who did not provide informed consent and patients whose lung tissue was unavailable for testing were excluded.We extracted genomic DNA from donor lymph nodes or spleen collected prior to organ procurement. Relative telomere length was determined using a high throughout monoplex quantitative real time polymerase chain reaction assay as previously described. Briefly2 sized specimen of the explanted whole lung tissue and determined relative telomere length in a similar fashion as donor lymphocyte telomere length. We were unable to isolate a specific type of lung tissue for this analysis and the reported recipient telomere length likely represented multiple tissue types. We divided recipients into those with short (first tertile) and long (other tertiles) telomeres.Pieces of explanted lungs were immediately snap frozen on dry ice and stored in liquid nitrogen. We extracted genomic DNA from a 1x1 cmst 2016, the time of data analysis. The primary predictor variables of interest were long donor telomere length and short recipient telomere length. We also collected data on clinical and demographic characteristics that may impact survival following transplant including recipient and donor age, native lung disease (IPF vs. non-IPF), lung allocation score (LAS) at transplant, and most recent percent predicted forced expiratory volume in 1 second (FEV1).The primary outcome was survival following transplant. Follow-up time started at date of transplant and ended at death or on May 1We collected data on secondary outcomes during the first year following transplantation including: 1) leukopenia requiring granulocyte colony stimulating factor (GCSF) or cessation of immunosuppression or antiviral medication; 2) acute kidney injury defined as a rise in creatinine 1.5 times baseline over one week; 3) need for dialysis; or 4) liver injury, defined as alanine aminotransferase or aspartate aminotransferase great than three times the upper limit of normal . Other secondary outcomes included cytomegalovirus (CMV) reactivation after stopping antiviral prophylaxis and acute cellular rejection (ACR) of any grade.We used descriptive statistics to identify percentages, medians and quartiles, and means and standard deviations for selected demographic and clinical variables. We used Pearson’s correlation coefficients to assess the relationship between donor age and donor telomere length and recipient age and recipient telomere length. We conducted two survival analyses using Cox proportional hazard models to determine the impact of: 1) long donor telomeres and 2) short recipient telomeres on survival following transplant. We included recipient and donor age, native lung disease, LAS, and most recent percent predicted FEV1 as covariates in both models. We examined Schoenfeld residuals to confirm the proportional hazard assumption. A joint donor and recipient telomere length analysis was not performed because of the small numbers of patients with donor-recipient length discordance.We used Fisher exact tests to compare the above secondary outcomes for recipients with short versus long lung tissue telomeres with 2-sided p<0.05 considered statistically significant. All analyses were performed using Stata .79 patients underwent lung transplant during the 3.1 year study enrollment period, of whom 54 (68.3%) underwent telomere analysis of explanted lung tissue. There was no significant difference in age, sex, native lung disease, FEV1 prior to transplant, or LAS in patients who were and were not included. Donor lymphocyte tissue was available for 45 (83.3%) of the included recipients. There was no significant difference in donor age or recipient characteristics for those for whom donor lymphocytes were and were not available. Demographic and clinical characteristics of the patients included in the cohort are in The median donor telomere length was 0.74 (interquartile range (IQR) = 0.65–0.85). There were 3 donors (6.7%) with T/S ratio <0.50. The median recipient telomere length was 0.56 (IQR = 0.48–0.70). There were 15 recipients (27.8%) with a T/S ratio <0.50. There was no difference in telomere length in recipients who were and were not on chronic steroids—defined as >5 mg prednisone daily—prior to transplant, , even after adjusting for age and native lung disease. There was an inverse correlation between telomere length and age in both recipients  and donors  . Donor lWe classified donor telomere lengths into those with long (third tertile) and short (other tertiles) telomeres as previously described ,14. We uThe mean length of follow-up from transplantation was 2.1 years. Long donor lymphocyte telomeres were not associated with survival following lung transplant, neither in unadjusted models  = 0.08–1.58, p = 0.17) nor when adjusted for donor and recipient age, native lung disease, LAS, and percent predict FEV1  . SimilarShort recipient telomere length was not associated with leukopenia requiring GCSF or cessation of immunosuppression/antiviral medication, liver injury, acute kidney injury, kidney injury requiring dialysis, or CMV reactivation after stopping antivirals . RecipieTelomere shortening has been described in all the advanced lung diseases—pulmonary fibrosis, bronchiectasis, and emphysema—represented in our cohort , 16, 17.The inverse correlation between recipient and donor telomere length and age was consistent with prior studies in the HCT literature. For example, Gadalla et al found similar correlation coefficients in their cohort  . They alOur decision to measure telomere length in recipient explant lung tissue was partly a matter of necessity, as we had limited access to recipient leukocytes before transplant and measurement of leukocyte telomeres post-transplant may be confounded by immunosuppressive medications. NeverthThe lack of association between recipient lung telomeres and survival or extra-pulmonary outcomes may be because of poor correlation between recipient lung telomeres and telomere length and cellular replicative reserve in other organs. Our study was underpowered to detect small or moderate effect sizes and it is possible that larger cohort would identify a relationship that we did not detect. Previous studies of recipient leukocyte telomere length in HCT, however, have not shown an impact on survival, which is consistent with our findings. It may Increased donor age is associated with worse survival following lung transplant in some but not all studies , 23. In Our study has several limitations. First, although this is the first study to report specifically on recipient lung tissue telomere length, this was a relatively small cohort with a limited follow-up period. Second, we do not know to what extent recipient donor lymphocyte telomere length correlated with lung telomere length. Among older patients, there is little correlation between leukocyte telomere length and telomere length in other tissues, but it is unknown whether this is true for donors in the age range in our cohort. MeasureIn this small observational cohort study, a significant portion of donors and recipients had relatively short telomeres in lymphocytes and lung tissue, respectively. Neither long donor telomeres nor short recipient telomeres, however, were associated with adjusted survival after lung transplantation. Short recipient telomeres were associated with decreased rates of acute cellular rejection but not extra-pulmonary organ dysfunction after transplantation. Larger cohort studies are needed to confirm these findings.


HIV-associated neurocognitive disorder (HAND) remains common, despite antiretroviral therapy (ART). HIV dysregulates iron metabolism, but cerebrospinal fluid (CSF) levels of iron and iron-transport proteins in HIV-infected (HIV+) persons are largely unknown. The objectives of this study were to characterize CSF iron-related biomarkers in HIV+ adults and explore their relationships to known predictors of HAND.Alb) measurements, QAlb and comorbidity severity were also included as covariates to account for variability in the blood–CSF-barrier.We quantified total iron, transferrin and heavy-chain (H)-ferritin by immunoassay in CSF sampled by lumbar puncture in 403 HIV+ participants in a multi-center, observational study and evaluated biomarker associations with demographic and HIV-related correlates of HAND  by multivariable regression. In a subset (N = 110) with existing CSF: serum albumin . CSF iron was 41% higher in self-reported Hispanics and 27% higher in (non-Hispanic) Whites than in (non-Hispanic) Blacks ; these findings persisted after adjustment for age, sex, and HIV-specific factors. Median H-ferritin was 25% higher (p < 0.05), and transferrin 14% higher (p = 0.06), in Whites than Blacks. Transferrin and H-ferritin were 33 and 50% higher, respectively, in older (age > 50 years) than in younger persons , but these findings lost statistical significance in subset analyses that adjusted for QAlb and comorbidity. After these additional adjustments, associations were observed for CSF iron and transferrin with race/ethnicity as well as CSF VL, for transferrin with sex and ART, and for H-ferritin with plasma virus detectability and significant comorbidity .Among 403 individuals , men had 25% higher CSF transferrin (median 18.1 vs. 14.5 µg/mL), and 71% higher H-ferritin (median 2.9 vs. 1.7 ng/mL) than women  contains supplementary material, which is available to authorized users. HIV affects an estimated 40 million people worldwide , and owiIron homeostasis is essential for normal brain function: iron is required for the synthesis of monoamine neurotransmitters, for mitochondrial function, myelin synthesis, and the catalytic function of a large number of metabolic enzymes , 24. AltTransferrin, the chief iron-transporter in the circulation, binds and transports ferric iron in a soluble, nonreactive state to metabolically active cells throughout the body, whereupon transferrin-bound iron is internalized by receptor-mediated endocytosis . TransfeSerum is the most convenient source for routine, noninvasive testing of iron parameters, but the CSF is more likely to provide an accurate reflection of iron transport in the CNS. There is also considerable precedent for the use of CSF biomarkers as surrogate markers of iron metabolism and/or neuronal injury in the brain –59. ReceIt is of interest to evaluate associations of known predictors of neurocognitive decline in HIV infection with levels of CSF iron and iron-transport proteins, because such associations would suggest a possible novel role for dysregulation of iron metabolism or iron transport pathways in the pathogenesis of HAND that might be targeted therapeutically. Analogous approaches have proven instructive in achieving a better understanding of other chronic disease phenotypes linked to inflammation, such as cancer . The purThe CNS HIV Antiretroviral Therapy Effects Research (CHARTER) Study is an observational, prospective cohort study that enrolled ambulatory, HIV+ adults at six large U.S. Medical Centers in the U.S., including Johns Hopkins University School of Medicine , the University of California , University of Texas Medical Branch , Washington University School of Medicine , University of Washington , and the Icahn School of Medicine of Mount Sinai . CHARTER was designed specifically to evaluate neurological outcomes in HIV+ individuals; hence, all participants underwent detailed neuromedical, neuropsychological, and cognitive evaluations at baseline and 6-month follow-up visits, as previously described . ComorbiParticipants in this study of CSF iron biomarkers were selected based on availability of at least 1.0 mL of CSF at both baseline (N = 403) and 6-month (N = 100) follow-up visits. Individuals with comorbid conditions deemed by expert clinicians to be severe  were excluded, and the remainder were classified as having minimal or mild-moderate comorbidity following extensive chart review . Non-HisAll CHARTER study participants provided written informed consent for the study, and the CHARTER study adheres to the ethical principles set forth in the Declaration of Helsinki. The present study was also approved by the institutional review boards of all participating institutions.Iron content within the CSF was evaluated using the Quantichrom iron assay  and the manufacturer’s protocol. Briefly, 50 μL of standards or patient CSF samples were mixed with 200 μL Quantichrom Working Reagent in a 96-well plate (in duplicate) and incubated at room temperature for 40 min. The iron concentration in experimental samples was determined by comparison of the optical density at 590 nm with the standard curve.Transferrin content within sampled CSF was determined by enzyme-linked immunosorbent assay (Human Transferrin ELISA kit (ab108902)  performed according to the manufacturer’s protocol. After the CSF was collected and centrifuged, the samples (in duplicate) were diluted 1:1000 with mix diluent. Briefly, transferrin standard or diluted CSF sample (50 μL) was added to each well and the solution was incubated for 2 h. Following incubation, the plate was washed five times with 200 μL of 1× Wash Buffer. Following the wash step, 1× Biotinylated transferrin antibody (50 μL) was added to each well and the solution was incubated for 1 h. The plate was washed again as described above. 1× SP Conjugate (50 μL) was added to each well and the solution was incubated for 30 min. The microplate was washed again as described above. Chromogen substrate (50 μL) was added to each well and the solution was incubated for 12 min. Stop solution (50 μL) was added to each well and the absorbance was read immediately on a microplate reader at 450 nm. Transferrin concentration was then determined by comparison to the standard curve.The H-ferritin content of CSF was determined by an enzyme-linked immunosorbent assay  performed according to the supplier’s instructions. Briefly, 20 µL of standards, subject samples (in duplicate), and controls were combined with 100 µL of enzyme conjugate reagent. The solution was mixed for 30 s and incubated at room temperature for 45 min. The incubation mixture was removed and the plate was rinsed five times with distilled water. The TMB Reagent (100 µL) was added into each well, the solution was mixed for 10 s, and the solution was incubated at room temperature in the dark for 20 min. Stop Solution (100 µL) was added to each well and the solution was mixed for 30 s. The ferritin concentration in the experimental samples was determined by comparison of the optical density at 450 nm with the standard curve within 15 min.Iron, ferritin, and transferrin were also quantified in serum using routine, commercially available, automated assays in a subset of study participants (N = 11) who had sufficient serum available at the 6-month follow-up visit. Circulating ferritin, which consists primarily of the L subunit, was quantified by electrochemiluminescence assay (“ECLIA”), using a biotinylated, monoclonal ferritin-specific antibody and a ruthenium-labeled monoclonal ferritin antibody to form a sandwich complex. Upon addition of streptavidin-coated microparticles, chemiluminescence emission was measured by a photomultiplier  and ferritin concentration determined from a standard curve. Serum iron and transferrin were measured on a Roche c311 automated chemistry analyzer , iron by a colorimetric method based on the FerroZine reaction (without deproteinization), which captures unbound and transferrin-bound iron, and transferrin by immunoturbidimetry .Alb) was calculated as QAlb = albumin(CSF)/albumin(serum) , p < 0.05) persisted following adjustment, but the association with ancestry lost significance. With regard to HIV-related factors, CSF transferrin levels remained negatively associated with ZDV use  and increased with CSF VL . H-ferritin levels in the CSF also remained positively associated with age and negatively associated with female sex after multivariable adjustment. Study participants with detectable plasma virus had lower CSF ferritin levels , and those who were currently on ZDV also tended to have lower levels ; the association of transferrin with plasma VL detectability, though in the same direction, was non-significant. Higher H-ferritin levels were associated with increasing CSF VL . Neither CSF transferrin nor H-ferritin was significantly related to race/ethnicity in multivariable-adjusted analyses. Estimates of association with age, sex, and ancestry did not appreciably change for any biomarker with full adjustment, including HIV disease factors.In unadjusted analyses (Alb data (N = 110), similar multivariable regression analyses were performed. In these models, QAlb as well as comorbidity were included as additional covariates to adjust for likely changes in the functional integrity of the blood-CSF barrier that occur with age as well as HIV infection, and for comorbidity-related inflammation, which can influence iron biomarkers [β = 0.732, p = 0.02; higher iron in Hispanics and Whites than in Blacks), as well as with plasma virus detectability  but lower CSF VL . Higher transferrin levels were associated with male sex , non-White race/ethnicity , but not with comorbidity; the associations with age and ZDV use lost significance. Finally, levels of H-ferritin in CSF were significantly higher among subjects with clinically mild-to-moderate comorbidity  and when plasma virus was undetectable ; CSF VL, age, and gender were no longer significant factors, though the direction of the point estimate for age remained the same. Associations for all iron biomarkers with blood–CSF barrier integrity (mean QAlb) were statistically significant with the exception of H-ferritin . None of the CSF iron biomarkers was associated with nadir CD4+ T-cell count, hemoglobin level, or anemia (data not shown).In the subset of individuals in this study who had available Qomarkers . These mIron and biomarkers of iron transport were quantified in serum in 11 individuals with available serum as well as measured CSF biomarkers at 6 months, and we evaluated correlations of iron, ferritin and transferrin levels in serum to levels of the same analytes in CSF -ferritin, in the CSF of HIV+ persons and to evaluate their associations with known predictors of neurocognitive impairment in this population. Due to the invasive nature of lumbar puncture, published data for CSF iron biomarkers have been scarce even in HIV-negative individuals, in whom data are available only in small numbers of individuals with specific disorders and at different stages of neurodegenerative disease , 67–69. Both nonhuman primate and human studies have shown that systemic iron deficiency and anemia have lasting impact on brain iron homeostasis and neuronal health, influencing memory and cognition , 71–73. Alb, a measure reflecting variability in integrity of the blood–CSF barrier. This apparent discordance suggests that either (1) more iron fails to cross the CSF–brain barrier when HIV levels in the brain or CSF are low, leading to higher levels in the CSF, or (2) higher amounts of virus in the brain or CSF are associated with greater extraction of iron from the CSF, because HIV requires iron for its replication [Alb were also in accord with the corresponding observed CSF iron differences. Even when accounting for the effects of ZDV, an antiretroviral drug that can affect iron transport, ART was independently associated with higher CSF transferrin, raising the possibility that other commonly used antiretroviral drugs may impact transferrin levels in the CNS, possibly by inducing a functionally iron-deficient state. Initiation of ART has been temporally linked to an increase in soluble transferrin receptors, a sensitive indicator of tissue iron deficiency [Alb adjustment. Interestingly, HIV+ individuals are at increased risk of RLS, a condition associated with iron deficiency in the brain [In the present study, higher CSF iron content tended to be associated with detectable virus in the plasma but with lower CSF VL, when adjusting for the Qlication . The sigficiency , 79. Othficiency , 76, 80.he brain , 82.Alb adjustment; this association persisted following adjustment only for transferrin, and the direction of the estimate remained the same for H-ferritin although significance was lost. That higher H-ferritin levels were associated with undetectable plasma virus but a higher quantity of CSF virus suggests that induction of H-ferritin in the CNS may be a physiological (cytoprotective) response to viral compartmentalization and replication in the CNS. HIV-induced oxidative stress is manifested by upregulation of heme-oxygenase-1 in the brain [Alb but lost significance after this adjustment; this begs the question whether the loss of iron regulation  in the CSF due to age- or HIV-related damage to the blood–CSF barrier may in part mediate age-related neurocognitive decline.Although men are known to have significantly higher systemic iron stores than pre-menopausal women, differences in CSF levels of iron, transferrin, or H-ferritin levels have not been documented, and they may have clinical relevance . Women whe brain , and thehe brain . Due to he brain , 86–88. he brain  previoushe brain –91. One he brain , and stuhe brain , 83. It he brain . Mechanihe brain , 94. FinLevels of CSF iron biomarkers in this HIV+ population were not dramatically different from levels previously reported in small numbers of healthy individuals, but the different assay methods used complicate any comparisons: CSF ferritin levels in this study were lower than levels in individuals either with or without RLS that were reported by Mizuno et al. and they were also lower than levels observed in chronic demyelinating disorders like chronic progressive multiple sclerosis , 38, 67.We acknowledge the limitations of this study, including its cross-sectional design, which limits interpretation, the lack of an HIV-negative comparison group, and absence of matched serum iron biomarker data for most participants. Moderately correlated, within-individual values of iron, transferrin and H-ferritin obtained at baseline and 6 months in 100 of the study participants, however, support the use of single CSF biomarker measurements for our analyses and favor their stability in HIV+ persons over short periods of time. Regarding the lack of HIV-negative controls, we would stress that our objective was not to determine the impact of HIV on CSF iron biomarker levels, but to ascertain their associations with key demographic, HIV disease- and treatment-related predictors of neurocognitive decline, since measures of HAND are often imprecise and fluctuate in individuals over time . Better In HIV+ adults, CSF levels of iron and major biomarkers of iron-transport are independently associated with sex, self-reported race/ethnicity, ART, plasma virus detectability and/or CSF VL. Associations of CSF iron biomarkers with age, which disappeared following adjustment for a measure of blood-CSF barrier function, suggests the possibility that age- or HIV-related declines in the integrity of the blood-CSF barrier may contribute to changes in CNS iron balance as HIV+ individuals age. Furthermore, this study raises the intriguing possibility that consistent correlates of HAND such as race/ethnicity, ART, and HIV RNA in the CSF, may be linked to HAND in part via changes in CNS iron homeostasis. Additional studies, preferably including longitudinal samples and a higher number of participants over 50 years of age, as well as matched HIV-negative controls, are needed to further investigate these questions.Additional file 1: Figure S1. Scatter plots of iron biomarker values in CSF at baseline (N=403) and 6 months (N=100). P-values shown are for corresponding Spearman correlations.Additional file 2: Table S1. Iron biomarker values for CHARTER study participants at baseline and 6-months. Units of measurement: (CSF) iron, µg/dL; transferrin, µg/mL; ferritin, ng/mL.Additional file 3. Visit-matched (6-month) CSF and serum iron biomarker values for 11 CHARTER study participants. Units of measurement: (CSF) iron, µg/dL; transferrin, µg/mL; ferritin, ng/mL; (Serum) iron, µg/dL; transferrin, mg/dL; ferritin, ng/mL.


It was shown that DT and TT were successfully loaded into the tips of the ceramic nanoporous microneedles, and by using near-infrared fluorescently labeled antigens, we found that DT and TT were released following piercing of the antigen-loaded npMNAs into ex vivo murine skin. Finally, the application of DT- and TT-loaded npMNAs onto mouse skin in vivo led to the induction of antigen-specific antibodies, with titers similar to those obtained upon subcutaneous immunization with a similar dose. In conclusion, we show for the first time, the potential of npMNAs for intradermal (ID) immunization with subunit vaccines, which opens possibilities for future ID vaccination designs.The skin is immunologically very potent because of the high number of antigen-presenting cells in the dermis and epidermis, and is therefore considered to be very suitable for vaccination. However, the skin’s physical barrier, the stratum corneum, prevents foreign substances, including vaccines, from entering the skin. Microneedles, which are needle-like structures with dimensions in the micrometer range, form a relatively new approach to circumvent the stratum corneum, allowing for minimally invasive and pain-free vaccination. In this study, we tested ceramic nanoporous microneedle arrays (npMNAs), representing a novel microneedle-based drug delivery technology, for their ability to deliver the subunit vaccines diphtheria toxoid (DT) and tetanus toxoid (TT) intradermally. First, the piercing ability of the ceramic  npMNAs, which contained over 100 microneedles per array, a length of 475 µm, and an average pore size of 80 nm, was evaluated in mouse skin. Then, the hydrodynamic diameters of DT and TT and the loading of DT, TT, and imiquimod into, and subsequent release from the npMNAs were assessed  The npMNAs had sufficient strength to reproducibly pierce ex vivo human skin without breaking  , were te2O3) , 13. In breaking  and havebreaking . Howeverex vivo, and in vivo immunogenicity of npMNA-delivered antigens were examined. We show that npMNA-mediated vaccine delivery elicits TT- and DT-specific antibody responses in mice, comparable to those induced by subcutaneous (SC) immunization with a similar dose. This is the first report showing the potential of porous microneedles in dermal immunization with subunit vaccines.In this study, characterization and application of alumina npMNAs are described. Loading of npMNAs with diphtheria toxoid (DT) and tetanus toxoid (TT), antigen release in murine skin in vitro assays) were obtained from Staten Serum Institute  and imiquimod (IMQ) Vaccigrade was obtained from Invivogen. Trifluoroacetic acid (TFA), 3,3′,5,5′-tetramethylbenzidine (TMB) and bovine serum albumin (BSA), and 0.4% (w/v) were obtained from Sigma Aldrich. High-performance liquid chromatography (HPLC)-R grade acetonitrile (ACN) was from Biosolve and phosphate-buffered saline  was from Gibco (ThermoFisher Scientific). IRdye800cw carboxylic acid N-hydroxy succinimide ester (IRdye800cw-NHS) was purchased from Li-cor . Dexdomitor was purchased from Orion Corporation, Narketan ketamine from Vétoquinol, and Atipam was purchased from Dechra. Goat anti-mouse immune globulin G (IgG) total horseradish peroxidase (HRP) , goat anti-mouse IgG1 HRP (GaM-IgG1 HRP), and goat anti-mouse IgG2a HRP (GaM-IgG2a HRP) were obtained from Southern Biotech and microtiter plates 9610 used for ELISA were from Corning Costar.Diphtheria toxoid and TT  by using a UAFM-V1 electrical applicator (uPRAX Microsolutions) at a velocity of 65 cm/s. Next, a drop of 5 µL drug formulation was applied onto the foil-pierced npMNAs to absorb a drug/vaccine formulation into the microneedle tips. After 5 s, the surplus drop of drug formulation and the foil were sequentially removed from the npMNAs. To confirm that only the microneedle tips can be loaded with a drug formulation, the tips of npMNAs were loaded with a 0.4% trypan blue solution as described earlier.To only load the tips of the microneedles, microneedles were pierced through a foil , which were collected from surplus mice, by using 3D-printed uPRAX impact applicators . Subsequently, the three pierced ex vivo mouse ears were incubated with 50 µL of a 0.4% trypan blue solution at room temperature. After 30 min, the trypan blue was removed, and the ears were washed in 10 mL PBS. Finally, the blue dots (piercings) were counted, from which the penetration efficiency was calculated.To test the piercing ability, a npMNA was applied trice on the dorsal side of s Figure C having To determine whether the npMNAs are suitable devices to load and release subunit antigens DT and TT, the hydrodynamic diameter and size distribution of DT and TT were determined by using dynamic light scattering on a Zetasizer Nano . For these measurements, DT and TT were at a concentration of 0.8 and 0.4 mg/mL, respectively.Imiquimod has the potential to enhance the immunogenicity of antigens in the skin . To evalTo assess the release of antigen from antigen-loaded nanoporous material, npMNAs (that were not pierced through a foil) were loaded with 15 µL PBS that contained 5 Lf DT or TT as described earlier. Next, antigen-loaded npMNAs were incubated in 4 mL release buffer while shaking at 300 rpm. At different time points , samples of 500 µL were taken in which the released amount of antigen was quantified by measuring the intrinsic fluorescence (emission wavelength of 348 nm) at an excitation wavelength of 280 nm and using standard concentrations of DT and TT ranging from 0.01 to 50 ng/mL on a Tecan Infinite M1000 plate reader. The release of antigen in the presence of IMQ was not investigated, because IMQ is fluorescent at similar wavelengths (excitation at 260 nm and emission at 340 nm) that are used to measure the intrinsic fluorescence of the antigens . To this end, 1 mg/mL solutions of DT and TT in a 100 mM carbonate buffer pH 8.5 were prepared. Subsequently, 1 mL of each of these solutions was added to 500 µg of IRdye800cw-NHS. After 1 h shaking (300 rpm) at 37°C, the free dye was removed and the carbonate buffer was exchanged by PBS using a Zeba™ spin desalting column with a molecular weight cutoff (MWCO) of 7 kDa (Thermo Fisher Scientific). Next, IRdye800cw-labeled antigens were concentrated approximately 50 times by using 0.5 mL Amicon (Millipore) 10 kDa MWCO filters. Finally, the concentration of IRdye800cw-labeled DT and TT was determined by using a calibration curve of non-labeled antigens and measuring the intrinsic fluorescence (as described earlier).® lumina II equipped with an ICG filter set. The intradermally delivered amounts of DT and TT were quantified by using Living Image® software (version 4.3.1).To quantify the delivered amount of DT and TT into murine skin, npMNAs of which only the tips were loaded (using foil piercing) with fluorescently labeled antigens were prepared by using 5 µL of 12 Lf/μL (DT) and 6 Lf/μL (TT), as described earlier. The microneedles were applied by impact application and retained onto the skin by using a uPRAX 3D-printed applicator. After 30 min at room temperature, the antigen-loaded npMNAs were removed from the ears, and their fluorescence was compared with the fluorescence of standard solutions having known amounts of fluorescently labeled DT and TT, by using a IVISSubunit vaccine formulations of DT and TT for loading the microneedle tips were prepared from antigen stock solutions . Antigen stock solutions were concentrated (6–30×) by using 0.5 mL Amicon (Millipore) 10 kDa MWCO filters. Next, the concentration of the concentrates was determined by measuring the intrinsic fluorescence as described earlier. Finally, the antigen concentration was adjusted by diluting the concentrates in PBS to a concentration of 12 and 6 Lf/μL for DT and TT, respectively.via a SC injection of 100 µL in the neck using traditional hypodermic needles, or by dermal administration in the ear pinnae by using microneedles of which only the tips were loaded with vaccine formulation. Before each microneedle-based immunization, mice were anesthetized with 30 mg/kg ketamine and 0.1 mg/kg Dexdomitor by intraperitoneal injection. After the microneedles were removed, the anesthetic was antagonized with 0.4 mg/kg Atipam. On each ear, a DT- and TT-loaded npMNA was applied for 30 min by using a uPRAX applicator  obtained from Charles River (France) were immunized with 1.2 Lf (~0.50 μg) DT and 1.5 Lf (~0.77 μg) TT, or with 0.6 Lf DT and 0.75 Lf TT adjuvanted with 0.5 µg IMQ, on days 0, 21, and 42. The vaccine was administered 2SO4, and the absorption was measured at a wavelength of 450 nm, with a reference wavelength of 650 nm, on a Microplate reader 550 (Bio-Rad). Titers of all animals at all time points for each isotype were measured in one experiment per antigen.Diphtheria toxoid- and TT-specific antibody titers were determined using ELISA. ELISA plates were coated with 0.2 µg DT or 0.2 µg TT for 30 min and then blocked with 1% BSA in PBS for 15 min. Thereafter, 50 µL of serum sample at dilutions ranging from 1:25 until 1:200 (day 0), or from 1:200 until 1:25,000  were added, for 30 min. After extensive washing with 0.01% Tween 20 in PBS (PBST), wells were incubated for 30 min with GaM-IgG total HRP , GaM-IgG1 HRP , or GaM-IgG2a HRP . After extensive washing with PBST, antibody titers were quantified by adding 50 µL of stock TMB. Reactions were stopped after 60 s, with 100 µL of 1 M H50 midpoint titers were determined using GraphPad Prism . Immunized mice showing OD values below the mean OD values + three times the SD measured for PBS treated mice were considered as non-responders and were given an arbitrary value of 0, equal to 10Log value of 1. Statistical differences between immunization groups were determined using a non-parametric one-way ANOVA Kruskal–Wallis test with a Dunn’s test for multiple comparisons, and statistical significance was presented as follows: *p < 0.05; **p < 0.01; ***p < 0.001, and ns, not significant. Ratio of IgG1:IgG2a was determined by dividing midpoint titers of individual isotypes, and if animals were considered as non-responders for one of the isotypes (see above), they were excluded from ratio analysis.From 4 optical density (OD) values (at a wavelength of 450 nm) of diluted serum samples, the ECvia surface Bruker analysis, which showed that the ceramic microneedles had an average length of 475 µm and a needle shaft diameter of 275 µm . No visual breakage or reduction in microneedle strength or sharpness was observed. Together, these data show that the developed npMNA can be used to repeatedly penetrate the skin without breakage. To determine whether the npMNAs, having an average pore size of 80 nm, are suitable to be loaded with subunit vaccine antigens, the hydrodynamic diameter of DT and TT were determined by using dynamic light scatter. This revealed that DT and TT had a hydrodynamic diameter of 8.7 ± 2.8 nm  and 13.5 ± 5.6 nm , respectively,  DT and a delivery of 1.53 Lf (~0.77 μg) TT. These values of delivered doses correspond with 26 and 31%, respectively, of the currently used human vaccination dose (5 Lf).To determine whether npMNA-mediated ID delivery induces antigen-specific immunity, mice were immunized with both DT 1.2 Lf) and TT 1.5 Lf) using antigen-loaded npMNAs (ID administration) or using a needle and a syringe (SC injection). After each immunization, antibody titers in the serum were determined  7 agonist IMQ were tested to determine whether adjuvants may modify the quality of vaccination-induced antibody responses. Antigen quantities loaded in combination with IMQ were half the antigen dose loaded into npMNAs in the absence of this TLR7 agonist . Despite the lower antigen dose, similar antibody titers were detected in mice immunized with adjuvanted compared with unadjuvanted vaccine, with the exception of DT-specific responses measured at day 41 where 3/10 in the adjuvanted group compared with 1/10 mice in the unadjuvanted group had failed to respond  AKP30 particles for the production of npMNAs results in approximately 40% porosity, with an average pore size of 80 nm  could be favorable over adsorption onto (or coating of) the surface of, for instance, solid microneedles, because (1) the microneedle tip sharpness is retained and (2) potentially less excipients are required to retain the immunogenicity of a vaccine. Coating of solid MNAs generally requires a thick drug-containing layer to achieve the required amounts of drug/vaccine loading, and this could reduce the sharpness of the tips and thereby their skin piercing ability  adjuvant for the induction of humoral immune responses, but it cannot fit into the nanopores of our npMNAs. Besides, alum causes granuloma formation and should therefore not be used as an ID adjuvant .In this study, IMQ, which is a toll-like receptor 7 agonist with a molecular weight of 240 g/mol, was chosen as an adjuvant. IMQ is extensively researched for its adjuvanticity , 29, 30,Finally, in this study, it was shown that strong antibody responses were induced without using an adjuvant. The antibody responses obtained in our study are in line with the ones described in literature , 38. ForTaken together, in this study we show that ceramic nanoporous microneedles are strong enough to repeatedly penetrate the skin and that they can be loaded with protein subunit vaccines such as DT and TT. After skin piercing with antigen-loaded npMNAs, the antigens are intradermally delivered, which resulted in an induction of antigen-specific antibody responses. In conclusion, we show for the first time the potential of npMNAs for ID immunization with subunit vaccines, which opens possibilities for future ID vaccination designs.Ethical approval was obtained from Animal Ethics Committee from Utrecht University, the Netherlands (DEC #2014.II.11.080).Substantial contributions to the conception was done by KM, PV, and AS and design of the work by KM, AS, and AP. Acquisition, analysis, and interpretation of data by AP, KM, AG, NK, and PK. Drafting the manuscript by AG and KM. All the authors revised the work and gave final approval of the version to be published.KM is co-owner of uPRAX Microsolutions. The other authors declare that they have no commercial or financial relationships that could be construed as a potential conflict of interest.


Semenogelins (SEMGs) are major components of human seminal vesicle secretions. Due to SEMG’s sperm-motility inhibitor, a significant negative correlation between sperm motility and the proportion of SEMG-bound spermatozoa (SEMG+) was found in asthenozoospermic patients. SEMGs also show intrinsic inhibitory capability for sperm capacitation; however, studies on actual clinical specimens have not been conducted.To reveal the relationship between SEMGs and the fertilizing capacity of sperm from male infertile patients who are not restricted to asthenozoospermia, we measured the proportion of SEMG+ in the spermatozoa of 142 male infertile patients. The pregnancy outcomes in partners of these patients were retrospectively analyzed using questionnaires.Among examined semen parameters, only the total SEMG-unbound sperm count showed a tendency to be different between the spontaneous pregnancy or intra-uterine-insemination-pregnancy groups and in-vitro-fertilization- or intracytoplasmic-sperm-injection-pregnancy groups. It was elevated in the former group, which includes patients who used in vivo fertilization.The total SEMG-unbound sperm count would be a relevant parameter for in vivo fertilization. This result suggests that SEMGs inhibit ectopic capacitation before sperm reach the fertilization site and that the number of total SEMG-unbound sperm is a parameter directly linked to the possibility of in vivo fertilization.The online version of this article (doi:10.1186/s12610-017-0059-6) contains supplementary material, which is available to authorized users. Globally, approximately 15% of couples of reproductive-ages do not achieve pregnancy within 1 year of regular unprotected sexual intercourse and seek fertility assessment . These dTo overcome these insufficiencies of routine semen analysis for diagnosis, specialized andrology tests have emerged to account for sperm dysfunction. Formerly, particularly before the advent of intracytoplasmic sperm injection (ICSI), tests that assessed antisperm antibodies, sperm hyperactivation, and acrosome reaction; sperm binding; and penetration to the human zona pellucida were used to investigate males with unexplained infertility . These tSemenogelins (SEMGs) are major components of human seminal vesicle secretions and are comprised of two closely-related proteins: SEMG 1 and 2 . These tBased on the clinical relevance of SEMGs to male factor infertility and their putative physiologic role, we hypothesized that SEMGs might be targets for developing new sperm function tests that can guide couples with male factor infertility to select the most appropriate treatment. In this study, we examined the condition of SEMGs bound to sperm from infertile patients and retrospectively analyzed the pregnancy outcomes of these patients’ partners using questionnaires. These results can provide information on the new sperm function tests using SEMGs by indicating whether they are practical for the evaluation of sperm function beyond motility.One hundred and forty-two cases of male infertile patients who had visited the reproduction center of the International University of Health and Welfare Hospital (IUHW) from August 2012 to March 2013 were included in this study. All infertile couples had undergone treatments at IUHW. The policy of infertility treatment at IUHW, according to the semen condition of the male partner, is as follows: for normal findings or in the case of mild oligozoospermia and /or asthenozoospermia, natural pregnancy or intra-uterine insemination (IUI) using the timing method are considered as the first choice, followed by a “step-up” to IVF/ICSI when pregnancy is not achieved; for severe oligozoospermia and/or asthenozoospermia, IVF/ICSI is conducted from the beginning. Furthermore, partners of patients with psychogenic erectile dysfunction or intravaginal ejaculation disorder are initially treated with IUI.As part of this study, a physical examination and evaluation of male infertility was performed for each subject. During the period of follow-up until the end of 2014, we sent questionnaires to all male infertile patients to obtain data of pregnancy outcomes and collected the answers from 96 of them, which were accompanied by semen analysis data and SEMG+ measurements taken at the first medical examination. Of these 96 partners of patients, 36 were confirmed to achieve pregnancy by any means of IUI, IVF, ICSI or spontaneous conception (“Pregnancy group”), while remaining 60 partners of patients kept unpregnant by the end of the follow-up period (“Non-pregnancy group”). Thirteen normal healthy male subjects whose wives were in the twentieth week or later of spontaneous pregnancy and who received prenatal examinations in the hospital were recruited to participate as “control” subjects in this study after it was confirmed that they did not have any types of intrascrotum abnormality, including varicocele. Informed consent was obtained from all participants for the use of the data obtained from a physical examination, semen, and blood samples. The study protocol was approved by the Institutional Review Board at IUHW and at Toin University of Yokohama in Japan.Semen samples were obtained via masturbation following sexual abstinence for at least 48 h. Specimens were allowed to liquefy for up to 1 h after ejaculation at room temperature. Complete liquefaction was certified macroscopically according to the WHO manual by confirming that the semen becomes homogeneous and quite watery, and only small areas of coagulation remain. Manual semen analysis was performed according to the WHO manual to determine semen volume and sperm concentration. For analysis of sperm motility, the SMAS™  system was used. Additionally, the serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) were measured using a chemiluminescent immunoassay . The reference ranges for LH, FSH, and T were 0.79–5.72 mIU/ml, 2.00–8.30 mIU/ml, and 1.31–8.71 ng/ml, respectively. The intra-assay coefficient of variation (CV) for LH, FSH, and T were 3.03%, 3.74%, and 5.13%, respectively. The inter-assay CV for LH, FSH, and T were 1.84%, 0.43%, and 3.99%, respectively.2, 0.5 mM MgCl2, 14 mM fructose and 10 mM HEPES, pH 8.0) and centrifuged at 1200 g for 30 min at 25 °C. The resulting pellet was fixed with 2% paraformaldehyde phosphate buffer, pH 7.4. The fixed sperm were then washed twice with HBS, mixed with 25% block-Ace  and then incubated at 37 °C for 1 h, then washed with HBS at 3300 g for 10 min at 25 °C. An aliquot of the washed sperm was incubated with 1 μg/mL anti-seminal plasma motility inhibitor (SPMI) mouse immunoglobulin G (IgG) that recognized a part of the SPMI region (138–154: GTQNPSQDSGNSPSGKG) of SEMG  or mouse IgG isotype control  at 37 °C for 1 h [Following semen analysis, semen samples were refrigerated until use. Pretreatment and staining methods were the same as described in the previous study . BrieflyFlow cytometric analysis was performed as described in the previous study . Brieflywww.cran.r-project.org) [Statistical analyses comparing mean values of each parameter for patients and control subjects were performed with JMP software  using Wilcoxon rank sum test. Additionally, the Spearman’s rank correlation coefficient was applied between the results for the proportion of Sg + and the results of standard semen analysis. Statistical analyses comparing differences in the continuous variables were performed with the “R” statistical software system (ect.org)  using anect.org) .P < 0.001). Notably, the number of control subjects was small. This is a reasonable result given that our control subjects were men who were confirmed not to be infertile. The correlation between the proportion of SEMG+ and the results of standard semen analysis among male infertile patients (Table P = 0.05), while no significant correlations were observed between the proportion of SEMG+ and sperm concentration, or between SEMG+ and sperm viability. None of the other parameters such as age, serum T level, serum FSH level, and/or serum LH level were significantly correlated with the proportion of SEMG+ in both infertile patients and in control subjects.The clinical characteristics and proportions of SEMG+ and SEMG− were examined retrospectively Table . Statistts Table  and cont6 vs. 73.4 × 106, P = 0.06).Next, we compared the pregnancy and non-pregnancy groups in patients to check if there were any differences in parameters. No parameters, including the proportion of SEMG+/− and total SEMG+/− count, demonstrated significant differences between the two groups Table . Then, wR = − 0.68) and intensity (R = − 0.38) of anti-SPMI labeling, implying that the SPMI, and eventually SEMGs, binding to the sperm surface might account for some disorders in sperm motility that are observed in infertile men with asthenozoospermia.Previously, our group reported the association between SEMGs and spermatozoa in infertile men with asthenozoospermia . A signiIn this study, we expanded the pool of potential subjects to include infertile patients whose infertility was caused not only by asthenozoospermia but also by oligozoospermia and normozoospermia . We further explored the possibility of SEMGs bound to spermatozoa being a predictor of infertility treatment for such a variety of infertile patients in the future. We confirmed a negative correlation between sperm motility and the proportion of SEMG+ in this study, although it was a comparatively weak correlation since the subjects included in this study were not restricted to those with asthenozoospermia Table . For thiThe proportion of SEMG+, as well as any other parameters, including sperm motility, did not show a significant difference between the pregnancy and non-pregnancy groups, which indicated that it might not be a general predictor of ability to bear children in an infertile subject. Naturally, the link between SEMGs and the success of pregnancy requires further support. This is because, in addition to factors associated with the male, factors associated with the female, the fertilized egg, and the early development of the embryo are all involved in the success of pregnancy. To conduct an analysis while considering these confounding factors, it is necessary to further investigate increasing the number of specimens.Next, based on the hypothesis that the condition of SEMGs bound to spermatozoa differs between pregnancy achieved through IUI or spontaneous pregnancy and that achieved through IVF or ICSI, we focused on the result of 36 cases in which conception was achieved. When the summed data of spontaneous pregnancy and IUI was compared to the summed data of IVF and ICSI, the total SEMG− count was apparently higher in the former. Such a tendency was not observed in any of the other parameters, including the proportion of SEMG+. Although the precise molecular mechanism underlying the controlling fertilizing capacity of sperm in vivo have not been revealed, the role of SEMGs on sperm were well studied and proved to control motility and capacitation , 16–18. While results from this study provide a minimum-requirement analysis of the SEMGs bound to spermatozoa and conventional semen analysis focused on pregnancy outcomes, this study also provides the need for further large-scale studies to establish diagnostic certainty. In addition to this study having a small patient population, this study has a limitation that the characteristics of the female partners, such as age and oocyte quality, are not considered. Pregnancy outcomes of a couple are usually strongly affected by the condition of the female partner. Thus, it is possible that the influence of male factors on pregnancy outcomes is importantly obscured under such uncontrolled conditions of the female. Surprisingly, SEMGs were found to be potential biomarkers that can guide a couple to select a suitable method of assisted reproductive technology. They were also found to be particularly valuable for the decision to use IVF or ICSI rather than IUI. Usually, natural pregnancy or IUI using the timing method are used in infertile couples with no particular female factors when the male partner is normozoospermic or mildly oligozoospermic or asthenozoospermic. According to the findings of this study, it is likely that even such a couple should consider early step-up using the data of total SEMG− count. More data must be collected to evidence this hypothesis and to determine any cut-off value. In the future, SEMGs bound to sperm may be used to determine whether IUI, IVF, or ICSI treatment should be administered promptly, without waiting for spontaneous pregnancy, in case of couples who desire to have their own child.Regarding the partner of patient pregnancy subgroup, the total SEMG− count tended to be high in patients whose partners had spontaneous pregnancy or who underwent IUI treatment, compared with partners of patients who used other treatments. This result suggests that SEMGs inhibit ectopic capacitation before sperm reach the fertilization site and that the number of total SEMG-unbound sperm is a parameter directly linked to the possibility of in vivo fertilization. It is possible that total SEMG− count might be a tool for determining whether IUI, spontaneous conception, IVF, or ICSI should be used for the partners’ treatment of ART. It is necessary to perform further large-scale studies to establish the diagnostic certainty of SEMGs bound to spermatozoa.Additional file 1: Figure S1.Representative histograms obtained using flow cytometry. Subpopulation of SEMG+ was determind by compearing to unstained sperm (mIgG isotype control). (PDF 1133 kb)Additional file 2: Table S1.N = 13). (DOCX 10 kb)Correlation between the proportion of SEMG+ and the result of standard semen analysis among control subjects (


Patients who suffer from subarachnoid hemorrhage (SAH) usually have long-term neurological impairments. Endogenous neurogenesis might play a potential role in functional recovery after SAH; however, the underlying neurogenesis mechanism is still unclear. We assessed the extent of neurogenesis in the subventricular zone (SVZ) to better understand the neurogenesis mechanism after SAH. We performed a rat model of SAH to examine the extent of neurogenesis in the SVZ and assessed functional effects of the neurotrophic factors in the cerebrospinal fluid (CSF) on neural stem cells (NSCs) after SAH. In this study, the proliferation, differentiation, and migratory capacities of NSCs in the SVZ were significantly increased on days 5 and 7 post SAH. Furthermore, treatment of cultured rat fetal NSCs with the CSF collected from rats on days 5 and 7 post SAH enhanced their proliferation, differentiation, and migration. Enzyme-linked immunosorbent assay (ELISA) of the CSF detected a marked increase in the concentration of brain-derived neurotrophic factor (BDNF). Treating the cultured NSCs with recombinant BDNF (at the same concentration as that in the CSF) or with CSF from SAH rats, directly, stimulated proliferation, differentiation, and migration to a similar extent. BDNF expression was upregulated in the SVZ of rats on days 5 and 7 post SAH, and BDNF release occurred from NSCs, astrocytes, and microglia in the SVZ. These results indicate that SAH triggers the expression of BDNF, which promotes the proliferation, differentiation, and migration of NSCs in the SVZ after SAH. Subarachnoid hemorrhage (SAH) is a fatal type of stroke and survivors usually have long-term physical, neurocognitive, psychological, and/or psychiatric impairments . CurrentPrevious studies have noted the higher proliferation capacity of NSCs in the SVZ of rats with SAH and the activation of endogenous NSCs in the brains of adult humans with SAH , 9. HoweThis research program using the animal experiment protocols specified below was approved by the Utilization Committee and the National Taiwan University Institutional Laboratory Animal Care committee . All procedures met the requirements of the Animal Welfare Protection Act of the Department of Agriculture, Executive Yuan, Taiwan. All surgery was performed under anesthesia by using 2.5% isoflurane with 70% nitrous oxide and 27.5% oxygen. Animals were sacrificed by using overdose of sodium pentobarbital and all efforts were made to minimize suffering.Super Glue®  to prevent fistula formation. After suturing the skin, rats were placed in a cage under an infrared heating lamp until recovery from anesthesia. The incision site was examined daily for evidence of wound dehiscence or infection until it is completely healed. In general, the animals showed drowsiness immediately after SAH induction. In rare cases mild paresis of hind limbs could be observed.Adult male Wistar rats weighing from 280 to 300 g were anesthetized by using 2.5% isoflurane with 70% nitrous oxide and 27.5% oxygen. A small suboccipital incision was made to expose the arch of the atlas, the occipital bone, and the atlantooccipital membrane overlying the cisterna magna. The cisterna magna was tapped using a U-100 insulin syringe with 28G x 1/2 inch needle , and 0.1 ml of CSF was then gently aspirated. The femoral artery was exposed and a PE-50 tube connected with 0.5-ml syringe was introduced into the artery. Approximately 0.2 ml of blood drawn from femoral artery was injected into the cisterna magna over a period of 2 to 3 minutes. In sham-operated controls, normal saline was injected into the cisterna magna. Immediately after the injection of blood, the hole was sealed with In this study 60 rats were totally used, including 44 male rats for SAH animal model  and 16 female pregnant Wistar rats for primary NSC culture. The mortality rate was 25% and the animals were randomized to experimental conditions.Immunohistochemistry was performed on cryostat brain sections that were collected from rats 1, 3, 5, and 7 days after SAH induction and sham induction. Sections were permeabilized with 1% Triton X100-PBS, incubated with 3% fetal bovine serum (FBS) in 0.1% Triton X100-PBS at room temperature for 2 hours to block nonspecific binding, incubated with primary antibodies in blocking solution  at 4°C overnight, incubated with secondary antibodies and Hoechst 33342  for 1 hour at room temperature, and mounted with Fluoro-Gel . Detailed information of antibodies used in this experiment were provided in the supporting information . All ima2 for 6 days by which time primary neurospheres had formed. Detailed procedure were provided in supporting information , incubated with 1% FBS in 1% Triton X100-PBS for 1 hour at room temperature to block nonspecific binding, incubated with primary antibodies diluted in blocking solution overnight at 4°C, incubated with secondary antibodies and Hoechst 33342 (diluted in blocking solution) for 1 hour at room temperature, and mounted on microscope slides  with Fluoro-Gel. Detailed information of antibodies used in this experiment were provided in the supporting information .The levels of erythropoietin (EPO), BDNF, beta-neural growth factor (NGF), and epidermal growth factor (EGF) protein were determined (detailed information of ELISA kits were provided in the supporting information ). All pr®). Immunoreactivity was then expressed as the total area of specific immunoreactivity within the total dorsolateral, lateral, and striatum areas. In images of the immunocytochemically stained neurospheres, neurospheres immunoreactive for Ki67 were counted. ImagePro Plus 6.0 was used to count the number of differentiated cells . Detailed information of live cell imaging by time-lapse microscopy were provided in the supporting information . P <0.05 was considered statistically significant.Values are expressed mean values ± SD. The image quantification and ELISA data were statistically processed using one way ANOVA followed by Tukey's multiple comparisons test or Dunnett's multiple comparisons test and plotted by GraphPadPrismvs. 55.5%, P< 0.001), returned to the baseline level (51.4%) on post-SAH day 5  and the percentage of these cells was similar in rats without SAH and rats with SAH at different times after SAH . Using KAH day 5 , and incvs. 8%, P < 0.001)   (We further used DCX (a neuroblast marker) and GFAP (an astrocyte marker) to determine the differentiation and migratory capacity of NSCs in the SVZ after SAH. DCX immunoreactivity in the SVZ was significantly above baseline (the sham control level) on 5 and 7 days post SAH (25.8% and 26% < 0.001) , indicat < 0.01) , indicat< 0.001) . Taken tvs. 21.7%, P< 0.001) . CSF from rats at 7 days post SAH was first serially diluted to determine the optimal conditions for further analysis. Primary cultured neurospheres treated with CSF  for 24 hours were double immunostained with anti-nestin and Ki67 antibodies. The percentage of Ki67-positive cells was significantly higher at the concentrations of 0.5%, 1%, and 2% in SAH neurospheres than control neurospheres  . The convs. 29.3%; P< 0.001), indicating post-SAH CSF could stimulate proliferation of cultured NSCs  in different groups . CSF colred NSCs .vs. 10.9%, P< 0.001; Tuj-1, 41.5% and 43.7% vs. 10.3%, P< 0.001), implying that post-SAH CSF can stimulate the neuronal differentiation of NSCs , indicating post-SAH CSF could trigger astrocyte differentiation of NSCs  control values, respectively , and migratory capacity of NSCs in cultured neurospheres.vs. 46 pg/ml, P = 0.95) and increased markedly by 5 or 7 days post SAH  , EPO , and bNGF  relative to their respective control values were modestly increased in CSF on day 3 or 5 post SAH , we proposed that BDNF might be the major trophic factor stimulating neurogenesis of NSCs after SAH.Enhanced expression of BDNF, EGF, EPO, and bNGF after cerebral ischemia or traumatic brain injuries may contribute to the activation of neurogenesis in the SVZ. We thus analyzed the concentrations of these neurotrophic factors in CSF collected before and after SAH using ELISA. The concentration of BDNF in CSF decreased relative to the baseline (control) value 3 days post SAH (5 P <0.01) . On the post SAH , but notvs. 21.7%, P <0.001), and was comparable to that (48.4%) in group of 0.5% CSF (CSF collected on 7 days post SAH)  and the increase was similar to that in cells treated with CSF   and the increase was similar between the BDNF and CSF (29.6%) treatments   (CSF notost SAH)  and 4. B-1, 42%) . BDNF treatments . In the eatments  than coneatments , and waseatments . Collectvs. 33.6%, P < 0.05)    . At 7 da < 0.05) , indicat < 0.01) . The per < 0.01) . The aboAdult neurogenesis is sensitive to pathologic stimuli, such as ischemic stroke and traumatic brain injury, which promote proliferation, differentiation, and migration of NSCs in the SVZ . InvestiAfter SAH, NSC proliferation in the SVZ declined in the first 3 days and then increased from days 5 to 7. The initial decline may result from a high concentration of free radicals derived from the degradation of hemoglobin and/or profound inflammation caused by the sudden increase of intracranial pressure , 15. Notin vivo and in vitro matching the time course of NSC activation and BDNF upregulation suggests possible neuronal recovery and reconstruction of neural circuitry. Infusion or viral overexpression of BDNF in the cerebroventricles of adult rats can cause marked neurogenesis in the olfactory bulbs and striatum [The activity of neurogenesis is mainly mediated by growth factors and/or neurotrophic factors and growth/neurotrophic factors differ in their relationship to particular pathological conditions . Among tstriatum , 30–32. striatum –41 or knstriatum , 43 willFrom post SAH day 5 to 7, the expression of BDNF increased in NSCs, astrocytes, and microglia in the SVZ and was especially high in NSCs on day 7 post SAH. BDNF from NSCs might further trigger neurogenesis in an autocrine and/or paracrine manner. Reacting to neural damage, astrocytes secrete many trophic factors such as BDNF –46. ActiAt present, there is no neuroprotective treatment for stroke, including subtypes of cerebral infarct, intracerebral hemorrhage, and SAH. Transgenic ablation of neurogenesis has been shown to impede motor function recovery after cerebral ischemia in mice, indicating the great importance of endogenous neurogenesis in the repair process following brain damage , 54. In In summary, we demonstrated that neurogenesis  was enhanced on days 5 and 7 post SAH. This enhancement was likely mediated by up-regulation of BDNF expression in NSCs, astrocytes, and microglia of the SVZ. This study may provide a rationale for the use of BDNF as a targeted SAH therapy, considering no curative or neuroprotective therapy for SAH exists.S1 Fig+  and nestin+  signals and as merged images  in the neurospheres of CSF treatment or control. Scale bar = 50 μm. Images of Ki67+ /nestin+ neural stem cells in the neurospheres treated with 0.5% CSF showing Ki67 (U) and nestin (V) immunoreactivity separately or as merged image (X). Scale bar = 16 μm (Y): Percentage of proliferating cells (Ki67+ of Hoechst+ cells) in the neurospheres. Means ± SD, 12 neurospheres were counted for each concentration. ns: non-significant; ***, P < 0.001, one way ANOVA with Tukey's multiple comparisons test.Primary cultured neurospheres treated with 0.25, 0.5, 1, and 2% CSF collected from rats on day 7 post SAH were immunostained with anti-nestin antibody (green: NSCs), anti-Ki67 antibody (red: proliferating cell) and Hoechst 33342 (blue: nucleus). (A)-(T): photomicrograph showing the distribution of Ki67(TIF)Click here for additional data file.S2 Fig+  signals and as merged images  in the neurospheres treated with or without CSF. Scale bar = 50 μm. (P): Percentage of Tuj-1+ area . Means ± SD, 12 neurospheres were counted for each condition. ns: non-significant; ***, P < 0.001, one way ANOVA with Tukey's multiple comparisons test.Neuronal differentiation were determined by immunocytostaining of cultured neurospheres with anti-Tuj-1 antibody (green: mature neuron) and Hoechst 33342 (blue: nucleus). (A)-(O): photomicrograph showing the distribution of Tuj-1(TIF)Click here for additional data file.S3 Fig+  signals and as merged images with Hoechst 33342 (blue: nucleus) . Scale bar = 50 μm. L (J): Percentage of Tuj-1+ area . Means ± SD, 12 neurospheres were counted for each condition. ns: non-significant; ***, P < 0.001, one way ANOVA with Tukey's multiple comparisons test.Neurospheres were immunostained with anti-Tuj-1 antibody (green: mature neuron) to determine the effects of recombinant BDNF (5 pg/ml) on neuronal differentiation of cultured neurospheres. The groups of neurospheres treated with 0.5% CSF (collected from rats on day 7 post SAH) and without treatment (control) were used for comparison. (A)-(I): photomicrograph showing the distribution of Tuj-1(TIF)Click here for additional data file.S4 FigMicroglial activation in the SVZ was determined by double immunostaining of the forebrain sections with anti-Iba-1 antibody (green: microglia), ED-1 antibody (red: activated microglia), and Hoechst 33342 (blue: nucleus). (A)-(T): photomicrograph showing the distribution of Iba-1+  and ED-1+  signals and as merged images  in the SVZ of animals with different SAH time course  and sham control. Scale bar = 50 μm. (U)-(X): Images of ED-1+ /Iba-1+ activated microglia in the SVZ on day 7 post SAH showing Iba-1+ (U) and ED-1+ (V) immunoreactivity separately or as merged image (X). Scale bar = 16 μm. (Y): Percentage of Iba-1+ of Hoechst+ cells in the SVZ; (Z): Percentage of ED-1+ of Hoechst+ cells in the SVZ; (Aa) Percentage of activated microglia (ED1+ of Iba-1+ cells) in the SVZ. Means ± SD, n = 6 for each time-point. *, P < 0.05;**, P < 0.01; ***, P < 0.001, one way ANOVA with Dunnett's multiple comparisons test.(TIF)Click here for additional data file.S1 Movie(MPG)Click here for additional data file.S2 Movie(MPG)Click here for additional data file.S3 Movie(MPG)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


R) genes is an effective and economical strategy to control the fungal pathogen Magnaporthe oryzae, the causal agent of the rice blast disease. Among the cloned blast resistance genes, Pi9, Pi2 and Piz-t confer broad-spectrum resistance to diverse M. oryzae isolates and were isolated from the Pi2/9 locus on chromosome 6. Identification and isolation of additional R genes with different resistance spectra from this locus will provide novel genetic resources for better control of this important rice disease.Utilization of broad-spectrum resistance  genes in the Nipponbare genome. Five bacterial artificial chromosome (BAC) clones spanning the region were identified, and a BAC contig covering the Pi2-2 locus was constructed.In this study, we identified a dominant Pi2/9 locus and fine-mapped the gene within a 270-kb region. Our results provide essential information for the isolation of the Pi2-2 gene and tightly linked DNA markers for rice blast resistance breeding.We identified a new allelic gene at the The online version of this article (doi:10.1186/1939-8433-5-29) contains supplementary material, which is available to authorized users. Magnaporthe oryzae, is one of the major limitations, and usually causes 10-30% yield loss in rice production when a rice blast epidemic occurs . Interestingly, most of them are NBS-LRR genes except Pi-d2 and pi21 ([Pi-d2 encodes a receptor-like kinase protein with a predicted extracellular domain of a bulb-type mannose-specific binding lectin (B-lectin) and an intracellular serine-threonine kinase domain  ([Pigm(t) ([Piz(t) ([Pi40(t) ([Pi50(t)  is incompatible to 97.7% of the 523 isolates from different regions of China  , Pigm(tPigm(t) , Piz(t)[Piz(t) , Pi40(tPi40(t)  and Pi5Pi50(t)  are in m China , 1999). . Pi2/9 luntries . Piz-t hailand . The nef China . HowevePiz(t), Pi-d(t) and Pi-kh(t), based on its disease reactions . To determine the genetic basis of broad-spectrum resistance in Jefferson, we performed greenhouse inoculations with individual isolates and genetic analysis using an F2 population derived from a cross between Jefferson and the susceptible cultivar CO39. We identified a dominant R gene in Jefferson on chromosome 6 at the Pi2/9 locus, named Pi2-2. Allelism analysis indicated that Pi2-2 is tightly linked or allelic to Pi9. We constructed a BAC contig in the genomic region and fine-mapped the gene within a region approximately 270 kb. These data will facilitate both the positional cloning of the R gene and molecular breeding programs of rice blast resistance.Jefferson, a long-grain tropical japonica cultivar grown in the southern U.S., has retained its resistance to blast since its first use in 1997 . It wasactions . Our prM. oryzae isolates collected from six countries, and the inoculation results are summarized in Additional filePi2-1 and Pi51 , indicating that the dominant inheritance of the R gene in Jefferson. The segregation of resistant and susceptible individuals in the F2 population fitted a ratio of 3:1 , suggesting that the resistance to 318–2 is controlled by a single dominant R gene in Jefferson. We designated this R gene in Jefferson as Pi2-2.The Piz(t), Pi-d(t) and Pi-kh(t), in Jefferson ([Piz(t) is located on chromosome 6 near the Pi2/9 locus ([Pi-d(t) and Pi-kh(t) are located on chromosome 11. Therefore, we selected 25 SSR markers around the Pi2/9 and Pi-kh loci for linkage analysis. Twenty highly resistant and twenty highly susceptible individuals from the F2 population of the Jefferson×CO39 cross were genotyped with the polymorphic markers. No marker around the Pi-kh locus co-segregated with the resistance to 318–2. But two polymorphic SSR markers around Pi2/9, RM7178 and RM7311 9 locus . Pi-d(tPi2 and Piz-t are tightly linked to Pi9 ([Piz(t) is allelic or tightly linked to Piz-t ([Piz(t) has not been determined yet. To understand the linkage relationship between Pi2-2 and the R genes in the same region, we developed an F2 population from a cross between Jefferson and Pi9- carrying line 75-1-127 for allelism test. A total of 637 F2 individuals were inoculated with M. oryzae isolate 318–2, which was incompatible to both Jefferson and 75-1-127, to observe the phenotype segregation. No susceptible plant was found in 637 F2 individuals, suggesting that Pi2-2 is tightly linked or allelic to the Pi9 gene.Previous studies showed that  to Pi9 , 2007) a aPi2 ando Piz-t . HoweveR genes at Pi2/9 locus have different resistance spectra. 75-1-127 (Pi9) was susceptible to ROR1, a M. oryzae strain from Korea. The isolate CHNOS60-2-3 from China could distinguish C101A51 (Pi2) and Toride (Piz-t) resistance specificities ([Piz-t) or 5173 (Pi2). These results suggest that Pi2-2 is a different R gene at the Pi2/9 locus. However, isogenic lines with all the R genes at the Pi2/9 locus should be used in inoculations with different isolates to confirm the conclusion.Previous research showed that the three cloned icities . HowevePi2-2 gene, another 14 SSR markers were used, and four of them exhibited polymorphism between the two parental lines  was constructed  and PK (protein kinase) genes are the 5′ and 3′ boundaries of the Pi2/9 locus, respectively, these are highly conserved in different haplotypes  was constructed that covered both Pi2-2 and the whole Pi2/9 locus in Jefferson , Pi-d(t) and Pik-h(t), were reported in Jefferson ([Pi-d(t) and Pik-h(t) are tightly linked on chromosome 11. Piz(t) was originally reported in the U.S. rice cultivar Zenith ([Piz(t) was mapped on the short arm of chromosome 6, close to the centromere, by several groups using different cultivars ([Pi2-2 is likely Piz(t). Our on-going cloning effort of the Pi2-2 gene will provide us the answer in the near future.Three blast resistance genes, fferson . Pi-dltivars , Toride (Piz-t), 75-1-127 (Pi9) and CO39, were used in this study. F1 and F2 populations from a cross between Jefferson and highly susceptible cultivar CO39 were constructed for genetic analysis. The F2 population derived from a cross between Jefferson and 75-1-127 was constructed for allelism tests.Seven rice cultivars, Jefferson, Tianye, XZ3150, 5173  and then kept in darkness at 25°C-27°C and over 90% relative humidity for 24 h. The inoculated plants were subsequently kept under a 12/12 (day/night) photoperiod at the same temperature and relative humidity. Disease reaction evaluation was carried out 7 days after inoculation according to the 0–5 scoring system described by[The 28 cribed by.2 population was inoculated with the M. oryzae isolate 318–2, which is avirulent to Jefferson and virulent to CO39. 318–2, which is also avirulent to 75-1-127, was employed to inoculate the Jefferson×75-1-127 F2 population for allelism analysis.The Jefferson×CO39 FPi2/9 and Pik loci were used for the polymorphism survey between Jefferson and CO39. Six polymorphic SSR markers spanning the Pi2/9 locus were used for preliminary and fine mapping of the R gene in Jefferson  ([http://rice.plantbiology.msu.edu/) ([http://ricegaas.dna.affrc.go.jp/)  . The geu.edu/)  and Ricgo.jp/) .Pi2-2 were used for screening of positive clones from the BAC pools. The contig map spanning the Pi2-2 locus was constructed based on the end sequencing results of the positive BAC clones.The genomic BAC library of Jefferson was constructed using the method described by. The marTable S1. Disease reaction of Jefferson and other 4 cultivars to 28 M. oryzaeisolates collected from different regions. (DOC 203 KB)Additional file 1:Authors’ original file for figure 1Authors’ original file for figure 2Below are the links to the authors’ original submitted files for images.