Conformationally selective multidimensional chemical shift ranges in proteins from a PACSY database purged using intrinsic quality criteria

We have determined refined multidimensional chemical shift ranges for intra-residue correlations ([superscript 13]C–[superscript 13]C, [superscript 15]N–[superscript 13]C, etc.) in proteins, which can be used to gain type-assignment and/or secondary-structure information from experimental NMR spectra. The chemical-shift ranges are the result of a statistical analysis of the PACSY database of >3000 proteins with 3D structures (1,200,207 [superscript 13]C chemical shifts and >3 million chemical shifts in total); these data were originally derived from the Biological Magnetic Resonance Data Bank. Using relatively simple non-parametric statistics to find peak maxima in the distributions of helix, sheet, coil and turn chemical shifts, and without the use of limited “hand-picked” data sets, we show that ~94 % of the [superscript 13]C NMR data and almost all [superscript 15]N data are quite accurately referenced and assigned, with smaller standard deviations (0.2 and 0.8 ppm, respectively) than recognized previously. On the other hand, approximately 6 % of the [superscript 13]C chemical shift data in the PACSY database are shown to be clearly misreferenced, mostly by ca. −2.4 ppm. The removal of the misreferenced data and other outliers by this purging by intrinsic quality criteria (PIQC) allows for reliable identification of secondary maxima in the two-dimensional chemical-shift distributions already pre-separated by secondary structure. We demonstrate that some of these correspond to specific regions in the Ramachandran plot, including left-handed helix dihedral angles, reflect unusual hydrogen bonding, or are due to the influence of a following proline residue. With appropriate smoothing, significantly more tightly defined chemical shift ranges are obtained for each amino acid type in the different secondary structures. These chemical shift ranges, which may be defined at any statistical threshold, can be used for amino-acid type assignment and secondary-structure analysis of chemical shifts from intra-residue cross peaks by inspection or by using a provided command-line Python script (PLUQin), which should be useful in protein structure determination. The refined chemical shift distributions are utilized in a simple quality test (SQAT) that should be applied to new protein NMR data before deposition in a databank, and they could benefit many other chemical-shift based tools.National Institutes of Health (U.S.) (Grant GM066976

Real-world, long-term effectiveness of allergy immunotherapy in allergic rhinitis: Subgroup analyses of the REACT study

Background: Randomized controlled trials have demonstrated the efficacy of allergy immunotherapy (AIT) in allergic rhinitis (AR) and the disease-modifying effects of the SQ grass sublingual immunotherapy (SLIT) tablet. // Objective: We sought to assess real-world, long-term effectiveness and safety across AIT subgroups: route of administration, therapeutic allergen, persistence to AIT, and SQ grass SLIT tablet. // Methods: The primary outcome of AR prescriptions from a retrospective cohort study (REAl-world effeCtiveness in allergy immunoTherapy; 2007-2017) was assessed across prespecified AIT subgroups in subjects with AR with and without AIT prescriptions (controls). Safety was assessed as anaphylaxis for 2 days or less of the first AIT prescription. Subgroup follow-up continued until samples were fewer than 200 subjects. // Results: Subcutaneous immunotherapy (SCIT) and SLIT tablets showed similarly greater reductions in AR prescriptions than controls (SCIT vs SLIT tablets: year 3, P = .15; year 5, P = .43). Comparably greater reductions in AR prescriptions were observed for grass- and house dust mite–specific AIT than for controls, but significantly smaller reductions were observed for tree-specific AIT (tree vs house dust mite, and vs grass: years 3 and 5, P < .0001). Persistence to AIT was associated with greater reductions in AR prescriptions versus nonpersistence (persistence vs nonpersistence: year 3, P = .09; year 5, P = .006). SQ grass SLIT tablet showed sustained reductions versus controls for up to 7 years (year 3, P = .002; year 5, P = .03). Rates of anaphylactic shock were low (0.000%-0.092%), with no events for SQ SLIT tablets. // Conclusions: These results demonstrate real-world, long-term effectiveness of AIT, complement disease-modifying effects observed in SQ grass SLIT-tablet randomized controlled trials, and highlight the importance of using newer evidence-based AIT products for tree pollen AR

Intracerebral Human Regulatory T Cells: Analysis of CD4+CD25+FOXP3+ T Cells in Brain Lesions and Cerebrospinal Fluid of Multiple Sclerosis Patients

Impaired suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45ROhiCD95hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3+ and CD4+ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4+ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45ROhiCD95hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion

Regulatory T cells inhibit Fas ligand-induced innate and adaptive tumour immunity

CD4+CD25+ regulatory T cells (Treg) are known to influence T cell responses to tumours. Here we have explored the role of Treg in inhibiting not only adaptive, but also innate immune responses to tumours. To this end we used a Fas ligand (FasL)-expressing melanoma cell line in a mouse model. In this system, innate immunity is sufficient to reject the tumour. All mice depleted of Treg and challenged with FasL-expressing melanoma remained tumour-free. Investigation of the underlying cellular effector mechanisms revealed that depletion of Treg enhanced an NK cell response capable of tumour lysis. Furthermore, this initial innate immune response primed mice to make an effective adaptive immune response leading to complete rejection of challenge with the parental melanoma. Both antigen-specific antibody and CD4+ T cells were implicated in protection via adaptive immunity. We conclude that removal of Treg and vaccination with whole tumour cells expressing FasL activates multiple arms of the immune system, leading to efficient tumour rejection. These findings highlight a novel role for FasL in inducing innate immune responses that are normally inhibited by Treg and uncover an adjuvant effect of FasL that can be used to stimulate tumour immunity after depletion of Treg

Resistance of Foxp3+ Regulatory T Cells to Nur77-Induced Apoptosis Promotes Allograft Survival

The NR4A nuclear receptor family member Nur77 (NR4A1) promotes thymocyte apoptosis during negative selection of autoreactive thymocytes, but may also function in mature extrathymic T cells. We studied the effects of over-expression of Nur77 on the apoptosis of murine peripheral T cells, including thymic-derived Foxp3+ regulatory (Treg) cells. Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by ∼80% compared to wild-type (WT) mice. However, the proportions of Treg cells were markedly increased in the thymus (61% of CD4+Foxp3+ singly positive thymocytes vs. 8% in WT) and secondary lymphoid organs (40–50% of CD4+Foxp3+ T cells vs. 7–8% in WT) of Nur77 transgenic (Nur77Tg) mice, and immunoprecipitation studies showed Nur77 was associated with a recently identified HDAC7/Foxp3 transcriptional complex. Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis. Fully allogeneic cardiac grafts transplanted to Nur77Tg mice survived long-term with well-preserved structure, and recipient splenocytes showed markedly enhanced apoptosis and greatly reduced anti-donor recall responses. Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9. Allograft rejection was restored by CD25 monoclonal antibody therapy, indicating that allograft acceptance was dependent upon Treg function in Nur77Tg recipients. These data show that compared to conventional CD4 and CD8 T cells, Foxp3+ Tregs are relatively resistant to Nur77-mediated apoptosis, and that tipping the balance between the numbers of Tregs and responder T cells in the early period post-transplantation can determine the fate of the allograft. Hence, induced expression of Nur77 might be a novel means to achieve long-term allograft survival

Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.

BACKGROUND: In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment. METHODS: Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis. RESULTS: More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method. CONCLUSION: Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations

Impaired Thymic Export and Apoptosis Contribute to Regulatory T-Cell Defects in Patients with Chronic Heart Failure

Animal studies suggest that regulatory T (T(reg)) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of T(reg) cells in patients with chronic heart failure (CHF). However, the mechanisms behind T(reg-)cell defects remained unknown. We here sought to elucidate the mechanism of T(reg-)cell defects in CHF patients.We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4(+)CD25(+)FOXP3(+)CD45RO(-)CD45RA(+) naïve T(reg) (nT(reg)) cells and CD4(+)CD25(+)FOXP3(+)CD45RO(+)CD45RA(-) memory T(reg) (mT(reg)) cells in CHF patients as compared with non-CHF controls. Moreover, the nT(reg)/mT(reg) ratio (p<0.01), CD4(+)CD25(+)FOXP3(+)CD45RO(-) CD45RA(+)CD31(+) recent thymic emigrant T(reg) cell (RTE-T(reg)) frequency (p<0.01), and T-cell receptor excision circle levels in T(reg) cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral T(reg) cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that T(reg)-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls.Our data suggested that the T(reg)-cell defects of CHF patients were likely caused by decreased thymic output of nascent T(reg) cells and increased susceptibility to apoptosis in the periphery

Effector and Naturally Occurring Regulatory T Cells Display No Abnormalities in Activation Induced Cell Death in NOD Mice

BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-), FoxP3(-)) and suppressor (CD25(+), FoxP3(+)) CD4(+) T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4(+) subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+)CD25(-) T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis

Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles

Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21–24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches