Cyclization of PLP139-151 peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis.

We report the novel synthesis of cyclic PLP139-151 (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H147 replaces W144 and F151-K150 replace H147 as TCR contacts, which may explain the difference on each peptide's response.

Imputation-powered whole-exome analysis identifies genes associated with kidney function and disease in the UK Biobank.

Genome-wide association studies have discovered hundreds of associations between common genotypes and kidney function but cannot comprehensively investigate rare coding variants. Here, we apply a genotype imputation approach to whole exome sequencing data from the UK Biobank to increase sample size from 166,891 to 408,511. We detect 158 rare variants and 105 genes significantly associated with one or more of five kidney function traits, including genes not previously linked to kidney disease in humans. The imputation-powered findings derive support from clinical record-based kidney disease information, such as for a previously unreported splice allele in PKD2, and from functional studies of a previously unreported frameshift allele in CLDN10. This cost-efficient approach boosts statistical power to detect and characterize both known and novel disease susceptibility variants and genes, can be generalized to larger future studies, and generates a comprehensive resource ( https://ckdgen-ukbb.gm.eurac.edu/ ) to direct experimental and clinical studies of kidney disease.

A complementary role for the tetraspanins CD37 and Tssc6 in cellular immunity.

The cooperative nature of tetraspanin-tetraspanin interactions in membrane organization suggests functional overlap is likely to be important in tetraspanin biology. Previous functional studies of the tetraspanins CD37 and Tssc6 in the immune system found that both CD37 and Tssc6 regulate T cell proliferative responses in vitro. CD37(-/-) mice also displayed a hyper-stimulatory dendritic cell phenotype and dysregulated humoral responses. In this study, we characterize "double knockout" mice (CD37(-/-)Tssc6(-/-)) generated to investigate functional overlap between these tetraspanins. Strong evidence for a cooperative role for these two proteins was identified in cellular immunity, where both in vitro T cell proliferative responses and dendritic cell stimulation capacity are significantly exaggerated in CD37(-/-)Tssc6(-/-) mice when compared with single knockout counterparts. Despite these exaggerated cellular responses in vitro, CD37(-/-)Tssc6(-/-) mice are not more susceptible to autoimmune induction. However, in vivo responses to pathogens appear poor in CD37(-/-)Tssc6(-/-) mice, which showed a reduced ability to produce influenza-specific T cells and displayed a rapid onset hyper-parasitemia when infected with Plasmodium yoelii. Therefore, in the absence of both CD37 and Tssc6, immune function is further altered when compared with CD37(-/-) or Tssc6(-/-) mice, demonstrating a complementary role for these two molecules in cellular immunity.

Conformational studies of immunodominant myelin basic protein 1-11 analogues using NMR and molecular modeling.

Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP(1-11)) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP(1-11)[4A]) or a tyrosine residue (Ac-MBP(1-11)[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-A(u) was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln(3) residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-A(u) complex, has a different orientation in the mutated analogues especially in the Ac-MBP(1-11)[4A] peptide. In particular the side chain of Gln(3) is not solvent exposed as for the native Ac-MBP(1-11) and it is not available for interaction with the TCR.

Evaluation of supervised machine-learning methods for predicting appearance traits from DNA.

The prediction of human externally visible characteristics (EVCs) based solely on DNA information has become an established approach in forensic and anthropological genetics in recent years. While for a large set of EVCs, predictive models have already been established using multinomial logistic regression (MLR), the prediction performances of other possible classification methods have not been thoroughly investigated thus far. Motivated by the question to identify a potential classifier that outperforms these specific trait models, we conducted a systematic comparison between the widely used MLR and three popular machine learning (ML) classifiers, namely support vector machines (SVM), random forest (RF) and artificial neural networks (ANN), that have shown good performance outside EVC prediction. As examples, we used eye, hair and skin color categories as phenotypes and genotypes based on the previously established IrisPlex, HIrisPlex, and HIrisPlex-S DNA markers. We compared and assessed the performances of each of the four methods, complemented by detailed hyperparameter tuning that was applied to some of the methods in order to maximize their performance. Overall, we observed that all four classification methods showed rather similar performance, with no method being substantially superior to the others for any of the traits, although performances varied slightly across the different traits and more so across the trait categories. Hence, based on our findings, none of the ML methods applied here provide any advantage on appearance prediction, at least when it comes to the categorical pigmentation traits and the selected DNA markers used here.

Testing the impact of trait prevalence priors in Bayesian-based genetic prediction modeling of human appearance traits.

The prediction of appearance traits by use of solely genetic information has become an established approach and a number of statistical prediction models have already been developed for this purpose. However, given limited knowledge on appearance genetics, currently available models are incomplete and do not include all causal genetic variants as predictors. Therefore such prediction models may benefit from the inclusion of additional information that acts as a proxy for this unknown genetic background. Use of priors, possibly informed by trait category prevalence values in biogeographic ancestry groups, in a Bayesian framework may thus improve the prediction accuracy of previously predicted externally visible characteristics, but has not been investigated as of yet. In this study, we assessed the impact of using trait prevalence-informed priors on the prediction performance in Bayesian models for eye, hair and skin color as well as hair structure and freckles in comparison to the respective prior-free models. Those prior-free models were either similarly defined either very close to the already established ones by using a reduced predictive marker set. However, these differences in the number of the predictive markers should not affect significantly our main outcomes. We observed that such priors often had a strong effect on the prediction performance, but to varying degrees between different traits and also different trait categories, with some categories barely showing an effect. While we found potential for improving the prediction accuracy of many of the appearance trait categories tested by using priors, our analyses also showed that misspecification of those prior values often severely diminished the accuracy compared to the respective prior-free approach. This emphasizes the importance of accurate specification of prevalence-informed priors in Bayesian prediction modeling of appearance traits. However, the existing literature knowledge on spatial prevalence is sparse for most appearance traits, including those investigated here. Due to the limitations in appearance trait prevalence knowledge, our results render the use of trait prevalence-informed priors in DNA-based appearance trait prediction currently infeasible.

Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation.

Leuprolide [DLeu6, NHEt10]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr5(OMe), DLeu6, Aze9, NHEt10]GnRH (1) and [Tyr5(OMe), DLeu6, NHEt10]GnRH (2) have been studied in DMSO and H2O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a ß-turn type I in the 2-5 segments of Leuprolide and its linear analogues in the case of using DMSO-d6 as solvent, whereas a ß-turn type II in the 3-6 segments is indicated using D2O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.

Anti-CD20 Agents for Multiple Sclerosis: Spotlight on Ocrelizumab and Ofatumumab.

Until recently, in the pathogenesis of Multiple Sclerosis (MS), the contribution of B cells has been largely underestimated, and the disease was considered a T-cell-mediated disorder. However, newer evidence shows that B cells play a crucial role in the pathogenesis of MS via antigen-driven autoantibody responses and through the cross regulation of T-helper cells. As B cells express the surface molecule CD20 at all points of differentiation, it provides a specific target for monoclonal antibodies, and the development and clinical testing of anti-CD20 antibody treatments for MS have been successful. After some observations, some small clinical trials found positive effects for the first anti-CD20 therapeutic rituximab in MS; newer agents have been specifically evaluated, resulting in the development of ocrelizumab and ofatumumab. Ocrelizumab, a humanized anti-CD20 monoclonal antibody, was approved in March 2017 by the Food and Drug Administration (FDA) and is also the first proven therapy to reduce disability progression in primary progressive MS. This is particularly significant considering that disease-modifying treatment options are few for both primary and secondary progressive MS. Ofatumumab, a fully human anti-CD20 monoclonal antibody, that binds a distinct epitope, has been further investigated in phase 3 trials for relapsing forms of MS. In this review, we discuss in detail these two anti-CD20 agents and their advent for treatment of MS.

Altered peptide ligands of myelin basic protein ( MBP87-99 ) conjugated to reduced mannan modulate immune responses in mice.

Mutations of peptides to generate altered peptide ligands, capable of switching immune responses from T helper 1 (Th1) to T helper 2 (Th2), are promising candidates for the immunotherapy of autoimmune diseases such as multiple sclerosis (MS). We synthesized two mutant peptides from myelin basic protein 87-99 (MBP(87-99)), an immunodominant peptide epitope identified in MS. Mutations of residues K(91) and P(96), known to be critical T-cell receptor (TCR) contact sites, resulted in the mutant peptides [R(91), A(96)]MBP(87-99) and [A(91), A(96)]MBP(87-99). Immunization of mice with these altered peptide ligands emulsified in complete Freund's adjuvant induced both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) responses compared with only IFN-gamma responses induced to the native MBP(87-99) peptide. It was of interest that [R(91), A(96)]MBP(87-99) conjugated to reduced mannan induced 70% less IFN-gamma compared with the native MBP(87-99) peptide. However, [A(91), A(96)]MBP(87-99) conjugated to reduced mannan did not induce IFN-gamma-secreting T cells, but elicited very high levels of interleukin-4 (IL-4). Furthermore, antibodies generated to [A(91), A(96)]MBP(87-99) peptide conjugated to reduced mannan did not cross-react with the native MBP(87-99) peptide. By molecular modelling of the mutant peptides in complex with major histocompatibility complex (MHC) class II, I-A(s), novel interactions were noted. It is clear that the double-mutant peptide analogue [A(91), A(96)]MBP(87-99) conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies investigating potential treatments for MS.

Mannosylation of mutated MBP83-99 peptides diverts immune responses from Th1 to Th2.

Multiple sclerosis (MS) is an autoimmune demyelinating disease mediated primarily by CD4+ T cells. The design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of MS. We designed and synthesized a number of peptide analogs by mutating the principal TCR contact residue based on MBP83-99 epitope and these peptides were conjugated to reduced mannan. Immune responses were diverted from Th1 to Th2 in SJL/J mice and generated antibodies which did not cross-react with native MBP protein. Peptide [Y91]MBP83-99 gave the best cytokine and antibody profile and constitutes a promising candidate peptide for immunotherapy of MS. Structural alignment of existing crystal structures revealed the peptide binding motif of I-As. Molecular modeling was used to identify H-bonding and van der Waals interactions between peptides and MHC (I-A(s)).

True colors: A literature review on the spatial distribution of eye and hair pigmentation.

DNA-based prediction of externally visible characteristics has become an established approach in forensic genetics, with the aim of tracing individuals who are potentially unknown to the investigating authorities but without using this prediction as evidence in court. While a number of prediction models have been proposed, use of prior probabilities in those models has largely been absent. Here, we aim at compiling information on the spatial distribution of eye and hair coloration in order to use this as prior knowledge to improve prediction accuracy. To this end, we conducted a detailed literature review and created maps showing the eye and hair pigmentation prevalence both by countries with available information and by interpolation in order to obtain prior estimates for populations without available data. Furthermore, we assessed the association between these two traits in a very large data set. A strong limitation was the quite low amount of available data, especially outside Europe. We hope that our results will facilitate the improvement of already existing and of novel prediction methods for pigmentation traits and induce further studies on the spatial distribution of these traits.

A double mutation of MBP(83-99) peptide induces IL-4 responses and antagonizes IFN-gamma responses.

A number of treatment options are available to multiple sclerosis patients, however this needs to be improved. Herein, we designed and synthesized a number of peptides by mutating principal TCR contact residues based on MBP(83-99) peptide epitope. Immunization of SJL/J mice with MBP(83-99) and mutant [A(91)]MBP(83-99), [E(91)]MBP(83-99), [F(91)]MBP(83-99), [Y(91)]MBP(83-99), and [R(91), A(96)]MBP(83-99) peptides, induced IFN-gamma, and only [R(91), A(96)]MBP(83-99) mutant peptide was able to induce IL-4 secretion by T cells. T cells against the native MBP(83-99) peptide cross-reacted with all peptides except [Y(91)]MBP(83-99) and [R(91),A(96)]MBP(83-99). The double mutant [R(91), A(96)]MBP(83-99) was able to antagonize IFN-gamma production in vitro by T cells against the native MBP(83-99) peptide. Antibodies generated to [R(91), A(96)]MBP(83-99) did not cross-react with whole MBP protein. Molecular modeling between peptide analogs and H2 I-A(s) demonstrated novel interactions. The [R(91), A(96)]MBP(83-99) double mutant peptide analog is the most promising for further therapeutic studies.

Towards immunotherapeutic drugs and vaccines against multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. Numerous treatment options are available to MS patients; however, these options need to be improved. Herein, we review the current drugs and therapeutic approaches available to MS patients, preclinical trial interventions and recent animal model studies for the potential therapy of MS. Since the current treatment of MS remains elusive and is limited, animal studies and clinical research offers an optimistic outlook.

Multiple Sclerosis: Immunopathology and Treatment Update.

The treatment of multiple sclerosis (MS) has changed over the last 20 years. All immunotherapeutic drugs target relapsing remitting MS (RRMS) and it still remains a medical challenge in MS to develop a treatment for progressive forms. The most common injectable disease-modifying therapies in RRMS include ß-interferons 1a or 1b and glatiramer acetate. However, one of the major challenges of injectable disease-modifying therapies has been poor treatment adherence with approximately 50% of patients discontinuing the therapy within the first year. Herein, we go back to the basics to understand the immunopathophysiology of MS to gain insights in the development of new improved drug treatments. We present current disease-modifying therapies (interferons, glatiramer acetate, dimethyl fumarate, teriflunomide, fingolimod, mitoxantrone), humanized monoclonal antibodies (natalizumab, ofatumumb, ocrelizumab, alentuzumab, daclizumab) and emerging immune modulating approaches (stem cells, DNA vaccines, nanoparticles, altered peptide ligands) for the treatment of MS.

Round and round we go: cyclic peptides in disease.

There is a need for novel drugs for the treatment of infectious diseases, autoimmunity and cancer. Cyclic peptides constitute a class of compounds that have made crucial contributions to the treatment of certain diseases. Penicillin, Vancomycin, Cyclosporin, the Echinocandins and Bleomycin are well-known cyclic peptides. Cyclic peptides, compared to linear peptides, have been considered to have greater potential as therapeutic agents due to their increased chemical and enzymatic stability, receptor selectively, and improved pharmacodynamic properties. They have been used as synthetic immunogens, transmembrane ion channels, antigens for Herpes Simplex Virus, potential immunotherapeutic vaccines for diabetes and Experimental Autoimmune Encephalomyelitis - an animal model of Multiple Sclerosis, as inhibitors against alpha-amylase and as protein stabilizers. Herein, we review important cyclic peptides as therapeutic agents in disease.

Relationship between exhaled nitric oxide levels and compliance with inhaled corticosteroids in asthmatic children.

Levels of exhaled nitric oxide (eNO) are elevated in subjects with asthma and fall in response to oral or inhaled steroids. This study explored the possibility the measurement of eNO levels could be used to identify subjects who were not adhering to their treatment regimen. Twenty children with asthma attending the respiratory clinic were recruited. Each attended on four occasions 1 month apart when eNO levels were measured. A data logger attached to a pressurised metered dose inhaler was used to objectively monitor use of inhaled corticosteroids (ICSs). The correlation between day and dose compliance with eNO was assessed. The data demonstrated a weak but non-significant correlation between eNO and both day (r = 0.055, P = 0.67) and dose (r = 0.153, P = 0.23). A recorded value of eNO less than 12 was associated with day compliance rates of 3-97%. Of the 19 recorded eNO values greater than 12 ppb almost 80% were from subjects with a day compliance of less than 50% during the preceding month. Of the four values greater than 12 ppb and day compliance > 60% one subject had a poor inhaler technique, one had a mild viral exacerbation and one appeared to be associated with increase pollen exposure. The measurement of eNO may prove to be a useful tool in helping to manage children with asthma but further work is required to define its precise role. Elevated eNO levels in asthmatic children taking ICSs are likely to reflect poor compliance but confounding factors such as disease activity and inhaler technique need to be carefully considered.

Immune responses of linear and cyclic PLP139-151 mutant peptides in SJL/J mice: peptides in their free state versus mannan conjugation.

BACKGROUND: The predominant proteins of the CNS are myelin basic protein, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein. PLP139-151 is one of the major encephalitogenic epitopes of PLP. The epitope PLP139-151 binds to MHC class II (I-A(s)) of SJL/J mice and induces Th1 responses. AIM: The aim was to synthesize and test the immunological activity and cyclic analogs of PLP139-151 peptide and determine the immunological differences between adjuvant and conjugation to mannan. Materials & methods: We designed and synthesized cyclic peptides based on the linear PLP139-151 epitope by mutating critical T-cell receptor contact sites of residues W(144) and H(147), resulting in the mutant peptides PLP139-151, [L(144), R(147)]PLP139-151 or cyclo(139-151)PLP139-151 and cyclo(139-151) [L(144), R(147)]PLP139-151. In this study, mice were immunized with mutant peptides either emulsified in complete Freund's adjuvant or conjugated to reduced mannan and responses were assessed. RESULTS: Linear double-mutant peptide [L(144), R(147)]PLP139-151 induced high levels of IL-4 responses and low levels of IgG total, and cyclization of this analog elicited low levels of IFN-γ. Moreover, linear [L(144), R(147)]PLP139-151 conjugated to reduced mannan did not induce IFN-γ, whilst both linear agonist PLP139-151 and cyclic agonist cyclo(139-151)PLP139-151 induced IFN-γ-secreting T cells. Molecular dynamics simulations of linear and cyclic(139-151)PLP139-151 analogs indicated the difference in topology of the most important for biological activity amino acids. CONCLUSION: Cyclic double-mutant analog cyclo(139-151) [L(144), R(147)]PLP139-151 has potential for further studies for the immunotherapy of multiple sclerosis.

Methods to measure T-cell responses.

A successful vaccine for immunotherapy, particularly for solid tumors or viral infections, requires a suitable target antigen and the production of a cytotoxic T-cell response. In addition, CD4 T cells play an important role in cellular immunity. Here, we briefly discuss methods by which T cells are measured in vitro after vaccination.

Design and synthesis of a cyclic double mutant peptide (cyclo(87-99)[A91,A96]MBP87-99) induces altered responses in mice after conjugation to mannan: implications in the immunotherapy of multiple sclerosis.

Altered peptide ligands that alter immune responses are a promising approach to the immunotherapy of multiple sclerosis. Cyclic peptides are of interest because the limited stability of linear peptides restricts their use in vivo. We designed and synthesized a cyclic double mutant peptide from MBP(87-99)-[cyclo(87-99)[A(91),A(96)]MBP(87-99)]. Immunization of mice, in CFA reduced Th1 responses. However, when conjugated to reduced mannan, a significant further reduction of Th1 responses and moderate Th2 responses were induced.