Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83–99)

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83–99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.     

Identification of sparse neural functional connectivity using penalized likelihood estimation and basis functions

One key problem in computational neuroscience and neural engineering is the identification and modeling of functional connectivity in the brain using spike train data. To reduce model complexity, alleviate overfitting, and thus facilitate model interpretation, sparse representation and estimation of functional connectivity is needed. Sparsities include global sparsity, which captures the sparse connectivities between neurons, and local sparsity, which reflects the active temporal ranges of the input-output dynamical interactions. In this paper, we formulate a generalized functional additive model (GFAM) and develop the associated penalized likelihood estimation methods for such a modeling problem. A GFAM consists of a set of basis functions convolving the input signals, and a link function generating the firing probability of the output neuron from the summation of the convolutions weighted by the sought model coefficients. Model sparsities are achieved by using various penalized likelihood estimations and basis functions. Specifically, we introduce two variations of the GFAM using a global basis (e.g., Laguerre basis) and group LASSO estimation, and a local basis (e.g., B-spline basis) and group bridge estimation, respectively. We further develop an optimization method based on quadratic approximation of the likelihood function for the estimation of these models. Simulation and experimental results show that both group-LASSO-Laguerre and group-bridge-B-spline can capture faithfully the global sparsities, while the latter can replicate accurately and simultaneously both global and local sparsities. The sparse models outperform the full models estimated with the standard maximum likelihood method in out-of-sample predictions.     

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14⁺ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.     

Omega-3 Eicosapentaenoic Acid Decreases CD133 Colon Cancer Stem-Like Cell Marker Expression While Increasing Sensitivity to Chemotherapy

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil.     

Proliferation-Linked Apoptosis of Adoptively Transferred T Cells after IL-15 Administration in Macaques

The adoptive transfer of antigen-specific effector T cells is being used to treat human infections and malignancy. T cell persistence is a prerequisite for therapeutic efficacy, but reliably establishing a high-level and durable T cell response by transferring cultured CD8⁺ T cells remains challenging. Thus, strategies that promote a transferred high-level T cell response may improve the efficacy of T cell therapy. Lymphodepletion enhances persistence of transferred T cells in mice in part by reducing competition for IL-15, a common γ-chain cytokine that promotes T cell memory, but lymphodepleting regimens have toxicity. IL-15 can be safely administered and has minimal effects on CD4⁺ regulatory T cells at low doses, making it an attractive adjunct in adoptive T cell therapy. Here, we show in lymphoreplete macaca nemestrina, that proliferation of adoptively transferred central memory-derived CD8⁺ effector T (T_CM/E) cells is enhanced in vivo by administering IL-15. T_CM/E cells migrated to memory niches, persisted, and acquired both central memory and effector memory phenotypes regardless of the cytokine treatment. Unexpectedly, despite maintaining T cell proliferation, IL-15 did not augment the magnitude of the transferred T cell response in blood, bone marrow, or lymph nodes. T cells induced to proliferate by IL-15 displayed increased apoptosis demonstrating that enhanced cycling was balanced by cell death. These results suggest that homeostatic mechanisms that regulate T cell numbers may interfere with strategies to augment a high-level T cell response by adoptive transfer of CD8⁺ T_CM/E cells in lymphoreplete hosts.     

Cecal Ligation and Puncture Results in Long-Term Central Nervous System Myeloid Inflammation

Survivors of sepsis often experience long-term cognitive and functional decline. Previous studies utilizing lipopolysaccharide injection and cecal ligation and puncture in rodent models of sepsis have demonstrated changes in depressive-like behavior and learning and memory after sepsis, as well as evidence of myeloid inflammation and cytokine expression in the brain, but the long-term course of neuroinflammation after sepsis remains unclear. Here, we utilize cecal ligation and puncture with greater than 80% survival as a model of sepsis. We found that sepsis survivor mice demonstrate deficits in extinction of conditioned fear, but no acquisition of fear conditioning, nearly two months after sepsis. These cognitive changes occur in the absence of neuronal loss or changes in synaptic density in the hippocampus. Sepsis also resulted in infiltration of monocytes and neutrophils into the CNS at least two weeks after sepsis in a CCR2 independent manner. Cellular inflammation is accompanied by long-term expression of pro-inflammatory cytokine and chemokine genes, including TNFα and CCR2 ligands, in whole brain homogenates. Gene expression analysis of microglia revealed that while microglia do express anti-microbial genes and damage-associated molecular pattern molecules of the S100A family of genes at least 2 weeks after sepsis, they do not express the cytokines observed in whole brain homogenates. Our results indicate that in a naturalistic model of infection, sepsis results in long-term neuroinflammation, and that this sustained inflammation is likely due to interactions among multiple cell types, including resident microglia and peripherally derived myeloid cells.     

Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease causing indefinite fatigue. ME/CFS has long been hypothesised to have an infectious cause; however, no specific infectious agent has been identified. We used metagenomics to analyse the RNA from plasma samples from 25 individuals with ME/CFS and compare their microbial content to technical controls as well as three control groups: individuals with alternatively diagnosed chronic Lyme syndrome (N = 13), systemic lupus erythematosus (N = 11), and healthy controls (N = 25). We found that the majority of sequencing reads were removed during host subtraction, thus there was very low microbial RNA content in the plasma. The effects of sample batching and contamination during sample processing proved to outweigh the effects of study group on microbial RNA content, as the few differences in bacterial or viral RNA abundance we did observe between study groups were most likely caused by contamination and batch effects. Our results highlight the importance of including negative controls in all metagenomic analyses, since there was considerable overlap between bacterial content identified in study samples and control samples. For example, Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodes were found in both study samples and plasma-free negative controls. Many of the taxonomic groups we saw in our plasma-free negative control samples have previously been associated with diseases, including ME/CFS, demonstrating how incorrect conclusions may arise if controls are not used and batch effects not accounted for.     

Serum 25-Hydroxyvitamin D Concentrations ≥40 ng/ml Are Associated with >65% Lower Cancer Risk: Pooled Analysis of Randomized Trial and Prospective Cohort Study

BackgroundHigher serum 25-hydroxyvitamin D [25(OH)D] concentrations have been associated with a lower risk of multiple cancer types across a range of 25(OH)D concentrations.ObjectivesTo investigate whether the previously reported inverse association between 25(OH)D and cancer risk could be replicated, and if a 25(OH)D response region could be identified among women aged 55 years and older across a broad range of 25(OH)D concentrations.MethodsData from two cohorts representing different median 25(OH)D concentrations were pooled to afford a broader range of 25(OH)D concentrations than either cohort alone: the Lappe cohort (N = 1,169), a randomized clinical trial cohort (median 25(OH)D = 30 ng/ml) and the GrassrootsHealth cohort (N = 1,135), a prospective cohort (median 25(OH)D = 48 ng/ml). Cancer incidence over a multi-year period (median: 3.9 years) was compared according to 25(OH)D concentration. Kaplan-Meier plots were developed and the association between 25(OH)D and cancer risk was examined with multivariate Cox regression using multiple 25(OH)D measurements and spline functions. The study included all invasive cancers excluding skin cancer.ResultsAge-adjusted cancer incidence across the combined cohort (N = 2,304) was 840 cases per 100,000 person-years (1,020 per 100,000 person-years in the Lappe cohort and 722 per 100,000 person-years in the GrassrootsHealth cohort). Incidence was lower at higher concentrations of 25(OH)D. Women with 25(OH)D concentrations ≥40 ng/ml had a 67% lower risk of cancer than women with concentrations <20 ng/ml (HR = 0.33, 95% CI = 0.12–0.90).Conclusions25(OH)D concentrations ≥40 ng/ml were associated with substantial reduction in risk of all invasive cancers combined.     

Glucocorticoid Receptor (NR3C1) Variants Associate with the Muscle Strength and Size Response to Resistance Training

Glucocorticoid receptor (NR3C1) polymorphisms associate with obesity, muscle strength, and cortisol sensitivity. We examined associations among four NR3C1 polymorphisms and the muscle response to resistance training (RT). European-American adults (n = 602, 23.8±0.4yr) completed a 12 week unilateral arm RT program. Maximum voluntary contraction (MVC) assessed isometric strength (kg) and MRI assessed biceps size (cm²) pre- and post-resistance training. Subjects were genotyped for NR3C1 -2722G>A, -1887G>A, -1017T>C, and +363A>G. Men carrying the -2722G allele gained less relative MVC (17.3±1.2vs33.5±6.1%) (p = 0.010) than AA homozygotes; men with -1887GG gained greater relative MVC than A allele carriers (19.6±1.4vs13.2±2.3%) (p = 0.016). Women carrying the -1017T allele gained greater relative size (18.7±0.5vs16.1±0.9%) (p = 0.016) than CC homozygotes. We found sex-specific NR3C1 associations with the muscle strength and size response to RT. Future studies should investigate whether these associations are partially explained by cortisol’s actions in muscle tissue as they interact with sex differences in cortisol production.