Chemical structures, extraction and analysis technologies, and bioactivities of edible fungal polysaccharides from Poria cocos: An updated review.

Poria cocos is a popular medicinal food. Polysaccharides are the key component of Poria cocos, forming 70-90 % of the dry sclerotia mass. Recent studies indicate that Poria cocos polysaccharides (PCP-Cs) have multiple beneficial functions and applications. A literature search was conducted using the Web of Science Core Collection and PubMed databases. For this review, we provided an updated research progress in chemical structures, various extraction and analysis technologies, bioactivities of PCP-Cs, and insights into the directions for future research. The main polysaccharides identified in Poria cocos are water-soluble polysaccharides and acidic polysaccharides. Hot water, alkali, supercritical fluid, ultrasonic, enzyme, and deep eutectic solvent-based methods are the most common methods for PCP-Cs extraction. Technologies such as near-infrared spectroscopy, high-performance liquid chromatography, and ultraviolet-visible spectrophotometry, are commonly used to evaluate the qualities of PCP-Cs. In addition, PCP-Cs have antioxidant, immunomodulatory, neuroregulatory, anticancer, hepatoprotective, and gut microbiota regulatory properties. Future research is needed to focus on scaling up extraction, enhancing quality control, elucidating mechanisms of bioactivities, and the utilisation of PCP-Cs in food industries. Overall, Poria cocos is a good source of edible fungi polysaccharides, which can be developed into functional foods with potential health benefits.

Spirulina platensis Suppressed iNOS and Proinflammatory Cytokines in Lipopolysaccharide-Induced BV2 Microglia.

The disease burden of neurodegenerative diseases is on the rise due to the aging population, and neuroinflammation is one of the underlying causes. Spirulina platensis is a well-known superfood with numerous reported bioactivities. However, the effect of S. platensis Universiti Malaya Algae Culture Collection 159 (UMACC 159) (a strain isolated from Israel) on proinflammatory mediators and cytokines remains unknown. In this study, we aimed to determine the anti-neuroinflammatory activity of S. platensis extracts and identify the potential bioactive compounds. S. platensis extracts (hexane, ethyl acetate, ethanol, and aqueous) were screened for phytochemical content and antioxidant activity. Ethanol extract was studied for its effect on proinflammatory mediators and cytokines in lipopolysaccharide (LPS)-induced BV2 microglia. The potential bioactive compounds were identified using liquid chromatography-mass spectrometric (LC-MS) analysis. Ethanol extract had the highest flavonoid content and antioxidant and nitric oxide (NO) inhibitory activity. Ethanol extract completely inhibited the production of NO via the downregulation of inducible NO synthase (iNOS) and significantly reduced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Emmotin A, palmitic amide, and 1-monopalmitin, which might play an important role in cell signaling, have been identified. In conclusion, S. platensis ethanol extract inhibited neuroinflammation through the downregulation of NO, TNF-α and IL-6. This preliminary study provided insight into compound(s) isolation, which could contribute to the development of precision nutrition for disease management.

Carrageenophyte Kappaphycus malesianus Inhibits Microglia-Mediated Neuroinflammation via Suppression of AKT/NF-κB and ERK Signaling Pathways.

Neuroinflammation is an inflammatory response in any part of the central nervous system triggered by the activation of microglia and astrocytes to produce proinflammatory cytokines in the brain. However, overproduction of proinflammatory cytokines further contributes to the development of neurodegenerative disorders. Red seaweed, Kappaphycus malesianus, is a predominant carrageenophyte commercially cultivated in Semporna, Sabah, Malaysia. It is an important source of raw material for kappa-carrageenan productions in the food, pharmaceutical and cosmetics industries. However, no studies have been conducted focusing on the antineuroinflammatory effects of K. malesianus. The aim of the present study was to investigate the effect of the antineuroinflammatory activity of K. malesianus extracts (ethyl acetate, ethanol and methanol) on lipopolysaccharide-stimulated BV2 microglia and the underlying mechanisms involved in the regulation of neuroinflammatory pathways. Extract with the most promising antineuroinflammatory activity was analyzed using liquid chromatography-mass spectrometry (LC-MS). Our results show that methanol extract has a convincing antineuroinflammatory effect by suppressing both AKT/NF-κB and ERK signaling pathways to inhibit the expression of all proinflammatory cytokines without causing a cytotoxicity effect. LC-MS analysis of methanol extract revealed two compounds: prosopinine and eplerenone. Our findings indicated that metabolites of K. malesianus are potent antineuroinflammatory agents with respect to prevention of neurological disorders.

Association of SPI1 Haplotypes with Altered SPI1 Gene Expression and Alzheimer's Disease Risk.

BACKGROUND: Genetic studies reveal that single-nucleotide polymorphisms (SNPs) of SPI1 are associated with Alzheimer's disease (AD), while their effects in the Chinese population remain unclear. OBJECTIVE: We aimed to examine the AD-association of SPI1 SNPs in the Chinese population and investigate the underlying mechanisms of these SNPs in modulating AD risk. METHODS: We conducted a genetic analysis of three SPI1 SNPs (i.e., rs1057233, rs3740688, and rs78245530) in a Chinese cohort (n = 333 patients with AD, n = 721 normal controls). We also probed public European-descent AD cohorts and gene expression datasets to investigate the putative functions of those SNPs. RESULTS: We showed that SPI1 SNP rs3740688 is significantly associated with AD in the Chinese population (odds ratio [OR] = 0.72 [0.58-0.89]) and identified AD-protective SPI1 haplotypes ß (tagged by rs1057233 and rs3740688) and γ (tagged by rs3740688 and rs78245530). Specifically, haplotypes ß and γ are associated with decreased SPI1 gene expression level in the blood and brain tissues, respectively. The regulatory roles of these haplotypes are potentially mediated by changes in miRNA binding and the epigenetic landscape. Our results suggest that the AD-protective SPI1 haplotypes regulate pathways involved in immune and neuronal functions. CONCLUSION: This study is the first to report a significant association of SPI1 with AD in the Chinese population. It also identifies SPI1 haplotypes that are associated with SPI1 gene expression and decreased AD risk.

An IL1RL1 genetic variant lowers soluble ST2 levels and the risk effects of APOE-ε4 in female patients with Alzheimer's disease.

Changes in the levels of circulating proteins are associated with Alzheimer's disease (AD), whereas their pathogenic roles in AD are unclear. Here, we identified soluble ST2 (sST2), a decoy receptor of interleukin-33-ST2 signaling, as a new disease-causing factor in AD. Increased circulating sST2 level is associated with more severe pathological changes in female individuals with AD. Genome-wide association analysis and CRISPR-Cas9 genome editing identified rs1921622 , a genetic variant in an enhancer element of IL1RL1, which downregulates gene and protein levels of sST2. Mendelian randomization analysis using genetic variants, including rs1921622 , demonstrated that decreased sST2 levels lower AD risk and related endophenotypes in females carrying the Apolipoprotein E (APOE)-ε4 genotype; the association is stronger in Chinese than in European-descent populations. Human and mouse transcriptome and immunohistochemical studies showed that rs1921622 /sST2 regulates amyloid-beta (Aß) pathology through the modulation of microglial activation and Aß clearance. These findings demonstrate how sST2 level is modulated by a genetic variation and plays a disease-causing role in females with AD.

Large-scale plasma proteomic profiling identifies a high-performance biomarker panel for Alzheimer's disease screening and staging.

INTRODUCTION: Blood proteins are emerging as candidate biomarkers for Alzheimer's disease (AD). We systematically profiled the plasma proteome to identify novel AD blood biomarkers and develop a high-performance, blood-based test for AD. METHODS: We quantified 1160 plasma proteins in a Hong Kong Chinese cohort by high-throughput proximity extension assay and validated the results in an independent cohort. In subgroup analyses, plasma biomarkers for amyloid, tau, phosphorylated tau, and neurodegeneration were used as endophenotypes of AD. RESULTS: We identified 429 proteins that were dysregulated in AD plasma. We selected 19 "hub proteins" representative of the AD plasma protein profile, which formed the basis of a scoring system that accurately classified clinical AD (area under the curve  = 0.9690-0.9816) and associated endophenotypes. Moreover, specific hub proteins exhibit disease stage-dependent dysregulation, which can delineate AD stages. DISCUSSION: This study comprehensively profiled the AD plasma proteome and serves as a foundation for a high-performance, blood-based test for clinical AD screening and staging.

Factors delaying chemotherapy in patients with breast cancer at a safety-net hospital.

BACKGROUND: Despite advances in healthcare and improved chemotherapy, disparities in breast cancer outcomes continue to persist. Our aim was to evaluate socioeconomic factors that may impact timing of treatment for patients receiving chemotherapy in underserved communities. METHODS: A review of patients with breast cancer who received neoadjuvant or adjuvant chemotherapy from 2015-2019 was conducted at a safety-net hospital. The primary outcomes were times from diagnosis to chemotherapy and surgery. Clinicodemographic factors including race, age, clinical stage, primary language, comorbidities, and median income by zip code were collected. Multivariable regression analysis was performed to evaluate for factors associated with the primary outcomes. RESULTS: One hundred patients were identified. For the neoadjuvant group, median time from diagnosis to chemotherapy and surgery was 52 ± 34 days and 256 ± 59 days, respectively. For the adjuvant group, median time from diagnosis to surgery and chemotherapy was 24.5 ± 18 days and 94.5 ± 53 days, respectively. Non-English language and older age were associated with increased time to chemotherapy in the adjuvant group (p < 0.05). Language and age were not associated with increased time to surgery in both groups. Race, age, comorbidities, and income were not associated with delay in treatment in either groups. CONCLUSIONS: Older age and non-English language were associated with prolonged time from surgery to adjuvant chemotherapy. Targeted interventions directed at patient education and decreasing language barriers especially post-operatively may decrease delays in treatment and subsequently reduce disparities seen in the breast cancer population.

CONJUGATION WITH SILVER NANOPARTICLES ENHANCES ANTI-ACANTHAMOEBIC ACTIVITY OF KAPPAPHYCUS ALVAREZII.

Nanomedicine has the potential in enhancing the efficacy and bioavailability of anti-infective agents. Here we determined whether conjugation of the Malaysian cultivated seaweed Kappaphycus alvarezii with silver-conjugated nanoparticles enhanced anti-acanthamoebic properties. Silver-conjugated K. alvarezii were successfully synthesized, followed by characterization with Fourier transform infrared spectroscopy, ultraviolet-visible spectrophotometry, and transmission electron microscopy. Amoebicidal effects were evaluated against Acanthamoeba castellanii, and cytotoxicity assays were performed using HaCaT cells. Viability assays revealed that silver nanoparticles conjugated with K. alvarezii extract exhibited significant antiamoebic properties (P < 0.05). Nano-conjugates induced the production of reactive oxygen species. Importantly, silver-conjugated extract inhibited amoeba-mediated host cell damage as established by lactate dehydrogenase release. Neither the nano-conjugates nor the extract showed cytotoxicity against human cells in vitro. Liquid chromatography and mass spectroscopy revealed several molecules, including 2,6-nonadien-1-ol, N-desmethyl trifluoperazine, dulciol B, lucidumol A, acetoxolone, 2-[4,6-bis(2,4-dimethylphenyl)-1,3,5-triazin-2-yl]-5-(octyloxy)phenol, C16 sphinganine, 22-tricosenoic acid, and ß-dihydrorotenone, of which dulciol B and C16 sphinganine are known to possess antimicrobial activities. In summary, marine organisms are an important source of bioactive molecules with anti-acanthamoebic properties that can be enhanced by conjugating with silver nanoparticles. Natural products combined with nanotechnology using multifunctional nanoparticle complexes can deliver therapeutic agents effectively and hold promise in the development of new formulations of anti-acanthamoebic agents.

Multiple behavior-specific cancellation signals contribute to suppressing predictable sensory reafference in a cerebellum-like structure.

Movement induces sensory stimulation of an animal's own sensory receptors, termed reafference. With a few exceptions, notably vestibular and proprioception, this reafference is unwanted sensory noise and must be selectively filtered in order to detect relevant external sensory signals. In the cerebellum-like electrosensory nucleus of elasmobranch fish, an adaptive filter preserves novel signals by generating cancellation signals that suppress predictable reafference. A parallel fiber network supplies the principal Purkinje-like neurons (called ascending efferent neurons, AENs) with behavior-associated internal reference signals, including motor corollary discharge and sensory feedback, from which predictive cancellation signals are formed. How distinct behavior-specific cancellation signals interact within AENs when multiple behaviors co-occur and produce complex, changing patterns of reafference is unknown. Here, we show that when multiple streams of internal reference signals are available, cancellation signals form that are specific to parallel fiber inputs temporally correlated with, and therefore predictive of, sensory reafference. A single AEN has the capacity to form more than one cancellation signal, and AENs form multiple cancellation signals simultaneously and modify them independently during co-occurring behaviors. Cancellation signals update incrementally during continuous behaviors, as well as episodic bouts of behavior that last minutes to hours. Finally, individual AENs, independently of their neighbors, form unique AEN-specific cancellation signals that depend on the particular sensory reafferent input it receives. Together, these results demonstrate the capacity of the adaptive filter to utilize multiple cancellation signals to suppress dynamic patterns of reafference arising from complex co-occurring and intermittently performed behaviors.

FcεR1-expressing nociceptors trigger allergic airway inflammation.

BACKGROUND: Lung nociceptor neurons amplify immune cell activity and mucus metaplasia in response to an inhaled allergen challenge in sensitized mice. OBJECTIVE: We sought to identify the cellular mechanisms by which these sensory neurons are activated subsequent to allergen exposure. METHODS: We used calcium microscopy and electrophysiologic recording to assess whether vagal neurons directly respond to the model allergen ovalbumin (OVA). Next, we generated the first nociceptor-specific FcεR1γ knockdown (TRPV1Cre::FcεR1γfl/fl) mice to assess whether this targeted invalidation would affect the severity of allergic inflammation in response to allergen challenges. RESULTS: Lung-innervating jugular nodose complex ganglion neurons express the high-affinity IgE receptor FcεR1, the levels of which increase in OVA-sensitized mice. FcεR1γ-expressing vagal nociceptor neurons respond directly to OVA complexed with IgE with depolarization, action potential firing, calcium influx, and neuropeptide release. Activation of vagal neurons by IgE-allergen immune complexes, through the release of substance P from their peripheral terminals, directly amplifies TH2 cell influx and polarization in the airways. Allergic airway inflammation is decreased in TRPV1Cre::FcεR1γfl/fl mice and in FcεR1α-/- mice into which bone marrow has been transplanted. Finally, increased in vivo circulating levels of IgE following allergen sensitization enhances the responsiveness of FcεR1 to immune complexes in both mouse jugular nodose complex ganglion neurons and human induced pluripotent stem cell-derived nociceptors. CONCLUSIONS: Allergen sensitization triggers a feedforward inflammatory loop between IgE-producing plasma cells, FcεR1-expressing vagal sensory neurons, and TH2 cells, which helps to both initiate and amplify allergic airway inflammation. These data highlight a novel target for reducing allergy, namely, FcεR1γ expressed by nociceptors.

Genetic and polygenic risk score analysis for Alzheimer's disease in the Chinese population.

INTRODUCTION: Dozens of Alzheimer's disease (AD)-associated loci have been identified in European-descent populations, but their effects have not been thoroughly investigated in the Hong Kong Chinese population. METHODS: TaqMan array genotyping was performed for known AD-associated variants in a Hong Kong Chinese cohort. Regression analysis was conducted to study the associations of variants with AD-associated traits and biomarkers. Lasso regression was applied to establish a polygenic risk score (PRS) model for AD risk prediction. RESULTS: SORL1 is associated with AD in the Hong Kong Chinese population. Meta-analysis corroborates the AD-protective effect of the SORL1 rs11218343 C allele. The PRS is developed and associated with AD risk, cognitive status, and AD-related endophenotypes. TREM2 H157Y might influence the amyloid beta 42/40 ratio and levels of immune-associated proteins in plasma. DISCUSSION: SORL1 is associated with AD in the Hong Kong Chinese population. The PRS model can predict AD risk and cognitive status in this population.

Neuronal, stromal, and T-regulatory cell crosstalk in murine skeletal muscle.

A distinct population of Foxp3+CD4+ regulatory T (Treg) cells promotes repair of acutely or chronically injured skeletal muscle. The accumulation of these cells depends critically on interleukin (IL)-33 produced by local mesenchymal stromal cells (mSCs). An intriguing physical association among muscle nerves, IL-33+ mSCs, and Tregs has been reported, and invites a deeper exploration of this cell triumvirate. Here we evidence a striking proximity between IL-33+ muscle mSCs and both large-fiber nerve bundles and small-fiber sensory neurons; report that muscle mSCs transcribe an array of genes encoding neuropeptides, neuropeptide receptors, and other nerve-related proteins; define muscle mSC subtypes that express both IL-33 and the receptor for the calcitonin-gene-related peptide (CGRP); and demonstrate that up- or down-tuning of CGRP signals augments or diminishes, respectively, IL-33 production by muscle mSCs and later accumulation of muscle Tregs. Indeed, a single injection of CGRP induced much of the genetic program elicited in mSCs early after acute skeletal muscle injury. These findings highlight neural/stromal/immune-cell crosstalk in tissue repair, suggesting future therapeutic approaches.

Gut-Innervating Nociceptor Neurons Regulate Peyer's Patch Microfold Cells and SFB Levels to Mediate Salmonella Host Defense.

Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.

An Intestinal Organ Culture System Uncovers a Role for the Nervous System in Microbe-Immune Crosstalk.

Investigation of host-environment interactions in the gut would benefit from a culture system that maintained tissue architecture yet allowed tight experimental control. We devised a microfabricated organ culture system that viably preserves the normal multicellular composition of the mouse intestine, with luminal flow to control perturbations (e.g., microbes, drugs). It enables studying short-term responses of diverse gut components (immune, neuronal, etc.). We focused on the early response to bacteria that induce either Th17 or RORg+ T-regulatory (Treg) cells in vivo. Transcriptional responses partially reproduced in vivo signatures, but these microbes elicited diametrically opposite changes in expression of a neuronal-specific gene set, notably nociceptive neuropeptides. We demonstrated activation of sensory neurons by microbes, correlating with RORg+ Treg induction. Colonic RORg+ Treg frequencies increased in mice lacking TAC1 neuropeptide precursor and decreased in capsaicin-diet fed mice. Thus, differential engagement of the enteric nervous system may partake in bifurcating pro- or anti-inflammatory responses to microbes.

Silencing Nociceptor Neurons Reduces Allergic Airway Inflammation.

Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8(+) sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4(+) and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma.

Variable processing and cross-presentation of HIV by dendritic cells and macrophages shapes CTL immunodominance and immune escape.

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.

Different antigen-processing activities in dendritic cells, macrophages, and monocytes lead to uneven production of HIV epitopes and affect CTL recognition.

Dendritic cells (DCs), macrophages (MPs), and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous Ags preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum translocation, trimming, and MHC-I presentation. In this study, we compared the capacity of DCs, MPs, and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848, and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs, and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs, and monocytes. Differences in Ag-processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load.

Metabolic profiling of plasma amino acids shows that histidine increases following the consumption of pork.

Amino acid (AA) status is determined by factors including nutrition, metabolic rate, and interactions between the metabolism of AA, carbohydrates, and lipids. Analysis of the plasma AA profile, together with markers of glucose and lipid metabolism, will shed light on metabolic regulation. The objectives of this study were to investigate the acute responses to the consumption of meals containing either pork (PM) or chicken (CM), and to identify relationships between plasma AA and markers of glycemic and lipemic control. A secondary aim was to explore AA predictors of plasma zinc concentrations. Ten healthy adults participated in a postprandial study on two separate occasions. In a randomized cross-over design, participants consumed PM or CM. The concentrations of 21 AA, glucose, insulin, triglycerides, nonesterified fatty acids, and zinc were determined over 5 hours postprandially. The meal composition did not influence glucose, insulin, triglyceride, nonesterified fatty acid, or zinc concentrations. Plasma histidine was higher following the consumption of PM (P=0.014), with consistently higher changes observed after 60 minutes (P<0.001). Greater percentage increases were noted at limited time points for valine and leucine + isoleucine in those who consumed CM compared to PM. In linear regression, some AAs emerged as predictors of the metabolic responses, irrespective of the meal that was consumed. The present study demonstrates that a single meal of PM or CM produces a differential profile of AA in the postprandial state. The sustained increase in histidine following the consumption of a PM is consistent with the reported effects of lean pork on cardiometabolic risk factors.

A real-time killing assay to follow viral epitope presentation to CD8 T cells.

The ability of cytotoxic T lymphocytes (CTL) to clear virus-infected cells requires the presentation of viral peptides intracellularly processed and displayed by major histocompatibility complex class I. Assays to measure CTL-mediated killing often use peptides exogenously added onto target cells--which does not account for epitope processing--or follow killing of infected cells at a single time point. In this study we established a real-time fluorogenic cytotoxic assay that measures the release of the Glucose-6-phosphate-dehydrogenase by dying target cells every 5 min after addition of CTL. It has comparable sensitivity to (51)chromium-based killing assay with the additional advantage of incorporating the kinetics of epitope presentation. We showed that HIV infection of immortalized or primary CD4 T cells leads to asynchronous killing by two CTL clones specific for epitopes located in different proteins. Real-time monitoring of killing of virus-infected cells will enable identification of immune responses efficiently preventing virus dissemination.

A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells.

BACKGROUND: Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. RESULTS: In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. CONCLUSION: By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.