Necrosis Factor Receptor Superfamily Member Research Articles

In silico molecular docking of marchantins and tumor necrosis factor receptor superfamily member 6 for apoptosis agonist activity

One of the peculiar properties of cancer cells is low incidence of apoptosis which leads the cells to divide in an uncontrollable manner. The present study aimed to predict the anticancer and apoptosis agonist activity of several marchantins active compounds present in Marchantia polymorpha retrieved from a database. In silico molecular docking was performed between marchantins and apoptosis induction receptor, tumor necrosis factor receptor superfamily member 6 (TNFRSF6). Additionally, screening on prediction of activity spectra for substances (PASS) was performed to predict apoptosis agonist activity of marchantins. A total of 11 marchnatins were docked with TNFRSF6. All marchantins showed docking score more negative than native ligand, and marchantin F was found to be the most effective with docking score of -64.8342. The results indicated the potency of marchantins to act as anticancer agents via induction of apoptosis by binding to TNF receptor. Thus, investigations focusing on isolation of these bioactive compounds might be useful to explore marchantins as novel drug candidates for cancer treatment.

Epidemiology of Amyloidosis and Genetic Pathways to Diagnosis and Typing

We reviewed our studies on epidemiology and germline genetics of amyloidosis. In epidemiology, we considered both hereditary and non-hereditary amyloidosis. As the source of data, we used the nationwide Swedish hospital discharge register. We estimated the incidence of hereditary ATTR amyloidosis, for which Sweden is a global endemic area, at 2/million. Surprisingly, the disease was also endemic within Sweden; the incidence in the province with the highest incidence was 100 times higher than in the rest of Sweden. Risk of non-Hodgkin lymphoma increased five-fold in the affected individuals. Among non-hereditary amyloidosis, the incidence for AL amyloidosis (abbreviated as AL) was estimated at 3.2/million, with a median survival time of 3 years. Secondary systemic amyloidosis (most likely AA amyloidosis) showed an incidence of 1.15/million for combined sexes. The female rate was two times higher than the male rate, probably relating to the higher female prevalence of rheumatoid arthritis. The median survival time was 4 years. We also identified patients who likely had familial autoinflammatory disease, characterized by early onset and immigrant background from the Eastern Mediterranean area. Young Syrian descendants had the highest incidence rate, which was over 500 times higher than that in individuals with Swedish parents. Germline genetics focused on AL on which we carried out a genome-wide association study (GWAS) in three AL cohorts (N = 1129) from Germany, UK, and Italy. Single nucleotide polymorphisms (SNPs) at 10 loci showed evidence of an association at p < 10−5; some of these were previously documented to influence multiple myeloma (MM) risk, including the SNP at the IRF4 binding site. In AL, SNP rs9344 at the splice site of cyclin D1, influencing translocation (11;14), reached the highest significance, p = 7.80 × 10−11; the SNP was only marginally significant in MM. The locus close to gene SMARCD3, involved in chromatin remodeling, was also significant. These data provide evidence for common genetic susceptibility to AL and MM. We continued by analyzing genetic associations in nine clinical profiles, characterized by organ involvement or Ig profiles. The light chain only (LCO) profile associated with the SNP at the splice site of cyclin D1 with p = 1.99 × 10−12. Even for the other profiles, distinct genetic associations were found. It was concluded that the strong association of rs9344 with LCO and t(11;14) amyloidosis offer attractive mechanistic clues to AL causation. Mendelian randomization analysis identified associations of AL with increased blood monocyte counts and the tumor necrosis factor receptor superfamily member 17 (TNFRSF17 alias BCMA) protein. Two other associations with the TNFRSF members were found. We discuss the corollaries of the findings with the recent success of treating t(11;14) AL with a novel drug venetoclax, and the application of BCMA as the common target of plasma cell immunotherapies.

Search for AL amyloidosis risk factors using Mendelian randomization

In amyloid light chain (AL) amyloidosis, amyloid fibrils derived from immunoglobulin light chain are deposited in many organs, interfering with their function. The etiology of AL amyloidosis is poorly understood. Summary data from genome-wide association studies (GWASs) of multiple phenotypes can be exploited by Mendelian randomization (MR) methodology to search for factors influencing AL amyloidosis risk. We performed a 2-sample MR analyzing 72 phenotypes, proxied by 3461 genetic variants, and summary genetic data from a GWAS of 1129 AL amyloidosis cases and 7589 controls. Associations with a Bonferroni-defined significance level were observed for genetically predicted increased monocyte counts (P = 3.8 × 10-4) and the tumor necrosis factor receptor superfamily member 17 (TNFRSF17) gene (P = 3.4 × 10-5). Two other associations with the TNFRSF (members 6 and 19L) reached a nominal significance level. The association between genetically predicted decreased fibrinogen levels may be related to roles of fibrinogen other than blood clotting. be related to its nonhemostatic role. It is plausible that a causal relationship with monocyte concentration could be explained by selection of a light chain-producing clone during progression of monoclonal gammopathy of unknown significance toward AL amyloidosis. Because TNFRSF proteins have key functions in lymphocyte biology, it is entirely plausible that they offer a potential link to AL amyloidosis pathophysiology. Our study provides insight into AL amyloidosis etiology, suggesting high circulating levels of monocytes and TNFRSF proteins as risk factors.

Raspberry Extract With Potential Antitumor Activity Against Cervical Cancer.

Cervical cancer (CC) is one of the leading causes of mortality worldwide. Previously, we reported that blueberry extract constrains the growth of CC. Raspberry is a widely consumed fruit that exhibits antitumor properties against several cancer types but little is known about its direct effect on CC. This study was designed to investigate the potential antitumor effect of raspberry extract (RE) on CC cells and to elucidate the possible mechanisms behind it. Clonogenic survival assay and caspase-3 activity kits were used to evaluate the effects of RE on cell survival, proliferation, and apoptosis of a widely used CC cell line, HeLa. Possible molecular mechanisms were investigated using reverse transcription-polymerase chain reaction. The percentage of colonies and optic density value of HeLa cells decreased in the presence of RE in comparison to controls. Relative caspase-3 activity in cancer cells increased in the presence of RE in comparison to controls. The antitumor effect displayed on HeLa cells by RE was associated with the increased expression of antiproliferative molecule P53 and the increased expression of pro-apoptotic molecule tumor necrosis factor receptor superfamily member 6 (FAS). RE displays anticancer activity against CC HeLa cells. The mechanism behind this is by up-regulation of anti-proliferative molecule P53 and pro-apoptotic molecule FAS.

Abstract 495: TNFR2 and PD-1/PD-L1/TNFR2 humanized mice aiding exploration of combination strategies to overcome tumor immune escape mechanisms

Abstract Tumor necrosis factor receptor 2 (TNFR2), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), is a transmembrane protein, which can mediate both pro- and anti-inflammatory activities of T cells. TNFR2 is mainly expressed on the surface of activated effector T cells and regulatory T cells (Tregs) and promotes proliferation and survival of Tregs through nuclear factor kappa B (NF-κB). Blockade of TNF-TNFR2 interaction with monoclonal antibodies can inhibit the activation of Tregs, possibly resulting in inhibition of their function and/or reduction of their numbers. Yet, until recently the potential of TNFR2 as a therapeutic target for cancer therapy has been underappreciated. Discovery of anti-TNFR2 antibodies, developed to inhibit NF-κB driven growth, has revived excitement for the use of TNFR2as a cancer therapy target and stressed the demand for suitable pre-clinical models to evaluate the safety and efficacy of TNFR2-targeted therapeutics. Biocytogen has generated a humanized TNFR2 mouse model for both in vitro function validation of signaling pathways and in vivo efficacy evaluation of TNFR2 antibodies. In this model, the exons 2~6 of mouse Tnfrsf1b gene which encode the extracellular domain were replaced by human TNFRSF1B counterparts. In the humanized mouse, human TNFR2 was detectable on Tregs in the spleen and the anti-human TNFR2 antibodies associated well with the splenocytes. In addition, anti-human TNFR2 antibodies bound well to CD3+ T cells and inhibited tumor growth. Furthermore, Biocytogen has also generated PD-1/PD-L1/TNFR2 multi-gene humanized mice, in which blood cell composition, morphology and the levels of ALT, AST and other indicators were similar to the wild-type counterpart. Basal leukocyte subpopulations including T/B/NK cells, DC, granulocytes, and monocytes/macrophages, were similar between humanized and wild-type mice. Moreover, using the MC38 tumor model, the combination of anti-TNFR2 antibody and anti-PD-1 (or anti-PD-L1) shows enhanced effect compared to the individual groups, demonstrating that the PD-1/PD-L1/TNFR2 humanized mice have a potential to provide a powerful preclinical model for in vivo evaluation of combination therapies. In conclusion, TNFR2 and PD-1/PD-L1/TNFR2 humanized mice are a useful tool to explore anti-PD-1, anti-PD-L1 and anti-TNFR2 combination therapy strategies with promising activity and low cytotoxicity. Citation Format: Huilin Li, Xiaofei Zhou, Jing Zhang, Veronika Chromikova, Qingcong Lin. TNFR2 and PD-1/PD-L1/TNFR2 humanized mice aiding exploration of combination strategies to overcome tumor immune escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 495.

Plasma Fms-Related Tyrosine Kinase 3 Ligand (Flt3L) Levels at Diagnosis Correlate with Treatment Response in Chronic Myeloid Leukemia

Background: Proteins such as cytokines and growth factors are important in cellular communication. They are also known to mediate messages between cancer cells and the immune system. CML is known to be an immunogenic cancer and function of the immune system has been linked to good treatment responses and successful treatment discontinuation. To understand the crosstalk between leukemic cells and the microenvironment we analyzed the plasma cytokine profile of CML patients with multiplex proteomic array at diagnosis and during treatment.Methods: Peripheral blood (PB) samples were collected from 33 CML patients at diagnosis and during treatment (3 and 12 months after treatment initiation). Patients received either bosutinib, imatinib or dasatinib as first-line therapy. The dasatinib treated patients received low-dose IFN-a after 3 months of dasatinib monotherapy. In addition, PB of 17 healthy volunteers was collected. The plasma cytokine profile was studied with multiplexed cytokine and growth factor panel that consists of 92 proteins (Proseek Multiplex Inflammation I96×96, Olink). Wilcox test was used to determine significant differences between patient and healthy volunteers, and p<0.05 was used as a cut-off value. Ingenuity pathway analysis (IPA) was performed to characterize aberrant disease associated pathways in CML.Results: The analysis of the cytokine panel showed that 43 proteins were upregulated in CML patients at diagnosis compared to the healthy. The IPA demonstrated that these cytokines and growth factors were related to the granulocyte adhesion and diapedesis and Th1 pathway. Top upstream regulators of these cytokines were NFKB1, RELA, STAT3, NFKBIA and RELB. The most pronounced differences were observed in the following proteins: Interleukin 10 receptor, beta subunit (IL-10RB), tumor necrosis factor receptor superfamily member 9 (TNFRSF9), CD5, Chemokine (C-C motif) ligand 23 (CCL23), Caspase-8 (CASP-8), CD244, adenosine deaminase (ADA), tumor growth factor a (TGFA), hepatocyte growth factor (HGF), tumor necrosis factor superfamily member 14 (TNFSF14), S100 calcium-binding protein A12 (EN-RAGE), urokinase-type plasminogen activator (uPA), vascular endothelial growth factor A (VEGFA) and oncostatin M (OSM). The levels of most proteins normalized to the levels of the healthy controls already after 3 months of treatment initiation regardless of the tyrosine kinase inhibitor (TKI) used suggesting that these proteins were related to the leukemic growth. Only the levels of IL-10RB and TNFRSF9 remained high compared to the healthy even during treatment.Interestingly, some of the protein levels at diagnosis correlated with BCR-ABL1 values (IS) at 3 months. Proteins such as Flt3L (r=-0.45, p=0.01), MCP-4 (r=-0.42, p=0.02), AXIN1 (r=-0.39, p=0.03), CXCL10 (r=-0.38, p=0.04) and CCL19 (r=-0.44, p=0.01) had a negative correlation and IL-4 (r=0.40, p=0.03) had a positive correlation, but only the Flt3L (r=-0.44, p=0.01) correlated also with the BCR-ABL1 values at 12 months. Patients who were treated with dasatinib therapy had increased levels of Flt3L at 3 months (p=0.008) and the level further increased at 12 months after the addition of IFN-a compared to the patients who received only imatinib or bosutinib (p=0.01).Conclusions: Multiplex plasma proteomic analysis provides tools for biomarker discovery and treatment monitoring. During first 3 months of TKI therapy most of the plasma cytokines and growth factors which were upregulated at the time of diagnosis returned to the levels of healthy controls. Flt3L levels at the diagnosis correlated negatively with the treatment response during the TKI therapy. As Flt3L is known to increase stem cell proliferation, low levels of Flt3L may lead to more quiescent stem cells and possible therapy resistance. During dasatinib and IFN-a treatment Flt3L levels increased above the levels noted in healthy controls. DisclosuresMustjoki:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria.

57-OR: Predictive Role of Atherosclerosis Biomarkers on Carotid Plaque Extension in Type 2 Diabetes

Matrix metalloproteinases (MMP) and tumor necrosis factor receptor superfamily members have been recently proposed as surrogate markers of atherosclerosis in the general population. The aim of this study was to examine their predictive role in type 2 diabetes (T2D). Plasma levels of MMP-12, MMP-7, CD40 and its ligand (CD40-L) were measured by Proximity Extension Assay in a well-characterized cohort of 1,144 patients with carotid atherosclerosis from the SUMMIT study (age= 69 [64 - 75], males 66.7%, T2D 66.5%). Carotid plaque area was measured by plaque contours using the ultrasound trace function. The associations were adjusted for age, sex, waist, hypertension, history of cardiovascular diseases (CVD), smoke load, plasma triglycerides, LDL-cholesterol (LDL-c) and HDL-cholesterol (HDL-c), and serum creatinine levels. All measured biomarkers were associated with carotid plaque area, independently of potential confounders, with CD40 showing the strongest effect and MMP12 showing a significant interaction with T2D. In subgroup analyses, the effect of MMP-12 and MMP-7 remained significant only in T2D, while the effect of CD40 and CD40-L was similar in both subgroups. This study demonstrates the ability of MMP-12, MMP-7, CD40, and CD40-L in predicting carotid atherosclerotic burden in a population at high cardiovascular risk and identifies MMP-12 as a specific biomarker for T2D. Disclosure S. Leonetti: None. D. Trico: None. I. Goncalves: None. A. Shore: None. F. Khan: None. J. Nilsson: None. A. Natali: None. Funding Innovative Medicines Initiative (IMI-2008/115006)

Proteomic profiling for detection of early-stage heart failure in the community.

AimsBiomarkers may provide insights into molecular mechanisms underlying heart remodelling and dysfunction. Using a targeted proteomic approach, we aimed to identify circulating biomarkers associated with early stages of heart failure.Methods and resultsA total of 575 community‐based participants (mean age, 57 years; 51.7% women) underwent echocardiography and proteomic profiling (CVD II panel, Olink Proteomics). We applied partial least squares‐discriminant analysis (PLS‐DA) and a machine learning algorithm [eXtreme Gradient Boosting (XGBoost)] to identify key proteins associated with echocardiographic abnormalities. We used Gaussian mixture modelling for unbiased clustering to construct phenogroups based on influential proteins in PLS‐DA and XGBoost. Of 87 proteins, 13 were important in PLS‐DA and XGBoost modelling for detection of left ventricular remodelling, left ventricular diastolic dysfunction, and/or left atrial reservoir dysfunction: placental growth factor, kidney injury molecule‐1, prostasin, angiotensin‐converting enzyme‐2, galectin‐9, cathepsin L1, matrix metalloproteinase‐7, tumour necrosis factor receptor superfamily members 10A, 10B, and 11A, interleukins 6 and 16, and α1‐microglobulin/bikunin precursor. Based on these proteins, the clustering algorithm divided the cohort into two distinct phenogroups, with each cluster grouping individuals with a similar protein profile. Participants belonging to the second cluster (n = 118) were characterized by an unfavourable cardiovascular risk profile and adverse cardiac structure and function. The adjusted risk of presenting echocardiographic abnormalities was higher in this phenogroup than in the other (P < 0.0001).ConclusionsWe identified proteins related to renal function, extracellular matrix remodelling, angiogenesis, and inflammation to be associated with echocardiographic signs of early‐stage heart failure. Proteomic phenomapping discriminated individuals at high risk for cardiac remodelling and dysfunction.

Immunoglobulin A Autoreactivity toward Brain Enriched and Apoptosis-Regulating Proteins in Saliva of Athletes after Acute Concussion and Subconcussive Impacts.

The diagnosis and management of concussion is hindered by its diverse clinical presentation and assessment tools reliant on subjectively experienced symptoms. The biomechanical threshold of concussion is also not well understood, and asymptomatic concussion or "subconcussive impacts" of variable magnitudes are common in contact sports. Concerns have risen because athletes returning to activity too soon have an increased risk of prolonged recovery or long-term adverse health consequences. To date, little is understood on a molecular level regarding concussion and subconcussive impacts. Recent research suggests that neuroinflammatory mechanisms may serve an important role subsequent to concussion and possibly to subconcussive impacts. These studies suggest that autoantibodies may be a valuable tool for detection of acute concussion and monitoring for changes caused by cumulative exposure to subconcussive impacts. Hence, we aimed to profile the immunoglobulin (Ig)A autoantibody repertoire in saliva by screening a unique sport-related head trauma biobank. Saliva samples (n = 167) were donated by male and female participants enrolled in either the concussion (24-48 h post-injury) or subconcussion (non-concussed participants having moderate or high cumulative subconcussive impact exposure) cohorts. Study design included discovery and verification phases. Discovery aimed to identify new candidate autoimmune targets of IgA. Verification tested whether concussion and subconcussion cohorts increased IgA reactivity and whether cohorts showed similarities. The results show a significant increase in the prevalence of IgA toward protein fragments representing 5-hydroxytryptamine receptor 1A (HTR1A), serine/arginine repetitive matrix 4 (SRRM4) and FAS (tumor necrosis factor receptor superfamily member 6) after concussion and subconcussion. These results may suggest that concussion and subconcussion induce similar physiological effects, especially in terms of immune response. Our study demonstrates that saliva is a potential biofluid for autoantibody detection in concussion and subconcussion. After rigorous confirmation in much larger independent study sets, a validated salivary autoantibody assay could provide a non-subjective quantitative means of assessing concussive and subconcussive events.

Risk prediction models for colorectal cancer: Evaluating the discrimination due to added biomarkers.

Most risk prediction models for colorectal cancer (CRC) are based on questionnaires and show a modest discriminatory ability. Therefore, we aim to develop risk prediction models incorporating plasma biomarkers for CRC to improve discrimination. We assessed the predictivity of 11 biomarkers in 736 men in the Health Professionals Follow-up Study and 639 women in the Nurses' Health Study. We used stepwise logistic regression to examine whether a set of biomarkers improved the predictivity on the basis of predictors in the National Cancer Institute's (NCI) Colorectal Cancer Risk Assessment Tool. Model discrimination was assessed using C-statistics. Bootstrap with 500 randomly sampled replicates was used for internal validation. The models containing each biomarker generated a C-statistic ranging from 0.50 to 0.59 in men and 0.50 to 0.54 in women. The NCI model demonstrated a C-statistic (95% CI) of 0.67 (0.62-0.71) in men and 0.58 (0.54-0.63) in women. Through stepwise selection of biomarkers, the C-statistic increased to 0.70 (0.66-0.74) in men after adding growth/differentiation factor 15, total adiponectin, sex hormone binding globulin and tumor necrosis factor receptor superfamily member 1B (P for difference=0.008); and increased to 0.62 (0.57-0.66) in women after further including insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 (P for difference=.06). The NCI + selected biomarkers model was internally validated with a C-statistic (95% CI) of 0.73 (0.70-0.77) in men and 0.66 (0.61-0.70) in women. Circulating plasma biomarkers may improve the performance of risk factor-based prediction model for CRC.

Empagliflozin Disrupts a Tnfrsf12a-Mediated Feed Forward Loop That Promotes Left Ventricular Hypertrophy

Although the cardioprotective benefits of sodium-glucose cotransporter 2 (SGLT2) inhibitors are now widely appreciated, the mechanisms underlying these benefits remain unresolved. Tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) is a receptor for tumor necrosis factor superfamily member 12 (Tnfsf12). Tnfrsf12a is highly inducible and plays a key role in the development of cardiac hypertrophy and heart failure. Here we set out to determine if SGLT2 inhibition affects the Tnfsf12/Tnfrsf12a system in the stressed myocardium. C57BL/6N mice that had undergone sham or transverse aortic constriction (TAC) surgery were treated with either the SGLT2 inhibitor empagliflozin (400 mg/kg diet; 60-65 mg/kg/day) or standard chow alone and were followed for 8 weeks. Tnfrsf12a expression in mouse hearts was assessed by in situ hybridization, qRT-PCR, and immunoblotting. Left ventricular (LV) mass, end-systolic volume, and end-diastolic volume were all increased in TAC mice and were significantly lower with empagliflozin. Myocyte hypertrophy and interstitial fibrosis in TAC hearts were similarly attenuated with empagliflozin. Tnfrsf12a expression was upregulated in mouse hearts following TAC surgery but not in the hearts of empagliflozin-treated mice. In cultured cardiomyocytes, Tnfrsf12a antagonism attenuated the increase in cardiomyocyte size that was induced by phenylephrine. Empagliflozin attenuates LV enlargement in mice with hypertrophic heart failure. This effect may be mediated, at least in part, by a reduction in loading conditions which limits upregulation of the inducible, proinflammatory, and prohypertrophic TNF superfamily receptor, Tnfrsf12a. Disruption of the Tnfsf12/Tnfrsf12a feed forward system may contribute to the cardioprotective benefits of SGLT2 inhibition.

RANK promotes colorectal cancer migration and invasion by activating the Ca2+-calcineurin/NFATC1-ACP5 axis

The tumor necrosis factor (TNF) receptor superfamily member 11a (TNFRSF11a, also known as RANK) was demonstrated to play an important role in tumor metastasis. However, the specific function of RANK in colorectal cancer (CRC) metastasis and the underlying mechanism are unknown. In this study, we found that RANK expression was markedly upregulated in CRC tissues compared with that in matched noncancerous tissues. Increased RANK expression correlated positively with metastasis, higher TNM stage, and worse prognosis in patients with CRC. Overexpression of RANK promoted CRC cell metastasis in vitro and in vivo, while knockdown of RANK decreased cell migration and invasion. Mechanistically, RANK overexpression significantly upregulated the expression of tartrate-resistant acid phosphatase 5 (TRAP/ACP5) in CRC cells. Silencing of ACP5 in RANK-overexpressing CRC cells attenuated RANK-induced migration and invasion, whereas overexpression of ACP5 increased the migration and invasion of RANK-silencing cells. The ACP5 expression was transcriptionally regulated by calcineurin/nuclear factor of activated T cells c1 (NFATC1) axis. The inhibition of calcineurin/NFATC1 significantly decreased ACP5 expression, and attenuated RANK-induced cell migration and invasion. Furthermore, RANK induced phospholipase C-gamma (PLCγ)-mediated inositol-1,4,5-trisphosphate receptor (IP3R) axis and stromal interaction molecule 1 (STIM1) to evoke calcium (Ca2+) oscillation. The RANK-mediated intracellular Ca2+ mobilization stimulated calcineurin to dephosphorylate NFATC1 and induce NFATC1 nuclear translocation. Both blockage of PLCγ-IP3R axis and STIM1 rescued RANK-induced NFATC1 nuclear translocation, ACP5 expression, and cell metastasis. Our study revealed the functional expression of RANK in human CRC cells and demonstrated that RANK induced the Ca2+-calcineurin/NFATC1-ACP5 axis in the regulation of CRC metastasis, that might be amenable to therapeutic targeting.

Single-Cell Deconvolution of Head and Neck Squamous Cell Carcinoma.

Simple SummaryTumors are not composed of a uniform ball of cells, but rather, a complex set of diverse cells. Unfortunately, most transcriptomic techniques analyze the entire tumor (bulk), and thus represent an average profile of genes expressed across heterogeneous cells. To estimate tumor composition from bulk data, many algorithms have been developed—broadly termed deconvolution. However, with the advent of single-cell RNA sequencing (scRNA-seq), which provides gene expression data for individual cells, a few deconvolution algorithms are now more nuanced. We have used our scRNA-seq data from head and neck tumors along with two cutting-edge deconvolution algorithms to analyze bulk expression data from >500 tumors. With this approach, we find that higher proportions of a class of immune cells (tumor-infiltrating regulatory T-cells) are associated with improved survival in head and neck cancer. Our findings and data establish a generalizable approach that can be applied across oncology to study tumor composition.Complexities in cell-type composition have rightfully led to skepticism and caution in the interpretation of bulk transcriptomic analyses. Recent studies have shown that deconvolution algorithms can be utilized to computationally estimate cell-type proportions from the gene expression data of bulk blood samples, but their performance when applied to tumor tissues, including those from head and neck, remains poorly characterized. Here, we use single-cell data (~6000 single cells) collected from 21 head and neck squamous cell carcinoma (HNSCC) samples to generate cell-type-specific gene expression signatures. We leverage bulk RNA-seq data from >500 HNSCC samples profiled by The Cancer Genome Atlas (TCGA), and using single-cell data as a reference, apply two newly developed deconvolution algorithms (CIBERSORTx and MuSiC) to the bulk transcriptome data to quantitatively estimate cell-type proportions for each tumor in TCGA. We show that these two algorithms produce similar estimates of constituent/major cell-type proportions and that a high T-cell fraction correlates with improved survival. By further characterizing T-cell subpopulations, we identify that regulatory T-cells (Tregs) were the major contributor to this improved survival. Lastly, we assessed gene expression, specifically in the Treg population, and found that TNFRSF4 (Tumor Necrosis Factor Receptor Superfamily Member 4) was differentially expressed in the core Treg subpopulation. Moreover, higher TNFRSF4 expression was associated with greater survival, suggesting that TNFRSF4 could play a key role in mechanisms underlying the contribution of Treg in HNSCC outcomes.

Albumin Urinary Excretion Is Associated with Increased Levels of Urinary Chemokines, Cytokines, and Growth Factors Levels in Humans.

The aim of the present study was to study the associations between urine albumin excretion, and a large number of urinary chemokines, cytokines, and growth factors in a normal population. We selected 90 urine samples from individuals without CVD, diabetes, stroke or kidney disease belonging to the Prospective Investigation of the Vasculature in Uppsala Seniors Study (41 males and 49 females, all aged 75 years). Urinary cytokine levels were analyzed with two multiplex assays (proximity extension assays) and the cytokine levels were correlated with urine albumin. After adjustment for sex, body mass index (BMI), estimated glomerular filtration rate (eGFR), smoking and multiplicity testing, 11 biomarkers remained significantly associated with urine albumin: thrombospondin 2, interleukin 6, interleukin 8, hepatocyte growth factor, matrix metalloproteinase-12 (MMP-12), C-X-C motif chemokine 9, tumor necrosis factor receptor superfamily member 11B, osteoprotegerin, growth-regulated alpha protein, C-X-C motif chemokine 6, oncostatin-M (OSM) and fatty acid-binding protein, intestinal, despite large differences in molecular weights. In this study, we found associations between urinary albumin and both small and large urine proteins. Additional studies are warranted to identify cytokine patterns and potential progression markers in various renal diseases.

Sodium acetate, propionate, and butyrate reduce fat accumulation in mice via modulating appetite and relevant genes

ObjectivesAcetate, propionate, and butyrate, three of the most common short-chain fatty acids (SCFAs), can be produced when some non-digestible carbohydrates enter the large intestine and undergo bacterial fermentation. The aim of this study was to investigate the effects of these three SCFAs on appetite regulation and lipid metabolism and to determine the extent that appetite contributes to the beneficial influences of SCFAs. MethodsIn a 35-d study, 48 C57BL/6J male mice were randomly allocated to six groups: control; 5% sodium acetate; 5% sodium propionate; 5% sodium butyrate; pair fed 1; and pair fed 2. ResultsThe study showed that dietary supplementation of sodium acetate reduced serum triacylglycerol, free fatty acids, glucose, and interleukin (IL)-6 levels (P < 0.05), increased serum glucagon-like peptide 1, and leptin levels (P < 0.05), downregulated the mRNA expressions of fatty acid synthase, peroxisome proliferator-activated receptor, and lipoprotein lipase (P < 0.05), and upregulated the mRNA expressions of fasting-induced adipose factor, nuclear respiratory factor 1, mitochondrial transcription factor A, tumor necrosis factor receptor superfamily member 9, cytochrome-C oxidase IV and free fatty acid receptor 2 (P < 0.05). Sodium propionate also reduced serum IL-1β level (P < 0.05), increased serum peptide YY level (P < 0.05), downregulated the mRNA expressions of acetyl-coenzyme A carboxylase and sterol regulatory element-binding protein 1c (P < 0.05), and upregulated the mRNA expression of transmembrane protein 26 (P < 0.05). Additionally, sodium butyrate decreased average daily feed intake (P < 0.05) downregulated the mRNA expression of myosin heavy-chain (MyHc) Ⅱb (P < 0.05), and upregulated the mRNA expressions of lipase hormone-sensitive, MyHC Ⅱa and carnitine palmitoyltransferase-1α (P < 0.05). Moreover, the metabolic benefits of SCFAs were partly attributed to the reduction of feed intake. ConclusionTaken together, SCFAs could reduce appetite and fat accumulation via modulating relevant genes and hormones, which might further illustrate the potential mechanisms that underlay the effects of SCFAs on lipid homeostasis and control of body weight.

Characterization of a novel OX40 ligand and CD40 ligand-expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform

Oncolytic viruses (OVs) have been shown to induce anti-cancer immunity and enhance cancer immunotherapies, such as immune checkpoint inhibitor therapies. OV therapies can be further improved by arming OVs with immunostimulatory molecules, including various cytokines or chemokines. Here, we have developed a novel adenovirus encoding two immunostimulatory molecules: cluster of differentiation 40 ligand (CD40L) and tumor necrosis factor receptor superfamily member 4 ligand (OX40L). This novel virus, designated VALO-D102, is designed to activate both innate and adaptive immune responses against tumors. CD40L affects the innate side by licensing antigen-presenting cells to drive CD8+ T cell responses, and OX40L increases clonal expansion and survival of CD8+ T cells and formation of a larger pool of memory T cells. VALO-D102 and its murine surrogate VALO-mD901, expressing murine OX40L and CD40L, were used in our previously developed PeptiCRAd cancer vaccine platform. Intratumoral administration of PeptiCRAd significantly increased tumor-specific T cell responses, reduced tumor growth, and induced systemic anti-cancer immunity in two mouse models of melanoma. In addition, PeptiCRAd therapy, in combination with anti-PD-1 immune checkpoint inhibitor therapy, significantly improved tumor growth control as compared to either monotherapy alone.

Immune profiling of pituitary tumors reveals variations in immune infiltration and checkpoint molecule expression.

Pituitary tumors are the second most common primary brain tumors. Functional tumors demonstrate increased PD-L1 expression, but expression of other checkpoint regulators has not been characterized. We sought to characterize the immune microenvironment of human pituitary tumors to identify new treatment opportunities. 72 pituitary tumors were evaluated for expression of the immune regulatory markers programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), V-domain Ig suppressor of T cell activation (VISTA), lymphocyte activation gene 3 (LAG3) and tumor necrosis factor receptor superfamily member 4 (OX40) by immunohistochemistry (IHC). Lymphocyte infiltration, macrophage infiltration, and angiogenesis were analyzed using IHC. Expression of pituitary tumor initiating cell marker CD15 and mismatch repair proteins MutS protein homolog 2 (MSH2) and MutS protein homolog 6 (MSH6) was also assessed. Pituitary tumors were infiltrated by macrophages and T cells, and they expressed varying levels of PD-L1, PD-L2, VISTA, LAG3, and OX40. Functional tumors and tumors with high expression of tumor stem cell markers had higher immune cell infiltration and greater expression of immunosuppressive checkpoint regulators. Increased PD-L1 and LAG3 and reduced VISTA were observed in primary tumors compared to recurrent tumors. Immune cell infiltration and checkpoint regulator expression vary depending on functional status and presence of pituitary tumor initiating cells. Functional tumors may have a particularly immunosuppressive microenvironment. Further studies of immune checkpoint blockade of pituitary tumors, particularly functional tumors, are warranted, though combination therapy may be required.

RANK Signaling in the Differentiation and Regeneration of Thymic Epithelial Cells.

Thymic epithelial cells (TECs) provide essential clues for the proliferation, survival, migration, and differentiation of thymocytes. Recent advances in mouse and human have revealed that TECs constitute a highly heterogeneous cell population with distinct functional properties. Importantly, TECs are sensitive to thymic damages engendered by myeloablative conditioning regimen used for bone marrow transplantation. These detrimental effects on TECs delay de novo T-cell production, which can increase the risk of morbidity and mortality in many patients. Alike that TECs guide the development of thymocytes, reciprocally thymocytes control the differentiation and organization of TECs. These bidirectional interactions are referred to as thymic crosstalk. The tumor necrosis factor receptor superfamily (TNFRSF) member, receptor activator of nuclear factor kappa-B (RANK) and its cognate ligand RANKL have emerged as key players of the crosstalk between TECs and thymocytes. RANKL, mainly provided by positively selected CD4+ thymocytes and a subset of group 3 innate lymphoid cells, controls mTEC proliferation/differentiation and TEC regeneration. In this review, I discuss recent advances that have unraveled the high heterogeneity of TECs and the implication of the RANK-RANKL signaling axis in TEC differentiation and regeneration. Targeting this cell-signaling pathway opens novel therapeutic perspectives to recover TEC function and T-cell production.

Factors associated with progression to death in patients with Lassa fever in Nigeria: an observational study

Lassa fever is endemic in several west African countries. Case-fatality rates ranging from 21% to 69% have been reported. The pathophysiology of the disease in humans and determinants of mortality remain poorly understood. We aimed to determine host protein biomarkers capable of determining disease outcome. In this observational study, we analysed left-over blood samples from patients who tested positive for Lassa fever at Irrua Specialist Teaching Hospital, Nigeria, between January, 2014, and April, 2017. We measured viral load, concentrations of clinical chemistry parameters, and levels of 62 circulating proteins involved in inflammation, immune response, and haemostasis. Patients with a known outcome (survival or death) and at least 200 μL of good-quality diagnostic sample were included in logistic regression modelling to assess the correlation of parameters with Lassa fever outcome. Individuals who gave consent could further be enrolled into a longitudinal analysis to assess the association of parameters with Lassa fever outcome over time. Participants were divided into two datasets for the statistical analysis: a primary dataset (samples taken between Jan 1, 2014, and April 1, 2016), and a secondary dataset (samples taken between April 1, 2016, and April 1, 2017). Biomarkers were ranked by area under the receiver operating characteristic curve (AUC) from highest (most predictive) to lowest (least predictive). Of 554 patients who tested positive for Lassa fever during the study period, 201 (131 in the primary dataset and 70 in the secondary dataset) were included in the biomarker analysis, of whom 74 (49 in the primary dataset and 25 in the secondary dataset) had died and 127 (82 in the primary dataset and 45 in the secondary dataset) had survived. Cycle threshold values (indicating viral load) and levels of 18 host proteins at the time of admission to hospital were significantly correlated with fatal outcome. The best predictors of outcome in both datasets were plasminogen activator inhibitor-1 (PAI-1; AUC 0·878 in the primary dataset and 0·876 in the secondary dataset), soluble thrombomodulin (TM; 0·839 in the primary dataset and 0·875 in the secondary dataset), and soluble tumour necrosis factor receptor superfamily member 1A (TNF-R1; 0·807 in the primary dataset and 0·851 in the secondary dataset), all of which had higher prediction accuracy than viral load (0·774 in the primary dataset and 0·837 in the secondary dataset). Longitudinal analysis (150 patients, of whom 36 died) showed that of the biomarkers that were predictive at admission, PAI-1 levels consistently decreased to normal levels in survivors but not in those who died. The identification of PAI-1 and soluble TM as markers of fatal Lassa fever at admission, and of PAI-1 as a marker of fatal Lassa fever over time, suggests that dysregulated coagulation and fibrinolysis and endothelial damage have roles in the pathophysiology of Lassa fever, providing a mechanistic explanation for the association of Lassa fever with oedema and bleeding. These novel markers might aid in clinical risk stratification and disease monitoring. German Research Foundation, Leibniz Association, and US National Institutes of Health.

RELT stains prominently in B-cell lymphomas and binds the hematopoietic transcription factor MDFIC

Receptor Expressed in Lymphoid Tissues (RELT) is a human tumor necrosis factor receptor superfamily member (TNFRSF) that is expressed most prominently in cells and tissues of the hematopoietic system. RELL1 and RELL2 are two homologs that physically interact with RELT and co-localize with RELT at the plasma membrane. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members. The transcription factor MyoD family inhibitor domain-containing (MDFIC) was identified in a yeast two-hybrid genetic screen using RELL1 as bait. MDFIC co-localizes with RELT family members at the plasma membrane; this co-localization was most prominently observed with RELL1 and RELL2. In vitro co-immunoprecipitation (Co-IP) was utilized to demonstrate that MDFIC physically interacts with RELT, RELL1, and RELL2. Co-IP using deletion mutants of MDFIC and RELT identified regions important for physical association between MDFIC and RELT family members and a computational analysis revealed that RELT family members are highly disordered proteins. Immunohistochemistry of normal human lymph nodes revealed RELT staining that was most prominent in macrophages. Interestingly, the level of RELT staining significantly increased progressively in low and high-grade B-cell lymphomas versus normal lymph nodes. RELT co-staining with CD20 was observed in B-cell lymphomas, indicating that RELT is expressed in malignant B cells. Collectively, these results further our understanding of RELT-associated signaling pathways, the protein structure of RELT family members, and provide preliminary evidence indicating an association of RELT with B-cell lymphomas.