Tumor Necrosis Factor Receptor Type Research Articles

IMMU-05. TNFR2 IS A NOVEL MARKER OF EXHAUSTION AND TNFR2 BLOCKADE IMPROVES SUBCUTANEOUS TUMOR CONTROL

Abstract Checkpoint inhibitors have been successful in various tumors. However, these treatments have failed in glioblastoma. Approaches harnessing the immune response are hindered by multiple factors, including T cell exhaustion. TOX is particularly important for the transcriptional and epigenetic reprogramming of exhausted T cells. While it is known that NFAT is upstream of TOX, our understanding of the upregulation of TOX remains incomplete. We hypothesized that tumor necrosis factor (TNF) could be involved in the upregulation of TOX as TNF can lead to the translocation of NFAT into the nucleus. We observed significant upregulation of the anti-inflammatory TNF receptor type II (TNFR2) in tumor-infiltrating T cells. This local upregulation mimics expression patterns of canonical exhaustion markers. TNFR2 expression is correlated with markers of exhaustion, including PD1, TIM3, and TOX. Furthermore, TNFR2 knock out (KO) CD8 T cells have significantly lower TOX expression, without the concomitant decrease of TIM3. Whereas previous studies have linked TIM3 and TOX expression, these data suggest that TIM3 and TOX are regulated independently. We utilized bulk RNA-sequencing to assess transcriptional regulation of WT and KO T cells and detected a significant reduction of T cell exhaustion and immune checkpoint pathways in TNFR2 KO T cells. Various exhaustion-related transcription factors and coinhibitory markers were significantly reduced. In contrast, a significant increase in AP1 transcription factors, commonly associated with T cell effector functions, was detected. Given this reduced exhaustion profile, we subsequently investigated the influence of TNFR2 on tumor burden. TNFR2 KO mice had significantly lower tumor burdens following subcutaneous tumor challenges. We subsequently treated mice with a TNFR2 antagonist. While the antagonist alone was not sufficient to improve tumor control, combination with anti-PD1 significantly reduced tumor volumes. These data provide evidence for a novel marker of exhaustion that could result in a unique therapeutic strategy.

Inactivation of Tnf-α/Tnfr signaling attenuates progression of intervertebral disc degeneration in mice.

Intervertebral disc degeneration (IVDD) is a major cause of low back pain (LBP), worsened by chronic inflammatory processes associated with aging. Tumor necrosis factor alpha (Tnf-α) and its receptors, Tnf receptor type 1 (Tnfr1) and Tnf receptor type 2 (Tnfr2), are upregulated in IVDD. However, its pathologic mechanisms remain poorly defined. To investigate the role of Tnfr in IVDD, we generated global Tnfr1/2 double knockout (KO) mice and age-matched control C57BL/6 male mice, and analyzed intervertebral disc (IVD)-related phenotypes of both genotypes under physiological conditions, aging, and lumbar spine instability (LSI) model through histological and immunofluorescence analyses and μCT imaging. Expression levels of key extracellular matrix (ECM) proteins in aged and LSI mice, especially markers of cell proliferation and apoptosis, were evaluated in aged (21-month-old) mice. At 4 months, KO and control mice showed no marked differences of IVDD-related parameters. However, at 21 months of age, the loss of Tnfr expression significantly alleviated IVDD-like phenotypes, including a significant increase in height of the nucleus pulposus (NPs) and reductions of endplates (EPs) porosity and histopathological scores, when compared to controls. Tnfr deficiency promoted anabolic metabolism of the ECM proteins and suppressed ECM catabolism. Tnfr loss largely inhibited hypertrophic differentiation, and, in the meantime, suppressed cell apoptosis and cellular senescence in the annulus fibrosis, NP, and EP tissues without affecting cell proliferation. Similar results were observed in the LSI model, where Tnfr deficiency significantly alleviated IVDD and enhanced ECM anabolic metabolism while suppressing catabolism. The deletion of Tnfr mitigates age-related and LSI-induced IVDD, as evidenced by preserved IVD structure, and improved ECM integrity. These findings suggest a crucial role of Tnf-α/Tnfr signaling in IVDD pathogenesis in mice. Targeting this pathway may be a novel strategy for IVDD prevention and treatment.

Ovalbumin-induced food allergy suppression via regulatory T cell expansion mediated by a TNFR2 agonist in mice

Food allergies represent a growing health concern worldwide, characterized by abnormal immune responses to specific dietary antigens. This condition is often associated with a dysregulation of immune tolerance, especially within the intestinal mucosa. Regulatory T cells (Tregs), a crucial subset of lymphocytes, play a central role in maintaining peripheral immune tolerance and are abundant in the intestinal lamina propria. Recent studies have highlighted Treg dysfunction in patients with food allergies, suggesting a potential connection between impaired Treg function and allergy onset. Therefore, strategies to adequately control and activate Tregs could offer new avenues for the prevention and treatment of food allergies. Our research focuses on targeting the regulatory molecule, tumor necrosis factor receptor type 2 (TNFR2), a key modulator of Treg function. We have developed a TNFR2 agonist, scR2agoTNF-Fc, characterized by high TNFR2-stimulating activity and enhanced blood retention in vivo for Treg expansion. In this study, we utilized an ovalbumin (OVA)-induced food allergy mouse model to verify the therapeutic potential of scR2agoTNF-Fc in modulating allergic responses and restoring immune balance. The results showed that scR2agoTNF-Fc promoted the expansion of Treg population in vivo in mice. In addition, scR2agoTNF-Fc reduced diarrhea caused by the food allergy. This was consistent with the molecular mechanisms of suppression of blood immunoglobulins and Th2 cells. Therefore, it was shown that quantitative and functional enhancement of Tregs by the TNFR2 agonist, scR2agoTNF-Fc, may be effective in treating food allergies.

A randomized proof-of-mechanism trial of TNF antagonism for motivational deficits and related corticostriatal circuitry in depressed patients with high inflammation.

Chronic, low-grade inflammation has been associated with motivational deficits in patients with major depression (MD). In turn, impaired motivation has been linked to poor quality of life across psychiatric disorders. We thus determined effects of the anti-inflammatory drug infliximab-a potent tumor necrosis factor (TNF) antagonist-on behavioral and neural measures of motivation in 42 medically stable, unmedicated MD patients with a C-reactive protein >3 mg/L. All patients underwent a double-blind, placebo-controlled, single-dose, randomized clinical trial with infliximab (5 mg/kg) versus placebo. Behavioral performance on an effort-based decision-making task, self-report questionnaires, and neural responses during event-related functional magnetic resonance imaging were assessed at baseline and 2 weeks following infusion. We found that relative to placebo, patients receiving infliximab were more willing to expend effort for rewards. Moreover, increase in effortful choices was associated with reduced TNF signaling as indexed by decreased soluble TNF receptor type 2 (sTNFR2). Changes in effort-based decision-making and sTNFR2 were also associated with changes in task-related activity in a network of brain areas, including dorsomedial prefrontal cortex (dmPFC), ventral striatum, and putamen, as well as the functional connectivity between these regions. Changes in sTNFR2 also mediated the relationships between drug condition and behavioral and neuroimaging measures. Finally, changes in self-reported anhedonia symptoms and effort-discounting behavior were associated with greater responses of an independently validated whole-brain predictive model (aka "neural signature") sensitive to monetary rewards. Taken together, these data support the use of anti-inflammatory treatment to improve effort-based decision-making and associated brain circuitry in depressed patients with high inflammation.

Anti-TNFR2 Antibody-Conjugated PLGA Nanoparticles for Targeted Delivery of Adriamycin in Mouse Colon Cancer.

High levels of tumor necrosis factor receptor type II (TNFR2) are preferentially expressed by immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in the tumor microenvironment, as initially reported by us. There is compelling evidence that targeting TNFR2 markedly enhances antitumor immune responses. Furthermore, a broad spectrum of human cancers also expresses TNFR2, while its expression by normal tissue is very limited. We thus hypothesized that TNFR2 may be harnessed for tumor-targeted delivery of chemotherapeutic agents. In this study, we performed a proof-of-concept study by constructing a TNFR2-targeted PEGylated poly(dl-lactic-co-glycolic acid) (PLGA-PEG) nanodrug delivery system [designated as TNFR2-PLGA-ADR (Adriamycin)]. The results of invitro study showed that this TNFR2-targeted delivery system had the properties in cellular binding and cytotoxicity toward mouse colon cancer cells. Further, upon intravenous injection, TNFR2-PLGA-ADR could efficiently accumulate in MC38 and CT26 mouse colon tumor tissues and preferentially bind with tumor-infiltrating Tregs. Compared with ADR and ISO-PLGA-ADR, the invivo antitumor effect of TNFR2-PLGA-ADR was markedly enhanced, which was associated with a decrease of TNFR2+ Tregs and an increase of IFNγ+CD8+ cytotoxic T lymphocytes in the tumor tissue. Therefore, our results clearly show that targeting TNFR2 is a promising strategy for designing tumor-specific chemoimmunotherapeutic agent delivery system.

Understanding the role of TNFR2 signaling in CD8 T cell exhaustion.

Abstract Glioblastoma (GBM) is the most common primary brain cancer in adults with a median survival less than 15 months. Approaches harnessing the immune response are hindered by multiple factors, including T cell exhaustion. Exhausted T cells are less capable of limiting tumor progression. As cells progress along the exhaustion pathway, SLAM Family Member 6 (SLAMF6) gets downregulated and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) and TOX are upregulated. TOX is both necessary and sufficient to initiate the terminal exhaustion program. However, our understanding of the pathways that lead to the upregulation of TOX remains incomplete. We hypothesized that tumor necrosis factor (TNF) could be involved in the upregulation of TOX, as TNF has been shown to induce exhaustion. We observed upregulation of the TNF receptor type II (TNFR2) in tumor-infiltrating T cells, but not in lymphoid organs. Furthermore, TNFR2 expression is correlated with canonical exhaustion markers. Interestingly, TNFR2 knock out (KO) CD8 T cells express TIM3 but exhibit significantly less TOX. These data suggest that TIM3 and TOX are regulated independently. Finally, TNFR2 KO mice have significantly better CD8-mediated tumor control against subcutaneous GBM tumors. Further studies will need to be performed to determine whether TNFR2 expression on CD8 T cells is sufficient to mediate this anti-tumor effect and whether TNFR2 blockade could improve the efficacy of current treatments.

Apoptosis as a therapeutic strategy for breast cancer: the role of Thymax, a gross thymic extract, in modulating cell death pathways

Background Breast cancer is a prevalent disease in women and a leading cause of cancer-related health issues. Thymax, a thymic extract, has shown potential for inducing breast cancer cell apoptosis in vitro. Objective This study aims to investigate how Thymax induces apoptosis and inhibits breast cancer growth and metastasis in vivo. Materials and methods Thymax treatment was divided into five groups: the first group (negative control) − normal rats without tumors. In the second group (positive control), rats were injected subcutaneously in the mammary gland with a single dose of 50 mg/kg b.w. of 7,12-Dimethylbenz(a)anthracene (in 2 ml of corn oil) and allowed to develop tumors for 120 days. Group 3: Thymax was orally administered 6 days a week to tumor-bearing rats (0.4 mg/rat) and continued for 5 weeks. Tumor-bearing rats in group 4 (Thymax injection) received 0.1 ml of Thymax solution through intraperitoneal injection twice weekly for 5 weeks. The last group was Thymax mix (oral and injection); tumor-bearing rats received Thymax solution by dual routes: orally with 0.4 ml six times per week and intraperitoneally with 0.1 ml twice weekly for 5 weeks. Thymax treatment, beginning after 120 days of tumor induction, continued for 5 weeks. Results and conclusion Thymax- induced apoptosis in breast cancer cells by increasing cytochrome c, tumor necrosis factor receptor type 1-associated death domain protein (TRADD), and Fas associated death domain (FADD) levels. It also activated the mitochondrial-dependent pathway with up-regulation of tumor protein gene (P53) expression and cysteine-dependent, aspartate-specific peptidase (caspase-8) activation. Thymax restored normal renal and hepatic cell function and enhanced the immune system by improving total antioxidant levels and inhibiting malondialdehyde levels in treated animals. Histopathological results showed a significant apoptotic effect in the group receiving Thymax injections, demonstrating its capability to induce apoptosis without tumors or atypia in mammary glands. Our findings indicate that Thymax has a significant effect on enhancing tumor cell death and inducing apoptosis in vivo. Thymax may also modulate proapoptotic and antiapoptotic protein expression and activity, regulate the penetrability of the mitochondrial membrane, and release cytochrome c. Furthermore, our findings show that the injection route of Thymax is the fastest and most efficient method to deliver the extract to the tumor site and exert its antitumor effects. These results suggest that Thymax has the potential to be a novel adjuvant in the treatment of breast cancer, as it can enhance the efficacy of conventional therapies and reduce the risk of recurrence and metastasis.

Soluble tumor necrosis factor receptor type 1 is an alternative marker of urinary albumin-creatinine ratio and estimated glomerular filtration rate for predicting the decline of renal function in subjects with type 2 diabetes mellitus

BackgroundUrinary albumin-creatinine ratio (UACR) and estimated glomerular filtration rates (eGFR) help predict worsening diabetic kidney disease (DKD) but have their limitations. Soluble tumor necrosis factor receptor type 1 (sTNFR1) is a biomarker of DKD. The predictive abilities of sTNFR1 and UACR plus eGFR have not been compared. MethodsThis prospective cohort study included patients with type 2 diabetes (T2D) to identify the risk factors of worsening DKD. Renal events were defined as > 30 % loss in eGFR based on consecutive tests after 6 months. The associations of sTNFR1, UACR, and eGFR levels and the risks of renal events were tested using a Cox regression model and the area under the curve (AUC) was compared between sTNFR1 levels and UACR plus eGFR using receiver-operating characteristic (ROC) analysis. The accuracy of stratification was evaluated using Kaplan–Meier analysis. ResultsLevels of sTNFR1 and UACR were associated with risks of > 30 % decline in eGFR after adjusting for relevant factors. The association between sTNFR1 levels and renal outcomes was independent of UACR and eGFR at baseline. The AUC of sTNFR1 level was comparable with that of combined UACR and eGFR (0.73 vs. 0.71, respectively, p = 0.72) and the results persisted for quartile groups of sTNFR1 and risk categories of Kidney Disease: Improving Global Outcomes (KDIGO) (0.70 vs. 0.71, respectively, p = 0.84). Both stratifications by sTNFR1 levels and KDIGO were accurate. ConclusionsTNFR1 could be an alternative marker for identifying patients with diabetes at risk of declining renal function.

TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation.

Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.

The strong inverse association between plasma concentrations of soluble tumor necrosis factor receptors type 1 with adiponectin/leptin ratio in older women

Complex inflammatory crosstalk between muscular and adipose organs during ageing is controlled by adipokines and myokines. The Adiponectin/Leptin ratio (A/L ratio) has proven to be a promising biomarker for identifying insulin sensitivity, cardiovascular risk and adipose tissue inflammation. Although the A/L ratio has been related to inflammatory conditions, its ability to associate with or indicate the behavior of other inflammatory mediators remains unknown. The present study aimed to verify the association between the A/L ratio and a panel of inflammatory biomarkers in community-dwelling older women. The plasmatic concentrations of adiponectin, leptin, resistin, brain-derived neurotrophic factor (BDNF), interferon-gamma (IFN-γ), interleukins 2, 4, 5, 6, 8 and 10, tumour necrosis factor (TNF) and its soluble receptors (sTNF-r) 1 and 2 were evaluated in 71 community-dwelling older women with 75 (±7) years. The A/L ratio was negative and inverse correlated with BNDF (r = −0.29; p = 0.01), IL-8 (r = −0.37; p = 0.001) and sTNFr- 1 (r = −0.98; p < 0.001) levels. A strong and inverse association, with proportional effect, between A/L ratio and sTNFr-1 concentrations was found (Adjusted R2 = 0.22; β = −0.48; p > 0.001). It suggests that the presence of sTNFr-1 causes an inflammatory effect that affect cross-talk between muscle and adipose tissue, contributing to pro-inflammatory imbalance, which may have molecular and functional consequences. In addition, we provide insights into diagnostic biomarkers for inflammation, especially related to muscle wasting and intrinsic capacity in older people.

An immunocytokine consisting of a TNFR2 agonist and TNFR2 scFv enhances the expansion of regulatory T cells through TNFR2 clustering

Regulatory T cells (Tregs) are lymphocytes that play a central role in peripheral immune tolerance. Tregs are promising targets for the prevention and suppression of autoimmune diseases, allergies, and graft-versus-host disease, and treatments aimed at regulating their functions are being developed. In this study, we created a new modality consisting of a protein molecule that suppressed excessive immune responses by effectively and preferentially expanding Tregs. Recent studies reported that tumor necrosis factor receptor type 2 (TNFR2) expressed on Tregs is involved in the proliferation and activation of Tregs. Therefore, we created a functional immunocytokine, named TNFR2-ICK-Ig, consisting of a fusion protein of an anti-TNFR2 single-chain Fv (scFv) and a TNFR2 agonist TNF-α mutant protein, as a new modality that strongly enhances TNFR2 signaling. The formation of agonist-receptor multimerization (TNFR2 cluster) is effective for the induction of a strong TNFR2 signal, similar to the TNFR2 signaling mechanism exhibited by membrane-bound TNF. TNFR2-ICK-Ig improved the TNFR2 signaling activity and promoted TNFR2 cluster formation compared to a TNFR2 agonist TNF-α mutant protein that did not have an immunocytokine structure. Furthermore, the Treg expansion efficiency was enhanced. TNFR2-ICK-Ig promotes its effects via scFv, which crosslinks receptors whereas the agonists transmit stimulatory signals. Therefore, this novel molecule expands Tregs via strong TNFR2 signaling by the formation of TNFR2 clustering.

Peptidomimetics for CVD screened via TRADD-TRAF2 complex interface assessments.

The main aim of this study is to screen and develop Peptidomimetics to treat atherosclerosis (AS) which is a Cardio Vascular Disease (CVD). Peptidomimetics were obtained from the protein-protein interaction interface of TRADD (Tumor necrosis factor receptor type 1-associated DEATH domain protein) and TRAF2 (TNF receptor-associated factor 2) complex. TRADD-TRAF2 interaction is critical in AS pathogenesis since it assists a series of signal transducers that activate NF-κB. Conceptually, the triggered NF-κB makes an extensive amount of nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS), which boons the progress of AS. The examined TRADD-TRAF2 complex (PDB ID: 1F3V) and its interaction details revealed that the sequence range W11-G165 of TRADD highly interacts with TRAF2. The sequence range W11-G165 was selected for the design and preparation of the inhibitory peptide in silico. The selected sequence was mutated with the alanine scanning method to have a range of inhibitory peptides. With the help of different in silico tools, the top three, namely MIP11-25L, MIP131-143h, and MIP149-164m peptides showed the best interaction with the critical residues of TRAF2. Thus, these three peptides were used for generating peptidomimetics using pepMMsMIMIC, a peptidomimetics virtual screening tool. Around 600 peptidomimetics were identified & and retrieved for further screening by employing molecular docking tools and MD (Molecular Dynamics) simulations. Density Functional Theory (DFT) and ADMET predictions were applied to validate the screened peptidomimetics druggability. In the results, peptidomimic compounds MMs03918858 and MMs03927281 with binding energy values of -9.6kcal/mol and - 9.1kcal/mol respectively were screened as the best and are proposed for further pre-clinical studies.

Identifying prognostic genes related PANoptosis in lung adenocarcinoma and developing prediction model based on bioinformatics analysis

Cell death-related genes indicate prognosis in cancer patients. PANoptosis is a newly observed form of cell death that researchers have linked to cancer cell death and antitumor immunity. Even so, its significance in lung adenocarcinomas (LUADs) has yet to be elucidated. We extracted and analyzed data on mRNA gene expression and clinical information from public databases in a systematic manner. These data were utilized to construct a reliable risk prediction model for six regulators of PANoptosis. The Gene Expression Omnibus (GEO) database validated six genes with risk characteristics. The prognosis of LUAD patients could be accurately estimated by the six-gene-based model: NLR family CARD domain-containing protein 4 (NLRC4), FAS-associated death domain protein (FADD), Tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), Receptor-interacting serine/threonine-protein kinase 1 (RIPK1), Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), and Mixed lineage kinase domain-like protein (MLKL). Group of higher risk and Cluster 2 indicated a poor prognosis as well as the reduced expression of immune infiltrate molecules and human leukocyte antigen. Distinct expression of PANoptosis-related genes (PRGs) in lung cancer cells was verified using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we evaluated the relationship between PRGs and somatic mutations, tumor immune dysfunction exclusion, tumor stemness indices, and immune infiltration. Using the risk signature, we conducted analyses including nomogram construction, stratification, prediction of small-molecule drug response, somatic mutations, and chemotherapeutic response.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

Inflammation-related proteins as biomarkers of treatment-related behavioral symptoms: A longitudinal study of breast cancer patients and age-matched controls

BackgroundBehavioral symptoms in breast cancer (BC) survivors have been attributed to cancer treatment and resulting inflammation. However, studies linking behavioral symptoms to BC treatment have observed patients only after some treatment. Our prospective study with pre-treatment baseline investigates post-treatment changes in inflammation-related biomarkers and whether those changes correlate with changes in symptoms. MethodsParticipants were postmenopausal women, newly-diagnosed with stage 0–3 BC before any treatment (n = 173 “patients”), and age-matched women without cancer (n = 77 “controls”), who were assessed on plasma markers [soluble tumor necrosis factor receptor type 2 (sTNF-RII), interleukin (IL)-6, IL-1 receptor antagonist (IL-1RA), C-reactive protein (CRP)]) and symptoms (Physical Functioning, Pain, Attention/concentration, Perceived Cognitive Problems, Fatigue, Sleep Insufficiency, Depression). Participants were assessed again 1 month, 1 year, and 2 years after completing primary treatment or similar interval in controls. Generalized linear mixed models tested 4 treatments (surgery alone or with chemotherapy, radiation, or both) for association with change per marker. Joint models tested change per marker for association with change per symptom. Models considered demographic, socioeconomic, and clinical covariates. False Discovery Rate method controlled risk of error from multiple hypotheses. ResultsAt one month post-completion of treatment, sTNF-RII and IL-6 were elevated by all BC treatments, as were IL-1RA and CRP after surgery alone (all, p < 0.05). By 1 year, markers’ average values returned to baseline. Throughout 2-year follow-up, increase-from-baseline in sTNF-RII, IL-1RA, and IL-6 coincided with worsened Physical Functioning, and increase-from-baseline in sTNF-RII coincided with increased Pain (all, p < 0.01). These biomarker-symptom associations (excepting IL-6) were exclusive to patients. No other symptoms worsened, and baseline Fatigue and Depression improved in all participants. ConclusionsBC treatment, even surgery, is associated with transient elevation in inflammatory markers. In patients post-treatment, increase-from-baseline in sTNF-RII accompanies increased Pain and decreased Physical Functioning, suggesting that sTNF-RII merits development as a clinical biomarker in BC patients.

Clinical significance of serum cytokine profiles for differentiating between Kawasaki disease and its mimickers

ObjectivesTo investigate the clinical significance of serum cytokine profiles for differentiating between Kawasaki disease (KD) and its mimickers. MethodsPatients with KD, including complete KD, KD shock syndrome (KDSS), and KD with macrophage activation syndrome (KD-MAS), and its mimickers, including multisystem inflammatory syndrome in children, toxic shock syndrome, and Yersinia pseudotuberculosis infection, were enrolled. Serum levels of interleukin (IL)-6, soluble tumor necrosis factor receptor type II (sTNF-RII), IL-10, IL-18, and chemokine (C-X-C motif) ligand 9 (CXCL9) were measured using enzyme-linked immunosorbent assay and compared them with clinical manifestations. ResultsSerum IL-6, sTNF-RII, and IL-10 levels were significantly elevated in patients with KDSS. Serum IL-18 levels were substantially elevated in patients with KD-MAS. Patients with KD-MAS and KD mimickers had significantly elevated serum CXCL9 levels compared with those with complete KD. Area under the receiver operating characteristic curve analysis showed that serum IL-6 was the most useful for differentiating KDSS from the others, IL-18 and CXCL9 for KD-MAS from complete KD, and CXCL9 for KD mimickers from complete KD and KD-MAS. ConclusionSerum cytokine profiles may be useful for differentiating between KD and its mimickers.

The Anti-inflammatory and Anti-arthritis Activity of Garcinia travancorica in a Rat Model: A Proof of Concept with Computational Studies.

Background Rheumatoid arthritis is a progressive disease of human joints characterized by severe pain, stiffness, and tissue damage at the local site. Bone and cartilaginous tissue damage at the synovial joints is initiated by the production of autoantibody induced by inflammatory signaling through cytokines. Objective This study aimed to evaluate the efficacy of Garcinia travancorica against acute and chronic inflammation in a rat model after designing the ligand library and target identification using computational analysis. Methods Acute inflammation was induced by carrageen, and chronic inflammation was induced by Freund's complete adjuvant in the plantar surface of the rats. The petroleum ether, ethanolic, and aqueous extracts in three divided doses (75 mg/kg, 150 mg/kg, and 300 mg/kg) were administered orally. Diclofenac sodium (10 mg/kg), prednisolone (5 mg/kg), and methotrexate (0.5 mg/kg) were used as standard. Various parameters were evaluated, and statistical significance between means was analyzed using one-way ANOVA, followed by Dunnett's multiple range test. Results: Docking-based in-silico screening of the ligand library has revealed the potential of Polyanxanthone-C as an anti-rheumatoid agent, which is supposed to deliver its therapeutic effect by synergistic targeting of interleukin-1, interleukin-6, and tumor necrosis factor receptor type-1. Conclusion: This plant has the potential to be used in the treatment of arthritis-related disorders.

TNFR2-targeting peptide for cancer immunotherapy

Abstract There is compelling evidence that tumor necrosis factor receptor type II (TNFR2) is preferentially expressed by highly immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in tumor-infiltrating lymphocytes (TILs). These findings led us and others to propose and experimentally proved that targeting TNFR2 with antibodies could induce anti-tumor immune responses. In this study, we identified a TNFR2-targeting peptide P20 through phage display. P20 had in vitro activity to block TNF-TNFR2 interaction and inhibit TNFR2-mediated activation/expansion of Treg cells. The in vivo effect of P20 on tumor growth was examined with mouse tumor models including CT26 colon cancer and OVA-EG7 lymphoma. The results showed that the treatment with P20 (i.p.) resulted in a marked inhibition of tumor growth, accompanied by a reduced number of Tregs and an increased number of IFN-γ+ CD8+ cytotoxic T lymphocytes in the tumor microenvironment. Furthermore, P20 could markedly enhance the efficacy of anti-PD-L1 in the inhibition of tumor growth in a syngeneic MC38 mouse colon cancer model. Thus, P20 may be a useful anti-tumor immunotherapeutic agent and merits further investigation. (Funded by FDCT 0099/2021/A2, UM MYRG2022-00260-ICMS, UM CPG2023-00025-ICMS) There is compelling evidence that tumor necrosis factor receptor type II (TNFR2) is preferentially expressed by highly immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in tumor-infiltrating lymphocytes (TILs). These findings led us and others to propose and experimentally proved that targeting TNFR2 with antibodies could induce anti-tumor immune responses. In this study, we identified a TNFR2-targeting peptide P20 through phage display. P20 had in vitro activity to block TNF-TNFR2 interaction and inhibit TNFR2-mediated activation/expansion of Treg cells. The in vivo effect of P20 on tumor growth was examined with mouse tumor models including CT26 colon cancer and OVA-EG7 lymphoma. The results showed that the treatment with P20 (i.p.) resulted in a marked inhibition of tumor growth, accompanied by a reduced number of Tregs and an increased number of IFN-γ+ CD8+ cytotoxic T lymphocytes in the tumor microenvironment. Furthermore, P20 could markedly enhance the efficacy of anti-PD-L1 in the inhibition of tumor growth in a syngeneic MC38 mouse colon cancer model. Thus, P20 may be a useful anti-tumor immunotherapeutic agent and merits further investigation

Optimized reagents for immunopotency assays on mesenchymal stromal cells for clinical use

Multipotent mesenchymal stromal cells (MSC) offer new therapeutic opportunities based on their ability to modulate an imbalanced immune system. Immunomodulatory potency is typically demonstrated in vitro by measuring the presence of surrogate markers (i.e., indoleamine-2,3-dioxygenase, IDO; tumor necrosis factor receptor type 1, TNFR1) and/or functional assays in co-cultures (i.e., inhibition of lymphoproliferation, polarization of macrophages). However, the biological variability of reagents used in the latter type of assays leads to unreliable and difficult to reproduce data therefore making cross-comparison between batches difficult, both at the intra- and inter-laboratory levels. Herein, we describe a set of experiments aiming at the definition and validation of reliable biological reagents as a first step towards standardization of a potency assay. This approach is based on the co-culture of Wharton’s jelly (WJ)-derived MSC and cryopreserved pooled peripheral blood mononuclear cells. Altogether, we successfully defined a robust and reproducible immunopotency assay based on previously described methods incorporating substantial improvements such as cryopreservation of multiple vials of pooled peripheral blood mononuclear cells (PBMC) from 5 individual donors that enable a number of tests with same reagents, also reducing waste of PBMC from individual donors and therefore contributing to a more efficient and ethical method to use substances of human origin (SoHO). The new methodology was successfully validated using 11 batches of clinical grade MSC,WJ. Methods described here contribute to minimize PBMC donor variability while reducing costs, streamlining assay setup and convenience and laying the foundations for harmonization of biological reagents usage in standardized immunopotency assays for MSC.Highlights• The use of pools of peripheral blood mononuclear cells (PBMCs) in potency assays contributes to robust and reproducible results, which is key in the assessment of mesenchymal stroma cells (MSC) potency for batch release.• Cryopreservation of PBMCs does not impact negatively on their activation and proliferation abilities.• Cryopreserved pools of PBMC constitutes convenient off-the-shelf reagents for potency assays.• Cryopreservation of pooled PBMCs from multiple donors is a way to reduce waste of donated PBMC and its associated costs, as well as reducing the impact of individual donor variability of substances of human origin (SoHO).

Contribution of Type H Blood Vessels to Pathologic Osteogenesis and Inflammation in an Experimental Spondyloarthritis Model.

Spondyloarthritis (SpA) is characterized by pathologic osteogenesis, inflammation, and extensive angiogenesis in axial and peripheral tissues. Current therapies effectively target inflammation, but these therapies lack efficacy in preventing pathologic osteogenesis. Transgenic mice overexpressing transmembrane tumor necrosis factor (tmTNF-Tg mice) exhibit SpA-like features. We hypothesized that type H blood vessels, which are implicated in osteogenesis, are increased and contribute to pathology in this experimental SpA model. We analyzed ankles, femora, and vertebrae of tmTNF-Tg mice and nontransgenic littermates and tmTNF-Tg mice on either a TNF receptor type I (TNFRI)-deficient or TNF receptor type II (TNFRII)-deficient background for osteogenesis, angiogenesis, and inflammation using advanced imaging technologies at various stages of disease. Compared to nontransgenic littermates, tmTNF-Tg mice exhibited an increase in vertebral type H vessels and osteoprogenitor cells in subchondral bone. These features of increased angiogenesis and osteogenesis were already present before onset of clinical disease symptoms. Type H vessels and osteoprogenitor cells were in close proximity to inflammatory lesions and ectopic lymphoid structures. The tmTNF-Tg mice also showed perivertebral ectopic type H vessels and osteogenesis, an increased number of vertebral transcortical vessels, and enhanced entheseal angiogenesis. In tmTNF-Tg mice crossed on a TNFRI- or TNFRII-deficient background, no clear reduction in type H vessels was shown, suggesting that type H vessel formation is not exclusively mediated via TNFRI or TNFRII. The contribution of type H vessels to pathologic osteogenesis in experimental SpA advances our knowledge of the pathophysiology of this disease and may also provide a novel opportunity for targeted intervention.