Pincer type coumarin based N-substituted semicarbazone ligands HL1-4 and their corresponding ruthenium(II) complexes (1-4) were synthesized, analyzed and confirmed by various spectro analytical techniques. The molecular structure of the ligand HL3 and complex 3 was confirmed by single crystal X-ray diffraction analysis. The stoichiometry of complexes 1, 2 and 4 was confirmed by high resolution mass spectroscopy (HRMS). The binding affinity of the compounds with CT-DNA (Calf Thymus DNA) and BSA (Bovine Serum Albumin) was established by absorption and emission titration methods. The results of In vitro cytotoxicity showed the significant cytotoxic potential of the complexes against MDA-MB-231 cells (TNBC- Triple-negative breast cancer). Among the complexes, 1 and 4 have shown appreciable results. Further, antimigratory activity against the MDA-MB-231 cells was studied for the complexes 1 and 4. The percentage cell cycle arrest, apoptosis and necrosis were explored by flow cytometry. The in vivo anti-tumor activity of the complexes 1 and 4 using C. elegans as model organism was established by using the tumoral C. elegans strain JK1466 (gld-1(q485)), which bears a mutation in the gld-1 tumor suppressor gene. We have determined the effect of our complexes on tumor gonad reduction and found to be non toxic to the JK1466 worms and they have prolonged their mean lifespan with potential antioxidant ability by overcoming stress responses. Overall, our study reported herein demonstrated that the complexes 1 and 4 could be established as potential metallo-drugs substantiating further exploration.

BACKGROUND: As the largest organ of the body, the skin is constantly subjected to ultraviolet radiation (UVR), leading to inflammations and changes that mirror those seen in chronological aging. Although various small molecule drugs have been explored for treating skin photoaging, they typically suffer from low stability and a high incidence of adverse reactions. Consequently, the continued investigation of photoaging treatments, particularly those utilizing herbal products, remains a critical clinical endeavor. One such herbal product, Lapagyl, is derived from the bark of the lapacho tree and possesses antioxidant efficacies that could be beneficial in combating skin photoaging.
OBJECTIVE: This research aimed to evaluate the efficacy of the herbal product Lapagyl in combating UVR-induced skin photoaging. Additionally, it sought to unravel the mechanisms by which Lapagyl promotes the regeneration of the skin extracellular matrix.
METHODS: To investigate whether Lapagyl can alleviate skin aging and damage, a UVR radiation model was established using SKH-1 hairless mice. The dorsal skins of these mice were evaluated for wrinkle formation, texture, moisture, transepidermal water loss (TEWL), and elasticity. Pathological assessments were conducted to determine Lapagyl\'s efficacy. Additionally, single-cell sequencing and spectrum analysis were employed to elucidate the working mechanisms and primary components of Lapagyl in addressing UVR-induced skin aging and injury.
RESULTS: Lapagyl markedly reduced UVR-induced wrinkles, moisture loss, and elasticity decrease in SKH-1 mice. Single-cell sequencing demonstrated that Lapagyl corrected the imbalance in cell proportions caused by UVR, decreased UVR-induced ROS expression, and protected basal and spinous cells from skin damage. Additionally, Lapagyl effectively prevented the entry of inflammatory cells into the skin by reducing CCL8 expression and curtailed the UVR-induced formation of Foxp3+ regulatory T cells (Tregs) in the skin. Both pathological assessments and ex vivo skin model results demonstrated that Lapagyl effectively reduced UVR-induced damage to collagen and elastin. Spectrum analysis identified Salidroside as the primary compound remaining in the skin following Lapagyl treatment. Taken together, our study elucidated the skin protection mechanism of the herbal product Lapagyl against UVR damage at the cellular level, revealing its immunomodulatory effects, with salidroside identified as the primary active compound for skin.
CONCLUSIONS: Our study provided a thorough evaluation of Lapagyl\'s protective effects on skin against UVR damage, delving into the mechanisms at the cellular level. We discovered that Lapagyl mitigates skin inflammation and immunosuppression by regulating Foxp3+ Tregs and the CCL pathway. These insights indicate that Lapagyl has potential as a novel therapeutic option for addressing skin photoaging.

BACKGROUND: Pi-pa-run-fei-tang (PPRFT), a traditional Chinese medicine formula with long-standing history, demonstrated beneficial effect on chronic cough. However, the mechanism underlying efficacy unclear. In current research, we explored the impact and molecular mechanism of chronic cough mouse stimulating with capsaicin combined with ammonia.
OBJECTIVE: To investigate the metabolic modulating effects, and potential mechanisms underlying the therapeutic effect of PPRFT in chronic cough.
METHODS: Chronic cough mouse models were created by stimulating mice by capsaicin combined with ammonia. Number of coughs and cough latency within 2 min were recorded. With lung tissue and serum samples collected for histopathology, metabolomics, RT-qPCR, immunohistochemistry, and WB analysis. Lymphocytes were isolated and flow cytometric assays were conducted to evaluate the differentiation between Th17 and Treg cell among CD4+ cells.
RESULTS: Results indicated that PPRFT obviously reduced the number of coughs, prolonged cough latency, reduced inflammatory cell infiltration and lung tissues damage, and decreased the serum level of IL-6, IL-1β, TNF-α, and IL-17 while increasing IL-10 levels. Notably, PPRFT suppressed Th17 cell divergence and promoted Treg cell divergence. Furthermore, serum metabolomic assays showed that 46 metabolites differed significantly between group, with 35 pathways involved. Moreover, mRNA levels of IL-6, NF-κB, IL-17, RORγT, JAK2, STAT3, PI3K and AKT in lung tissues remarkably reduced and mRNA levels of IL-10 and FOXP3 were elevated after PPRFT pretreatment. Additionally, PPRFT treatments decreased the protein levels of IL-6, NF-κB, IL-17, RORγT, p-JAK2, p-STAT3, p-PI3K, and p-AKT and increased the protein levels of IL-10 and FOXP3, but no significantly effects to the levels on JAK2, STAT3, PI3K, and AKT in the lungs.
CONCLUSIONS: Conclusively, our result suggested the effect with PPRFT on chronic cough may be mediated through IL-6/JAK2/STAT3 and PI3K/AKT/NF-κB pathway, which regulate the differentiation between Th17 and Treg cell. This beneficial effect of PPRFT in capsaicin and ammonia-stimulated chronic cough mice indicates its potential application in treating chronic cough.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype, with dismal prognosis and limited option in advanced settings, yet stromal tumor infiltrating lymphocytes (sTILs) in this subtype has a predictive role.
METHODS: The International Breast Cancer Study Group (IBCSG) Trial 22-00 is a randomized phase III clinical trial testing the efficacy of low-dose metronomic oral Cyclophosphamide-Methotrexate (CM) maintenance following standard adjuvant chemotherapy treatment for early-stage hormone receptor-negative breast cancer patients. A case-cohort sampling was used. We characterized immune cells infiltrates in patients with TNBC by 6 plex immunofluorescence (IF) staining for CD4, FOXP3, CD3, cytokeratine and CD8 RESULTS: We confirmed that high immune CD3+ T cells as well as stromal and intra-epithelial Tregs (CD4+Foxp3+ T cells) infiltrates were associated with a better Distant Recurrence-Free Interval (DRFI), especially in LN+ patient, regardless of the treatment. More importantly, we showed that the spatial distribution of immune cells at baseline is crucial, as CM maintenance was detrimental for T cells excluded LN+ TNBC patients.
CONCLUSIONS: immune spatial classification on immune cells infiltrates seems crucial and could help patients\' selection in clinical trial and greatly improve responses to specific therapies.

BACKGROUND: Obstructive sleep apnea (OSA) can be considered a chronic inflammatory disease that impacts all bodily systems, including the immune system. This study aims to assess the Th17/Treg pattern in patients with OSA and the effect of continuous positive airway pressure (CPAP) treatment.
METHODS: OSA patients and healthy controls were recruited. OSA patients recommended for CPAP treatment were followed up for three months. Flow cytometry was employed to determine the proportion of Th17 and Treg cells. Real-time quantitative polymerase chain reaction (PCR) and western blotting were utilized to detect the mRNA and protein levels of receptor-related orphan receptor γt (RORγt) and forkhead/winged helix transcription factor (Foxp3), respectively, in peripheral blood mononuclear cells (PBMCs). Enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum levels of interleukin-17 (IL-17), IL-6, transforming growth factor-β1 (TGF-β1), and hypoxia-induced factor-1α (HIF-1α).
RESULTS: A total of 56 OSA patients and 40 healthy controls were recruited. The proportion of Th17 cells, Th17/Treg ratio, mRNA and protein levels of RORγt, and serum IL-17, IL-6, and HIF-1α levels were higher in OSA patients. Conversely, the proportion of Treg cells, mRNA and protein levels of Foxp3, and serum TGF-β1 levels were decreased in OSA patients. The proportion of Th17 and Treg cells in OSA can be predicted by the apnea hypopnea index (AHI), IL-6, TGF-β1 and, HIF-1α. 30 moderate-to-severe OSA patients were adherent to three-month CPAP treatment, with improved Th17/Treg imbalance, IL-17, IL-6, TGF-β1, and HIF-1α levels compared to pre-treatment values.
CONCLUSIONS: There was a Th17/Treg imbalance in OSA patients. The prediction of Th17 and Treg cell proportions in OSA can be facilitated by AHI, as well as serum IL-6, TGF-β1, and HIF-1α levels. Furthermore, CPAP treatment can potentially improve the Th17/Treg imbalance and reduce proinflammatory cytokines in OSA patients.

BACKGROUND: Triple-negative breast cancer (TNBC) has been documented as the most aggressive subtype of breast cancer. This study aimed to analyze antitumor and protumor immune activities, and their ratios as significant prognostic biomarkers in metastatic TNBC (mTNBC).
METHODS: A multicenter cohort study was conducted among 103 de novo mTNBC patients. The expression of CD8 and CD163 was evaluated using immunohistochemistry staining, CD4 and FOXP3 using double-staining immunohistochemistry, and PD-L1 using immunohistochemistry and RT-PCR.
RESULTS: Multivariate analysis revealed that high CD4/FOXP3 (HR 1.857; 95% CI 1.049-3.288; p = 0.034) and the CD8/CD163 ratio (HR 2.089; 95% CI 1.174-3.717; p = 0.012) yield significantly improved 1 year overall survival (OS). Kaplan-Meier analysis showed that high levels of CD4 (p = 0.023), CD8 (p = 0.043), CD4/FOXP3 (p = 0.016), CD8/FOXP3 (p = 0.005), CD8/CD163 (p = 0.005) ratios were significantly associated with higher rate of 1 year OS. Furthermore, 1 year OS was directly correlated with antitumor CD4 (R = 0.233; p = 0.018) and CD8 (R = 0.219; p = 0.026) and was indirectly correlated with protumor CD163 and FOXP3 through CD4/FOXP3 (R = 0.282; p = 0.006), CD4/CD163 (R = 0.239; p = 0.015), CD8/FOXP3 (R = 0.260; p = 0.008), and CD8/CD163 (R = 0.258; p = 0.009).
CONCLUSIONS: This is the first study to demonstrate that high levels of CD4/FOXP3 and CD8/CD163 significantly improved the 1 year OS in de novo mTNBC patients. Thus, we recommend the application of these markers as prognosis determination and individual treatment decision.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive cancer subtype, with limited treatments and a high metastasis risk. The varying location of metastasis in TNBC patients often leads to in prognosis in breast cancer. Therefore, this study aimed to investigate the potential association between immune cells profiles in the tumor microenvironment and metastatic patterns.
METHODS: We conducted a multicenter cross-sectional study in 2022 to examine formalin-fixed paraffin-embedded (FFPE) and medical record data from 2015 to 2020 in de novo metastatic TNBC patients. The medical records provided crucial information about the sites of metastasis. Immunohistochemistry (IHC) analysis was carried out on primary breast tumor tissues to evaluate the expressions of cluster of differentiation (CD)4 T-cells, CD8 T-cells, CD163, FOXP3 Tregs, and programmed death-ligand 1 (PD-L1), along with immune cells ratios showing antitumor-to-protumor activity (CD4/FOXP3, CD8/FOXP3, CD4/CD163, CD8/CD163). Metastatic locations were grouped into bone-only, visceral, lung, liver, and brain metastasis.  Results: A total of 120 metastatic TNBC patients were documented for their metastatic location and IHC report. The clinical and histopathological characteristics showed that the majority of the patients were within the 40-65 years old group, and 34.2% had standard body mass index (BMI). Furthermore, the majority (89.22%) of the patients showed No Special Type (NST), (56.7%) had histopathology grade III, high Ki-67 ≥20% (85.8%), and positive PD-L1 expression (30.8%), with visceral metastasis indicating the highest proportion of 75.8%. Patients with a high CD8/FOXP3 and CD4/FOXP3 ratio were significantly prone to have bone-only metastasis compared to visceral metastasis (p= 0.028 and p=0.024, respectively).  Conclusion: The ratio of antitumor to protumor T-lymphocytes had a significant relevance in the metastatic location patterns in TNBC.

Immune checkpoint therapy (ICT) provides a new idea for the treatment of advanced clear cell renal cell carcinoma (ccRCC), which can bring significant benefits to patients. However, the clinical application of ICT is limited because of the lack of predictive biomarkers to select potential responders. This study aims to propose a new biomarker to predict the response to Nivolumab in patients with ccRCC.
The genes that significantly improve the prognosis of ccRCC were retrieved from The Cancer Genome Atlas (TCGA) database. The genomic and clinical data were from patients that had been registered in prospective clinical trials (CheckMate 009, CheckMate 010 and CheckMate 025). TCGA, Gene Expression Omnibus (GEO), and The Human Protein Atlas database were used to analyze the gene and protein expression of WD repeat-containing protein 72 (WDR72) in ccRCC. Gene Ontology (GO) & The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to dig relevant mechanisms of WDR72. Single sample gene set enrichment analysis (ssGSEA) was conducted to evaluate the role of WDR72 in immune infiltration. Cell proliferation assay, FAO and ATP quantification were used to explore and verify the molecular mechanisms. The expression of WDR72, FOXP3, CD8, and CPT1A was examined by IHC in 20 advanced ccRCC tissue samples at the Urology Department of our hospital. The MethSurv was used to identify PBRM1 and WDR72 gene methylation and its effect on prognosis of ccRCC.
WDR72 is the most significant gene for improving overall survival (OS) in ccRCC. In all three checkmates, OS and progression free survival (PFS) were found to be significantly higher in WDR72 high expression group than that in WDR72 low expression group (P=0.040 and P=0.012, respectively), and similar conclusions could be drawn from the PBRM1-mutation (MUT) compared with the PBRM1-wildtype (WT) (P=0.007 and P=0.006, respectively). What\'s more, high expression of WDR72 plus PBRM1-MUT as a combinatorial biomarker showed improved OS (HR=0.388, P=0.0026) and PFS (HR=0.39, P=0.0066) compared to low expression of WDR72 plus PBRM1-WT. Functional enrichment analysis showed that WDR72 was closely positively related to fatty acid degradation and fatty acid beta oxidation pathway in ccRCC. In vitro experiments showed that high expression of WDR72 can promote fatty acids oxidation and inhibit the proliferation of ccRCC cells. Immune analysis revealed that WDR72 high expression was associated with decreased infiltration of Treg cells and low ssGSEA score of check-point. IHC results showed that WDR72 was negatively correlated with FOXP3 expression (r=-0.506, P=0.023) and positively correlated with CPT1A expression (r=0.529, P=0.017).
The present study indicated that high expression of WDR72 may indicate a good prognosis of patients treated with Nivolumab and WDR72 expression combined with PBRM1 mutation could be more persuasive to predict the response for ICT in ccRCC patients.

BACKGROUND: Regulatory T cells (Tregs) are involved in the systemic immune response after ischemic stroke. However, their role remains unclear, and the effect appears to be both neuroprotective and detrimental. Treg suppressor function may result in immunodepression and promote stroke-associated infection (SAI). Thus we assume that the bidirectional effects of Tregs may be in part attributed to the intracellular transcription factor Helios. Tregs with Helios expression (H+ Tregs) constitute 70-90% of all Treg cells and more frequently than Helios-negative Tregs (H- Tregs) express molecules recognized as markers of Tregs with suppressor abilities.
RESULTS: We prospectively assessed the circulating Treg population with flow cytometry in 52 subjects on days 1, 3, 10 and 90 after ischemic stroke and we compared the results with those obtained in concurrent age-, sex- and vascular risk factor-matched controls. At all studied time points the percentage of H+ Tregs decreased in stroke subjects-D1: 69.1% p < 0.0001; D3: 62.5% (49.6-76.6), p < 0.0001; D10: 60.9% (56.5-72.9), p < 0.0001; D90: 79.2% (50.2-91.7), p = 0.014 vs. controls: 92.7% (81.9-97.0) and the percentage of H- Tregs increased accordingly. In patients with SAI the percentage of pro-suppressor H+ Tregs on post-stroke day 3 was higher than in those without infection (p = 0.03). After adjustment for confounders, the percentage of H+ Tregs on day 3 independently correlated with SAI [OR 1.29; CI 95%: 1.08-1.27); p = 0.02]. Although the percentage of H+ Tregs on day 3 correlated positively with NIHSS score on day 90 (rS = 0.62; p < 0.01) and the infarct volume at day 90 (rS = 0.58; p < 0.05), in regression analysis it was not an independent risk factor.
CONCLUSIONS: On the first day after stroke the proportion of H+ vs. H- Tregs changes in favor of pro-inflammatory H- Tregs, and this shift continues toward normalization when assessed on day 90. A higher percentage of pro-suppressive H+ Tregs on day 3 independently correlates with SAI and is associated positively with NIHSS score, but it does not independently affect the outcome and stroke area in the convalescent phase of stroke.

Immune checkpoint inhibitor (ICI) combination therapies have shown promise in the first-line treatment of advanced biliary tract cancer (BTC). However, the best partner remains to be validated. Moreover, progress on biomarkers predicting the efficacy of ICI in BTC is slow. This study aimed to assess the efficacy and investigate reliable predictive biomarkers of programmed cell death protein-1 (PD-1) antibody combination therapy in the first-line treatment of advanced BTC.
Clinical data from patients with advanced BTC who received chemotherapy or anti-PD-1 combination therapy as first-line were collected. The primary outcome was overall survival (OS). Biomarkers, including peripheral blood inflammation scores, genetic alterations, and tumor microenvironment were investigated.
Sixty-four patients were recruited and divided into four treatment groups: chemotherapy, anti-PD-1 plus chemotherapy, anti-PD-1 plus targeted therapy, and triple group (anti-PD-1 plus chemotherapy and targeted therapy). The median OS was 7.9, 11.3, 12.8, and 28.7 months, respectively. Compared to chemotherapy, mOS significantly prolonged in the triple group (p = 0.031). It showed that patients with five different peripheral blood inflammation scores had significantly prolonged mOS (p < 0.05). Genetic testing results suggested that patients with poor survival all had TP53 mutations and higher levels of KRAS and ERBB2 mutations. Low FOXP3/CD8 ratio was associated with prolonged OS (p = 0.029). With CD4-low, CD8-high, CD56-positive, CD163-high, FOXP3-high and MPO-high in TME as one factor, we calculated PLUS score according to the number of factors. The high-PLUS (>2) group showed significantly superior OS (p = 0.003).
First-line anti-PD-1 combination therapy was superior to chemotherapy, and triple therapy significantly improved survival. Peripheral blood immune-inflammation score, FOXP3/CD8 ratio, and PLUS have potential as biomarkers for predicting the efficacy of first-line anti-PD-1 therapy in advanced BTC.