Innate Lymphocyte Mechanisms in Skin Diseases.

Innate lymphocyte populations are emerging as key effectors in tissue homeostasis, microbial defense, and inflammatory skin disease. The cells are evolutionarily ancient and carry conserved principles of function, which can be achieved through shared or unique specific mechanisms. Recent technological and treatment advances have provided insight into heterogeneity within and between individuals and species. Similar pathways can extend through to adaptive lymphocytes, which softens the margins with innate lymphocyte populations and allows investigation of nonredundant pathways of immunity and inflammation that might be amenable to therapeutic intervention. Here, we review advances in understanding of innate lymphocyte biology with a focus on skin disease and the roles of commensal and pathogen responses and tissue homeostasis.

CD1 lipidomes reveal lipid-binding motifs and size-based antigen-display mechanisms.

The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid display. Answering a basic question, the detection of >2,000 CD1-lipid complexes demonstrates broad presentation of self-sphingolipids and phospholipids. Whereas peptide antigens are chemically processed, many lipids are presented in an unaltered form. However, each type of CD1 protein differentially edits the self-lipidome to show distinct capture motifs based on lipid length and chemical composition, suggesting general antigen display mechanisms. For CD1a and CD1d, lipid size matches the CD1 cleft volume. CD1c cleft size is more variable, and CD1b is the outlier, where ligands and clefts show an extreme size mismatch that is explained by uniformly seating two small lipids in one cleft. Furthermore, the list of compounds that comprise the integrated CD1 lipidome supports the ongoing discovery of lipid blockers and antigens for T cells.

Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity.

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.

Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a.

Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a-PG complex showed that the acyl chains were buried within the A'- and F'-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F'-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer+CD4+ T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a+ dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4+ CD1a-(lysyl)PG tetramer+ T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis.

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.

Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI. METHODS: A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-ß)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI. RESULTS: The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-ß-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function. CONCLUSION: This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

CD1a and skin T cells: a pathway for therapeutic intervention.

The CD1 and MR1 protein families present lipid antigens and small molecules to T cells, complementing well-studied major histocompatibility complex-peptide mechanisms. The CD1a subtype is highly and continuously expressed within the skin, most notably on Langerhans cells, and has been demonstrated to present self and foreign lipids to T cells, highlighting its cutaneous sentinel role. Alteration of CD1a-dependent T-cell responses has recently been discovered to contribute to the pathogenesis of several inflammatory skin diseases. In this review, we overview the structure and role of CD1a and outline the current evidence implicating CD1a in the development of psoriasis, atopic dermatitis and allergic contact dermatitis.

Jagged/Notch proteins promote endothelial-mesenchymal transition-mediated pulmonary arterial hypertension via upregulation of the expression of GATAs.

This study tested the hypothesis that Jagged2/Notches promoted the endothelial-mesenchymal transition (endMT)-mediated pulmonary arterial hypertension (PAH) (i.e. induction by monocrotaline [MCT]/63 mg/kg/subcutaneous injection) through increasing the expression of GATA-binding factors which were inhibited by propylthiouracil (PTU) (i.e. 0.1% in water for daily drinking since Day 5 after PAH induction) in rodent. As compared with the control (i.e. HUVECs), the protein expressions of GATAs (3/4/6) and endMT markers (Snail/Zeb1/N-cadherin/vimentin/fibronectin/α-SMA/p-Smad2) were significantly reduced, whereas the endothelial-phenotype markers (CD31/E-cadherin) were significantly increased in silenced JAG2 gene or in silenced GATA3 gene of HUVECs (all p < 0.001). As compared with the control, the protein expressions of intercellular signallings (GATAs [3/4/6], Jagged1/2, notch1/2 and Snail/Zeb1/N-cadherin/vimentin/fibronectin/α-SMA/p-Smad2) were significantly upregulated in TGF-ß/monocrotaline-treated HUVECs that were significantly reversed by PTU treatment (all p < 0.001). By Day 42, the results of animal study demonstrated that the right-ventricular systolic-blood-pressure (RVSBP), RV weight (RVW) and lung injury/fibrotic scores were significantly increased in MCT group than sham-control (SC) that were reversed in MCT + PTU groups, whereas arterial oxygen saturation (%) and vasorelaxation/nitric oxide production of PA exhibited an opposite pattern of RVW among the groups (all p < 0.0001). The protein expressions of hypertrophic (ß-MHC)/pressure-overload (BNP)/oxidative-stress (NOX-1/NOX-2) biomarkers in RV and the protein expressions of intercellular signalling (GATAs3/4/6, Jagged1/2, notch1/2) and endMT markers (Snail/Zeb1/N-cadherin/vimentin/fibronectin/TGF-ß/α-SMA/p-Smad2) in lung parenchyma displayed an identical pattern of RVW among the groups (all p < 0.0001). Jagged-Notch-GATAs signalling, endMT markers and RVSBP that were increased in PAH were suppressed by PTU.

Different treatment efficacies and T790M acquisition of EGFR-TKIs on NSCLC patients with variable Del-19 subtypes of EGFR.

EGFR exon 19 deletion (Del-19) comprises multiple advanced NSCLC subtypes. EGFR-tyrosine kinase inhibitor (TKI) efficacy and T790M acquisition in various Del-19 subtypes is unknown. We prospectively collected tissue samples from patients harboring NSCLC with Del-19 between 2006 and 2020. We evaluated EGFR-TKI treatment effectiveness among the different Del-19 subtypes. We collected 1391 NSCLC samples from 892 patients with Del-19, and the most common subtype was del E746-A750 (67.5%). 741 patients had taken first- or second-generation EGFR-TKIs. There were no significant differences in response rates between patients with different Del-19 subtypes (P = .630). Patients with indel E746 had the longest median PFS (14.6 months), but those with non-LRE deletions had the shortest PFS (8.9 months; P = .002). For OS analysis, patients with indel E746 also had the longest OS (34.1 months), but those with non-LRE deletions had the shortest OS (21.1 months; P = .046). Patients with different Del-19 subtypes showed no significant differences in the T790M acquisition rates (P = .443). Among the 151 patients with acquired T790M who received third-generation EGFR-TKIs, the Del-19 subtype was not associated with different RR and PFS. In vitro cellular viability and activation of the EGFR pathway analysis were consistent with the clinical findings. In conclusion, compared with del E746-A750, indel E746 was associated with longer PFS and OS, but the non-LRE subtype was correlated with shorter survival prognosis. There were no significant differences in the acquired T790M rate and treatment effectiveness of subsequent third-generation EGFR-TKIs between various Del-19 subgroups.

Competitive Assay for the Ultrasensitive Detection of Organophosphate Pesticides Based on a Fiber-Optic Particle Plasmon Resonance Biosensor and an Acetylcholinesterase Binding Peptide.

An acetylcholinesterase (AChE) binding-based biosensor was developed for the ultrasensitive detection of organophosphate (OP) pesticides. The biosensor integrates the technique based on fiber-optic particle plasmon resonance detection and a synthetic AChE binding peptide conjugated with gold nanoparticles on the optical fiber surface via an AChE competitive binding assay. The OP pesticides present in the solution hinder the binding of AChE to the peptide on the biosensor by competing for the binding sites present in AChE. The limit of detection obtained for parathion using this method was observed to be 0.66 ppt (2.3 pM). This method shows a wide linear dynamic range of 6 orders. Furthermore, the use of the AChE binding peptide in the biosensor can better discriminate OPs against carbamates by using only a single biosensor. The practical application of this method was tested using spiked samples, which yielded good recovery and reproducibility. The spiked sample required minimal pretreatment before analysis; hence, this biosensor may also be used in the field.

Phospholipase activity of acyloxyacyl hydrolase induces IL-22-producing CD1a-autoreactive T cells in individuals with psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by Th17 responses. Recent evidence has identified Langerhans cells to have a key role in disease pathogenesis, with constitutive high expression of CD1a and capacity to present lipid antigens to T cells. Phospholipase A2 enzymes generate neolipid antigens for recognition by CD1a-reactive T cells; however, the broader enzymatic pathways of CD1a lipid ligand generation have not been thoroughly investigated. In this study, we used immunofluorescence of skin and ELISpot analyses of CD1a-reactive T cells to investigate the role of the lipase acyloxyacyl hydrolase (AOAH) in CD1a ligand generation with relevance to the pathogenesis of psoriasis. We found that the PLA2 activity of rAOAH leads to the activation of circulating CD1a auto-reactive T cells, leading to the production of IFN-γ and IL-22. Circulating AOAH-responsive CD1a-reactive T cells from patients with psoriasis showed elevated IL-22 production. We observed that AOAH is highly expressed in psoriatic lesions compared to healthy skin. Overall, these data present a role for AOAH in generating antigens that activate circulating lipid-specific CD1a-restricted T cells and, thus, contribute to psoriatic inflammation. These findings suggest that inhibition of PLA2 activity of AOAH may have therapeutic potential for individuals with psoriasis.

Identification of immune correlates of fatal outcomes in critically ill COVID-19 patients.

Prior studies have demonstrated that immunologic dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of the immunologic drivers of death in the most critically ill patients. We performed immunophenotyping of viral antigen-specific and unconventional T cell responses, neutralizing antibodies, and serum proteins in critically ill patients with SARS-CoV-2 infection, using influenza infection, SARS-CoV-2-convalescent health care workers, and healthy adults as controls. We identify mucosal-associated invariant T (MAIT) cell activation as an independent and significant predictor of death in COVID-19 (HR = 5.92, 95% CI = 2.49-14.1). MAIT cell activation correlates with several other mortality-associated immunologic measures including broad activation of CD8+ T cells and non-Vδ2 γδT cells, and elevated levels of cytokines and chemokines, including GM-CSF, CXCL10, CCL2, and IL-6. MAIT cell activation is also a predictor of disease severity in influenza (ECMO/death HR = 4.43, 95% CI = 1.08-18.2). Single-cell RNA-sequencing reveals a shift from focused IFNα-driven signals in COVID-19 ICU patients who survive to broad pro-inflammatory responses in fatal COVID-19 -a feature not observed in severe influenza. We conclude that fatal COVID-19 infection is driven by uncoordinated inflammatory responses that drive a hierarchy of T cell activation, elements of which can serve as prognostic indicators and potential targets for immune intervention.

LncRNA SLCO4A1-AS1 suppresses lung cancer progression by sequestering the TOX4-NTSR1 signaling axis.

BACKGROUND: Metastasis is a multistep process involving the migration and invasion of cancer cells and is a hallmark of cancer malignancy. Long non-coding RNAs (lncRNAs) play critical roles in the regulation of metastasis. This study aims to elucidate the role of the lncRNA solute carrier organic anion transporter family member 4A1-antisense 1 (SLCO4A1-AS1) in metastasis and its underlying regulatory mechanisms. METHODS: A comprehensive analysis of the Gene Expression Omnibus (GEO) database were used to identify metastasis-associated lncRNAs. Transwell migration and invasion assays, and a tail vein-injection mouse model were used to assess the migration and invasion of cancer cells in vitro and in vivo, respectively. High-throughput screening methods, including MASS Spectrometry and RNA sequencing (RNA-seq), were used to identify the downstream targets of SLCO4A1-AS1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blotting, RNA pull-down, RNA immunoprecipitation (RIP), fluorescence in situ hybridization (FISH), and chromatin immunoprecipitation (ChIp) assays were conducted to identify and validate the underlying regulatory mechanisms of SLCO4A1-AS1. RESULTS: SLCO4A1-AS1 reduced cancer cell migration and invasion by disrupting cytoskeleton filaments, and was associated with longer overall survival in patients with lung adenocarcinoma. SLCO4A1-AS1 directly interacted with the DNA-binding protein, TOX High Mobility Group Box Family Member 4 (TOX4), to inhibit TOX4-induced migration and invasion. Furthermore, RNA-seq revealed that neurotensin receptor 1 (NTSR1) is a novel and convergent downstream target of SLCO4A1-AS1 and TOX4. Mechanistically, SLCO4A1-AS1 functions as a decoy of TOX4 by interrupting its interaction with the NTSR1 promoter and preventing NTSR1 transcription. Functionally, NTSR1 promotes cancer cell migration and invasion through cytoskeletal remodeling, and knockdown of NTSR1 significantly inhibits TOX4-induced migration and invasion. CONCLUSION: These findings demonstrated that SLCO4A1-AS1 antagonizes TOX4/NTSR1 signaling, underscoring its pivotal role in lung cancer cell migration and invasion. These findings hold promise for the development of novel therapeutic strategies targeting the SLCO4A1-AS1/TOX4/NTSR1 axis as a potential avenue for effective therapeutic intervention in lung cancer.

The outcomes of thoracoscopic decortication between fungal empyema and bacterial empyema.

BACKGROUND: Fungal empyema is an uncommon disease and is associated with a high mortality rate. Surgical intervention is suggested in stage II and III empyema. However, there were no studies that reported the outcomes of surgery for fungal empyema. METHODS: This study is a retrospective analysis in a single institute. Patients with empyema thoracis who underwent thoracoscopic decortication between January 2012 and December 2021 were included in the study. We separated the patients into a fungal empyema group and a bacterial empyema group according to culture results. We used 1:3 propensity score matching to reduce selection bias. RESULTS: There were 1197 empyema patients who received surgery. Of these, 575 patients showed positive culture results and were enrolled. Twenty-eight patients were allocated to the fungal empyema group, and the other 547 patients were placed in the bacterial empyema group. Fungal empyema showed significantly longer intensive care unit stay (16 days vs. 3 days, p = 0.002), longer median ventilator usage duration (20.5 days vs. 3 days, p = 0.002), longer hospital stay duration (40 days vs. 17.5 days, p < 0.001) and a higher 30-day mortality rate (21.4% vs. 5.9%, p < 0.001). Fungal empyema revealed significantly poorer 1-year survival rate than bacterial empyema before matching (p < 0.001) but without significant difference after matching. CONCLUSIONS: The fungal empyema patients had much worse surgical outcomes than the bacterial empyema patients. Advanced age and high Charlson Comorbidity Index score are independent predictors for poor prognosis. Prompt surgical intervention combined with the use of antifungal agents was the treatment choice for fungal empyema.

Dual-task multicomponent exercise-cognitive intervention improved cognitive function and functional fitness in older adults.

BACKGROUND: Maintaining cognitive and physical function is essential to healthy aging. AIM: This study aims to investigate the effect of an exercise-cognitive dual-task program in Chinese language on cognitive function and functional fitness in older adults. METHODS: Seventy adults aged 60-84 years were conveniently assigned to one of the three groups: exercise-cognitive dual-task (EC) group (n = 28), exercise group (n = 22), and control group (n = 20). The EC group received a 90-min class including multicomponent exercise-cognitive dual-task twice a week. The exercise group received a 90-min class including multicomponent exercise twice a week. The control group maintained their regular level of physical activity and general lifestyle. Cognitive functions and functional fitness were measured before and after the 12-week intervention. RESULTS: The participants in the EC and exercise group significantly improved scores in Taiwanese Frontal Assessment Battery, the Chang Gung University Orthographical Fluency Test, and the Mini-Mental State Examination; however, those in the control group did not. The participants in the EC and exercise group showed significant increases in almost all functional fitness tests. The participants in the EC group showed significantly higher improvements in Chang Gung University Orthographical Fluency Test score and aerobic endurance than the exercise group and in Chang Gung University Orthographical Fluency Test score and lower body strength than the control group. In addition, the changes in Taiwanese Frontal Assessment Battery and Mini-Mental State Examination scores were significantly correlated with the changes in functional fitness. CONCLUSION: The dual-task intervention resulted in greater improvements in verbal fluency, endurance, and muscular strength than exercise alone and control.

A Robust GPS Navigation Filter Based on Maximum Correntropy Criterion with Adaptive Kernel Bandwidth.

Multiple forms of interference and noise that impact the receiver's capacity to receive and interpret satellite signals, and consequently the preciseness of positioning and navigation, may be present during the processing of Global Positioning System (GPS) navigation. The non-Gaussian noise predominates in the signal owing to the fluctuating character of both natural and artificial electromagnetic interference, and the algorithm based on the minimum mean-square error (MMSE) criterion performs well when assuming Gaussian noise, but drops when assuming non-Gaussian noise. The maximum correntropy criteria (MCC) adaptive filtering technique efficiently reduces pulse noise and has adequate performance in heavy-tailed noise, which addresses the issue of filter performance caused by the presence of non-Gaussian or heavy-tailed unusual noise values in the localizing measurement noise. The adaptive kernel bandwidth (AKB) technique employed in this paper applies the calculated adaptive variables to generate the kernel function matrix, in which the adaptive factor can modify the size of the kernel width across a reasonably appropriate spectrum, substituting the fixed kernel width for the conventional MCC to enhance the performance. The conventional maximum correntropy criterion-based extended Kalman filter (MCCEKF) algorithm's performance is significantly impacted by the value of the kernel width, and there are certain predetermined conditions in the selection based on experience. The MCCEKF with a fixed adaptive kernel bandwidth (MCCEKF-AKB) has several advantages due to its novel concept and computational simplicity, and gives a qualitative solution for the study of random structures for generalized noise. Additionally, it can effectively achieve the robust state estimation of outliers with anomalous values while guaranteeing the accuracy of the filtering.

How Cultural Behaviors and Superstitions Associate the Willingness to Undergo Cataract Surgery in Taiwan: A Nationwide Survey.

Background and Objectives: Cultural beliefs influence the conceptualization, adaptation, and coping strategies for diseases. This study aimed to investigate the impact of cultural beliefs and customs on the willingness to undergo cataract surgery in Taiwan. Materials and Methods: The data were retrospectively retrieved from the national Longitudinal Health Insurance Database 2000 (LHID2000). From the national database, we enrolled patients that were diagnosed with cataracts and underwent cataract surgery from 2001 to 2010. All the patients were stratified according to their gender and living area. Gender was categorized as male or female, and the living area was classified as urban or rural. We compared the difference in the number of surgeries between stratified patient groups in each Chinese lunar month. Results: The number of cataract surgeries decreased significantly in the seventh and twelfth lunar months for both genders. There was a significant reduction in cataract surgeries in both the urban and rural groups during the seventh lunar month. Interestingly, only the seventh lunar month had an association with sex in different living areas, which meant that in the seventh month, there was a gender-specific difference in the surgical numbers. Conclusions: The Taiwanese population holds a belief that surgical procedures, including cataract surgery, during the lunar ghost month is inauspicious. Citizens tend to avoid elective surgery due to cultural practice, resulting in a decrease in surgical numbers during the period of the Chinese New Year. The authorities should consider these cultural behaviors when developing medical policies and allocating resources. Healthcare providers should be aware of these superstitions and take them into account when providing medical care and advice to patients.

Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization.

BACKGROUND: Asymmetric dimethylarginine (ADMA), which is significantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. However, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known. METHODS: Three mouse breast cell lines (normal breast epithelial HC11, breast cancer EMT6 and triple negative breast cancer 4T1) and their equivalent 3D stem cell culture were used to analyze the secretion of ADMA using ELISA and their responses to ADMA. Bone marrow-derived macrophages and/or RAW264.7 cells were used to determine the impact of increased extracellular ADMA on macrophage-tumor interactions. Gene/protein expression was analyzed through RNAseq, qPCR and flow cytometry. Protein functional analyses were conducted via fluorescent imaging (arginine uptake, tumor phagocytosis) and enzymatic assay (arginase activity). Cell viability was measured via MTS assay and/or direct cell counting using Countess III FL system. RESULTS: For macrophages, ADMA impaired proliferation and phagocytosis of tumor cells, and even caused death in cultures incubated without arginine. ADMA also led to an unusual macrophage phenotype, with increased expression of arginase, cd163 and cd206 but decreased expression of il10 and dectin-1. In contrast to the severely negative impacts on macrophages, ADMA had relatively minor effects on proliferation and survival of mouse normal epithelial HC11 cells, mouse breast cancer EMT6 and 4T1 cells, but there was increased expression of the mesenchymal markers, vimentin and snail2, and decreased expression of the epithelial marker, mucin-1 in EMT6 cells. When tumor cells were co-cultured ex vivo with tumor antigen in vivo-primed splenocytes, the tumor cells secreted more ADMA and there were alterations in the tumor cell arginine metabolic landscape, including increased expression of genes involved in arginine uptake, metabolism and methylation, and decreased expression of a gene that is responsible for arginine demethylation. Additionally, interferon-gamma, a cytokine involved in immune challenge, increased secretion of ADMA in tumor cells, a process attenuated by an autophagy inhibitor. CONCLUSION: Our results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.

Synergic Effect of Early Administration of Probiotics and Adipose-Derived Mesenchymal Stem Cells on Alleviating Inflammation-Induced Chronic Neuropathic Pain in Rodents.

This study investigated the hypothesis that probiotics enhanced the therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on alleviating neuropathic pain (NP) due to chronic constriction injury (CCI) mainly through regulating the microbiota in rats. SD rats (n = 50) were categorized into group 1 (sham-control), group 2 (NP), group 3 (NP + probiotics (i.e., 1.5 billion C.F.U./day/rat, orally 3 h after NP procedure, followed by QOD 30 times)), group 4 (NP + ADMSCs (3.0 × 105 cells) 3 h after CCI procedure, followed by QOD six times (i.e., seven times in total, i.e., mimic a clinical setting of drug use) and group 5 (NP + probiotics + ADMSCs (3.0 × 105 cells)) and euthanized by day 60 after NP induction. By day 28 after NP induction, flow-cytometric analysis showed circulating levels of early (AN-V+/PI−) and late (AN-V+/PI+) apoptotic, and three inflammatory (CD11b-c+, Ly6G+ and MPO+) cells were lowest in group 1 and significantly progressively reduced in groups 2 to 5 (all p < 0.0001). By days 7, 14, 21, 28, and 60 after CCI, the thresholds of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT) were highest in group 1 and significantly progressively increased in groups 2 to 5 (all p < 0.0001). Numbers of pain-connived cells (Nav1.8+/peripherin+, p-ERK+/peripherin+, p-p38+/peripherin+ and p-p38+/NF200+) and protein expressions of inflammatory (p-NF-κB, IL-1ß, TNF-α and MMP-9), apoptotic (cleaved-caspase-3, cleaved-PARP), oxidative-stress (NOX-1, NOX-2), DNA-damaged (γ-H2AX) and MAPK-family (p-P38, p-JNK, p-ERK1/2) biomarkers as well as the protein levels of Nav.1.3, Nav.1.8, and Nav.1.9 in L4-L5 in dorsal root ganglia displayed an opposite pattern of mechanical PWT among the groups (all p < 0.0001). In conclusion, combined probiotic and ADMSC therapy was superior to merely one for alleviating CCI-induced NP mainly through suppressing inflammation and oxidative stress.

Upregulation of peroxisome proliferator-activated receptor-α and the lipid metabolism pathway promotes carcinogenesis of ampullary cancer.

Ampullary cancer is a rare periampullary cancer currently with no targeted therapeutic agent. It is important to develop a deeper understanding of the carcinogenesis of ampullary cancer. We attempted to explore the characteristics of ampullary cancer in our dataset and a public database, followed by a search for potential drugs. We used a bioinformatics pipeline to analyze complementary (c)DNA microarray data of ampullary cancer and surrounding normal duodenal tissues from five patients. A public database from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) was applied for external validation. Bioinformatics tools used included the Gene Set Enrichment Analysis (GSEA), Database for Annotation, Visualization and Integrated Discovery (DAVID), MetaCore, Kyoto Encyclopedia of Genes and Genomes (KEGG), Hallmark, BioCarta, Reactome, and Connectivity Map (CMap). In total, 9097 genes were upregulated in the five ampullary cancer samples compared to normal duodenal tissues. From the MetaCore analysis, genes of peroxisome proliferator-activated receptor alpha (PPARA) and retinoid X receptor (RXR)-regulated lipid metabolism were overexpressed in ampullary cancer tissues. Further a GSEA of the KEGG, Hallmark, Reactome, and Gene Ontology databases revealed that PPARA and lipid metabolism-related genes were enriched in our specimens of ampullary cancer and in the NCBI GSE39409 database. Expressions of PPARA messenger (m)RNA and the PPAR-α protein were higher in clinical samples and cell lines of ampullary cancer. US Food and Drug Administration (FDA)-approved drugs, including alvespimycin, trichostatin A (a histone deacetylase inhibitor), and cytochalasin B, may have novel therapeutic effects in ampullary cancer patients as predicted by the CMap analysis. Trichostatin A was the most potent agent for ampullary cancer with a half maximal inhibitory concentration of < 0.3 µM. According to our results, upregulation of PPARA and lipid metabolism-related genes are potential pathways in the carcinogenesis and development of ampullary cancer. Results from the CMap analysis suggested potential drugs for patients with ampullary cancer.