Peripheral Blood Of Patients Research Articles

Dl-3-n-butylphthalide improves stroke outcomes after focal ischemic stroke in mouse model by inhibiting the pyroptosis-regulated cell death and ameliorating neuroinflammation

Recent studies have highlighted the involvement of pyroptosis-mediated cell death and neuroinflammation in ischemic stroke (IS) pathogenesis. DL-3-n-butylphthalide (NBP), a synthesized compound based on an extract from seeds of Apium graveolens, possesses a broad range of biological effects. However, the efficacy and the underlying mechanisms of NBP in IS remain contentious. Herein, we investigated the therapeutic effects of NBP and elucidated its potential mechanisms in neuronal cell pyroptosis and microglia inflammatory responses. Adult male mice underwent permanent distal middle cerebral artery occlusion (dMCAO), followed by daily oral gavage of NBP (80 mg/kg) for 1, 7, or 21 consecutive days. Gene Expression Omnibus (GEO) dataset of IS patients peripheral blood RNA sequencing was analyzed to identify differentially expressed pyroptosis-related genes (PRGs) during the ischemic process. Our results suggested that NBP treatment effectively alleviated brain ischemic damage, resulting in decreased neurological deficit scores, reduced infarct volume, and improved neurological and behavioral functions. RNA sequence data from human unveiled upregulated PRGs in IS. Subsequently, we observed that NBP downregulated pyroptosis-associated markers at days 7 and 21 post-modeling, at both the protein and mRNA levels. Additionally, NBP suppressed the co-localization of pyroptosis markers with neuronal cells to variable degrees and simultaneously mitigated the accumulation of activated microglia. Overall, our data provide novel evidence that NBP treatment significantly attenuates ischemic brain damage and promotes recovery of neurological function in the early and recovery phases after IS, probably by negatively regulating the pyroptosis cell death of neuronal cells and inhibiting toxic neuroinflammation in the central nervous system.

Two missense STK11 gene variations impaired LKB1/adenosine monophosphate-activated protein kinase signaling in Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11 (STK11/LKB1) gene mutations. Preimplantation genetic testing can protect a patient's offspring from mutated genes; however, some variations in this gene have been interpreted as variants of uncertain significance (VUS), which complicate reproductive decision-making in genetic counseling. To identify the pathogenicity of two missense variants and provide clinical guidance. Whole exome gene sequencing and Sanger sequencing were performed on the peripheral blood of patients with PJS treated at the Reproductive and Genetic Hospital of Citic-Xiangya. Software was employed to predict the protein structure, conservation, and pathogenicity of the two missense variation sites in patients with PJS. Additionally, plasmids were constructed and transfected into HeLa cells to observe cell growth. The differences in signal pathway expression between the variant group and the wild-type group were compared using western blot and immunohistochemistry. Statistical analysis was performed using one-way analysis of variance. P < 0.05 was considered statistically significant. We identified two missense STK11 gene VUS [c.889A>G (p.Arg297Gly) and c.733C>T (p.Leu245Phe)] in 9 unrelated PJS families who were seeking reproductive assistance. The two missense VUS were located in the catalytic domain of serine/threonine kinase, which is a key structure of the liver kinase B1 (LKB1) protein. In vitro experiments showed that the phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) at Thr172 and LKB1 at Ser428 were significantly higher in transfected variation-type cells than in wild-type cells. In addition, the two missense STK11 variants promoted the proliferation of HeLa cells. Subsequent immunohistochemical analysis showed that phosphorylated-AMPK (Thr172) expression was significantly lower in gastric, colonic, and uterine polyps from PJS patients with missense variations than in non-PJS patients. Our findings indicate that these two missense STK11 variants are likely pathogenic and inactivate the STK11 gene, causing it to lose its function of regulating downstream phosphorylated-AMPK (Thr172), which may lead to the development of PJS. The identification of the pathogenic mutations in these two clinically characterized PJS patients has been helpful in guiding them toward the most appropriate mode of pregnancy assistance. These two missense variants can be interpreted as likely pathogenic variants that mediated the onset of PJS in the two patients. These findings not only offer insights for clinical decision-making, but also serve as a foundation for further research and reanalysis of missense VUS in rare diseases.

Altered Expression of B Cell Receptor Signaling Pathway Genes in Peripheral Blood of Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease and has adverse implications. The exact mechanism of its pathogenesis is not fully understood and remains to be elucidated. In the current study we aimed to identify key genes that can serve as potential biomarkers and therapeutic targets for MS and shed light on pathogenesis mechanisms involved in MS. We analyzed a gene expression dataset (GES21942) and found 266 differentially expressed genes (DEGs) including 183 upregulated and 83 downregulated genes in MS patients compared to controls. Then we conducted pathway enrichment on DEGs and selected the top enriched pathway i.e., B cell receptor signaling pathway, and 5 genes of this pathway (CR2, BLK, BLNK, RASGRP3, and KRAS) for further investigation in our clinical samples. We recruited 50 MS patients and 50 controls and assessed the expression of selected genes in the circulation of patients versus controls. Expression of CR2, BLK, BLNK, and RASGRP3 were significantly higher in MS cases compared with controls. There was no significant difference in expression of KRAS between patients and controls. All of the selected genes with differential expression had noticeable diagnostic power and CR2 was the most robust gene in differentiating MS cases from controls. Additionally, a combination of genes resulted in enhanced diagnostic power. Collectively our results suggest that the B cell receptor signaling pathway and the selected genes from this pathway may be implicated in the pathogenesis of MS and each of these genes can be considered as potential diagnostic biomarkers and therapeutic targets.

Analysis of the expression level and predictive value of CLEC16A|miR-654-5p|RARA regulatory axis in the peripheral blood of patients with ischemic stroke based on biosignature analysis.

Ischemic stroke (IS) is a cerebrovascular disease that can be disabling and fatal, and there are limitations in the clinical treatment and prognosis of IS. It has been reported that changes in the expression profile of circRNAs have been found during injury in ischemic stroke, and circRNAs play an important role in the IS cascade response. However, the specific mechanisms involved in the pathogenesis of IS are not yet fully understood, and thus in-depth studies are needed. In this study, one circRNA dataset (GSE161913), one miRNA dataset (GSE60319) and one mRNA dataset (GSE180470) were retrieved from the Gene Expression Omnibus (GEO) database and included, and the datasets were differentially expressed analyzed by GEO2R and easyGEO to get the DEcircRNA, DEmiRNA and DEmRNA, and DEmRNA was enriched using ImageGP, binding sites were predicted in the ENCORI database, respectively, and the competitive endogenous RNA (ceRNA) regulatory network was visualized by the cytoscape software, and then selected by MCC scoring in the cytoHubba plugin Hub genes. In addition, this study conducted a case-control study in which blood samples were collected from stroke patients and healthy medical examiners to validate the core network of ceRNAs constructed by biosignature analysis by real-time fluorescence quantitative qRT-PCR experiments. A total of 233 DEcircRNAs, 132 DEmiRNAs and 72 DEmRNAs were screened by bioinformatics analysis. circRNA-mediated ceRNA regulatory network was constructed, including 148 circRNAs, 43 miRNAs and 44 mRNAs. Finally, CLEC16A|miR-654-5p|RARA competitive endogenous regulatory axis was selected for validation by qRT-PCR, and the validation results were consistent with the bioinformatics analysis. In conclusion, the present study establishes a new axis of regulation associated with IS, providing new insights into the pathogenesis of IS.

Analysis of the value of potential biomarker S100-A8 protein in the diagnosis and pathogenesis of spinal tuberculosis.

The objective of this study is to evaluate the value of S100-A8 protein as a diagnostic marker for spinal tuberculosis and to explore its role in the potential pathogenesis of spinal tuberculosis (STB). The peripheral blood of 100 spinal tuberculosis patients admitted to the General Hospital of Ningxia Medical University from September 2018 to June 2021 were collected as the observation group, and the peripheral blood of 30 healthy medical examiners were collected as the control group. Three samples from the observation group and three samples from the control group were selected for proteomics detection and screening of differential proteins. Kyoto Encyclopedia of Genes (KEGG) was used to enrich and analyze related signaling pathways to confirm the target protein. The serum expression levels of the target proteins were determined and compared between the two groups using enzyme-linked immunosorbent assay (ELISA). Statistical methods were used to evaluate the value of target protein as a diagnostic marker for STB. A macrophage model of Mycobacterium tuberculosis infection was constructed and S100-A8 small interfering RNA was used to investigate the molecular mechanism of the target protein. S100-A8 protein has the value of diagnosing spinal tuberculosis (AUC = 0.931, p < 0.001), and the expression level in the peripheral blood of the observation group (59.04 ± 19.37 ng/mL) was significantly higher than that of the control group (43.16 ± 10.07 ng/mL) (p < 0.05). S100-A8 protein expression showed a significant positive correlation with both CRP and ESR values (p < 0.01). Its AUCs for combined bacteriological detection, T-SPOT results, diagnostic imaging, antacid staining results, and pathological results were 0.705 (p < 0.05), 0.754 (p < 0.01), 0.716 (p < 0.01), 0.656 (p < 0.05), and 0.681 (p < 0.01), respectively. Lack of S100-A8 leads to a significant decrease in the expression levels of TLR4 and IL-17A in infected macrophages. S100-A8 protein is differentially expressed in the peripheral blood of patients with spinal tuberculosis and healthy individuals and may be a novel candidate biomarker for the diagnosis of spinal tuberculosis. The feedback loop on the S100-A8-TLR4-IL-17A axis may play an important role in the inflammatory mechanism of spinal tuberculosis.

Single-cell characterisation of tissue homing CD4 + and CD8 + T cell clones in immune-mediated refractory arthritis

BackgroundImmune-mediated arthritis is a group of autoinflammatory diseases, where the patient’s own immune system attacks and destroys synovial joints. Sustained remission is not always achieved with available immunosuppressive treatments, warranting more detailed studies of T cell responses that perpetuate synovial inflammation in treatment-refractory patients.MethodsIn this study, we investigated CD4 + and CD8 + T lymphocytes from the synovial tissue and peripheral blood of patients with treatment-resistant immune-mediated arthritis using paired single-cell RNA and TCR-sequencing. To gain insights into the trafficking of clonal families, we compared the phenotypes of clones with the exact same TCRß amino acid sequence between the two tissues.ResultsOur results show that both CD4 + and CD8 + T cells display a more activated and inflamed phenotype in the synovial tissue compared to peripheral blood both at the population level and within individual T cell families. Furthermore, we found that both cell subtypes exhibited clonal expansion in the synovial tissue.ConclusionsOur findings suggest that the local environment in the synovium drives the proliferation of activated cytotoxic T cells, and both CD4 + and CD8 + T cells may contribute to tissue destruction and disease pathogenesis.

TNF-α: A serological marker for evaluating the severity of hippocampal sclerosis in medial temporal lobe epilepsy?

ObjectiveBy analysing the difference in TNF-α levels in the peripheral blood of patients with medial temporal lobe epilepsy (mTLE) with or without hippocampal sclerosis and the correlation between TNF-α and N-acetylaspartate levels in the hippocampus, we explored the relationship between TNF-α and the degree of damage to hippocampal sclerosis neurons in medial temporal lobe epilepsy. MethodsThis is a prospective, population-based study. A total of 71 Patients with medial temporal lobe epilepsy diagnosed by clinical seizures, video-EEG, epileptic sequence MRI, and other imaging examinations were recruited from October 2020 to July 2022 in the Department of Neurology, Affiliated Hospital of Xuzhou Medical University. Twenty age-matched healthy subjects were selected as the control group. The patients were divided into two groups: the medial temporal epilepsy with hippocampal sclerosis group (positive group, mTLE-HS-P group) and the medial temporal epilepsy without hippocampal sclerosis group (negative group, mTLE-HS-N group). The levels of IL-1β, IL-5, IL-6, IL-8, IL-17, IFN-γ and TNF-α in the peripheral blood of the patients in the three groups were detected by multimicrosphere flow immunofluorescence assay. The level of N-acetylaspartate (NAA) in the hippocampus was measured by 1H-MRS. The differences in cytokine levels among the three groups were analysed, and the correlation between cytokine and NAA levels was analysed. ResultsThe level of TNF-α in the peripheral blood of the patients in the mTLE-HS-P group was significantly higher than that of the patients in the mTLE-HS-N and healthy control groups, and the level of TNF-α in the patients in the mTLE-HS-N group was significantly higher than that of the patients in the healthy control group. The NAA level in mTLE-HS-P group patients was significantly lower than that of mTLE-HS-N patients and healthy controls, but there was no significant difference between mTLE-HS-N patients and healthy controls (P > 0.05). Spearman correlation analysis showed that TNF-α level (rs = -0.437, P < 0.05) and the longest duration of a single seizure (rs = -0.398, P < 0.05) were negatively correlated with NAA level. Logistic regression analysis showed that there was no significant correlation between the longest duration of a single seizure and hippocampal sclerosis, but TNF-α level was closely related to hippocampal sclerosis in patients with mTLE (OR = 1.315, 95 % CI 1.084–1.595, P = 0.005). ConclusionThe level of TNF-α in the peripheral blood of patients with medial temporal lobe epilepsy with hippocampal sclerosis was higher, and it was correlated with NAA and hippocampal sclerosis. The high expression of TNF-α may be of important value in the evaluation of hippocampal sclerosis patients.

Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

Analysis of mononuclear leukocyte functional activity in peripheral blood of patients with varying clinical severity of acute Lyme disease

The aim of the study is to assess spontaneous and phytohemagglutinin (PHA)-induced proliferative and cytokine secretory activity in mononuclear blood cell cultures of patients with acute Lyme borreliosis (LB) over times before and after antibiotic therapy as well as depending on clinical severity. Materials and methods. Activity of blast transformation reaction (BTR) of lymphocytes and the secretion levels of interleukins (IL)-1β, IL-2, IL-6, IL-4, IL-10, IL-8, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-γ and IFN-α were assessed twice over time in the cultures of mononuclear blood cells of 18 and 14 patients with mild and moderate severity of acute LB. Results. It was determined that the moderate clinical course of acute LB as compared with the mild course was associated with the significant suppression of BTR levels, the increases in spontaneous and/or PHA-stimulated production levels of proinflammatory cytokines IL-1β, IL-6, IL-8, TNF-α and MCP-1, which were balanced by the increases in secretion levels of anti-inflammatory cytokines IL-10 and IL-4. The initial suppression of IFN-γ secretion in this group gave way to an increase in its production over time, which generally corresponded to the formation of an immune response of the mixed Th1/Th2 type.

Use of circulating tumor cells and microemboli to predict diagnosis and prognosis in diffuse glioma.

Circulating tumor cell (CTC) detection is a promising noninvasive technique that can be used to diagnose cancer, monitor progression, and predict prognosis. In this study, the authors aimed to investigate the clinical utility of CTCs in the management of diffuse glioma. Sixty-three patients with newly diagnosed diffuse glioma were included in this multicenter clinical cohort. The authors used a platform based on isolation by size of epithelial tumor cells (ISET) to detect and analyze CTCs and circulating tumor microemboli (CTMs) in the peripheral blood of patients both before and after surgery. Least absolute shrinkage and selector operation (LASSO) and Cox regression analyses were used to verify whether CTCs and CTMs are independent prognostic factors for diffuse glioma. CTC levels were closely related to the degree of malignancy, WHO grade, and pathological subtypes. Receiver operating characteristic curve analysis revealed that a high CTC level was a predictor for glioblastoma. The results also showed that CTMs originate from the parental tumor rather than from the circulation and are an independent prognostic factor for diffuse glioma. The postoperative CTC level is related to the peripheral immune system and patient survival. Cox regression analysis showed that postoperative CTC levels and CTM status are independent prognostic factors for diffuse glioma, and CTC- and CTM-based survival models had high accuracy in internal validation. The authors revealed a correlation between CTCs and clinical characteristics and demonstrated that CTCs and CTMs are independent predictors for the diagnosis and prognosis of diffuse glioma. Their CTC- and CTM-based survival models can enable clinicians to evaluate patients' response to surgery as well as their outcomes.

Autologous human preclinical modeling of melanoma interpatient clinical responses to immunotherapeutics

BackgroundDespite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge...

Study of the Activity of Indicators of Systemic Inflammation and Surfactant Protein D in Chronic Obstructive Pulmonary Disease, their Relationship with the Nutritional Status of Patients

Abstract. Studying the levels of biomarkers of inflammation and surfactant protein D (SDP) in the peripheral blood of patients with chronic obstructive pulmonary disease (COPD) depending on the nutritional status (NS) of patients is relevant, since they are important factors in the progression of the disease. Aim. To study the relationship between tumor necrosis factor-α (TNF-α), transforming growth factor β1 (TGFβ1), C-reactive protein (CRP), surfactant protein D (SPD) and NS in patients with COPD. Materials and methods. 210 patients with COPD with different body mass index (BMI) were examined. Laboratory, instrumental (spirography, bioimpedansometry, pulse oximetry), spectrophotometric and immunoenzyme research methods were used. Results. It is shown that in patients with COPD during an exacerbation of the disease, regardless of NS, there is an increase in the levels of TNFα, TGFβ1, CRP, as well as SDP in the blood serum, which is most pronounced when COPD is combined with obesity. A moderate correlation has been established between the level of SDP in the blood serum and the duration of smoking, as well as between SDP and BMI in patients with COPD; a negative correlation has been established between the level of SDP and indicators of external respiratory function. Conclusion. The degree of increase in serum levels of TNF-α, TGFβ1 and CRP directly correlates with concomitant obesity in patients with COPD. The level of SDP in COPD depends on the patient’s NS and can serve as a diagnostic marker of the disease. Multivariate analysis confirms the dependence of the level of SDP in the blood serum on the parameters of bioimpedance analysis in COPD.

ADCY4 promotes brain metastasis in small cell lung cancer and is associated with energy metabolism

Brain metastasis (BMs) in small cell lung cancer (SCLC) has a very poor prognosis. This study combined WGCNA with the mfuzz algorithm to identify potential biomarkers in the peripheral blood of patients with BMs. By comparing the significantly differentially expressed genes present in BMs samples, we identified ADCY4 as a target for further study. Expression of ADCY4 was used to cluster mfuzz expression pattern, and 28 hub genes for functional enrichment. PPI network analysis were obtained by comparing with differentially expressed genes in BMs. GABRE, NFE4 and LMOD2 are highly expressed in patients with BMs and have a good diagnostic effect. Immunoinfiltration analysis showed that SCLC patients with BMs may be associated with memory B cells, Tregs, NK cell activation, macrophage M0 and dendritic cell activation. prophytic was used to investigate the ADCY4-mediated anti-tumor drug response. In conclusion, ADCY4 can be used as a promising candidate biomarker for predicting BMs, molecular and immune features in SCLC. PCR showed that ADCY4 expression was increased in NCI–H209 and NCI–H526 SCLC cell lines. In vitro experiments confirmed that the expression of ADCY4 was significantly decreased after anti-PD1 antibody treatment, while the expression of energy metabolism factors were significantly different. This study reveals a potential mechanism by which ADCY4 mediates poor prognosis through energy metabolism -related pathways in SCLC.

Novel peripheral blood mononuclear cell mRNA signature for IFN-beta therapy responsiveness prediction in multiple sclerosis

Interferon-beta (IFN- β ) is one of the classical drugs for immunomodulatory therapy in relapsing-remitting multiple sclerosis (RRMS) patients, but the drug responsiveness of different patients varies. Currently, there is no valid model to predict IFN- β responsiveness. This research attempted to develop an IFN- β responsiveness prediction model based on mRNA expression in RRMS patient peripheral blood mononuclear cells. Peripheral blood mononuclear cell mRNA expression datasets including 50 RRMS patients receiving IFN- β treatment were obtained from GEO. Among the datasets, 24 cases from GSE24427 were included in a training set, and 18 and 9 cases from GSE19285 and GSE33464, respectively, were adopted as two independent test sets. In the training set, blood samples were collected immediately before first, second, month 1, 12, and 24 IFN- β injection, and the mRNA expression data at four time points, namely, two days, one month, one year and two years after the onset of IFN- β treatment, were compared with pre-treatment data to identify IFN-stimulated genes (ISGs). The ISGs at the one-month time point were used to construct the drug responsiveness prediction model. Next, the drug responsiveness model was verified in the two independent test sets to examine the performance of the model in predicting drug responsiveness. Finally, we used CIBERSORTx to estimate the content of cell subtypes in samples and evaluated whether differences in the proportions of cell subtypes were related to differences in IFN- β responsiveness. Among the four time points, one month was the time point when the training set GSE24427 and test set GSE33464 had the highest number of ISGs. Functional analysis showed that these one-month ISGs were enriched in biological functions such as the innate immune response, type-I interferon signalling pathway, and other IFN- β -associated functions. Based on these ISGs, we obtained a four-factor prediction model for IFN- β responsiveness including MX1, MX2, XAF1, and LAMP3. In addition, the model demonstrated favourable predictive performance within the training set and two external test sets. A higher proportion of activated NK cells and lower naive CD4/total CD4 ratio might indicate better drug responsiveness. This research developed a polygene-based biomarker model that could predict RRMS patient IFN- β responsiveness in the early treatment period. This model could probably help doctors screen out patients who would not benefit from IFN- β treatment early and determine whether a current treatment plan should be continued.

Causal role of myeloid cells in Parkinson's disease: Mendelian randomization study.

Previous studies have observed elevated myeloid cells in the peripheral blood of patients with Parkinson's disease (PD), but the causal relationship between them remains to be elucidated. We investigated whether there is a causal relationship between different subtypes of peripheral blood myeloid cells and PD using Mendelian randomization (MR) combined with bioinformatics analysis. Exploring the etiology of PD from the perspective of genetics can remove confounding factors and provide a more reliable theoretical basis for elucidating the pathogenesis of PD. Comprehensive two-sample MR analysis and sensitivity analyses were conducted to explore the causal associations between 64 myeloid cell signatures and PD risk. The Venn diagram and protein-protein interaction network analysis of instrumental variables (IV) corresponding genes were used to further investigate the potential mechanism of myeloid cells influencing the pathogenesis of PD. We investigated the impact of four immunophenotypes on the risk of PD, including Im MDSC% CD33dim HLA DR- CD66b- (relative count), CD33dim HLA DR+ CD11b+% CD33dim HLA DR+ (relative count), and CD11b on Mo MDSC (MFI) and CD11b on CD33br HLA DR+ CD14dim (MFI), while an immunophenotype's protective effect on PD was observed CD45 on Im MDSC (MFI). The results of bioinformatics analysis showed that CD33, NTRK2, PLD2, GRIK2 and RELN had protein interactions with the risk genes of PD. Our study has demonstrated a close genetic correlation between different subtypes of myeloid cells and PD, providing guidance for early identification and immunotherapeutic development in patients with PD.

P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

BackgroundGuillain–Barré syndrome (GBS), a post-infectious, immune-mediated, acute demyelinating disease of the peripheral nerves and nerve roots, represents the most prevalent and severe acute paralyzing neuropathy. Purinergic P2X7 receptors (P2X7R) play a crucial role in central nervous system inflammation. However, little is known about their role in the immune-inflammatory response within the peripheral nervous system.MethodsInitially, we assessed the expression of purinergic P2X7R in the peripheral blood of patients with GBS using flow cytometry and qRT-PCR. Next, we explored the expression of P2 X7R in CD4+ T cells, CD8+ T cells, and macrophages within the sciatic nerves and spleens of rats using immunofluorescence labeling and flow cytometry. The P2X7R antagonist brilliant blue G (BBG) was employed to examine its therapeutic impact on rats with experimental autoimmune neuritis (EAN) induced by immunization with the P0180 − 199 peptide. We analyzed CD4+ T cell differentiation in splenic mononuclear cells using flow cytometry, assessed Th17 cell differentiation in the sciatic nerve through immunofluorescence staining, and examined the expression of pro-inflammatory cytokine mRNA using RT-PCR. Additionally, we performed protein blotting to assess the expression of P2X7R and NLRP3-related inflammatory proteins within the sciatic nerve. Lastly, we utilized flow cytometry and immunofluorescence labeling to examine the expression of NLRP3 on CD4+ T cells in rats with EAN.ResultsP2X7R expression was elevated not only in the peripheral blood of patients with GBS but also in rats with EAN. In rats with EAN, inhibiting P2X7R with BBG alleviated neurological symptoms, reduced demyelination, decreased inflammatory cell infiltration of the peripheral nerves, and improved nerve conduction. BBG also limited the production of pro-inflammatory molecules, down-regulated the expression of P2X7R and NLRP3, and suppressed the differentiation of Th1 and Th17 cells, thus protecting against EAN. These effects collectively contribute to modifying the inflammatory environment and enhancing outcomes in EAN rats.ConclusionsSuppression of P2X7R relieved EAN manifestation by regulating CD4+ T cell differentiation and NLRP3 inflammasome activation. This finding underscores the potential significance of P2X7R as a target for anti-inflammatory treatments, advancing research and management of GBS.

Analysis of changes in high-mobility group box 1, receptor for advanced glycation endproducts, and T helper 17/regulatory T balance in severe preeclampsia with acute heart failure.

We measured the levels of High-Mobility Group Box 1 (HMGB1), Receptor for Advanced Glycation Endproducts (RAGE), T Helper 17 cells (Th17), Regulatory T cells (Treg), and related cytokines in the peripheral blood of patients with severe preeclampsia (SPE) complicated with acute heart failure (AHF) to explore the expression changes in these indicators. In total, 96 patients with SPE admitted to Gansu Provincial Maternity and Child-care Hospital between June 2020 and June 2022 were included in the study. The patients were divided into SPE+AHF (40 patients) and SPE (56 patients) groups based on whether they suffered from AHF. Additionally, 56 healthy pregnant women who either received prenatal examinations or were admitted to our hospital for delivery during the same period were selected as the healthy control group. An enzyme-linked immunosorbent assay was performed to detect the expression levels of HMGB1, RAGE, interleukin (IL)-17, IL-6, transforming growth factor β (TGF-β), IL-10, and NT-proBNP in plasma. Flow cytometry was employed to determine the percentages of Th17 and Treg cells. Compared to the healthy control group, the SPE+AHF and SPE groups had higher plasma levels of HMGB1 and RAGE expression, higher Th17 percentage and Th17/Treg ratio, and lower Treg percentage. Compared to the SPE group, the SPE+AHF group had higher plasma levels of HMGB1 and RAGE expression, higher Th17 percentage and Th17/Treg ratio, and lower Treg percentage (P<.05). In patients with SPE with AHF, plasma HMGB1 was positively correlated with RAGE, Th17, Th17/Treg, IL-17, and IL-6 and was negatively correlated with TGF-β and IL-10 (P<.05). Our findings revealed that patients with SPE with AHF had elevated levels of HMGB1 and RAGE while exhibiting Th17/Treg immune imbalance, suggesting that the abnormal expression of these indicators may be involved in the pathogenesis of SPE with AHF.

Expression of USP25 associates with fibrosis, inflammation and metabolism changes in IgG4-related disease

IgG4-related disease (IgG4-RD) has complex clinical manifestations ranging from fibrosis and inflammation to deregulated metabolism. The molecular mechanisms underpinning these phenotypes are unclear. In this study, by using IgG4-RD patient peripheral blood mononuclear cells (PBMCs), IgG4-RD cell lines and Usp25 knockout mice, we show that ubiquitin-specific protease 25 (USP25) engages in multiple pathways to regulate fibrotic and inflammatory pathways that are characteristic to IgG4-RD. Reduced USP25 expression in IgG4-RD leads to increased SMAD3 activation, which contributes to fibrosis and induces inflammation through the IL-1β inflammatory axis. Mechanistically, USP25 prevents ubiquitination of RAC1, thus, downregulation of USP25 leads to ubiquitination and degradation of RAC1. Decreased RAC1 levels result in reduced aldolase A release from the actin cytoskeleton, which then lowers glycolysis. The expression of LYN, a component of the B cell receptor signalosome is also reduced in USP25-deficient B cells, which might result in B cell activation deficiency. Altogether, our results indicate a potential anti-inflammatory and anti-fibrotic role for USP25 and make USP25 a promising diagnostic marker and potential therapeutic target in IgG4-RD.

Abstract 2303: Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes

Abstract Background: Circulating tumor cell clusters (CTCCs) are aggregated groups of tumor cells that detached from primary tumors and circulate the bloodstream. However, while Circulating Tumor Cells (CTCs) are a well studied phenomenon, CTCCs remain relatively unexplored and ill-defined, with only a few descriptive investigations evaluating their clinical utility. Adding to CTCC complexity is that various subtypes exist, including homotypic clusters consisting of a homogenous population of tumor cells and heterotypic CTCCs made of CTCs with immune/stromal white blood cells (WBCs). Furthermore, CTCs can undergo Epithelial-Mesenchymal Transition (EMT), a process characterized by loss of epithelial traits and upregulation of mesenchymal traits, and can form clustered EMTs (CEMTs). Moreover, CTCs can fuse with macrophages forming Tumor Macrophage Hybrid Cells, aka Cancer-Associated Macrophage Like cells (CAMLs) when in circulation. We enumerated all these types of cells and complex aggregates in the peripheral blood of patients with metastatic breast cancer (mBC). Methods: We enumerated the 6 populations from patient samples enrolled in IRB aproved prospective pilot studies. Whole peripheral blood (7.5mL) was filtered and stained with cytokeratin (CK) &amp; CD45/CD14 to identify CTCs. CTCs were defined as an intact DAPI nuclei and strong filamentous CK. Homotypic CTCCs were defined as ≥2 CTCs attached together. Heterotypic CTCCs were defined as ≥1 CTC attached to ≥1 WBC. EMTs were defined as having DAPI nuclei and weak non-filamentous CK. CEMTS were defined as ≥2 EMTs. CAMLs were defined as having an enlarged polynucleated DAPI, and positive for CD45/CD14 or non-filamentous CK. Median progression free survival (mPFS) and median overall survival (mOS) over 24 months (m) were evaluated. Results: We evaluated the blood of 79 mBC patients. Single CTCs were found in 57% of patients (n=34/79), homotypic CTCCs 15% (n=12/79), heterotypic CTCCs 66% (n=27/79), EMTs 56% (n=44/79), CEMTs 23% (n=18/79) and CAMLs 97% (n=77/79). Over 24 ms, patierns with detectable heterotypic CTCCs &amp; homotypic CTCCs had the shortest response times at mPFS=2.8 &amp; mOS=5.8 ms, or mPFS=2.1 &amp; mOS=4.3 ms, respectively. This was followed by CTCs mPFS=3.7 &amp; mOS=13.7 ms, EMTs mPFS=4.3 &amp; mOS=14.4 ms, CAMLs mPFS=4.3 &amp; mOS=14.4 ms, and CEMT mPFS=4.7 &amp; mOS=13.2 ms. Additionally, CTCCs were rare in HER2+ patients at 4.4% (n=1/23), but no associations to other hormone statuses were found. Conclusion: CTCCs appear to represent an array of subtypes with different biological and clinical meanings. Here we enumerated CTCC subtypes in the blood of mBC patients and stratified the various types to clinical outcomes. These data suggest CTCCs are clinically important and further understanding of their biology in the context of tumor pathogenesis is needed. Citation Format: Daniel L. Adams, Carolina Reduzzi, Aravind Aryasomayajula, R. Katherine Alpaugh, Saranya Chumsri, Cha-Mei Tang, Giuseppe Del Priore, William V. Williams, Massimo Cristofanilli. Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2303.

Abstract 3732: Peripheral pharmacodynamic activity of epcoritamab as monotherapy and in combination with rituximab + lenalidomide in patients with follicular lymphoma

Abstract Introduction: Epcoritamab (DuoBody®-CD3xCD20) is a subcutaneous IgG1 bispecific antibody that simultaneously binds to CD3 on T cells and CD20 on malignant B cells, inducing activation and cytotoxic activity of T cells to mediate killing of target lymphoma cells. Epcoritamab is approved in the US for the treatment of adults with relapsed/refractory (R/R) diffuse and high-grade large B-cell lymphoma after ≥2 lines of systemic therapy (also approved in Europe, the UK, Japan, and Canada). Here we present the pharmacodynamic effects of epcoritamab as monotherapy (EPCORE NHL-1: NCT03625037) or in combination with rituximab + lenalidomide (R2) (EPCORE NHL-2: NCT04663347) in peripheral blood of patients with follicular lymphoma (FL), a heterogeneous disease for which many patients have an unmet medical need for efficacious, well-tolerated, off-the-shelf therapies. Methods: In EPCORE NHL-1 expansion phase, adults with R/R FL received epcoritamab monotherapy; in EPCORE NHL-2, adults with R/R (arm 2) or newly diagnosed (ND) (arm 6) FL received epcoritamab combined with R2. B-cell markers and T-cell proliferation and activation markers in whole blood were assessed using validated flow cytometry assays. Cytokines in plasma were measured using a validated multiplex immunoassay. Results: Epcoritamab as monotherapy and in combination with R2 induced rapid depletion of circulating peripheral B cells (CD19+) in patients with detectable peripheral B cells at baseline. Additionally, within the first 2 cycles, epcoritamab monotherapy induced a transient elevation of peripheral CD8+ and CD4+ T cells expressing proliferation (Ki67+%) and activation (HLA-DR+% and PD-1+%) markers. R/R and ND patient groups treated with epcoritamab + R2 showed a trend for greater induction of proliferating and activated peripheral T cells. Likewise, elevation in T-cell absolute counts was observed from cycle 2 onward, with a trend of higher levels with epcoritamab + R2 vs epcoritamab monotherapy. In addition, a moderate but transient elevation of circulating cytokines including IFNγ, IL-6, and IL-10 during cycle 1 was observed with epcoritamab monotherapy and epcoritamab + R2. Importantly, IL-6 and IL-10 returned to approximate baseline levels prior to the subsequent dose. Compared with epcoritamab monotherapy, patients treated with epcoritamab + R2 showed a trend for lower median levels of IL-6 peaks 24 hours after the first full dose. Conclusion: The pharmacodynamic activity of epcoritamab combined with R2 in R/R and ND FL was consistent with epcoritamab’s mechanism of action as monotherapy. Epcoritamab + R2 showed a favorable pharmacodynamic profile with a trend toward increased T-cell activation and T-cell elevation, and lower IL-6 levels. These data support the ongoing phase 3 trial of epcoritamab + R2 compared with R2 alone in patients with R/R FL (EPCORE FL-1: NCT05409066). Citation Format: Han Si, Jimin Zhang, Monica Wielgos-Bonvallet, Lorenzo Falchi, Kim Linton, Wojciech Jurczak, David Belada, Işıl Altıntaş, Mariana Sacchi, Elena Favaro, Ali Rana, Daniela Hoehn, David Soong, Christopher W. Chiu, Maria Jure-Kunkel. Peripheral pharmacodynamic activity of epcoritamab as monotherapy and in combination with rituximab + lenalidomide in patients with follicular lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3732.