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The Fas/Fas ligand (FasL) system is a major apoptosis-regulating pathway with a key role in tumor immune surveillance and metastasis. The expression of Fas/FasL on mammary tumor tissues holds prognostic value for breast cancer (BC) patients. We herein assessed Fas/FasL expression on circulating tumor cells (CTCs) and matched peripheral blood mononuclear cells (PBMCs) from 98 patients with metastatic BC receiving first-line treatment. Fas+, FasL+, and Fas+/FasL+ CTCs were identified in 88.5%, 92.3%, and 84.6% of CTC-positive patients, respectively. In addition, Fas+/FasL+, Fas-/FasL+, and Fas-/FasL- PBMCs were identified in 70.3%, 24.2%, and 5.5% of patients, respectively. A reduced progression-free survival (PFS) was revealed among CTC-positive patients (median PFS: 9.5 versus 13.4 months; p = 0.004), and specifically among those harboring Fas+/FasL+ CTCs (median PFS: 9.5 vs. 13.4 months; p = 0.009). On the other hand, an increased overall survival (OS) was demonstrated among patients with Fas+/FasL+ PBMCs rather than those with Fas-/FasL+ and Fas-/FasL- PBMCs (median OS: 35.7 vs. 25.9 vs. 14.4 months, respectively; p = 0.008). These data provide for the first time evidence on Fas/FasL expression on CTCs and PBMCs with significant prognostic value for patients with metastatic BC, thus highlighting the role of the Fas/FasL system in the peripheral immune response and metastatic progression of BC.

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BACKGROUND: This study aimed to investigate the differential expression levels of the cGAS-STING pathway in peripheral blood mononuclear cells (PBMCs) of spinal tuberculosis (TB) patients with different progression and its feasibility as a diagnostic marker. METHODS: Peripheral blood and medical records of 25 patients with spinal TB and 10 healthy individuals, were prospectively collected and analyzed. PBMCs and serum were extracted from peripheral blood and the expression levels of the cGAS-STING pathway in PBMCs were measured by real-time PCR (RT-PCR) and serum interferon ß (IFN-ß) expression levels were measured by enzyme-linked immunosorbent assay (ELISA). The expression of Interferon regulatory Factor 3 (IRF3) in PBMCs was measured using western blot. Statistical analysis was performed using the SPSS 26.0 statistical package. RESULTS: The results showed that the expression level of the TANK-binding kinase 1 (TBK1) and IRF3 was significantly higher in PBMCs (P < 0.05), in patients with active lesions than in patients with stable lesions. The serum concentration of IFN-ß was significantly higher in patients with active lesions (P = 0.028). Compared with healthy individuals, the expression level of the cGAS-STING pathway was elevated in PBMCs of TB patients (P < 0.05), and the difference in the expression level of IFN-ß was not statistically significant (P > 0.05), and the serum IFN-ß concentration was elevated (P < 0.05). The calculated AUC values for TBK1 and IRF3 in PBMCs, IFN-ß in serum and erythrocyte sedimentation rate (ESR) to distinguish between patients with active and stable lesions were 0.732, 0.714, 0.839, and 0.714 respectively. CONCLUSIONS: The expression level of TBK1 and IRF3 in PBMCs, and IFN-ß in the serum of patients with spinal TB is positively correlated with disease activity. TBK1 has higher specificity and IFN-ß in serum has higher sensitivity when used to differentiate between patients with active and stable lesions.

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To explore molecular biomarkers associated with the pathophysiology and therapy of lupus nephritis (LN), we conducted a joint analysis of transcriptomic data from 40 PBMCs (GSE81622) and 21 kidney samples (GSE112943) from the GEO database using bioinformatics. 976 and 2427 differentially expressed genes (DEGs) were identified in PBMCs and renal tissues. Seven and two functional modules closely related to LN were identified. Further enrichment analysis revealed that the neutrophil activation pathway was highly active in both PBMC and kidney. Subsequently, 16 core genes closely associated with LN were verified by PPI screening and qPCR. In vitro cell models and MRL/lpr mouse models confirmed that the abnormal expression of these core genes was closely linked to neutrophil extracellular traps (NETs) generated by neutrophil activation, while degradation of NETs led to down-regulation of core gene expression, thereby improving pathological symptoms of LN. Therefore, identification of SLE patients exhibiting abnormal expression patterns for these core genes may serve as a useful indicator for kidney involvement. Additionally, targeting neutrophils to modulate their activation levels and inhibit aberrant expression of these genes represents a potential therapeutic strategy for treating LN.

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BACKGROUND: Pre-pregnancy overweight and obesity promote deleterious health impacts on both mothers during pregnancy and the offspring. Significant changes in the maternal peripheral blood mononuclear cells (PBMCs) gene expression due to obesity are well-known. However, the impact of pre-pregnancy overweight on immune cell gene expression during pregnancy and its association with maternal and infant outcomes is not well explored. METHODS: Blood samples were collected from healthy normal weight (NW, pre-pregnancy BMI 18.5-24.9) or overweight (OW, pre-pregnancy BMI 25-29.9) 2nd parity pregnant women at 12, 24 and 36 weeks of pregnancy. PBMCs were isolated from the blood and subjected to mRNA sequencing. Maternal and infant microbiota were analyzed by 16S rRNA gene sequencing. Integrative multi-omics data analysis was performed to evaluate the association of gene expression with maternal diet, gut microbiota, milk composition, and infant gut microbiota. RESULTS: Gene expression analysis revealed that 453 genes were differentially expressed in the OW women compared to NW women at 12 weeks of pregnancy, out of which 354 were upregulated and 99 were downregulated. Several up-regulated genes in the OW group were enriched in inflammatory, chemokine-mediated signaling and regulation of interleukin-8 production-related pathways. At 36 weeks of pregnancy healthy eating index score was positively associated with several genes that include, DTD1, ELOC, GALNT8, ITGA6-AS1, KRT17P2, NPW, POT1-AS1 and RPL26. In addition, at 36 weeks of pregnancy, genes involved in adipocyte functions, such as NG2 and SMTNL1, were negatively correlated to human milk 2'FL and total fucosylated oligosaccharides content collected at 1 month postnatally. Furthermore, infant Akkermansia was positively associated with maternal PBMC anti-inflammatory genes that include CPS1 and RAB7B, at 12 and 36 weeks of pregnancy. CONCLUSIONS: These findings suggest that prepregnancy overweight impacts the immune cell gene expression profile, particularly at 12 weeks of pregnancy. Furthermore, deciphering the complex association of PBMC's gene expression levels with maternal gut microbiome and milk composition and infant gut microbiome may aid in developing strategies to mitigate obesity-mediated effects.

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This Technical Note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cell and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates the reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24 min active gradient. In 200 ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45 min run covers ∼90% of the expressed proteome. This complete workflow allows for large swaths of the proteome to be identified and is compatible with diverse sample types.

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Dengue is a significant public health problem with no specific viral treatment. One of the main challenges in studying dengue is the lack of adequate animal models recapitulating human immune responses. Most studies on humanized mice use NOD-scid IL2R gamma null (NSG) mice, which exhibit poor hematopoiesis for some cell populations. This study compares three humanized (hu) NOD-derived mouse models for dengue virus-2 (DENV-2) infection in the context of human cytokine expression. Three mouse strains (hu-NSG, hu-EXL, and hu-SGM3) received xenotransplants of human CD34+ fetal cord blood cells from a single donor, and one mouse strain received human peripheral blood mononuclear cells (hu-SGM3-PBMCs). All models exhibited infectious viruses in blood confirmed by plaque assay, but mice expressing human cytokines showed higher viremia compared to conventional NSG mice. The hu-SGM3-PBMCs model developed lethal infections, showing a significant increase in viremia and clinical signs. A detectable human cytokine response was observed in all the DENV-2-infected humanized mouse models. In conclusion, humanized NOD-derived mouse models expressing human cytokines offer a relevant platform for the study of dengue pathogenesis and antiviral therapies.

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Objective: Silibinin has exhibited antitumor activities. However, there are few reports about the immunomodulatory properties of silibinin on T lymphocyte function in the tumor microenvironment. Here, we determined the effects of silibinin on T cells of peripheral blood mononuclear cells (PBMCs), cultivated alone or with a human cell line of glioblastoma (U-87 MG). Materials and Methods: The proliferation of T lymphocytes was assessed by MTT test in the presence of silibinin (15 and 45 µM). Also, total antioxidant capacity (TAC), the activity of superoxide dismutase-3 (SOD3), and the levels of two cytokines interferon gamma (IFN-γ) and tumor growth beta (TGF-ß) were compared between treated and untreated PBMCs alone or co-cultured with U-87 cells. Results: According to our results, silibinin raised the TAC levels and SOD3 activity in the PBMCs and in the co-culture condition. Moreover, silibinin-treated PBMCs showed higher IFN-γ levels and lower TGF-ß levels. Interestingly, silibinin protected PBMCs against the U-87-induced suppression. Conclusion: Altogether, these results proposed the immunomodulatory potential of silibinin on T cells of PBMCs, as well as its partially protective effects on PBMCs against the suppression induced by U-87 MG cells.

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Innate and adaptive immune responses at mucosal surfaces play a role in protection against most infectious diseases. However, the relative importance either of mucosal versus systemic, or of cellular versus humoral immunity in protection against such infections remains unclear. We aimed to determine the relative percentages and reproducibility of detection of five major T lymphocyte phenotypes in stimulated whole mouth fluid (SWMF); to compare matched mucosal and blood phenotypes; to evaluate the consistency of phenotypes in SWMF over time; and to determine any associations with age or gender. Peripheral blood and SWMF samples were collected from 194 participants and sequential concomitant samples were collected from 27 of those and from 12 subjects living with HIV. CD3, CD4, CD8, Th1 and Th2 T lymphocyte phenotypes were determined by FACS. All the five T lymphocyte phenotypes were detected consistently by FACS in PBMC and SWMF with experimental replicates (N = 10; PBMC CV: 3-30%; SWMF CV: 12-36%). In longitudinal samples detection rates were reproducible in both fluids but variations were higher in SWMF (CV: 23-79.6%) than PBMC (CV: 9.7-75%). Statistically significant correlations of the percentages of all the T lymphocyte phenotypes except CD8 was seen between the two fluids. In PBMCs a negative correlation with age was found with CD3, CD4 and CD8 phenotypes, whilst a positive correlation was found in both SWMF and PBMC with the Th2 phenotype. CD3, CD4 and CD8 phenotypes in SWMF and PBMCs from an HIV-positive cohort were not significantly correlated in contrast with the HIV-negative controls. Our study provides a robust FACS protocol for the detection of the five major T lymphocyte phenotypes in SWMF which should prove useful for research with other mucosal fluids.

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Growing evidence identifies extracellular vesicles (EVs) as important cell-to-cell signal transducers in autoimmune disorders, including multiple sclerosis (MS). If the etiology of MS still remains unknown, its molecular physiology has been well studied, indicating peripheral blood mononuclear cells (PBMCs) as the main pathologically relevant contributors to the disease and to neuroinflammation. Recently, several studies have suggested the involvement of EVs as key mediators of neuroimmune crosstalk in central nervous system (CNS) autoimmunity. To assess the role of EVs in MS, we applied electron microscopy (EM) techniques and Western blot analysis to study the morphology and content of plasma-derived EVs as well as the ultrastructure of PBMCs, considering four MS patients and four healthy controls. Through its exploratory nature, our study was able to detect significant differences between groups. Pseudopods and large vesicles were more numerous at the plasmalemma interface of cases, as were endoplasmic vesicles, resulting in an activated aspect of the PBMCs. Moreover, PBMCs from MS patients also showed an increased number of multivesicular bodies within the cytoplasm and amorphous material around the vesicles. In addition, we observed a high number of plasma-membrane-covered extensions, with multiple associated large vesicles and numerous autophagosomal vacuoles containing undigested cytoplasmic material. Finally, the study of EV cargo evidenced a number of dysregulated molecules in MS patients, including GANAB, IFI35, Cortactin, Septin 2, Cofilin 1, and ARHGDIA, that serve as inflammatory signals in a context of altered vesicular dynamics. We concluded that EM coupled with Western blot analysis applied to PBMCs and vesiculation can enhance our knowledge in the physiopathology of MS.

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Aim: This study aimed to explore using peripheral blood mononuclear cell (PBMC)-derived chimeric antigen receptor (CAR) NK cells targeting ROBO1 as a personalized medicine approach for ovarian cancer. Methods: A two-step strategy generated ROBO1-targeted CAR NK cells from PBMCs of ovarian cancer patients. Efficacy was evaluated using xCELLigence RTCA, CCK-8 and Live/Dead fluorescence assays. Results: ROBO1-NK cells exhibited higher efficiency in eradicating primary ovarian cancer cells and lysing ovarian tumor organoids compared with primary NK cells without ROBO1-CAR modification. Conclusion: These findings highlight the potential of developing ROBO1-targeted CAR-NK cells from patients' PBMCs as a personalized treatment option for ovarian cancer.

Ovarian cancer represents a formidable clinical challenge necessitating the urgent exploration of novel therapeutic approaches. In this study, the focus was directed toward ROBO1, a molecule known to play a pivotal role in cancer angiogenesis and metastasis, while limited investigation in the context of ovarian cancer. Leveraging this knowledge, we sought to construct ROBO1-targeting chimeric antigen receptor natural killer (CAR-NK) cells utilizing peripheral blood mononuclear cells derived from the patients themselves. The overarching goal of this investigation was to harness the potential of immunotherapy using autologous resources to realize personalized treatment strategies for ovarian cancer in clinical settings.

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COVID-19, caused by the SARS-CoV-2 virus, has caused a global health crisis, necessitating a deeper understanding of its pathophysiology. In this study, we explored the immune and hematological dynamics in COVID-19 patients to gain insights into disease severity and prognosis. Our findings revealed distinct cytokine profiles in moderate and severe cases. IL12A was significantly upregulated in peripheral blood mononuclear cells from moderate cases, suggesting a potential role in initiating an effective immune response. Conversely, severe cases exhibited downregulation of key pro-inflammatory cytokines (IL23A, TNFalpha, IL1B, and IFNG) alongside an upregulation of the immunosuppressive IL10, indicative of a dysregulated immune environment. Serum analysis showed elevated IL6 and IL10 levels in both moderate and severe cases, emphasizing their potential as markers for disease severity. Notably, no significant differences in serum cytokines were found between recovery and lethal cases. In lethal cases of COVID-19, elevated D-dimer, urea, and creatinine correlated with IL6 and IL10. This study contributes valuable information to the ongoing efforts to understand and manage the dysregulated immune responses underlying COVID-19 pathology.

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Liver cirrhosis is a condition with high mortality that poses a significant health and economic burden worldwide. The clinical characteristics of liver cirrhosis are complex and varied. Therefore, the evaluation of immune infiltration-involved genes incirrhosis has become mandatory in liver disease research, not only to identify the potential biomarkers but also to provide important insights into the underlying mechanisms of the disease. In this study, we aimed to investigate the expression profile of cytokine genes in peripheral blood mononuclear cells (PBMCs) of HCV patients and identify the gene expression signature associated with advanced cirrhosis. A cross-sectional study of 90 HCV genotype 4 patients, including no fibrosis patients (F0, n = 24), fibrotic patients (F1-F3, n = 36), and cirrhotic patients (F4, n = 30) has been conducted. The expression of cytokine genes was analyzed by quantitative real-time PCR in the subjects' PBMCs, and the serum level of TGFß2 was measured by ELISA. Our findings showed that the expression level of the TGIF1 transcript was lower in cirrhotic and fibrotic patients compared to no fibrosis patients (p = 0.046 and 0.022, respectively). Also, there was an upregulation of the TGFß1 gene in cirrhotic patients relative to fibrotic patients (p = 0.015). Additionally, the cirrhotic patients had higher expression levels of the TGF-ß2 transcript and elevated levels of the TGF-ß2 protein than patients with no cirrhosis or fibrosis. According to the ROC analysis, TGFß1, TGIF1 transcripts, and TGFß2 protein have a good discriminatory performance in distinguishing between cirrhotic, fibrotic, and non-fibrotic patients. Our results suggested that the expression of TGIF1, TGF-ß1, and TGF-ß2 genes in PBMCs may provide a valuable tool for identifying patients with advanced cirrhosis and that TGF-ß and TGIF1 may be potential biomarkers for cirrhosis. These findings may have implications for the diagnosis and treatment of cirrhosis in HCV patients.

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An important approach to stopping the AIDS epidemic is the development of a vaccine that elicits antibodies that block virus capture, the initial interactions of HIV-1 with the target cells, and replication. We utilized a previously developed qRT-PCR-based assay to examine the effects of broadly neutralizing antibodies (bNAbs), plasma from vaccine trials, and monoclonal antibodies (mAbs) on virus capture and replication. A panel of bNAbs inhibited primary HIV-1 replication in PBMCs but not virus capture. Plasma from RV144 and RV305 trial vaccinees demonstrated inhibition of virus capture with the HIV-1 subtype prevalent in Thailand. Several RV305 derived V2-specific mAbs inhibited virus replication. One of these RV305 derived V2-specific mAbs inhibited both virus capture and replication, demonstrating that it is possible to elicit antibodies by vaccination that inhibit virus capture and replication. Induction of a combination of such antibodies may be the key to protection from HIV-1 acquisition.

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Background: Klebsiella pneumoniae is a common Gram-negative bacterium. Blood infection caused by K. pneumoniae is one of the most common causes of human sepsis, which seriously threatens the life of patients. The immune status of peripheral blood mononuclear cells (PBMCs) based on single-cell RNA sequencing (scRNA-seq) in acute stage and recovery stage of sepsis caused by K. pneumoniae bloodstream infection has not been studied. Methods: A total of 13 subjects were included in this study, 3 healthy controls, 7 patients with K. pneumoniae bloodstream infection in the acute stage (4 patients died), and 3 patients in the recovery stage. Peripheral blood of all patients was collected and PBMCs were isolated for scRNA-seq analysis. We studied the changes of PBMCs components, signaling pathways, differential genes, and cytokines in acute and recovery stages. Results: During K. pneumoniae acute infection we observed a decrease in the proportion of T cells, most probably due to apoptosis and the function of T cell subtypes was disorder. The proportion of monocytes increased in acute stage. Although genes related to their phagocytosis function were upregulated, their antigen presentation capacity-associated genes were downregulated. The expression of IL-1ß, IL-18, IFNGR1 and IFNGR2 genes was also increased in monocytes. The proportion of DCs was depleted during the acute stage and did not recover during sepsis recovery. DCs antigen presentation was weakened during the acute stage but recovered fast during the recovery stage. pDCs response to MCP-1 chemokine was weakened, they recovered it quickly during the recovery stage. B cells showed apoptosis both in the acute stage and recovery stage. Their response to complement was weakened, but their antigen presentation function was enhanced. The proportion of NK cells stable during all disease's stages, and the expression of IFN-γ gene was upregulated. Conclusion: The proportion of PBMCs and their immune functions undergo variations throughout the course of the disease, spanning from the acute stage to recovery. These findings provide new insights into the mechanism of PBMCs immune function during K. pneumoniae bloodstream infection sepsis and recovery and sets the basis for further understanding and treatment.

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Special attention is given to cow's milk and its variants, with ongoing discussions about health-related impacts primarily focusing on the A1 variant in contrast to the A2 variant. The difference between these variants lies in a single amino acid alteration at position 67 of ß-casein. This alteration is presumed to make the A1 variant more susceptible to enzymatic breakdown during milk digestion, leading to an increased release of the peptide ß-casomorphin-7 (BCM-7). BCM-7 is hypothesized to interact with µ-opioid receptors on immune cells in humans. Although BCM-7 has demonstrated both immunosuppressive and inflammatory effects, its direct impact on the immune system remains unclear. Thus, we examined the influence of A1 and A2 milk on Concanavalin A (ConA)-stimulated human peripheral blood mononuclear cells (PBMCs), as well as the effect of experimentally digested A1 and A2 milk, containing different amounts of free BCM-7 from ß-casein cleavage. Additionally, we evaluated the effects of pure BCM-7 on the proliferation of ConA-stimulated PBMCs and purified CD4+ T cells. Milk fundamentally inhibited PBMC proliferation, independent of the ß-casein variant. In contrast, experimentally digested milk of both variants and pure BCM-7 showed no influence on the proliferation of PBMCs or isolated CD4+ T cells. Our results indicate that milk exerts an anti-inflammatory effect on PBMCs, regardless of the A1 or A2 ß-casein variant, which is nullified after in vitro digestion. Consequently, we deem BCM-7 unsuitable as a biomarker for food-induced inflammation.

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Type I interferons (IFN-Is) are pivotal in innate immunity against human immunodeficiency virus I (HIV-1) by eliciting the expression of IFN-stimulated genes (ISGs), which encompass potent host restriction factors. While ISGs restrict the viral replication within the host cell by targeting various stages of the viral life cycle, the lesser-known IFN-repressed genes (IRepGs), including RNA-binding proteins (RBPs), affect the viral replication by altering the expression of the host dependency factors that are essential for efficient HIV-1 gene expression. Both the host restriction and dependency factors determine the viral replication efficiency; however, the understanding of the IRepGs implicated in HIV-1 infection remains greatly limited at present. This review provides a comprehensive overview of the current understanding regarding the impact of the RNA-binding protein families, specifically the two families of splicing-associated proteins SRSF and hnRNP, on HIV-1 gene expression and viral replication. Since the recent findings show specifically that SRSF1 and hnRNP A0 are regulated by IFN-I in various cell lines and primary cells, including intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs), we particularly discuss their role in the context of the innate immunity affecting HIV-1 replication.

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Introduction: Circulating T follicular helper (cTfh) cells and circulating T peripheral helper (cTph) cells (which share common characteristics with the cTfh population) are implicated in the pathogenesis of immune-mediated and autoimmune diseases such as psoriasis (Ps). Their close interplay with the interleukin 17 (IL-17) axis and the ex vivo effect of IL-17-targeting biologic agents used to treat Ps on them are elusive. This study aimed to investigate the effect of biologics targeting IL-17 on cTfh and cTph cell subpopulations isolated from the blood of patients with Ps. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with Ps at treatment initiation and three months later. Samples were also collected from controls. Cells were stained using monoclonal antibodies. Flow cytometry assessed the fraction of cTfh (CD3+CD4+CXCR5+) and cTph (CD3+CD4+CXCR5-PD-1hi) cells.. Results: Flow cytometric analysis showed increased fractions of activated cTfh subsets including ICOS+ and ICOS+PD-1+ expressing cells, in patients compared to controls. Biologic blocking of IL-17A diminished the cTfh population. Furthermore, ICOS+ and ICOS+PD-1+ sub-populations were also inhibited. Finally, the cTph cell fraction significantly decreased after three months of successful treatment with biologics. Conclusion: Early anti-IL-17-mediated clinical remission in Ps is associated with decreased cTfh and cTph cell subpopulations.

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Infertility affects 15 % of couples in the US, and many turn to assisted reproductive technologies, including in vitro fertilization and subsequent frozen embryo transfer (FET) to become pregnant. This study aimed to perform a broad assessment of the maternal immune system to determine if there are systemic differences on the day of FET in cycles that result in a live birth compared to those that do not. Women undergoing FET of euploid embryos were recruited and blood was collected on the day of FET as well as at early timepoints in pregnancy. Sixty immune and angiogenic proteins were measured in plasma, and gene expression of 92 immune-response related genes were evaluated in peripheral blood mononuclear cells (PBMCs). We found plasma concentrations of interleukin-13 (IL-13) and macrophage derived chemokine (MDC) were significantly lower on the day of FET in cycles that resulted in a live birth. We also found genes encoding C-C chemokine receptor type 5 (CCR5), CD8 subunit alpha (CD8A) and SMAD family member 3 (SMAD3) were upregulated in PBMCs on the day of FET in cycles that resulted in live birth. Measurements of immune mediators from maternal blood could serve as prognostic markers during FET to guide clinical decision making and further our understanding of implantation failure.

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Background: Spexin is a novel peptide hormone and has shown antinociceptive effects in experimental mice. This study is aimed at evaluating the association of serum spexin level with diabetic peripheral neuropathy (DPN) and related pain in a Chinese population. Methods: We enrolled 167 type 2 diabetes mellitus (T2DM) including 56 patients without DPN (non-DPN), 67 painless DPN, and 44 painful DPN. Serum spexin was measured using ELISA. Logistic regression models were performed to analyze the independent effects of spexin on prevalence of DPN and painful DPN. In streptozotocin (STZ)-induced diabetic mice, mechanical pain threshold was measured using electronic von Frey aesthesiometer. Human peripheral blood mononuclear cells (PBMCs) were isolated and further stimulated with lipopolysaccharide without or with spexin. The gene expression was assayed by qPCR. Results: Compared with non-DPN, serum spexin level decreased in painless DPN and further decreased in painful DPN. The odds of DPN was associated with low spexin level in T2DM, which was similar by age, sex, BMI, and diabetes duration, but attenuated in smokers. The odds of having pain was associated with decreased spexin level in DPN, which was similar by age, sex, smoking status, and diabetes duration, but attenuated in normal weight. Furthermore, we observed that mechanical pain threshold increased in spexin-treated diabetic mice. We also found that lipopolysaccharide treatment increased the mRNA level of TNF-α, IL-6, and MCP-1 in human PBMCs, while spexin treatment prevented this increase. Conclusions: These results suggested that spexin might serve as a protective factor for diabetes against neuropathology and pain-related pathogenesis.

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Lipid nanoparticles (LNPs) tailored for mRNA delivery were optimized to serve as a platform for treating metabolic diseases. Four distinct lipid mixes (LMs) were formulated by modifying various components: LM1 (ALC-0315/DSPC/Cholesterol/ALC-0159), LM2 (ALC-0315/DOPE/Cholesterol/ALC-0159), LM3 (ALC-0315/DSPC/Cholesterol/DMG-PEG2k), and LM4 (DLin-MC3-DMA/DSPC/Cholesterol/ALC-0159). LNPs exhibited stability and homogeneity with a mean size of 75 to 90 nm, confirmed by cryo-TEM and SAXS studies. High mRNA encapsulation (95-100%) was achieved. LNPs effectively delivered EGFP-encoding mRNA to HepG2 and DC2.4 cell lines. LNPs induced cytokine secretion from human peripheral blood mononuclear cells (PBMCs), revealing that LM1, LM2, and LM4 induced 1.5- to 4-fold increases in IL-8, TNF-α, and MCP-1 levels, while LM3 showed minimal changes. Reporter mRNA expression was observed in LNP-treated PBMCs. Hemotoxicity studies confirmed formulation biocompatibility with values below 2%. In vivo biodistribution in mice post intramuscular injection showed significant mRNA expression, mainly in the liver. The modification of LNP components influenced reactogenicity, inflammatory response, and mRNA expression, offering a promising platform for selecting less reactogenic carriers suitable for repetitive dosing in metabolic disease treatment.