Group Of Healthy Donors Research Articles

P0070 Distinct Serum Proteome Alterations Following Vedolizumab and Ustekinumab Therapy in IBD Patients

Abstract Background Inflammatory bowel disease (IBD) is a chronic intestinal disorder characterized by relapse and remission. Dysregulated cytokine networks and excessive immune activation drive intestinal inflammation. Therapies targeting cytokines (e.g., TNF, IL-12/23) and immune cell trafficking (e.g., integrin inhibitors) have been effective. However, the impact of anti-integrin (vedolizumab) and anti-IL-12/23 (ustekinumab) therapies on systemic inflammation is poorly understood. This study compares serum proteome profiles of patients treated with vedolizumab or ustekinumab to elucidate their distinct effects on inflammatory pathways. Methods This study included patients with Crohn’s disease (CD), ulcerative colitis (UC), and age- and sex-matched healthy donors (HD), who received either ustekinumab or vedolizumab treatment. The ustekinumab cohort comprised 23 CD and 10 UC patients, while the vedolizumab cohort included 7 CD and 27 UC patients. Serum samples were collected from ustekinumab-treated patients at baseline (week 0) and 8 weeks post-treatment, and from vedolizumab-treated patients at baseline (week 0) and 6 weeks post-treatment. Additionally, serum samples were collected from 32 healthy donors. Proteomic analysis was performed using the Olink Inflammation Panel, measuring 92 inflammation-related proteins. Differential protein expression analysis was conducted to compare baseline and post-treatment samples within each cohort. Results Serum proteomic analysis using the Olink Inflammation panel revealed significant alterations in the levels of 15 proteins in IBD patients compared to healthy controls. Pro-inflammatory markers, including 4E-BP1, SIRT2 and TNF were significantly upregulated, indicating immune activation in IBD. Conversely, anti-inflammatory regulators, such as LIF-R and SCF, were significantly downregulated. Ustekinumab treatment led to upregulation of IL-12B and downregulation of 4E-BP1, SIRT2. Notably, IL-12B was upregulated in both CD and UC, while 4E-BP1 and SIRT2 were downregulated in CD (week 8 vs week 0). Conversely, vedolizumab treatment induced robust proteomic changes over time. Between week 0 and week 6, majority of proteins showed significant differential expression, more than the differences observed between IBD and HD groups. Conclusion Serum proteomic profiles of IBD patients differ from healthy donors. Ustekinumab induced minimal serum changes, indicating a limited systemic effect, while vedolizumab led to substantial proteomic shifts, suggesting broader modulation of inflammation. These distinct effects highlight the differing mechanisms of ustekinumab and vedolizumab on systemic inflammation in IBD, prompting further investigation to optimize therapeutic strategies.

CCR8/CCL1 and CXCR3/CXCL10 axis-mediated memory T-cell activation in patients with recalcitrant drug-induced hypersensitivity.

As a drug-induced hypersensitivity syndrome, drug reaction with eosinophilia and systemic symptoms (DRESS) is potentially fatal. Most patients with DRESS recover within a few weeks; however, some patients may suffer from a prolonged disease course and develop autoimmune sequelae. To investigate the immune mechanism and therapeutic targets of patients with recalcitrant DRESS with a prolonged disease course. Thirty-two patients with recalcitrant DRESS with a prolonged treatment course (≥ 8 weeks; 'prolonged DRESS'), 28 patients with DRESS with a short treatment course (< 2 weeks; 'short-duration DRESS') and 26 healthy donors (HDs) were enrolled. Bulk transcriptome results showed that the mRNA expression levels of CCR8 and CXCR3 were significantly increased in blood samples from patients in the acute stage of prolonged DRESS [Padj = 1.50 × 10-9 (CCR8) and Padj = 2.60 × 10-4 (CXCR3), patients with prolonged DRESS compared with the HD group]. Serum and skin lesion concentrations of CCL1 and CXCL10 (ligands of CCR8 and CXCR3, respectively) were significantly increased in patients with prolonged DRESS compared with patients with short-duration DRESS. The results from high-parameter flow cytometry and autoantibody screening also identified significant increases in CD8+ GNLY+ CXCR3+ effector memory T cells, CD8+ central memory T cells, CD4+ CCR8+ T helper 2 cells and IgG anti-HES-6 autoantibodies in patients with prolonged DRESS. Furthermore, in vitro blocking assays revealed that Janus kinase inhibitors (JAKi; mainly tofacitinib and upadacitinib) significantly decreased the release of CCL1 and CXCL10. Some patients with prolonged DRESS were successfully treated with JAKi. JAKi (tofacitinib and upadacitinib) were associated with decreased concentrations of CCL1 and CXCL10, suggesting that they may attenuate CCR8/CCL1 and CXCR3/CXCL10 axis-mediated memory T-cell activation, which contributes to disease pathogenesis in patients with recalcitrant DRESS and a long-term treatment course.

Male fertility is preserved following ixekizumab treatment-a real life pilot study.

Preserving fertility is crucial when managing male patients with spondyloarthritis (SpA) and/or psoriasis (PsO), especially in young men. Chronic inflammation, hormonal dysregulation, and immunosuppressive therapies can negatively impact male fertility. Over the past decades, positive data have emerged regarding the reproductive safety of various therapies in men. Ixekizumab (IXE), a high-affinity monoclonal antibody targeting IL-17A, has shown a safe profile for male fertility in small studies. This pilot study assesses the impact of IXE treatment on sperm parameters in SpA and/or PsO patients in a real-world setting. Consecutive adult male SpA and/or PsO patients eligible for or undergoing IXE treatment were prospectively enrolled. Demographic data, disease characteristics, laboratory assessments, comorbidities, and previous treatments were recorded. Sperm analysis was conducted after a minimum of 6 months of IXE treatment, and also before treatment inititation in a subgroup of patients. Parallel sperm evaluations were performed in a control group of healthy donors. Ten patients were enrolled: 8 with SpA and 2 with PsO. After 6 months of IXE treatment, all patients had normal sperm parameters. One SpA and PsO patient with baseline oligozoospermia showed normal parameters at follow-up and achieved a successful pregnancy post-treatment. Compared with controls, IXE-treated patients had lower sperm concentrations but higher vitality. In our limited size pilot study on SpA and PsO patients, Ixekizumab exposure did not impair male fertility. Sperm parameters remained within normal ranges after a minimum of 6 months of treatment. Early Ixekizumab treatment may preserve or potentially reverse fertility impairment.

Low CD46 expression on activated CD4+ T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors.

Previous research showed that the intracellular complement system, with CD46 as its central molecule, regulates the Th1 response associated with IFN-γ production and transition to a type 1 regulatory response (Tr1) characterized by IL-10 production. This transition can be influenced by a vitamin D (calcitriol), favouring a shift towards Tr1 cells and increased IL-10 production, as described in some autoimmune diseases. It is unknown whether calcitriol modulates CD46-induced Th1 response towards regulatory type 1 T cells (Tr1) in allergic eosinophilic asthma and its value in relation to reducing inflammatory response. CD4+ T cells from 58 patients with allergic eosinophilic asthma (AEA) and 49 healthy donors (HDs) were stimulated with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol in vitro for 60 h and analyzed by flow cytometry. IFN-γ and IL-10 levels in cell culture supernatants were measured using ELISA. CD4+ T cells from patients with AEA demonstrated elevated CD46 expression in both the non-activated state and under stimulation conditions with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol. Moreover, CD46 expression in AEA patients fluctuated with the pollen season, showing a significant increase during period of low pollen exposure. Calcitriol further induced CD4+Tr1 cells from in vitro generated CD4+Th1 cells in both HDs and AEA patients. However, in both cohorts were individuals (HDs: 35/49, AEA: 40/58) who responded to calcitriol with a more pronounced regulatory response. The calcitriol-induced regulatory effect manifested by a stronger surface decrease of CD46 on activated CD4+ T cells (by 40% in HDs and by 26% in AEA), accompanied by a significant inhibition of IFN-γ and increased IL-10 production (by 31% in HDs and by 85% in AEA). These individuals were termed as the CD46D group. Contrary to this, calcitriol induced an increase in CD46 expression at the CD4+ T cell surface in a minor group of HDs (14/49), and AEA patients (18/58), who were termed as the CD46I group. In CD46I group, CD4+ T cells produced less IFN-γ in comparison with CD46D group (by 33% in HDs and by 43% in AEA) and were unable to upregulate IL-10 production following stimulation with αCD3/αCD46/IL-2/Calcitriol. Our results suggest the potential existence of a key for stratifying individuals suitable for calcitriol treatment in the context of low serum vitamin D levels. After validation in clinical studies, this key could be used as an adjunctive therapy not only for patients with allergic eosinophilic asthma, but also for other diseases.

Screening for autoimmune diseases in apparently healthy antinuclear antibody positive individuals.

Anti-nuclear antibodies (ANA) assessed by immunofluorescence (IF) microscopy are associated with systemic autoimmune rheumatic diseases (SARD) and can be detected years before onset of clinical symptoms. Recent data indicate dysregulation of the immune system with increased levels of proinflammatory cytokines, including type I interferons (IFN), in ANA-positive versus ANA-negative individuals. Herein, the aims were to investigate IF-ANA, ANA fine specificities, and IFN-α protein levels in relation to self-reported symptoms, as well as clinical signs, of SARD in a large group of healthy blood donors (HBD). Sera from 825 HBD (48.8% females) were included. IF-ANA was assessed, using HEp-2 cells, according to the routine at the accredited laboratory of Clinical Immunology, Linköping University Hospital. All samples were analyzed for IgG-ANA fine specificities using addressable laser bead assay (ALBIA) at the same laboratory. IFN-α was determined using ELISA. Antibody-positive individuals, and their sex- and age-matched antibody-negative controls, were asked to fill a questionnaire regarding symptoms associated with SARD. In total, 130 HBD (15.8%) were positive with IF-ANA and/or ALBIA. Anti-U1RNP was significantly more common among women. Generally, self-reported symptoms correlated poorly with IF-ANA and/or ALBIA results. Two females with high levels of Ro60/SSA, Ro52/SSA and IFN-α reported mild sicca symptoms and were diagnosed with Sjögren's disease after clinical evaluation. A considerable proportion of apparently HBD are autoantibody positive, but without clear association to self-reported symptoms. Nevertheless, the combination of autoantibodies, relevant symptoms and high IFN-α levels identified the small proportion of individuals with SARD in the study population.

Free radical fragmentation and oxidation in the polar part of lysophospholipids: Results of the study of blood serum of healthy donors and patients with acute surgical pathology

The interaction of reactive oxygen species with cell membrane lipids is usually considered in the context of lipid peroxidation in the nonpolar component of the membrane. In this work, for the first time, data were obtained indicating that damage to human cell membranes can occur in the polar part of lysophospholipids at the interface with the aqueous environment due to free radical fragmentation (FRF) processes. FRF products, namely 1-hexadecanoyloxyacetone (PAc) and 1-octadecanoyloxyacetone (SAc), were identified in human serum, and a GC-MS method was developed to quantify PAc and SAc.The content of FRF products in serum samples of 52 healthy donors was found to be in the range of 1.98–4.75 μmol/L. A linear regression equation, CPAc&SAc (μmol/L) = 0.51 + 0.064 × years, was derived to describe the relationship between age and content of FRF products. In 70 patients with acute surgical pathology in comparison with the control group of healthy donors, two distinct clusters with different concentration levels of FRF products were revealed. The first cluster: groups of 43 patients with various localized inflammatory-destructive lesions of hollow organ walls and bacterial translocation (septic inflammation) of abdominal cavity organs. These patients showed a 1.5–1.9-fold (p = 0.012) decrease in the total concentration of PAc and SAc in serum. In the second cluster: groups of 27 patients with ischemia-reperfusion tissue damage (aseptic inflammation), – a statistically significant increase in the concentration of FRF products was observed: in 2.2–4.0 times (p = 0.0001).The obtained data allow us to further understand the role of free-radical processes in the damage of lipid molecules. FRF products can potentially be used as markers of the degree of free-radical damage of hydroxyl containing phospholipids.

Epigenetic and Molecular Alterations in Obesity: Linking CRP and DNA Methylation to Systemic Inflammation.

Obesity is marked by excessive fat accumulation in the adipose tissue, which disrupts metabolic processes and causes chronic systemic inflammation. Commonly, body mass index (BMI) is used to assess obesity-related risks, predicting potential metabolic disorders. However, for a better clustering of obese patients, we must consider molecular and epigenetic changes which may be responsible for inflammation and metabolic changes. Our study involved two groups of patients, obese and healthy donors, on which routine analysis were performed, focused on BMI, leukocytes count, and C-reactive protein (CRP) and completed with global DNA methylation and gene expression analysis for genes involved in inflammation and adipogenesis. Our results indicate that obese patients exhibited elevated leukocytes levels, along with increased BMI and CRP. The obese group revealed a global hypomethylation and upregulation of proinflammatory genes, with adipogenesis genes following the same trend of being overexpressed. The study confirms that obesity is linked to systematic inflammation and metabolic dysfunction through epigenetic and molecular alterations. The CRP was correlated with the hypomethylation status in obese patients, and this fact may contribute to a better understanding of the roles of specific genes in adipogenesis and inflammation, leading to a better personalized therapy.

Humoral and cellular immune response after mRNA SARS-CoV-2 vaccine in children on treatment for cancer: A pilot observational study

Immunocompromised children are at risk of developing severe COVID-19 infection. We conducted a pilot prospective study to evaluate the impact of cancer treatment and stem cell transplantation on immunogenicity of two doses of BNT162b2 vaccine in pediatric patients.Humoral, B- and T-cell responses to the BNT162b2 vaccine were assessed before, after the first and the second dose in patients aged 5–12 years (n = 35) and in a group of healthy donors (HD, n = 12). Patients were divided in three groups: solid tumors (ST, n = 11), hematological malignancies (HM, n = 14) and Hematopoietic Stem Cell Transplantation (HSCT) recipients (n = 10). After two vaccine doses, the seroconversion rate was 79.3 % (72.7 % in ST, 66.7 % in HM and 100 % in HSCT). The antibodies production was not associated to the presence of memory B and T-cells. Memory B-cells were measurable in 45.5 % ST, 66.6 % HSCT and in 22.0 % HM. The specific T-cell response was observed in most ST (81.8 %) and HSCT (85.7 %) patients and at lesser extent in those with HM (55.5 %). The combination of all immunological parameters (antibodies, memory B and T cells) showed that a significant fraction of HM (33.3 %) and ST (18.2 %) patients completely failed to respond to vaccination. Although able to produce antibodies, 11.1 % of HM and 27.3 % of ST had no B- and T-cell memory. HSCT subgroup showed the best immune function, with 80 % complete response and optimal T-cell function.Combination of anti-RBD antibody, and specific memory B- and T-cell responses represents a reliable read-out of vaccine immune efficacy in frail patients.

Insights on P2X7 in patients with abdominal aortic aneurysm and carotid atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero della Salute, Italia, Rete Cardiologica Nazionale Background Carotid atherosclerosis and abdominal aortic aneurysm (AAA) are co-morbidities [1]. P2X-purinoceptor-7 (P2X7) is expressed both in human AAA and atherosclerotic carotid plaque (CPL) and is instability-associated in CPL [2-4]. P2X7 is functionally related to miR-150 and miR-186 (two microRNA which modulate vascular cell proliferation) [5,6], and may be shed into the bloodstream in correlation with C-reactive protein increase [7]. Little is known about the possible association between AAA risk and circulating P2X7, miR-150 and miR-186 in patients with/without hemodinamically significant CPL. Purpose Our aim is exploring the possible relationship between circulating P2X7, target miRs clinical status and lesion dimensions in patients with CPL and/or AAA. Methods Male patients with AAA and/or CPL were characterized.CPL stenosis was assessed by Ecocolordoppler-TSA. Eligible cohort included 20 patients with AAA undergoing aneurysmectomy [10 with CPL stenosis ≤40% (group A), 10with CPL stenosis &amp;gt;40% (group B)], 10 patients with hemodinamically significant CPL and small or no AAA undergoing endarterectomy (group C) donating blood and vascular samples at surgery. Twelve healthy blood donors (group D) were enrolled as controls. P2X7 protein was evaluated by ELISA. P2X7 gene, miR-150 and miR-186 were determined by RT-qPCR. Results In group A, CPL appeared mainly localized at carotid bulb, in group B extended along branches. P2X7 gene was undetermined into serum and expressed without differences among groups in patient tissues. P2X7 protein was measurable in all serum samples and was lower in group B than in the others (p=0,0117, p=0,0009, p=0,0003 vs. group A, C, D, respectively) and in group C vs. D (p=0,0006). These data differentiated patients with hemodinamically significant CPLs with severe AAA from the other patients and all those with hemodinamically significant CPLs (irrespectively from AAA severity) from healthy individuals. In group B but not in the others, serum P2X7 was linearly associated to platelet concentration. In group C the amounts of P2X7 in CPL tissue and serum were negatively associated, whereas in group A and B the P2X7 levels in AAA tissue and serum were unrelated. The miRs were detected into both serum and tissue and not correlated among them nor with circulating P2X7 protein. The serum miR-186, but not miR-150, was differentially expressed in group A vs. B (p=0,0266) and linearly related to AAA diameter in group B (p=0.0351). P2X7 content and expressed gene (p=0,015, p= 0,023, respectively) and miR expressions (p=0,0418 for miR-186, p=0,0012 for miR-150) were more elevated in AAA vs. CPL tissues, highlighting the vascular heterogenicity. Conclusions Preliminary data suggest a role for P2X7 and miR-186 in profiling patients with hemodinamically significant CPL with/without high risk AAA, deserving further investigations.

Human leukocyte antigen class II (DRB1 and DQB1) alleles frequencies in patients with various forms of pemphigus among the Russian population

BACKGROUND: Autoimmune bullous dermatoses are known to be the most severe blistering conditions of skin. HLA-DRB1 and DQB1 alleles might play a crucial role in their onset. In pemphigus HLA class II molecules stimulate the division of T helper cells, which in turn stimulate B cells to produce antibodies to epidermal keratinocytes causing acantholysis. The HLA-DRB1 and DQB1 alleles’ frequencies studied in pemphigus in a vast variety of populations worldwide. However, as of yet, this mechanism was not investigated in Russian population. AIM: To estimate the prevalence of the HLA-DRB1 and DQB1 alleles at a low- and high-resolution levels in patients with various forms of pemphigus. We observed 86 patients with pemphigus vulgaris, 13 ― with pemphigus foliaceus, 6 patients with paraneoplastic pemphigus and 92 healthy volunteers. MATERIALS AND METHODS: HLA typing for DRB1 and DQB1 was performed with 50 nanogram DNA extraction and polymerase chain reaction. RESULTS: At a low-resolution level HLA-DRB1*4 and DRB1*14 alleles were statistically significant more frequent in pemphigus vulgaris and pemphigus foliaceus patients compared to those in control subjects, whereas HLA-DRB1*11, DRB*16, and DRB1*3 alleles were more frequent in healthy volunteers. At a high-resolution level, DRB1*04:02 allele was observed to show its statistically significant higher frequency in all variants of pemphigus, including paraneoplastic pemphigus. However, DRB1*14:05 HLA allele was more frequent in pemphigus vulgaris and pemphigus foliaceus patients, whereas DRB1*11:04 one was found to be 3.7 times more frequent in healthy controls. Additionally, at a low-resolution level for HLA-DQB1 alleles no statistically significant results were observed. However, at a high-resolution level the chances for more frequent indication of DQB1*03:02 allele were 7.09 times higher in pemphigus foliaceus group and 2.49 higher in pemphigus vulgaris patients compared to healthy volunteers. Moreover, DQB1*05:03 was identified more frequently in pemphigus vulgaris and paraneoplastic pemphigus groups of patients, whereas DQB1*03:01 allele was shown to be increased in the group of healthy donors. CONCLUSION: HLA-DRB1*4, DRB1*14, DRB1*04:02, DRB1*14:05, DQB1*03:02 and DQB1*05:03 alleles might be considered as the genetic markers for pemphigus vulgaris susceptibility, while HLA-DRB1*11, DRB*16, DRB1*3, DRB1*11:04 and DQB1*03:01 allelic groups appear to be protective for Russian population.

Safety and efficacy of faecal microbiota transplantation in patients with mild to moderate Parkinson's disease (GUT-PARFECT): a double-blind, placebo-controlled, randomised, phase 2 trial

Dysregulation of the gut microbiome has been implicated in Parkinson's disease (PD). This study aimed to evaluate the clinical effects and safety of a single faecal microbiota transplantation (FMT) in patients with early-stage PD. The GUT-PARFECT trial, a single-centre randomised, double-blind, placebo-controlled trial was conducted at Ghent University Hospital between December 01, 2020 and December 12, 2022. Participants (aged 50-65 years, Hoehn and Yahr stage 2) were randomly assigned to receive nasojejunal FMT with either healthy donor stool or their own stool. Computer-generated randomisation was done in a 1:1 ratio through permutated-block scheduling. Treatment allocation was concealed for participants and investigators. The primary outcome measure at 12 months was the change in the Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score obtained during off-medication evaluations. Intention-to-treat analysis was performed using a mixed model for repeated measures analysis. This completed trial is registered on ClinicalTrials.gov (NCT03808389). Between December 2020 and December 2021, FMT procedures were conducted on 46 patients with PD: 22 in the healthy donor group and 24 in the placebo group. Clinical evaluations were performed at baseline, 3, 6, and 12 months post-FMT. Full data analysis was possible for 21 participants in the healthy donor group and 22 in the placebo group. After 12 months, the MDS-UPDRS motor score significantly improved by a mean of 5.8 points (95% CI-11.4 to-0.2) in the healthy donor group and by 2.7 points (-8.3 to 2.9) in the placebo group (p=0.0235). Adverse events were limited to temporary abdominal discomfort. Our findings suggested a single FMT induced mild, but long-lasting beneficial effects on motor symptoms in patients with early-stage PD. These findings highlight the potential of modulating the gut microbiome as a therapeutic approach and warrant a further exploration of FMT in larger cohorts of patients with PD in various disease stages. Flemish PD patient organizations (VPL and Parkili), Research Foundation Flanders (FWO), Biocodex Microbiota Foundation.

Immunological Profile and Markers of Endothelial Dysfunction in Elderly Patients with Cognitive Impairments.

The process of aging is accompanied by a dynamic restructuring of the immune response, a phenomenon known as immunosenescence. Further, damage to the endothelium can be both a cause and a consequence of many diseases, especially in elderly people. The purpose of this study was to carry out immunological and biochemical profiling of elderly people with acute ischemic stroke (AIS), chronic cerebral circulation insufficiency (CCCI), prediabetes or newly diagnosed type II diabetes mellitus (DM), and subcortical ischemic vascular dementia (SIVD). Socio-demographic, lifestyle, and cognitive data were obtained. Biochemical, hematological, and immunological analyses were carried out, and extracellular vesicles (EVs) with endothelial CD markers were assessed. The greatest number of significant deviations from conditionally healthy donors (HDs) of the same age were registered in the SIVD group, a total of 20, of which 12 were specific and six were non-specific but with maximal differences (as compared to the other three groups) from the HDs group. The non-specific deviations were for the MOCA (Montreal Cognitive Impairment Scale), the MMSE (Mini Mental State Examination) and life satisfaction self-assessment scores, a decrease of albumin levels, and ADAMTS13 (a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13) activity, and an increase of the VWF (von Willebrand factor) level. Considering the significant changes in immunological parameters (mostly Th17-like cells) and endothelial CD markers (CD144 and CD34), vascular repair was impaired to the greatest extent in the DM group. The AIS patients showed 12 significant deviations from the HD controls, including three specific to this group. These were high NEFAs (non-esterified fatty acids) and CD31 and CD147 markers of EVs. The lowest number of deviations were registered in the CCCI group, nine in total. There were significant changes from the HD controls with no specifics to this group, and just one non-specific with a maximal difference from the control parameters, which was α1-AGP (alpha 1 acid glycoprotein, orosomucoid). Besides the DM patients, impairments of vascular repair were also registered in the CCCI and AIS patients, with a complete absence of such in patients with dementia (SIVD group). On the other hand, microvascular damage seemed to be maximal in the latter group, considering the biochemical indicators VWF and ADAMTS13. In the DM patients, a maximum immune response was registered, mainly with Th17-like cells. In the CCCI group, the reaction was not as pronounced compared to other groups of patients, which may indicate the initial stages and/or compensatory nature of organic changes (remodeling). At the same time, immunological and biochemical deviations in SIVD patients indicated a persistent remodeling in microvessels, chronic inflammation, and a significant decrease in the anabolic function of the liver and other tissues. The data obtained support two interrelated assumptions. Taking into account the primary biochemical factors that trigger the pathological processes associated with vascular pathology and related diseases, the first assumption is that purine degradation in skeletal muscle may be a major factor in the production of uric acid, followed by its production by non-muscle cells, the main of which are endothelial cells. Another assumption is that therapeutic factors that increase the levels of endothelial progenitor cells may have a therapeutic effect in reducing the risk of cerebrovascular disease and related neurodegenerative diseases.

Comparative proteomics reveals different protein expression in platelets in patients with alcoholic liver cirrhosis

BackgroundThe role of platelets in disease progression as well as the function of platelets as part of the haemostatic and immunological system in patients with liver cirrhosis is only incompletely understood. This is partly due to difficulties in assessing platelet function. Proteome analyses of platelets have been used to further investigate the role of platelets in other diseases.AimTo assess possible changes in the platelet proteome during different stages of alcohol induced liver cirrhosis compared to healthy donors.Patients and methodsA 45 ml blood sample was drawn from 18 participants aged 18–80 years evenly divided into three groups of healthy donors, patients with less advanced alcohol induced liver cirrhosis (Child-Pugh < 7) and patients with advanced liver cirrhosis (Child-Pugh > 10). The blood was processed to isolate platelets and perform subsequent two-dimensional gel-electrophoresis using a SYPRO™ Ruby dye. After computational analysation significantly in- or decreased protein spots (defined as a two-fold abundance change between different study cohorts and ANOVA < 0.05) were identified via liquid chromatography–mass spectrometry (LCMS) and searching against human protein databases.ResultsThe comparative analysis identified four platelet proteins with progressively decreased protein expression in patients with liver cirrhosis. More specifically Ras-related protein Rab-7a (Rab-7a), Ran-specific binding protein 1 (RANBP1), Rho GDP-dissociation inhibitor 1 (RhoGDI1), and 14–3-3 gamma.ConclusionThere is significant change in protein expression in the platelet proteome throughout the disease progression of alcohol induced liver cirrhosis. The identified proteins are possibly involved in haemostatic and immunoregulatory function of platelets.

Diminished Diversities and Clonally Expanded Sequences of T-Cell Receptors in Patients with Chronic Spontaneous Urticaria.

Studies establish a link between autoimmune factors and chronic spontaneous urticaria (CSU). T cells are crucial in immune-mediated diseases like CSU, and T-cell receptor (TCR) diversity could be pivotal in autoimmune responses. The clinical relevance of TCR variations in CSU is unknown, but understanding them may offer insights into CSU's pathogenesis and treatment. This cross-sectional study included 132 chronic urticaria (CU) patients versus 100 age-matched healthy donors (HD), with subgroup analyses on CU type, angioedema, allergic comorbidities, and anti-IgE therapy efficacy. Peripheral TCRβ repertoires were analyzed by high-throughput sequencing. CSU patients showed reduced TCR diversity (lower D50) and increased large clone proportions than HD. Moreover, TCR diversity in CSU patients was significantly lower than in those with Chronic Inducible Urticaria (ClndU). There were also differences in variable (V) and joining (J) gene usage between CU and HD groups as well as CSU and ClndU groups. However, in subgroup analyses regarding angioedema, allergic comorbidities, and the efficacy of anti-IgE treatment, no significant differences were found in TCR diversity or large TCRβ clones. Notably, patients with treatment relapse or poor response to anti-IgE therapy had a higher proportion of positively charged CDR3. Additionally, age affected TCR diversity, but TIgE value, EOS counts, CU duration, and UAS7 score did not associate significantly with D50. CSU patients exhibit reduced TCR diversity and increased large clone proportions, indicating abnormal T cell activation. The TCR diversity differences and distinct V and J gene usage between CSU and ClndU may indicate different mechanisms in T lymphocyte-associated immune responses for these two subtypes of CU. The higher positive charge in CDR3 of relapsed or poorly responsive patients to anti-IGE treatment may indicate more antigen charge involvement. These findings provide new insights into the pathogenesis of CSU and potential future treatments.

Research and Analysis of Molecules such as CD28, CD45RA, CD45RO, CD38, HLA-DR, and CD57 on T Cells in Multiple Myeloma.

For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC). For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells. We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group. Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Expression of genes potentially involved in loss of response in patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematological malignancy characterized by the presence of the BCR::ABL1 fusion gene, which leads to uncontrolled cell growth and survival. Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of CML, but a significant proportion of patients develop resistance or lose response to these drugs. Understanding the molecular mechanisms underlying treatment response and resistance is crucial for improving patient outcomes. This study aimed to analyze the expression patterns of genes involved in treatment response and resistance in CML patients receiving TKI therapy. The expression levels of MET, FOXO3, p15, p16, HCK, and FYN genes were examined in CML patients and compared to healthy donors. Gene expression levels were compared between optimal responders (OR) and resistant patients (R) vs. healthy donors (HD). The MET and FOXO3 OR group showed significant differences compared with the HD, (p < 0.0001) and (p = 0.0003), respectively. p15 expression showed significant differences between OR and HD groups (p = 0.0078), while no significant differences were found in p16 expression between the HD groups. FYN showed a statistically significant difference between R vs. HD (p = 0.0157). The results of HCK expression analysis revealed significant differences between OR and HD (p = 0.0041) and between R and HD (p = 0.0026). When we analyzed OR patients with undetectable BCR::ABL1 transcripts, a greater expression of HCK was observed in the R group. These findings suggest that monitoring the expression levels of MET and FOXO3 genes could be valuable in predicting treatment response and relapse in CML patients. Our study provides important insights into the potential use of gene expression analysis as a tool for predicting treatment response and guiding treatment decisions in CML patients. This knowledge may ultimately contribute to the development of personalized treatment strategies to improve patient outcomes in CML.

UHRF1-Mediated Epigenetic Reprogramming Regulates Glycolysis to Promote Progression of B-Cell Acute Lymphoblastic Leukemia

Background Acute lymphoblastic leukemia (ALL) is a hematologic malignant disease with high heterogeneity, including B-ALL and T-ALL. The 5-year overall survival rate of children ALL has been significantly improved, which is over 90%. However, the prognosis of adult patients is not satisfactory, and the 5-year OS is only 30%-50%. Among them, the prognosis of patients with relapsed/refractory B-ALL is very poor. Exploring the molecular mechanism of disease progression is of great significance to improve the outcome. Methods Application of 10× Genomics chromium sequencer reveals the single-cell transcriptome profiling of peripheral blood or bone marrow MNC cells, which were from 6 patients with B-ALL（3 new diagnosed patients and 3 relapsed patients） and 3 healthy donors. The expression of key genes in 84 clinical blood or bone marrow samples was detected, and the prognosis was analyzed in TARGET database by bioinformatics technology. The molecular mechanism was investigated by RNAseq, 850k methylation microarray, qRT-PCR, Western blot, BSP, CHIP-qPCR and flow cytometry. Additionally, we identified a UHRF1 inhibitor using virtual compounds screening, and demonstrated its therapeutic efficacy both in vitro and vivo. Results Using Seurat method to cluster and dimension reduction of scRNA-seq data, 29 subclusters were obtained from 9 clinical samples. It mainly includes eight progenitor B cells, one B cell, six T cells, two NK cells, three monocytes, two neutrophils and three primordial erythrocytes. Nine samples were divided into three groups: Newly Diagnosis group (ND), Relapsed/Refractory group (RR) and Healthy Donors group (HD). The proportion of three subpopulations (cluster 2,5,16) in progenitor B cell group showed a decreasing trend of RR group &amp;gt; ND group &amp;gt; HD group. The marker genes of these three subgroups were enriched by GO and KEGG using ClusterProfiler R package. Cluster 2 was enriched into cancer signal pathway, cluster 5 was enriched into DNA replication process, and the marker genes of cluster 16 was mainly involved in metabolic pathway. The differential expression of these three subgroups between RR group and ND group was analyzed by FindMarkers function. The results showed that cluster 16 was the main differential cluster. The intergroup differential genes of cluster 16 mainly involve metabolic pathway, cancer pathway, cell cycle and p53 signaling pathway (Figure 1). The three groups were compared in pairs. After the intersection of differential genes in cluster 16 and all progenitor B cell groups, four common differential genes (HSPB1, UHRF1, H1F0 and IGFBP2) were screened. The results showed that the expression of UHRF1 gene was up-regulated in acute lymphoblastic leukemia patients, especially in patients with relapsed/refractory. Moreover, UHRF1 overexpression is an independent prognostic risk factor for B-ALL patients in our clinical data, and it is also a molecular marker of poor prognosis in the TARGET dataset. In vitro experiments, knockout of gene UHRF1 promoted the cycle arrest and apoptosis of B-ALL tumor cell line (Reh and nalm6), inhibited the proliferation of cells, and improved the drug sensitivity of vindesine. RNAseq and methylation microarray showed that the gene SFRP5 was demethylated and the expression of mRNA was overexpressed in UHRF1 knockout cell lines. This was verified by subsequent qRT-PCR, Western Blot and Bisulfite sequencing. Further studies showed that UHRF1 was involved in glycolysis by regulating the SFRP5-p38 MAPK-HK2 signal axis. Subsequently, through overexpressing SFRP5, we confirmed that upregulating SFRP5 can inhibit the P38 MAPK-HK2 signaling pathway, thereby suppressing glycolytic levels. Moreover, virtual compound screening identified UM164 as an inhibitor of UHRF1, which significantly inhibits P38 protein phosphorylation, downregulates HK2 expression, and reduces the production of glycolysis-related metabolites. Furthermore, UM164 demonstrated the ability to inhibit the proliferation of B-ALL cells both in vitro and in vivo, leading to prolonged survival in a B-ALL mouse model. In conclusion, we have unraveled the molecular mechanisms of epigenetic and metabolic rearrangements in relapsed/refractory B-ALL, and have provided potential targeted therapeutic strategies to improve its unfavorable prognosis.

Partitioning of Circulating Monocyte Subsets in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an autoimmune disease characterized by accelerated platelet destruction with impaired platelet production leading to a platelet count decreased +/- bleeding manifestations, mostly caused by antiplatelet autoantibodies. The role of monocytes in ITP pathophysiology is not clearly established. Circulating monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 and of CD16 into classical (cMo), intermediate (iMo) and nonclassical (ncMo) monocytes. A new subpopulation, defined by the expression of the slan marker, has recently been described and represents a fraction of ncMo (ncMo slan +). Objectives The 1 st objective of the study was to analyze the partitioning of peripheral blood monocyte subsets by flow cytometry in ITP patients compared to a group of healthy blood donors (HD). Our second objective was to further characterize different phenotypic profiles of monocytes among ITP patients based on baseline characteristics, severity and treatment(s). Methods From April 2019 to January 2023, peripheral blood samples from ITP patients and HD were collected on EDTA and stained with the following antibodies: anti-CD45, CD2, CD56, CD24, CD14, CD16 and anti-slan. Acquisition analysis was performed either with a Navios or a DxFLEX cytometer. All files were analyzed with the previously described exclusion strategy of non-monocytes populations, using Kaluza™ analysis software, to characterize cMo, iMo, ncMo and ncMo slan + subsets. ITP patients were included regardless of their ITP status and/or phase of the disease. Results In total, 56 peripheral blood samples from 46 ITP patients and from 19 HD were analyzed. ITP patients had a median age of 50.5 years [19-91], 54.3% were females, whereas the median age of HD was 36 years [21-65] with 42.1% of females. ITP was defined as primary in 82.6% of the cases with a median disease duration of 3 years (&amp;lt;3 months or newly-diagnosed in 26.8% and &amp;gt;12 months or chronic ITP in 60.7%). Nine patients had undergone splenectomy (19.6%) at time of analysis. The platelet count was &amp;lt;50 x 10 9/L in 60.7% of ITP patients and absolute monocyte count ≥ 1 x 10 9/L was found in 21.4%. Regarding ITP treatment, 14 patients (25%) were receiving glucocorticoids (GC) at time of analysis whereas 9 (16.1%) had been exposed to GC within the previous month. Eleven patients (19.6%) had received intravenous immunoglobulin within a month prior to the analysis, 20 (35.7%) were on thrombopoietin receptor agonists, 9 (16.1%) had received rituximab in the previous year, 4 (7.1%) received another immunosuppressive therapy and 6 (10.7%) received another treatment. Eighteen patients (32.1%) were considered as “treatment-free” as they had not received any treatment for their ITP in the previous month or rituximab in the previous year. The latter group of “untreated” patients displayed a significant increase of the iMo relative fraction when compared to HD (4.92% vs 3.18%, p=0.018). There was no significant difference between the two groups regarding other monocytic subpopulations, including cMo, ncMo, ncMo slan+, the absolute and relative counts of monocytes. As for the treatment effect, the ongoing intake of GC therapy did not have a significant impact on monocytosis but was strongly associated with an increased proportion of cMo (94.2% vs 85.0%, p &amp;lt; 0.0001; Figure 1) and a decreased proportion of ncMo (2.31% vs 9.85%, p&amp;lt;0.0001) and ncMo slan+ (3.12% vs 7.46%, p&amp;lt;0.0001), mimicking the cMo accumulation characteristic of chronic myelomonocytic leukemia. Splenectomy and other treatments did not significantly interfere neither with monocyte count nor with the monocyte subpopulation partitioning. When excluding patients receiving GC, splenectomized patients had a higher monocyte count compared to non-splenectomized patients (1.0 vs 0.5G/L, p=0.008), with no difference in monocyte subset repartition. Conclusion Using multiparameter flow cytometry analysis, we demonstrated the expansion of iMo in untreated ITP patients. GC treatment led to the accumulation of cMo with reduced ncMo proportion, hence should be considered when interpreting the monocyte subset partitioning. These data suggest a potential role of monocytes in ITP pathogenesis and clinical response to GC therapy.

A third (booster) dose of the inactivated SARS-CoV-2 vaccine elicits immunogenicity and T follicular helper cell responses in people living with HIV.

This study sought to explore the immunogenicity of a booster dose of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in people living with human immunodeficiency virus (HIV) and identify the factors affecting the magnitude of anti-SARS-CoV-2 antibody levels. A total of 34 people living with HIV (PLWH) and 34 healthy donors (HD) were administered a booster dose of the same SARS-CoV-2 vaccine. Anti-SARS-CoV-2 antibody and immunoglobulin G (IgG) levels were measured using the SARS-CoV-2 S protein neutralizing antibody Enzyme-Linked Immunosorbent Assay (ELISA) and 2019-nCov IgG Chemiluminescent Immunoassay Microparticles, respectively. Spearman correlation analysis was used to measure the correlation between laboratory markers and neutralizing antibody and IgG levels. Peripheral blood mononuclear cells (PBMCs) were extracted from each subject using density gradient centrifugation and the numbers of memory T and T follicular helper (Tfh) cells were determined using flow cytometry. PLWH had a marked reduction in CD4 and B cell levels that was accompanied by a lower CD4/CD8 T cell ratio. However, those who received a supplementary dose of inactivated SARS-CoV-2 vaccines exhibited antibody positivity rates that were analogous to levels previously observed. The booster vaccine led to a reduction in IgG and neutralizing antibody levels and the amplitude of this decline was substantially higher in the PLWH than HD group. Correlation analyses revealed a strong correlation between neutralizing antibody levels and the count and proportion of CD4 cells. Anti-SARS-CoV-2 IgG antibody levels followed a similar trend. The expression of memory T and Tfh cells was considerably lower in the PLWH than in the HD group. PLWH had an attenuated immune response to a third (booster) administration of an inactivated SARS-CoV-2 vaccine, as shown by lower neutralizing antibody and IgG levels. This could be attributed to the reduced responsiveness of CD4 cells, particularly memory T and cTfh subsets. CD4 and cTfh cells may serve as pivotal markers of enduring and protective antibody levels. Vaccination dose recalibration may be critical for HIV-positive individuals, particularly those with a lower proportion of CD4 and Tfh cells.

Otoprotective and antioxidant activity of cytoflavin in pulmonary tuberculosis patients receiving amikacin

The objective: to study the potential otoprotective and antioxidant activity of cytoflavin in new pulmonary tuberculosis patients receiving aminoglycoside amikacin.Subjects and Methods. A randomized controlled trial was conducted and included 90 patients with new infiltrative pulmonary tuberculosis with decay, of both genders aged from 20 to 50 years old. Clinical, paraclinical and statistical methods were used in the study. Audiometric screening was carried out by Diagnostic Audiometer AD 226. The degree of antioxidant protection was determined by the oxidation reaction of ABTS in blood serum and plasma.Results. The study included 4 groups of tuberculosis patients and 1 group of healthy donors. Dissemination of pulmonary tuberculosis in 4 groups was approximately the same and was measured by scores. Prior to the start of treatment in all 4 groups (the group receiving standard chemotherapy (CTX) without amikacin, the group receiving «CTX+amikacin», the group receiving «CTX+cytofl avin», the group receiving «CTX+amikacin+cytofl avin») there was a decrease in blood antioxidant activity compared to Control Group. After 3 months of tuberculosis treatment in all 4 groups, the results improved, while in «CTX+Cytofl avin» Group they were similar to Control Group, and «CTX+Amikacin+Cytofl avin Group», they were only 4.8% less. The elevated level of leukocytes decreased by 10.6% in CTX Group, 16.9% in «CTX+Amikacin» Group, 38.3% in «CTX+Cytofl avin» Group, and 72.3% in «CTX+Amikacin+Cytofl avin» Group. In the study, manifestations of oto- and vestibulotoxicity were found only in the «CTX+Amikacin» Group (in 4/20 (5%)). There were no such cases in «Amikacin+Cytofl avin» Group consisting of 23 patients.