Cells In Peripheral Blood Samples Research Articles

Investigating Vα7.2+/CD161- T Cell and MAIT Cell Profiles Using Flow Cytometry in Healthy Subjects and Subjects with Atopic Dermatitis.

This study investigates the roles of mucosal-associated invariant T (MAIT) cells and Vα7.2+/CD161- T cells in skin diseases, focusing on atopic dermatitis. MAIT cells, crucial for bridging innate and adaptive immunity, were analyzed alongside Vα7.2+/CD161- T cells in peripheral blood samples from 14 atopic dermatitis patients and 10 healthy controls. Flow cytometry and machine learning algorithms were employed for a comprehensive analysis. The results indicate a significant decrease in MAIT cells and CD69 subsets in atopic dermatitis, coupled with elevated CD38 and polyfunctional MAIT cells producing TNFα and Granzyme B (TNFα+/GzB+). Vα7.2+/CD161- T cells in atopic dermatitis exhibited a decrease in CD8 and IFNγ-producing subsets but an increase in CD38 activated and IL-22-producing subsets. These results highlight the distinctive features of MAIT cells and Vα7.2+/CD161- T cells and their different roles in the pathogenesis of atopic dermatitis and provide insights into their potential roles in immune-mediated skin diseases.

Flow cytometric detection of leukemic blasts in Libyan pediatric patients with acute lymphoblastic leukemia

ABSTRACT The diagnosis of acute lymphoblastic leukemia (ALL), which is the most common type of cancer in children, has become more accurate with the use of flow cytometry. Here, this technology was used to immunophenotype leukemic cells in peripheral blood samples from Libyan pediatric ALL patients. We recruited 152 newly diagnosed patients at Tripoli Medical Center (Tripoli, Libya) by morphological examination of blood and bone marrow. Twenty-three surface and cytoplasmic antigen markers were used to characterize B and T cells in circulating blood cells by four-color flow cytometry. Six children (3.9%) turned out to have biphenotypic acute leukemia, 88 (57.9%) had B ALL, and 58 (38.1%) had T ALL. There were 68 cases of pro-B ALL CD10-positive (44.7%), 8 cases of pro-B ALL CD10-negative (5.2%), 6 cases of pre-B ALL (3.9%), and 6 of mature-B ALL (3.9%). CD13 was the most commonly expressed myeloid antigen in ALL. We present immunophenotypic data for the first time describing ALL cases in Libya. The reported results indicate that the most common subtype was pro-B ALL, and the frequency of T-ALL subtype was higher compared to previous studies. Six cases were positive for both myeloid and B lymphoid markers. Our findings may provide the basis for future studies to correlate immunophenotypic profile and genetic characteristics with treatment response among ALL patients.

Blockade of PD-1 and LAG-3 expression on CD8+ T cells promotes the tumoricidal effects of CD8+ T cells.

The diffuse large B-cell lymphoma (DLBCL) has the highest incidence of all lymphomas worldwide. To investigate the functions of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in tissues and peripheral blood of patients with DLBCL, the expression of LAG-3 and PD-1 genes in DLBCL-TCGA were analyzed. LAG-3 and PD-1 mRNA levels in DLBCL were analyzed using data from The Cancer Genome Atlas (TCGA) database. Utilize the Genotype-Tissue Expression (GTEx) database for assessing the variance in the expression of LAG-3, PD-1, and other associated factors between the tissues of DLBCL patients and healthy individuals. Immunohistochemistry was applied to detect the expression of LAG-3 and PD-1 levels in 137 cases of DLBCL tissues and 20 cases of reactive lymphoid hyperplasia. The prognostic value of LAG-3 and PD-1 were assessed using the Kaplan-Meier curve. The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and ssGSEA algorithm were used to explore the immune microenvironment of DLBCL. Additionally, the expression and co-expression of LAG-3 and PD-1 were detected on CD4 and CD8 T cells in peripheral blood samples from 100 cases of DLBCL tissues and 30 cases of healthy individuals using flow cytometry. According to TCGA database, LAG-3 and PD-1 gene expression levels were significantly up-regulated in DLBCL tissues. LAG-3 and PD-1 levels were also strongly positively correlated with those of most infiltrating immune cells. Overall survival of patients with high LAG-3 and PD-1 co-expression was significantly shorter than that of patients with low co-expression. In DLBCL patients, LAG-3 and PD-1 were highly expressed in peripheral blood CD8+ T cells. In addition, LAG-3 was highly expressed in CD4+ T cells, while the expression of PD-1 in CD4+ T cells of DLBCL patients showed no significant difference compared to healthy individuals. Additionally, CD8+ T cells and SU-DHL6/OCI-LY3 from patients with DLBCL were co-cultured in vitro; after addition of LAG-3 and/or PD-1 inhibitors alone, an increased perforin and granzyme B secretion levels by CD8+ T cells were detected, as well as an increase in the overall proportion of tumor cells undergoing apoptosis. High LAG-3 and PD-1 levels significantly inhibit CD8+ T cell function, resulting in weakened ability to kill tumor cells. Combined LAG-3 and PD-1 blockade can restore CD8+ T cell function and provides a potential avenue for development of personalized cellular immunotherapy for DLBCL.

Effect of thymectomy on the frequencies of peripheral regulatory B and T lymphocytes in patients with Myasthenia gravis-a pilot study

Aim We aimed to investigate the relationship between the peripheral lymphocyte subset frequency and thymectomy in patients with myasthenia gravis (MG). Materials and Methods The frequencies of regulatory B (Breg) and regulatory T (Treg) cells in peripheral blood samples obtained from 69 patients with MG and 10 healthy controls were analyzed using flow cytometry. Serum acetylcholine receptor antibodies (AchR-Ab) were measured. Patients with MG were subdivided into pre-thymectomy, post-thymectomy, and normal thymus control group. Results The percentage of Breg cells was significantly decreased in both the pre-thymectomy (7.92 ± 1.30%) and post-thymectomy (8.14 ± 1.34%) groups compared to healthy controls (16.02 ± 2.78%) and reduced in the exacerbation and relapse phase compared to the stable maintenance stage. The proportion of cluster of differentiation (CD) 4 + CD25 + T cells and CD4 + CD25 + CD127low/- Treg cells in MG patients were not significantly different than healthy controls. AchR-Ab titers in aggravating or recurrence patients after thymectomy were significantly higher than that of the stable remission patients (11.13 ± 0.70 and 6.03 ± 0.85 nmol/L, respectively; p < 0.001). Conclusion The frequency of Breg cells may serve as a potential indicator of MG prognosis, while Treg cell frequency did not demonstrate the same prognostic ability. The concentration of AchR-Ab can be used as a dynamic monitoring index of disease severity in patients with MG.

Functional analysis of a novel nonsense variant c.91A>T of the TRAPPC2 gene in a Chinese family with X-linked recessive autosomal spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDT) is a condition involving late-onset, X-linked recessive skeletal dysplasia caused by mutations in the TRAPPC2 gene. In this paper, we identified a novel nonsense variant in a SEDT pedigree and analyzed the function of the variant in an attempt to explain the new pathogenesis of the TRAPPC2 protein in SEDT. Briefly, DNA and RNA samples from the peripheral blood of SEDT individuals were prepared. The causative variant in the Chinese SEDT family was identified by clinic whole-exome sequencing analysis. Then, we observed the mRNA expression of TRAPPC2 in patients and the mutant TRAPPC2 level in vitro and analyzed the protein stability and subcellular distribution by cell fluorescence and Western blotting. We also investigated the effect of TRAPPC2 knockdown on the expression and secretion of COL2A1 in SW1353 cells or primary human chondrocytes. Herein, we found a nonsense variant, c.91A>T, of the TRAPPC2 gene in the pedigree. TRAPPC2 mRNA expression levels were significantly decreased in the available peripheral blood cell samples of two affected patients. An in vitro study showed that the mutant plasmid exhibited significantly lower mRNA and protein of TRAPPC2, and the mutant protein changed its membrane distribution. TRAPPC2 knockdown resulted in decreased COL2A1 expression and collagen II secretions. Our data indicate that the novel nonsense variant, c.91A>T, of the TRAPPC2 gene is the cause of SEDT in this pedigree. The variant results in a lowered expression of TRAPPC2 and then affects the COL2A1 expression and collagen II secretions, which may explain the mechanism of loss of function of the variant.

Transcriptomic network analysis reveals key drivers of response to anti-TNF biologics in patients with rheumatoid arthritis.

Anti-TNF biologics have been widely used to ameliorate disease activity in patients with RA. However, a large fraction of patients show a poor response to these agents. Moreover, no clinically applicable predictive biomarkers have been established. This study aimed to identify response-associated biomarkers using longitudinal transcriptomic data in two independent RA cohorts. RNA sequencing data from peripheral blood cell samples of Korean and Caucasian RA cohorts before and after initial treatment with anti-TNF biologics were analysed to assess treatment-induced expression changes that differed between highly reliable excellent responders and null responders. Weighted correlation network, immune cell composition, and key driver analyses were performed to understand response-associated transcriptomic networks and cell types and their correlation with disease activity indices. In total, 305 response-associated genes showed significantly different treatment-induced expression changes between excellent and null responders. Co-expression network construction and subsequent key driver analysis revealed that 41 response-associated genes played a crucial role as key drivers of transcriptomic alteration in four response-associated networks involved in various immune pathways: type I IFN signalling, myeloid leucocyte activation, B cell activation, and NK cell/lymphocyte-mediated cytotoxicity. Transcriptomic response scores that we developed to estimate the individual-level degree of expression changes in the response-associated key driver genes were significantly correlated with the changes in clinical indices in independent patients with moderate or ambiguous response outcomes. This study provides response-specific treatment-induced transcriptomic signatures by comparing the transcriptomic landscape between patients with excellent and null responses to anti-TNF drugs at both gene and network levels.

Upregulated complement receptors correlate with Fc gamma receptor 3A-positive natural killer cells (NK) and natural killer-T cells (NKT) in neuromyelitis optica spectrum disorder. (S50.003)

Upregulated complement receptors correlate with Fc gamma receptor 3A-positive natural killer cells (NK) and natural killer-T cells (NKT) in neuromyelitis optica spectrum disorder. (S50.003)

The Role of Follicular Regulatory T Cells/Follicular Helper T Cells in Primary Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by thrombocytopenia. Herein, we sought to identify potential immune-related therapeutic targets in ITP. Methods: The differentially expressed genes (DEGs) between ITP patients and controls in GSE43177 and PRJNA299534 were analyzed. The intersections of the two DEG groups were screened as common genes, and enrichment analysis was performed. Additionally, differential analysis of immune cell levels between ITP and controls was performed. Changes in the proportions of T follicular helper (Tfh) and follicular regulatory T (Tfr) cells in peripheral blood samples from ITP patients, ITP patients responding to therapy, and healthy controls were identified. The expression changes in B-cell lymphoma (Bcl)-6 and interleukin (IL)-21 were further evaluated. Results: A total of 76 common genes were identified, and enrichment analysis found that these genes were mainly associated with neutrophil-mediated immunity, the MAPK signaling pathway, and the FOXO signaling pathway. Furthermore, we found different levels of Tfh cells in patients with ITP and controls. The level of Tfh cells in the peripheral blood is significantly increased in ITP patients and declines after responding to therapy. The Tfr/Tfh ratio was reduced in ITP patients and increased after responding to therapy. IL-21 and Bcl-6 were more highly expressed in ITP patients than in controls. Conclusion: We identified abnormally expressed genes in ITP related to immune-related biological functions. We further identified the changes in Tfh and Tfr cells during ITP treatment. This provides a rationale for immunotherapy in ITP patients.

Imbalanced IL-1B and IL-18 Expression in Sézary Syndrome.

Sézary syndrome (SS) is a rare and aggressive type of cutaneous T-cell lymphoma, with an abnormal inflammatory response in affected skin. The cytokines IL-1B and IL-18, as key signaling molecules in the immune system, are produced in an inactive form and cleave to the active form by inflammasomes. In this study, we assessed the skin, serum, peripheral mononuclear blood cell (PBMC) and lymph-node samples of SS patients and control groups (healthy donors (HDs) and idiopathic erythroderma (IE) nodes) to investigate the inflammatory markers IL-1B and IL-18 at the protein and transcript expression levels, as potential markers of inflammasome activation. Our findings showed increased IL-1B and decreased IL-18 protein expression in the epidermis of SS patients; however, in the dermis layer, we detected increased IL-18 protein expression. In the lymph nodes of SS patients at advanced stages of the disease (N2/N3), we also detected an enhancement of IL-18 and a downregulation of IL-1B at the protein level. Moreover, the transcriptomic analysis of the SS and IE nodes confirmed the decreased expression of IL1B and NLRP3, whereas the pathway analysis indicated a further downregulation of IL1B-associated genes. Overall, the present findings showed compartmentalized expressions of IL-1B and IL-18 and provided the first evidence of their imbalance in patients with Sézary syndrome.

Label-Free Enrichment of Circulating Tumor Plasma Cells: Future Potential Applications of Dielectrophoresis in Multiple Myeloma.

In multiple myeloma (MM), circulating tumor plasma cells (CTPCs) are an emerging prognostic factor, offering a promising and minimally invasive means for longitudinal patient monitoring. Recent advances highlight the complex biology of plasma cell trafficking, highlighting the phenotypic and genetic signatures of intra- and extra-medullary MM onset, making CTPC enumeration and characterization a new frontier of precision medicine for MM patients, requiring novel technological platforms for their standardized and harmonized detection. Dielectrophoresis (DEP) is an emerging label-free cell manipulation technique to separate cancer cells from healthy cells in peripheral blood samples, based on phenotype and membrane capacitance that could be successfully tested to enumerate and isolate CTPCs. Herein, we summarize preclinical data on DEP development for CTPC detection, as well as their clinical and research potential.

T cell immunoglobulin and mucin domain-containing protein 3 is highly expressed in patients with acute decompensated heart failure and predicts mid-term prognosis.

Background and aimsT cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is mainly expressed by immune cells and plays an immunomodulatory role in cardiovascular disease. However, the prognostic value of Tim-3 in acute decompensated heart failure (ADHF) is unclear. This study aimed to investigate the expression profile of Tim-3 on CD4+ and CD8+ T cells in patients with ADHF and its impact on their prognosis.MethodsIn this prospective study, 84 patients who were hospitalized with ADHF and 83 patients without heart failure were enrolled. Main clinical data were collected during patient visits. The Tim-3 expression on CD4+ and CD8+ T cells in peripheral blood samples was assayed by flow cytometry. Long-term prognosis of the patients with ADHF was evaluated by major adverse cardiac and cerebrovascular events (MACCE) over a 12-month follow-up period.ResultsWe found that the Tim-3 expression on CD4+ T cells [2.08% (1.15–2.67%) vs. 0.88% (0.56–1.39%), p < 0.001] and CD8+ T cells [3.81% (2.24–6.03%) vs. 1.36% (0.76–3.00%), p < 0.001] in ADHF group were significantly increased vs. the non-ADHF group. Logistic analysis revealed that high levels of Tim-3 expressed on CD4+ and CD8+ T cells were independent risk factors of ADHF (OR: 2.76; 95% CI: 1.34–5.65, p = 0.006; OR: 2.58; 95% CI: 1.26–5.31, p = 0.010, respectively). ROC curve analysis showed that the high level of Tim-3 on CD4+ or CD8+ T cells as a biomarker has predictive performance for ADHF (AUC: 0.75; 95% CI: 0.68–0.83; AUC: 0.78, 95% CI: 0.71–0.85, respectively). During a median follow-up of 12 months, the Cox regression analysis revealed that higher Tim-3 on CD4+ and CD8+ T cells were strongly associated with increased risks of MACCE within 12 months after ADHF (HR: 2.613; 95% CI: 1.11–6.13, p = 0.027; HR: 2.762, 95% CI: 1.15–6.63, p = 0.023; respectively).ConclusionOur research indicated that the expression level of Tim-3 on CD4+ and CD8+ T cells, elevated in patients with ADHF, was an independent predictor of MACCE within 12 months after ADHF. It suggests a potential immunoregulatory role of Tim-3 signaling system in the mechanism of ADHF.

Prognostic value of minimal residual disease among patients with classical Hodgkin lymphoma undergoing autologous stem cell transplantation

Improved biomarkers are needed to guide patient selection for autologous stem cell transplantation (ASCT) and post-ASCT maintenance therapies in relapsed/refractory classical Hodgkin lymphoma (cHL). To assess the prognostic value of minimal residual disease (MRD) using immunoglobulin-based high-throughput sequencing (Ig-HTS), we analyzed pre- and post-ASCT peripheral blood and pre-ASCT apheresis stem cell (ASC) samples in 36 cHL patients. A tumor clonotype was detected in only 12 patients (33%). Among these patients, MRD within plasma samples was closely associated with impending relapse. All patients (n = 3) with detectable MRD in any post-ASCT plasma sample relapsed (100% specificity), and MRD was not detected in any patients in remission. MRD testing from cellular specimens (peripheral blood mononuclear cell or ASC samples) was not associated with relapse. In this small cohort, plasma-based MRD testing appeared to be a promising biomarker in cHL, but given low clonotype detection rates with Ig-HTS, alternative MRD approaches should be investigated.

Characteristics of changes in double positive CD4+CD8+ T cells in liver transplantation

Although double positive CD4+CD8+ T (DPT) cells has been reported to be involved in some diseases, their trajectory and function as associated with liver transplantation (LT) remain unclear. In the present study, we found that the number of DPT cells was increased in the blood and liver tissue of LT patients. Meanwhile, we compared the distribution of DPT cells in peripheral blood samples and in penetrating liver tissue between liver rejection versus non-rejection patients, as well as the proportion of DPT cells as a function of the extent of liver rejection. The number of DPT cells in the rejection group was significantly increased. An analysis of the spatial distance and correlations between DPT and Treg cells, revealed that these cells showed a high degree of contiguity. In a mouse liver transplant model, the number of DPT cells were significantly increased in liver tissue, and the number of CD8+ T cells gradually increased, while CD4+ T cells decreased as a function of time post-transplantation. Expression level of PD-1 in DPT cells also increased in a temporally-dependent manner post liver transplantation and the changes of PD-1+ DPT cells were related to the degree of liver transplant rejection. In DPT cells interacting with Treg, there was an increased expression of PD-1, which enhanced cellular exhaustion. In conclusion, the capacity for DPT cells to induce immune tolerance may represent a new and important protocol for use in targeting treatments for the prevention of liver transplant rejection.

Abstract 5118: Postoperative minimal residual disease to predict the risk of hepatocellular carcinoma (HCC) recurrence using plasma-only circulating tumor DNA assay

Abstract Background: Postoperative minimal residual diseases (MRD) are one of the main causes of postoperative short-term recurrence, which were difficult to be detected by conventional imaging examinations. Circulating tumor DNA (ctDNA) provides a novel approach for detecting the existence of MRD and predicting clinical outcomes of hepatocellular carcinoma (HCC). Most ctDNA MRD assays require tumor sequencing to identify tumor-derived mutations to facilitate ctDNA detection, requiring tumor and blood. Herein, we evaluated a plasma-only ctDNA assay integrating genomic signatures to enable tumor-uninformed MRD detection. Methods: 20 patients with hepatocellular carcinoma who underwent radical surgical resection were involved from July 2019 to October 2020. Blood samples were collected for next generation sequencing (NGS) within 7 days after surgery, and 10 of them had preoperatively matched plasma ctDNA results. NGS (381 genes panel) was performed on plasma samples to capture tumor somatic single-nucleotide variants (SNVs) and copy number variants (CNVs). Clinical information was obtained to evaluate the prognostic performance of ctDNA comparing with traditional clinical biomarkers. Plasma and peripheral blood cell samples were sequenced to a mean depth of 7396 and ctDNA results correlated with recurrence-free survival (RFS). Results: Characteristics of the 20 HCC patients were as follows: 85% age≥50 years, 70% HBV positive, 75% largest tumor diameter≥5cm, 55% liver cirrhosis, 85% AFP &amp;gt; 400ng/ml, 60% MVI, 55% PVTT, 50% BCLC C, 70% CNLC III. After a median follow-up of 13.60 months, 10 recurrence events were recorded. In order to evaluate the presence of MRD after radical surgery, at least one low frequency SNV detected was considered ctDNA-positive. Using stringent quality criteria 8/20 (20%) were ctDNA positive after resection. The median RFS for ctDNA-positive patients was 6.98 months versus 27.57 months for ctDNA-negative patients (HR 3.727, 95% CI 0.9461-14.65; p=0.0247). Combining postoperative ctDNA SNV and preoperatively ctDNA CNV features could increase the sensitivity for predicting prognosis. To further confirm the effect of MRD on the RFS of HCC patients, multiple Cox regression was used to assess the effect of multiple factors, including MRD, alpha-fetoprotein (AFP), HBV, Liver cirrhosis, macrovascular invasion, PVTT, Child-Pugh score and CNLC stage, on the RFS. The forest plot results showed that the postoperative ctDNA was an independent factor for predicting the RFS of patients with HCC (MRD: HR=14.02, p=0.018). Conclusion: These preliminary results suggest that ultra-deep sequencing of ctDNA analyses can detect somatic mutations to reflect postoperative MRD state and predict recurrence of HCC. Plasma-only MRD detection may be an effective and convenient approach for clinical prediction of HCC postoperative recurrence risk. Citation Format: Mingxin Pan, Jianpeng Cai, Yuyan Xu, Kaihang Zhong, Tingting Chen. Postoperative minimal residual disease to predict the risk of hepatocellular carcinoma (HCC) recurrence using plasma-only circulating tumor DNA assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5118.

Fast, Reusable, Cell Uniformly Distributed Membrane Filtration Device for Separation of Circulating Tumor Cells.

Isolation of circulating tumor cells (CTCs) is of great significance for the diagnosis, prognosis, and treatment of metastatic cancer. Among CTC capture methods independent of antibodies, membrane filtration-based methods have the advantages of simplicity, rapidity, and high throughput but usually have problems such as clogging, high pressure drop, and impaired cell viability. In this study, we designed and tested a reusable device that used horizontal rotor and fluid-assisted separation to capture CTCs by centrifugal membrane filtration, achieving simple, fast, highly efficient, and viable cell capture on traditional centrifuge. The average capture efficiency was 95.8% for different types of cancer cells with >90% survival, and the removal of white blood cells can reach 99.72% under four times cleaning of the membrane after filtration. A further clinic demo was performed using the device to detect residual leukemic cells in patients; the results showed a 10-fold enrichment of the leukemic cells in peripheral blood samples. Taken together, the simple, robust, and efficient CTC capture device may have the potential for clinic routine detection and analysis of circulating tumor cells.

Oxytocin receptor gene methylation as a molecular marker for severity of depressive symptoms in affective disorder patients

BackgroundOxytocin (OXT) is a neuropeptide and hormone involved in emotional functioning and also seems to play a role in moderating the stress response. Both preclinical and clinical studies point to an increased methylation status of the Oxytocin receptor (OXTR) promoter region with concomitant deficits in social, cognitive and emotional functioning. We hypothesize that methylation levels (%) of the oxytocin receptor promoter region correlate with the severity of depression symptoms and/or with the severity of childhood trauma within this present sample of affective disorder patients.MethodologyEight hundred forty six (846) affective disorder patients of Central European origin were recruited at the Department of Psychiatry and Psychotherapy of the Medical University Vienna, the Karl Landsteiner University for Health and Science and Zentren für seelische Gesundheit, BBRZ-Med Leopoldau. Psychiatric assessment included a semi-structured diagnostic interview (Schedules for Clinical Assessment in Neuropsychiatry), the Hamilton Depression Scale and the Childhood Trauma Questionnaire. Concomitantly DNA samples of peripheral blood cells were collected for Multiplexed and Sensitive DNA Methylation Testing.ResultsOur data suggests a positive but not significant association between OXTR promoter Exons 1–3 methylation levels and severity of depression symptoms as well as severity of emotional neglect in affective disorder patients and no association with childhood trauma.ConclusionsOur findings contribute to elucidate the role of OXTR in affective disorders, but further longitudinal studies in particular are necessary to broaden the current state of knowledge.

Topical Application of Methyl Nicotinate Solution Enhances Peripheral Blood Collection.

The purpose of this study was to investigate whether local application of methyl nicotinate solution can change the content and proportion of blood cells in peripheral blood samples and to determine whether this treatment is a safe and reliable method for improving peripheral blood collection. Routine blood analysis and flow cytometry were used to analyze the contents and proportions of blood cells and T lymphocyte subsets in peripheral blood samples. Experimental blood specimens were collected from earlobes treated with different concentrations of methyl nicotinate solution, and the control group consisted of blood specimens collected from untreated earlobes. The blood flow in the earlobe was significantly increased after methyl nicotinate solution stimulation, especially when the methyl nicotinate solution concentration was greater than 10-4 mol/L. There were no significant changes in the proportions of white blood cells, red blood cells, platelets, neutrophils, eosinophils, basophils, monocytes, or lymphocytes in the peripheral blood obtained from earlobes treated with methyl nicotinate solution. The proportion of T lymphocytes increased in the experimental group, but this difference was not significant. Local application of methyl nicotinate solution is a feasible method for improving peripheral blood collection, especially for patients with venous blood collection phobia or an inability to provide venous blood samples.

Effect of circRNA_FOXO3 rs12196996 polymorphism and FOXO3 rs2232365 polymorphism on survival rate and severity of intensive care unit-acquired sepsis

ABSTRACT The expression of circRNA_FOXO3 was found to be positively associated with the expression of Forkhead Box O3 (FOXO3), which is targeted and regulated by miR-23a. Polymorphisms in rs12196996 and rs2232365 have been reported in various diseases. In this study, we recruited intensive care unit (ICU)-acquired sepsis patients and grouped them according to their genotypes of rs12196996 and rs2232365. Quantitative real-time PCR was performed to analyze the expression of circRNA_FOXO3, FOXO3 mRNA, and miR-23a. ELISA was carried out to evaluate the abundance of cytokines and luciferase assay was used to explore the inhibitory role of miR-23a on circRNA_FOXO3 and FOXO3. Accordingly, we found that rs12196996 GG and rs2232365 AA were significantly correlated with prolonged survival of ICU-acquired sepsis patients. Rs12196996 GG and rs2232365 AA were also correlated with increased level of miR-23a, IL-10 and decreased level of TNF, IL-2, IFN, IL-6 and IL-1β in the peripheral blood cell samples of patients with ICU-acquired sepsis. The luciferase activity of wild-type (WT) circRNA_FOXO3 and FOXO3 were severely reduced by miR-23a. MiR-23a precursors could effectively suppress the expression of circRNA_FOXO3 and FOXO3 in the cells. Moreover, LPS-induced cell viability loss and dysregulation of cytokines were effectively restored by the knockdown of FOXO3 or circRNA_FOXO3 siRNA in the cells. This study revealed that the minor allele of rs12196996 polymorphism and rs2232365 polymorphism collaboratively contributed to the increased survival and suppressed severity of ICU-acquired sepsis.

Study on the expression level of NF-κB and clinical evaluations in spinal tuberculosis patients.

Spinal tuberculosis or tuberculous spondylitis is one of the most common types of skeletal tuberculosis. Complications of the spine and spinal cord tuberculosis include destruction of the vertebrae, deformity, and paraplegia. Since in some patients, the clinical manifestations of tuberculosis are unusual and timely diagnosis and treatment of this disease can prevent its serious consequences, so in the present study, some cases of rare manifestations of tuberculosis were investigated. The expression of the NF-κB gene in these patients was also evaluated. In this regard, 36 patients with spinal tuberculosis and 30 healthy individuals (as a control group) were assessed. Clinical symptoms, imaging, laboratory tests, pathology, and response to treatment related to patients with spinal tuberculosis and spinal cord tuberculosis were evaluated. NF-κB expression was also evaluated using the PCR technique in peripheral white blood cell samples. The obtained results were analyzed using SPSS ver. 16, χ2 and T-test statistical methods. Mann-Whitney U test and Kruskal-Wallis non-parametric tests were used to analyze non-parametric data. The results showed that out of 36 cases of spinal tuberculosis, 29 cases had spinal tuberculosis, five cases had tuberculous radiculomyelitis, one case had spinal intramedullary tuberculoma, and one case had syringomyelia. 52.78% of patients were male, and 70% of cases were observed between the ages of 35 and 55 years. Fever and back pain were seen in more than 80% of cases. The study of NF-κB expression in the control and case groups showed that the NF-κB expression in the case group increased compared to the control group. This increase was statistically significant (P = 0.0071). In general, in the present study, the methods of clinical diagnosis of spinal tuberculosis were evaluated. Also, the amount of NF-κB transcription factor was evaluated as an effective genetic factor in the diagnosis of this disease.

Low Mutational Burden of Extra Nodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in Patients with Primary Sjogren's Syndrome

Background: Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of the exocrine glands. Patients with pSS are at increased risk of developing extra nodal marginal zone lymphoma of the mucosa-associated lymphoid tissue (MALT) type, predominantly in the salivary glands. The presence of cryoglobulinemia, low C4 levels and salivary gland enlargement, are important risk factors for lymphoma development in pSS. Although lymphoma associated mutations have been described in circulating B-cells of patients with pSS associated cryoglobulinemia, it is unknown which somatic aberrations underlie the development of pSS-associated MALT lymphomas. The aim of the current study was to define the genomic landscape of pSS salivary gland MALT lymphomas. Identification of specific recurrent mutations or copy number aberrations and defining the affected pathways would help to understand the mechanisms by which intra-epithelial B-cells undergo malignant transformation.Methods: Whole exome sequencing was performed on 14 fresh frozen and 3 paraffin embedded MALT tissue samples of pSS patients at the University Medical Center Groningen (UMCG). Matched peripheral white blood cell samples were available for 12 patients and were used as germline controls. Fluorescence in situ hybridization was performed for the detection of MALT1 translocations.Results: Presence of a clonal B-cell population, indicative of a MALT lymphoma was confirmed by IgH PCR (BIOMED-2) for all patients. Whole exome sequencing resulted in a median target coverage of 130x, ranging from 84-163. More than 90% of the target regions had a coverage of >50x. In total we identified 232 somatic mutations in 184 genes. Three cases had a relatively high mutational load (>25 / case), while the median number of mutations in the remaining 14 cases was 7. Across the 17 cases there were 18 recurrently mutated genes. PRKD1, associated with malignant epithelial tumor of the salivary glands, was the most frequently mutated gene (six cases). Besides PRKD1, five additional recurrently mutated genes are also involved in epithelial surface and/or extracellular matrix (MAMDC4, COL14A1, CAMSAP3, TMEM2 and MUC4). Five genes, all with mutations in two cases, were shown to be associated with lymphomagenesis (ID3, TBL1XR1, PAX5, IGLL5, APC). A total of 18 copy number alterations (CNAs) were detected in 8 of the 14 evaluable cases, with gains of part of chromosomes 2 and 19, and loss of chromosome 6 each being detected in two cases. With respect to outcome, only 2 cases with high mutational load relapsed outside of the salivary glands, suggesting a more advanced stage of lymphoma.Conclusion: The low mutational load and lack of a clear lymphoma related gene signature suggests that localized pSS MALT lymphomas are genetically stable and most likely depend on the inflammatory state of the micro-environment. Only those cases characterized by higher mutational load showed a relapse-remitting disease, as is typical for indolent lymphoma. These observations could be translated to the clinical setting and potentially have value as biomarker for identification of patients with a more aggressive disease. Confirmation in larger cohorts are required to confirm this potential association. This study also paves way for treatment strategies targeting the micro-environment. DisclosuresNo relevant conflicts of interest to declare.