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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Structural insights into human MHC-II association with invariant chain.

The loading of processed peptides on to major histocompatibility complex II (MHC-II) molecules for recognition by T cells is vital to cell-mediated adaptive immunity. As part of this process, MHC-II associates with the invariant chain (Ii) during biosynthesis in the endoplasmic reticulum to prevent premature peptide loading and to serve as a scaffold for subsequent proteolytic processing into MHC-II-CLIP. Cryo-electron microscopy structures of full-length Human Leukocyte Antigen-DR (HLA-DR) and HLA-DQ complexes associated with Ii, resolved at 3.0 to 3.1 Å, elucidate the trimeric assembly of the HLA/Ii complex and define atomic-level interactions between HLA, Ii transmembrane domains, loop domains, and class II-associated invariant chain peptides (CLIP). Together with previous structures of MHC-II peptide loading intermediates DO and DM, our findings complete the structural path governing class II antigen presentation.

Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in tuberculosis patients.

BACKGROUND: Human T cells play an important role in immunity against tuberculosis (TB) infection. Activating receptor HLA-DR and inhibitory receptor KLRG1 are critical regulators of T cell function during viral infection and tumorigenesis, but they have been less studied in TB infection. METHODS: In this study, we explored the relationship between CD3+ T cell expression of HLA-DR and KLRG1 receptors and function against TB infection. Flow cytometry was conducted to assess the immunomodulatory effects of HLA-DR and KLRG1 receptors on CD3+ T cells in patients with different TB infection status. RESULTS: We found activating receptors HLA-DR, NKG2C, CD57 and NKP46, and inhibitory receptors KLRG1 and KIR on CD3+ T cells in different TB infection status showed different distribution patterns; the cytotoxic potential and cytokine secretion capacity of CD3+ T cells after Mtb-specific antigen stimulation were significantly enhanced in TB infection groups. Further studies revealed HLA-DR+ T and KLRG1+ T cells expressed higher activating and inhibitory receptors than the negative population. In addition, the expression of cytotoxic potential and cytokine secretion capacity of HLA-DR+ T and KLRG1+ T cells was significantly higher than that of HLA-DR- T and KLRG1- T cells. CONCLUSIONS: Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in TB patients, suggesting CD3+ T cells expressing HLA-DR and KLRG1 are important effector cell phenotypes involved in the host anti-TB infection. HLA-DR and KLRG1 expressed by CD3+ T cells may be potential predictive markers of TB disease progression and clinical immune assessment.

Identification of Antibodies to DQß:DRα Interisotypic Heterodimers in Human Sera.

BACKGROUND: HLA class II antigens, DR, DQ, and DP, comprised an α and ß chains, which typically combine, within the same isotype, to form the major histocompatibility complex:peptide complex. Interisotypic pairing is not commonly observed. Although reports of DQß:DRα heterodimers exist, the pairing was reported to be unstable and, therefore, not studied to any extent. METHODS: DQß:DRα single antigens were produced through transfectant cell lines and used to identify and characterize positive reactive human sera by a multiplex bead-based assay. RESULTS: Stable DQß:DRα transfectants were constructed. Cell surface staining with class II-specific monoclonal antibodies revealed that some DQB1 alleles appear to be more efficient in expressing DQß:DRα heterodimers. Interestingly, alleles within the same serological group varied in their efficiency of forming dimers on the cell surface. For example, DQß0601:DRα had the highest transfection and cell membrane expression efficiency among 16 common DQB1 alleles tested. In contrast, DQß0603:DRα-positive transfectants demonstrated minimal surface expression. Assembly of DQß0601:DRα was not affected by the presence of a DQα chain. DQß0601:DRα and DQß0603:DRα single-antigen beads were used to screen human sera. Positive sera were identified that reacted to the unique epitopes of DQß0601:DRα protein on the cell surface of the transfectants. CONCLUSIONS: Our studies have demonstrated that unique DQß:DRα heterodimers can be formed and are stably expressed on the cell surface. Such antigenic combinations, presented on single-antigen beads, demonstrated that patient sera can react with such heterodimers. Investigations on the potential clinical roles of antibodies against such interisotypic heterodimers are now possible.

New light on the HLA-DR immunopeptidomic landscape.

The set of peptides processed and presented by major histocompatibility complex class II molecules defines the immunopeptidome, and its characterization holds keys to understanding essential properties of the immune system. High-throughput mass spectrometry (MS) techniques enable interrogation of the diversity and complexity of the immunopeptidome at an unprecedented scale. Here, we analyzed a large set of MS immunopeptidomics data from 40 donors, 221 samples, covering 30 unique HLA-DR molecules. We identified likely co-immunoprecipitated HLA-DR irrelevant contaminants using state-of-the-art prediction methods and unveiled novel light on the properties of HLA antigen processing and presentation. The ligandome (HLA binders) was enriched in 15-mer peptides, and the contaminome (nonbinders) in longer peptides. Classification of singletons and nested sets showed that the first were enriched in contaminants. Investigating the source protein location of ligands revealed that only contaminants shared a positional bias. Regarding subcellular localization, nested peptides were found to be predominantly of endolysosomal origin, whereas singletons shared an equal distribution between the cytosolic and endolysosomal origin. According to antigen-processing signatures, no significant differences were observed between the cytosolic and endolysosomal ligands. Further, the sensitivity of MS immunopeptidomics was investigated by analyzing overlap and saturation between biological MS replicas, concluding that at least 5 replicas are needed to identify 80% of the immunopeptidome. Moreover, the overlap in immunopeptidome between donors was found to be very low both in terms of peptides and source proteins, the latter indicating a critical HLA bias in the antigen sampling in the HLA antigen presentation. Finally, the complementarity between MS and in silico approaches for comprehensively sampling the immunopeptidome was demonstrated.

Autoreactive T cells target peripheral nerves in Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a rare heterogenous disorder of the peripheral nervous system, which is usually triggered by a preceding infection, and causes a potentially life-threatening progressive muscle weakness1. Although GBS is considered an autoimmune disease, the mechanisms that underlie its distinct clinical subtypes remain largely unknown. Here, by combining in vitro T cell screening, single-cell RNA sequencing and T cell receptor (TCR) sequencing, we identify autoreactive memory CD4+ cells, that show a cytotoxic T helper 1 (TH1)-like phenotype, and rare CD8+ T cells that target myelin antigens of the peripheral nerves in patients with the demyelinating disease variant. We characterized more than 1,000 autoreactive single T cell clones, which revealed a polyclonal TCR repertoire, short CDR3ß lengths, preferential HLA-DR restrictions and recognition of immunodominant epitopes. We found that autoreactive TCRß clonotypes were expanded in the blood of the same patient at distinct disease stages and, notably, that they were shared in the blood and the cerebrospinal fluid across different patients with GBS, but not in control individuals. Finally, we identified myelin-reactive T cells in the nerve biopsy from one patient, which indicates that these cells contribute directly to disease pathophysiology. Collectively, our data provide clear evidence of autoreactive T cell immunity in a subset of patients with GBS, and open new perspectives in the field of inflammatory peripheral neuropathies, with potential impact for biomedical applications.

Dampened Inflammatory Signalling and Myeloid-Derived Suppressor-Like Cell Accumulation Reduces Circulating Monocytic HLA-DR Density and May Associate With Malignancy Risk in Long-Term Renal Transplant Recipients.

Background: Malignancy is a major cause of morbidity and mortality in transplant recipients. Identification of those at highest risk could facilitate pre-emptive intervention such as reduction of immunosuppression. Reduced circulating monocytic HLA-DR density is a marker of immune depression in the general population and associates with poorer outcome in critical illness. It has recently been used as a safety marker in adoptive cell therapy trials in renal transplantation. Despite its potential as a marker of dampened immune responses, factors that impact upon monocytic HLA-DR density and the long-term clinical sequelae of this have not been assessed in transplant recipients. Methods: A cohort study of stable long-term renal transplant recipients was undertaken. Serial circulating monocytic HLA-DR density and other leucocyte populations were quantified by flow cytometry. Gene expression of monocytes was performed using the Nanostring nCounter platform, and 13-plex cytokine bead array used to quantify serum concentrations. The primary outcome was malignancy development during one-year follow-up. Risk of malignancy was calculated by univariate and multivariate proportionate hazards modelling with and without adjustment for competing risks. Results: Monocytic HLA-DR density was stable in long-term renal transplant recipients (n=135) and similar to non-immunosuppressed controls (n=29), though was suppressed in recipients receiving prednisolone. Decreased mHLA-DRd was associated with accumulation of CD14+CD11b+CD33+HLA-DRlo monocytic myeloid-derived suppressor-like cells. Pathway analysis revealed downregulation of pathways relating to cytokine and chemokine signalling in monocytes with low HLA-DR density; however serum concentrations of major cytokines did not differ between these groups. There was an independent increase in malignancy risk during follow-up with decreased HLA-DR density. Conclusions: Dampened chemokine and cytokine signalling drives a stable reduction in monocytic HLA-DR density in long-term transplant recipients and associates with subsequent malignancy risk. This may function as a novel marker of excess immunosuppression. Further study is needed to understand the mechanism behind this association.

Accréditation de la mesure de l'expression des molécules HLA-DR à la surface des monocytes (mHLA-DR) par cytométrie en flux.

INTRODUCTION: Le niveau d'expression des molécules HLA-DR à la surface des monocytes (mHLA-DR) est un marqueur diagnostique utilisé pour évaluer l'immunité des patients en réanimation (choc septique, polytraumatisés, brulures, greffe et plus récemment Covid-19). Il est également utilisé comme un outil de stratification dans les essais cliniques utilisant des thérapies immunostimulantes chez ces patients. L'objectif de cette étude était d'évaluer les performances analytiques d'une méthode de cytométrie en flux pour mesurer mHLA-DR afin de répondre aux exigences de la norme NF EN ISO 15189 dans le cadre de l'accréditation des laboratoires de biologie médicale. Matériels et méthodes. L'évaluation (performances de la technique, étendue de la mesure, comparaison de méthode) a été menée en suivant le SH GTA 04, guide recommandé par le Comité français d'accréditation (COFRAC). En complément, certaines conditions pré analytiques ont été ré-évaluées. Résultats. L'ensemble des coefficients de variation évaluant les performances étaient inférieurs à 10 % (répétabilité, reproductibilité, variabilité interopérateur). Les limites de quantification et de linéarité étaient adaptées à l'utilisation clinique du paramètre. Les résultats étaient identiques quel que soit le type et le fournisseur de cytomètre en flux. Les contraintes de conservation pré-analytiques des échantillons ont été confirmées. CONCLUSION: Les résultats étaient conformes aux exigences de qualité recommandées par le COFRAC. Ils permettent l'accréditation de la mesure de mHLA-DR par cytométrie en flux et son utilisation en soins courants.

Accurate MHC Motif Deconvolution of Immunopeptidomics Data Reveals a Significant Contribution of DRB3, 4 and 5 to the Total DR Immunopeptidome.

Mass spectrometry (MS) based immunopeptidomics is used in several biomedical applications including neo-epitope discovery in oncology, next-generation vaccine development and protein-drug immunogenicity assessment. Immunopeptidome data are highly complex given the expression of multiple HLA alleles on the cell membrane and presence of co-immunoprecipitated contaminants. The absence of tools that deal with these challenges effectively and guide the analysis and interpretation of this complex type of data is currently a major bottleneck for the large-scale application of this technique. To resolve this, we here present the MHCMotifDecon that benefits from state-of-the-art HLA class-I and class-II predictions to accurately deconvolute immunopeptidome datasets and assign individual ligands to the most likely HLA molecule, allowing to identify and characterize HLA binding motifs while discarding co-purified contaminants. We have benchmarked the tool against other state-of-the-art methods and illustrated its application on experimental datasets for HLA-DR demonstrating a previously underappreciated role for HLA-DRB3/4/5 molecules in defining HLA class II immune repertoires. With its ease of use, MHCMotifDecon can efficiently guide interpretation of immunopeptidome datasets, serving the discovery of novel T cell targets. MHCMotifDecon is available at https://services.healthtech.dtu.dk/service.php?MHCMotifDecon-1.0.

Lymphocyte HLA-DR/CD-38 co-expression correlates with Hodgkin lymphoma cell cytotoxicity in vitro independent of PD-1/PD1-L pathway.

The interactions between Hodgkin and Reed Sternberg cells and tumor microenvironment, the changes that occur with therapy and, in particular, checkpoint inhibition are not fully understood. Understanding these is key to optimizing outcomes for patients with Hodgkin lymphoma (HL). We evaluated the immunophenotypic characteristics of cytotoxic, helper T and NK lymphocytes upon in vitro stimulation, cell-mediated cytotoxicity against HL cells, HDLM-2 and KM-H2, and the association with effector cell activation state, as well as changes in cytotoxicity following PD-1 or PDL-1 blockade. Higher HLA-DR/CD38 expression on effector cells was associated with increased cytotoxicity against HL cells. All effector cell types were cytotoxic of HL cells, though achieved maximum activation and cytotoxicity at variable timepoints. HLA-DR/CD38 co-expression correlated with cytotoxicity, but PD-1 expression did not. There was no significant change in cell-mediated cytotoxicity following PD-1/PDL-1 blockade. The mechanism of action of checkpoint inhibitors may not be limited to direct PD-1/PDL-1 blockade.

Clinical and prognostic significance of CD14 (+) HLA-DR (-/low) myeloid-derived suppressor cells in patients with hepatocellular carcinoma received transarterial radioembolization with Yttrium-90.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. For unresectable HCC, transarterial radioembolization (TARE) with Yttrium-90 is a widely used treatment. The aim of this study was to investigate whether monocytic myeloid-derived suppressor cells (M-MDSC) and CD39+ T cells can be non-invasive predictive biomarkers of radiological response and prognosis in patients with HCC treated with TARE. This study was conducted on 39 patients with HCC who were treated with TARE between August 2018 and December 2019 and the control group consisted of 23 healthy volunteers. CD4+, CD8+, CD39+ T cells, Natural killer (NK) cells, myeloid cells (MC) and M-MDSC parameters are examined in the course of TARE treatment with student t test and Kaplan-Meier method. There were statistically significant differences in M-MDSC, CD39+ T cells and MC values between healthy controls and HCC patients. A statistically significant difference was found in M-MDSC and CD4+ T cells values in the HCC patient group who responded to the treatment compared to those who did not. Survival analysis found that patients with lower frequencies (under 3.81%) of M-MDSC showed more prominent differences of overall survival (OS) compared to patients with all high groups. We found that M-MDSC in the peripheral blood might be a useful non-invasive biomarker to predict OS. We have shown for the first time that M-MDSC is correlated with treatment response in HCC patients treated with TARE. Additionally, we have found that the percentage of CD39+ T cells is high in HCC patients and these cells are positively correlated with M-MDSC.

Acute myeloid leukemia with cup-like blasts and FLT3-ITD and NPM1 mutations mimics features of acute promyelocytic leukemia: a case of durable remission after sorafenib and low-dose cytarabine.

Some previous researches raised the possibility of a novel acute myeloid leukemia (AML) entity presenting cup-like cytomorphology with mutations of both FLT3 and NPM1 or one of them. However, the clinical implications of this subtype remain unknown. We describe a 63-year-old patient belonging to this distinct AML subtype, who presented similar features of acute promyelocytic leukemia (APL) including nuclear morphology, negative for CD34 and HLA-DR, and abnormal coagulation. He had no response to both arsenic trioxide and CAG regimen (cytarabine, aclarubicin, and G-CSF). Given that the patient carried the FLT3-ITD mutation, we switched to a pilot treatment of FLT3 inhibitor sorafenib combined with low-dose cytarabine (LDAC). To date, the patient achieved durable complete remission over 58 months. These findings suggest that AML with cup-like blasts and FLT3-ITD and NPM1 mutations mimic APL, and the prognosis of this subtype may be improved by sorafenib combined with LDAC.

Combination of HLA-DR on Mycobacterium tuberculosis-Specific Cells and Tuberculosis Antigen/Phytohemagglutinin Ratio for Discriminating Active Tuberculosis From Latent Tuberculosis Infection.

Background: Novel approaches for tuberculosis (TB) diagnosis, especially for distinguishing active TB (ATB) from latent TB infection (LTBI), are urgently warranted. The present study aims to determine whether the combination of HLA-DR on Mycobacterium tuberculosis (MTB)-specific cells and TB antigen/phytohemagglutinin (TBAg/PHA) ratio could facilitate MTB infection status discrimination. Methods: Between June 2020 and June 2021, participants with ATB and LTBI were recruited from Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort), respectively. The detection of HLA-DR on MTB-specific cells upon TB antigen stimulation and T-SPOT assay were simultaneously performed on all subjects. Results: A total of 116 (54 ATB and 62 LTBI) and another 84 (43 ATB and 41 LTBI) cases were respectively enrolled from Qiaokou cohort and Caidian cohort. Both HLA-DR on IFN-γ+TNF-α+ cells and TBAg/PHA ratio showed discriminatory value in distinguishing between ATB and LTBI. Receiver operator characteristic (ROC) curve analysis showed that HLA-DR on IFN-γ+TNF-α+ cells produced an area under the ROC curve (AUC) of 0.886. Besides, TBAg/PHA ratio yield an AUC of 0.736. Furthermore, the combination of these two indicators resulted in the accurate discrimination with an AUC of 0.937. When the threshold was set as 0.36, the diagnostic model could differentiate ATB from LTBI with a sensitivity of 92.00% and a specificity of 81.82%. The performance obtained in Qiaokou cohort was further validated in Caidian cohort. Conclusions: The combination of HLA-DR on MTB-specific cells and TBAg/PHA ratio could serve as a robust tool to determine TB disease states.

Results of Complex Immunopharmacological Analysis of the Mechanisms of Action of Bioregulation Agents.

Elucidation of the pharmacodynamic mechanisms of drugs capable of potentiating the effects of non-steroidal anti-inflammatory drugs is an important task. In this in vitro study, the ability of Traumeel S to influence the innate and acquired immunity was evaluated. Traumeel S was found to reduce activities of NADPH oxidase and neutrophil extracellular traps, as well as to evoke anti-inflammatory activity of lymphocyte subpopulations.

The Ontogeny and Function of Placental Macrophages.

The placenta is a fetal-derived organ whose function is crucial for both maternal and fetal health. The human placenta contains a population of fetal macrophages termed Hofbauer cells. These macrophages play diverse roles, aiding in placental development, function and defence. The outer layer of the human placenta is formed by syncytiotrophoblast cells, that fuse to form the syncytium. Adhered to the syncytium at sites of damage, on the maternal side of the placenta, is a population of macrophages termed placenta associated maternal macrophages (PAMM1a). Here we discuss recent developments that have led to renewed insight into our understanding of the ontogeny, phenotype and function of placental macrophages. Finally, we discuss how the application of new technologies within placental research are helping us to further understand these cells.

Circulating Placental Vesicles Carry HLA-DR in Pre-Eclampsia: A New Potential Marker of the Syndrome.

Background: Pre-eclampsia (PE) is a common disorder of pregnancy that usually presents with hypertension and proteinuria. The clinical presentation arises from soluble factors released into the maternal circulation from the placenta owing to the stress of syncytiotrophoblast, consequence of defective placentation occurring in the first half of pregnancy. Reduced tolerance of the semiallogeneic fetus by the maternal immune system has been proposed as first trigger leading to poor placentation. We previously observed aberrant expression of human leukocyte antigen (HLA)-DR molecules in the syncytiotrophoblast of a subset of women with PE. Aim of this study was to investigate abnormal expression of circulating HLA-DR in syncytiotrophoblast-derived extracellular vesicles (STBEVs) in women with PE compared to normal pregnant women. Methods: peripheral venous blood was collected from 22 women with PE and 22 normal pregnant women. Circulating STBEVs were collected by ultra-centrifugation (120000 g) and analyzed for the expression of HLA-DR and placental alkaline phosphatase (PLAP), a specific marker of the placenta, by Western blot analysis and flow cytometry. Results: circulating STBEVs positive for HLA-DR were observed in 64% of PE women while no HLA-DR positivity was detected in any of the controls (P<0.01). Conclusions: Aberrant expression of HLA-DR in circulating STBEVs is specifically associated to PE. Further studies are required: a) to define the role of aberrant placental expression of HLA-DR molecules in the pathogenesis of PE; b) evaluate a possible application of detecting circulating HLA-DR positive STBEVs in the diagnosis and prediction of PE in the first and second trimester of pregnancy.

Persistent High Percentage of HLA-DR+CD38high CD8+ T Cells Associated With Immune Disorder and Disease Severity of COVID-19.

Background: The global outbreak of coronavirus disease 2019 (COVID-19) has turned into a worldwide public health crisis and caused more than 100,000,000 severe cases. Progressive lymphopenia, especially in T cells, was a prominent clinical feature of severe COVID-19. Activated HLA-DR+CD38+ CD8+ T cells were enriched over a prolonged period from the lymphopenia patients who died from Ebola and influenza infection and in severe patients infected with SARS-CoV-2. However, the CD38+HLA-DR+ CD8+ T population was reported to play contradictory roles in SARS-CoV-2 infection. Methods: A total of 42 COVID-19 patients, including 32 mild or moderate and 10 severe or critical cases, who received care at Beijing Ditan Hospital were recruited into this retrospective study. Blood samples were first collected within 3 days of the hospital admission and once every 3-7 days during hospitalization. The longitudinal flow cytometric data were examined during hospitalization. Moreover, we evaluated serum levels of 45 cytokines/chemokines/growth factors and 14 soluble checkpoints using Luminex multiplex assay longitudinally. Results: We revealed that the HLA-DR+CD38+ CD8+ T population was heterogeneous, and could be divided into two subsets with distinct characteristics: HLA-DR+CD38dim and HLA-DR+CD38hi. We observed a persistent accumulation of HLA-DR+CD38hi CD8+ T cells in severe COVID-19 patients. These HLA-DR+CD38hi CD8+ T cells were in a state of overactivation and consequent dysregulation manifested by expression of multiple inhibitory and stimulatory checkpoints, higher apoptotic sensitivity, impaired killing potential, and more exhausted transcriptional regulation compared to HLA-DR+CD38dim CD8+ T cells. Moreover, the clinical and laboratory data supported that only HLA-DR+CD38hi CD8+ T cells were associated with systemic inflammation, tissue injury, and immune disorders of severe COVID-19 patients. Conclusions: Our findings indicated that HLA-DR+CD38hi CD8+ T cells were correlated with disease severity of COVID-19 rather than HLA-DR+CD38dim population.

In-silico design of envelope based multi-epitope vaccine candidate against Kyasanur forest disease virus.

Kyasanur forest disease virus (KFDV) causing tick-borne hemorrhagic fever which was earlier endemic to western Ghats, southern India, it is now encroaching into new geographic regions, but there is no approved medicine or effective vaccine against this deadly disease. In this study, we did in-silico design of multi-epitope subunit vaccine for KFDV. B-cell and T-cell epitopes were predicted from conserved regions of KFDV envelope protein and two vaccine candidates (VC1 and VC2) were constructed, those were found to be non-allergic and possess good antigenic properties, also gives cross-protection against Alkhurma hemorrhagic fever virus. The 3D structures of vaccine candidates were built and validated. Docking analysis of vaccine candidates with toll-like receptor-2 (TLR-2) by Cluspro and PatchDock revealed strong affinity between VC1 and TLR2. Ligplot tool was identified the intermolecular hydrogen bonds between vaccine candidates and TLR-2, iMOD server confirmed the stability of the docking complexes. JCAT sever ensured cloning efficiency of both vaccine constructs and in-silico cloning into pET30a (+) vector by SnapGene showed successful translation of epitope region. IMMSIM server was identified increased immunological responses. Finally, multi-epitope vaccine candidates were designed and validated their efficiency, it may pave the way for up-coming vaccine and diagnostic kit development.

Identification of novel biomarkers in Hunner's interstitial cystitis using the CIBERSORT, an algorithm based on machine learning.

BACKGROUND:  Hunner's interstitial cystitis (HIC) is a complex disorder characterized by pelvic pain, disrupted urine storage, and Hunner lesions seen on cystoscopy. There are few effective diagnostic biomarkers. In the present study, we used the novel machine learning tool CIBERSORT to measure immune cell subset infiltration and potential novel diagnostic biomarkers for HIC. METHODS: The GSE11783 and GSE57560 datasets were downloaded from the Gene Expression Omnibus for analysis. Ten HIC and six healthy samples from GSE11783 were analyzed using the CIBERSORT algorithm. Gene Set Enrichment Analysis (GSEA) was performed to identify biological processes that occur during HIC pathogenesis. Finally, expression levels of 11 T cell follicular helper cell (Tfh) markers were compared between three healthy individuals and four patients from GSE57560. RESULTS: Six types of immune cells in HIC from GSE11783 showed significant differences, including resting mast cells, CD4+ memory-activated T cells (CD3+ CD4+ HLA-DR+ cells), M0 and M2 macrophages, Tfh cells, and activated natural killer cells. Except for plasma cells, there were no significant differences between Hunner's lesion and non-Hunner's lesion areas in HIC. The GSEA revealed significantly altered biological processes, including antigen-antibody reactions, autoimmune diseases, and infections of viruses, bacteria, and parasites. There were 11 Tfh cell markers with elevated expression in patients from GSE57560. CONCLUSION: This was the first demonstration of Tfh cells and CD3+ CD4+ HLA-DR+ cells with elevated expression in HIC. These cells might serve as novel diagnostic biomarkers.