Human Antigen Research Articles

Informing pregnancy dose via target-mediated drug disposition modeling and simulations for a recombinant human monoclonal antibody.

RLYB212 is a human monoclonal anti-human platelet antigen (HPA)-1a immunoglobulin gamma 1 in clinical development as a subcutaneous injection for the prevention of maternal alloimmunization to fetal HPA-1a leading to fetal and neonatal alloimmune thrombocytopenia (FNAIT). This analysis developed a target-mediated drug disposition (TMDD) model to simultaneously characterize RLYB212 pharmacokinetics (PK) and HPA-1a-positive platelet dynamics in HPA-1b/b (HPA-1a-negative) volunteers. The model was then used to perform simulations to inform a dosing regimen in a phase II clinical study in pregnant women, where simulations accounted for physiological changes throughout pregnancy. Allometric scaling (0.75) for clearance and intercompartment transfer rate and volume (1) was included in the base model to account for variations in body weight. A 0.06 mg RLYB212 dose with a loading dose of 0.12 mg was identified as the optimal dosing regimen of RLYB212, which maintained exposures below the target upper boundary of ~10 ng/mL throughout pregnancy. This work presents an application of the TMDD model that advances the quantitative clinical pharmacology toolkit to understand monoclonal antibody PK in pregnancy.

LncRNA Taurine Up-Regulated 1 Knockout Provides Neuroprotection in Ischemic Stroke Rats by Inhibiting Nuclear-Cytoplasmic Shuttling of HuR

Background: Long non-coding RNA taurine-upregulated gene 1 (TUG1) is involved in various cellular processes, but its role in cerebral ischemia–reperfusion injury remains unclear. This study investigated TUG1’s role in regulating the nucleocytoplasmic shuttling of human antigen R (HuR), a key apoptosis regulator under ischemic conditions. Methods: CRISPR-Cas9 technology was used to generate TUG1 knockout Sprague Dawley rats to assess TUG1’s impact on ischemic injury. The infarct area and neuronal apoptosis were evaluated using TUNEL, hematoxylin and eosin (HE), and TTC staining, while behavioral functions were assessed. Immunofluorescence staining with confocal microscopy was employed to examine TUG1-mediated HuR translocation and expression changes in the apoptosis-related proteins COX-2 and Bax. Results: TUG1 knockout rats showed significantly reduced cerebral infarct areas, decreased neuronal apoptosis, and improved neurological functions compared to controls. Immunofluorescence staining revealed that HuR translocation from the nucleus to the cytoplasm was inhibited, leading to decreased COX-2 and Bax expression levels. Conclusions: TUG1 knockout reduces ischemic damage and neuronal apoptosis by inhibiting HuR nucleocytoplasmic shuttling, making TUG1 a potential therapeutic target for ischemic stroke.

Modulating versatile pathways using a cleavable PEG shell and EGFR-targeted nanoparticles to deliver CRISPR-Cas9 and docetaxel for triple-negative breast cancer inhibition.

Human antigen R (HuR), an RNA-binding protein, is implicated in regulating mRNA stability and translation in cancer, especially in triple-negative breast cancer (TNBC), a highly aggressive form. CRISPR/Cas9-mediated HuR knockout (HuR CRISPR) presents a promising genetic therapeutic approach, but it encounters transfection limitations. Docetaxel (DTX), an effective cytotoxic agent against metastatic breast cancer (BC), faces challenges related to vehicle-associated adverse events in DTX formulations. Therefore, we designed multifunctional nanoparticles with pH-sensitive PEG derivatives and targeting peptides to enable efficient HuR CRISPR and DTX delivery to human TNBC MDA-MB-231 cells and tumor-bearing mice. Our findings indicated that these nanoparticles displayed pH-responsive cytotoxicity, precise EGFR targeting, efficient tumor penetration, successful endosomal escape, and accurate nuclear and cytoplasmic localization. They also demonstrated the ability to spare normal cells and prevent hemolysis. Our study concurrently modulated multiple pathways, including EGFR, Wnt/β-catenin, MDR, and EMT, through the regulation of EGFR/PI3K/AKT, HuR/galectin-3/GSK-3β/β-catenin, and P-gp/MRPs/BCRP, as well as YAP1/TGF-β/ZEB1/Slug/MMPs. The combined treatment arrested the cell cycle at the G2 phase and inhibited EMT, effectively impeding tumor progression. Tissue distribution, biochemical assays, and histological staining revealed the enhanced safety profile of pH-responsive PEG- and peptide-modified nanoformulations in TNBC mice. The DTX-embedded and peptide-modified nanoparticles mitigated the side effects of DTX, enhanced cytotoxicity in TNBC MDA-MB-231 cells, and exhibited remarkable antitumor efficacy and safety in TNBC-bearing mice with HuR CRISPR deletion. Collectively, the combination therapy of DTX and CRISPR/Cas9 offers an effective platform for delivering antineoplastic agents and gene-editing systems to combat tumor resistance and progression in TNBC.

Identification and validation of cross-reactivity of anti-Thailand orthohantavirus nucleocapsid peptides

A Thailand orthohantavirus (THAIV) is endemic in Southeast Asia. This assumption is supported by isolation of THAIV from local small mammals. Also, anti-orthohantavirus antibodies were detected in human serum. However, our understanding of THAIV cross-reactivity with antibodies against other orthohantaviruses remains largely unknown.We used the in-silico approach to identify the cross-reactive immunogenic peptides of THAIV. The immunogenicity of these peptides was tested using convalescent serum from patients infected with Puumala (PUUV), Hantaan (HNTV) and Dobrava (DOBV) orthohantaviruses.We identified three THAIV peptides reacting with orthohantavirus convalescent serum. P1 peptide was reactive with serum from patients infected with PUUV, HNTV and DOBV. These peptides were found to be non-allergenic. Molecular docking and population coverage analysis revealed the potential of selected peptides to interact with diverse HLA alleles worldwide.Our data indicate that THAIV peptides could be used to develop diagnostics for orthohantaviruses circulating in Southeast Asia.

Coxsackievirus B3-Induced m6A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

N6-methyladenosine (m6A) is the most prevalent internal RNA modification. Here, we demonstrate that coxsackievirus B3 (CVB3), a common causative agent of viral myocarditis, induces m6A modification primarily at the stop codon and 3′ untranslated regions of its genome. As a positive-sense single-stranded RNA virus, CVB3 replicates exclusively in the cytoplasm through a cap-independent translation initiation mechanism. Our study shows that CVB3 modulates the expression and nucleo-cytoplasmic transport of the m6A machinery components—METTL3, ALKBH5 and YTHDFs—resulting in increased m6A modifications that enhance viral replication. Mechanistically, this enhancement is mediated through YTHDF-driven stress granule (SG) formation. We observed that YTHDF proteins co-localize with human antigen R (HuR), a protein facilitating cap-independent translation, in SGs during early infection. Later in infection, YTHDFs are cleaved, suppressing SG formation. Notably, for the first time, we identified that during early infection CVB3’s RNA-dependent RNA polymerase (3D) and double-stranded RNA (dsRNA) are stored in SGs, co-localizing with HuR. This early-stage sequestration likely protects viral components for use in late-phase replication, when SGs are disrupted due to YTHDF cleavage. In summary, our findings reveal that CVB3-induced m6A modifications enhance viral replication by regulating YTHDF-mediated SG dynamics. This study provides a potential therapeutic strategy for CVB3-induced myocarditis.

HuR-dependent expression of RyR2 contributes to calcium-mediated thermogenesis in brown adipocytes.

Several uncoupling protein 1 (UCP1)-independent thermogenic pathways have been described in thermogenic adipose tissue, including calcium-mediated thermogenesis in beige adipocytes via sarco/endoplasmic reticulum ATPase (SERCA). We have previously shown that adipocyte-specific deletion of the RNA binding protein human antigen R (HuR) results in thermogenic dysfunction independent of UCP1 expression. RNA sequencing revealed the downregulation of several genes involved in calcium ion transport upon HuR deletion. The goal of this work was to define the HuR-dependent mechanisms of calcium driven thermogenesis in brown adipocytes. We generated (BAT)-specific HuR-deletion (BAT-HuR -/- ) mice and show that their body weight, glucose tolerance, brown and white adipose tissue weights, and total lipid droplet size were not significantly different compared to wild-type. Similar to our initial findings in Adipo-HuR -/- mice, mice with BAT-specific HuR deletion are cold intolerant following acute thermal challenge at 4°C, demonstrating specificity of acute HuR-dependent thermogenesis to BAT. We also found decreased expression of ryanodine receptor 2 (RyR2), but no changes in RyR2, SERCA1, SERCA2, or UCP1 expression, in BAT from BAT-HuR -/- mice. Next, we used Fluo-4 calcium indicator dye to show that genetic deletion or pharmacological inhibition of HuR blunts the increase in cytosolic calcium concentration in SVF-derived primary brown adipocytes. Moreover, we saw a similar blunting in β-adrenergic-mediated heat generation, as assessed by ERtherm AC fluorescence, in SVF-derived brown adipocytes following HuR inhibition or deletion. Mechanistically, we show that HuR directly binds and reduces the decay rate of RyR2 mRNA in brown adipocytes, and stabilization of RyR2 via S107 rescues β-adrenergic-mediated cytosolic calcium increase and heat generation in HuR deficient brown adipocytes. In conclusion, our results suggest that HuR-dependent control of RyR2 expression plays a significant role in the thermogenic function of brown adipose tissue through modulation of SR calcium cycling.

Predictive immunoinformatics reveal promising safety and anti-onchocerciasis protective immune response profiles to vaccine candidates (Ov-RAL-2 and Ov-103) in anticipation of phase I clinical trials.

Onchocerciasis (river blindness) is a debilitating tropical disease that causes significant eye and skin damage, afflicting millions worldwide. As global efforts shift from disease management to elimination, vaccines have become crucial supplementary tools. The Onchocerciasis Vaccine for Africa (TOVA) Initiative was established in 2015, to advance at least one vaccine candidate initially targeting onchocerciasis in infants and children below 5 years of age, through Phase I human trials by 2025. Notably, Ov-RAL-2 and Ov-103 antigens have shown great promise during pre-clinical development, however, the overall success rate of vaccine candidates during clinical development remains relatively low due to certain adverse effects and immunogenic limitations. This study, thus, aimed at predicting the safety and immunogenicity of Ov-RAL-2 and Ov-103 potential onchocerciasis vaccine candidates prior to clinical trials. Advanced molecular simulation models and analytical immunoinformatics algorithms were applied to predict potential adverse side effects and efficacy of these antigens in humans. The analyses revealed that both Ov-RAL-2 and Ov-103 demonstrate favourable safety profiles as toxicogenic and allergenic epitopes were found to be absent within each antigen. Also, both antigens were predicted to harbour substantial numbers of a wide range of distinct epitopes (antibodies, cytokines, and T- Cell epitopes) associated with protective immunity against onchocerciasis. In agreement, virtual vaccination simulation forecasted heightened, but sustained levels of primary and secondary protective immune responses to both vaccine candidates over time. Ov-103 was predicted to be non-camouflageable, as it lacked epitopes identical to protein sequences in the human proteome. Indeed, both antigens were able to bind with high affinity and activate the innate immune TLR4 receptor, implying efficient immune recognition. These findings suggest that Ov-RAL-2 and Ov-103 can induce sufficient protective responses through diverse humoral and cellular mechanisms. Overall, our study provides additional layer of evidence for advancing the clinical development of both vaccine candidates against onchocerciasis.

Association of HPA Antigens with Immune Thrombocytopenia: A Case-Control Study by PCR-SSP Method

Background: Human platelet antigens (HPAs) play a clinically significant role in alloimmunization and the development of immune-mediated disorders such as immune thrombocytopenia (ITP), fetal and neonatal alloimmune thrombocytopenia (FNAIT), and post-transfusion purpura (PTP). Understanding the genetic profiles of HPAs is critical for preventing and treating these conditions. Given the limitations of serological methods in determining HPA genotypes, this study aims to investigate the association between the genotypes of HPA1, HPA2, HPA3, HPA4, and HPA15 antigens and autoimmune thrombocytopenia in Lorestan Province, utilizing the PCR-SSP method. Materials and Methods: This case-control study involved 80 individuals diagnosed with ITP and 120 healthy controls. DNA samples were extracted using a commercial DNA extraction kit, with concentrations quantified via a Nanodrop spectrophotometer. Genotyping was performed using the PCR-SSP method with specific primers for each HPA gene. The genotype data were verified using previously established sample sets. The frequencies of each HPA genotype were recorded, and a comparative analysis was conducted between the patient and control groups to evaluate the study hypothesis. Results: The results revealed that individuals carrying the HPA2b allele had a 5.31-fold increased risk of developing ITP, a statistically significant finding (P < 0.05, OR = 5.31). Similarly, the presence of the HPA15b allele was associated with a 6.54-fold increased risk (P < 0.05, OR = 6.54). Conclusion: These findings, in conjunction with previous studies, suggest the need for larger-scale investigations across different populations. Such research could aid in the early diagnosis and prediction of thrombocytopenia severity, inform treatment strategies, and facilitate the removal of pathogenic antibodies from circulation.

Novel, soluble 3-heteroaryl-substituted tanshinone mimics attenuate the inflammatory response in murine macrophages

The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can lead to beneficial anti-inflammatory effects. To this aim, we previously synthesized a novel class of small molecules named Tanshinone Mimics (TMs) able to interfere with HuR-RNA binding, and that dampen the LPS-induced immune response. Herein, we present a novel series of TMs, encompassing thiophene 3/TM9 and 4/TM10, furan 5/TM11 and 6/TM12, pyrrole 7b/TM13, and pyrazole 8. The furan-containing 5(TM11) showed the greatest inhibitory effect of the series on HuR-RNA complex formation, as suggested by RNA Electromobility Shift Assay and Time-Resolved FRET. Molecular Dynamics Calculation of HuR − 5/TM11 interaction, quantum mechanics approaches and Surface Plasmon Resonance data, all indicates that, within the novel heteroaryl substituents, the furan ring better recapitulates the chemical features of the RNA bound to HuR. Compound 5/TM11 also showed improved aqueous solubility compared to previously reported TMs. Real-time monitoring of cell growth and flow cytometry analyses showed that 5/TM11 preferentially reduced cell proliferation rather than apoptosis in murine macrophages at immunomodulatory doses. We observed its effects on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages, showing that 5/TM11 significantly reduced the expression of proinflammatory cytokines as Cxcl10 and Il1b.

Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.

Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (VH, Vκ, and Vλ) fused to human constant domains. The human constant domains, CH1 of IgG1 and CL (Cκ or Cλ), not only provide established purification and detection handles but also facilitate higher expression in Escherichia coli compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.

Phase 1 first-in-human study of MEDI2228, a BCMA-targeted ADC, in patients with relapsed refractory multiple myeloma

MEDI2228 is an antibody drug conjugate (ADC) comprised of a fully human B-cell maturation antigen (BCMA) antibody conjugated to a pyrrolobenzodiazepine (PBD) dimer. This phase 1 trial evaluated MEDI2228 in patients with relapsed/refractory (R/R) multiple myeloma (MM), who received prior treatment with approved agents from 3 classes of antimyeloma drugs (proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies). Primary endpoint was safety and tolerability; secondary endpoints included efficacy, pharmacokinetics, and immunogenicity. A total of 107 patients were treated and the maximum tolerated dose (MTD) was 0.14 mg/kg Q3W. Two patients had dose-limiting toxicities (DLTs; thrombocytopenia; 0.20 mg/kg Q3W). The most frequent treatment-related adverse events were photophobia (43.9%), rash (29.0%), and thrombocytopenia (19.6%). In MTD cohort A (n = 41), the objective response rate (ORR) was 56.1%, with 1 stringent complete response, 9 very good partial responses, and 13 partial responses. ORR was 53.3% in triple refractory patients. In cohort B (n=25), ORR was 32%. Although MEDI2228 demonstrated efficacy in R/R MM, ocular toxicity precluded further development of this drug.

Two Concurrent Cases of NAIT in Pregnant Sisters

Abstract Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal antibodies directed against fetal platelet antigens inherited from the father. Approximately 80% of cases of NAIT are attributed to antibodies against human platelet antigen (HPA)-1a and less commonly to antibodies against HPA-5b. We present two cases of possible NAIT attributed to anti-HPA-5b that were identified in sisters requiring concurrent management. Patient #1 is a 32-year-old G9P4044 woman with one pregnancy complicated by intrauterine growth retardation and partial placental abruption, another pregnancy complicated by fetal anomaly (intracerebral hemorrhage) status post termination of pregnancy at 23 weeks’ gestation and three miscarriages (one with chromosomal abnormality). The patient is homozygous for HPA-1a and HPA-5a, and the partner is homozygous for HPA-5b. HPA-5b antibodies and HLA antibodies were identified in the patient’s serum. Deemed at high risk for subsequent NAIT, she was started on intravenous immunoglobulin twice weekly and steroids during her second trimester until delivery. The patient underwent weekly fetal sonograms during pregnancy which were normal. The patient’s platelet counts were stable. The patient refused caesarean delivery. At 38 weeks’ gestation, the patient gave birth to a healthy female via normal spontaneous vaginal delivery. HPA-5b negative platelets were procured from our blood supplier, but neither the mother nor neonate required transfusion. The neonate showed no signs of bleeding and had normal platelet counts. Ultrasound imaging of the head was unremarkable. The patient and neonate were discharged home. Patient #2 is a 33-year-old G13P6156 woman with a prior pregnancy complicated by stillbirth at 32 weeks’ gestation. Considering her personal and family history, HPA testing was performed and HPA-5b antibodies were identified in the patient’s serum. The patient’s partner is heterozygous for HPA-5b. Crossmatched platelets were procured from our blood supplier. The patient refused caesarean delivery. The patient gave birth at 37 weeks’ gestation to a healthy male via normal spontaneous vaginal delivery. The fetus had mild thrombocytopenia (107 x 103/uL), with no bleeding or bruising identified. Neither the mother nor neonate required transfusion. The patient and neonate were discharged home. Ultrasound imaging of the head was unremarkable, and his thrombocytopenia resolved. While some studies postulate that anti-HPA-5b identification may be coincidental rather contributory to the disease process, these cases re-emphasize the importance of obtaining pregnancy, neonatal and family history to best risk stratify patients and determine management from both the clinical and transfusion medicine perspectives. If transfusion is needed, transfusion options include HPA negative platelets, crossmatched platelets and random donor platelets depending on clinical urgency and platelet availability.

Molecular testing in a patient with multiple alloantibodies and warm autoimmune hemolytic anemia: A case study

Abstract Introduction/Objective We present a case of RBC alloimmunization and warm autoimmune hemolytic anemia (WAIHA) in a patient with a recent RBC transfusion. Serological testing followed by molecular testing revealed a rare D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/- predicted phenotype. Methods/Case Report A 75-year-old black male with secondary myelofibrosis presented with a sudden onset of chest pain, dyspnea and weakness during an RBC transfusion. The patient was subsequently admitted for further workup. Labs showed low hemoglobin, low haptoglobin, elevated LDH and a positive direct antiglobulin (DAT) test. The patient was started on intravenous immunoglobulin and steroids for suspected WAIHA. On immunohematology serologic testing, blood typed as B-positive and polyspecific DAT positive with anti-IgG positive and anti-C3 negative. Serum plasma studies showed probable warm autoimmune hemolytic anemia and allo anti-S, -E, and -C by previous history. Eluate studies showed panagglutinin and antibody with D-like specificity. Immucor PreciseTypeTM Human Erythrocyte Antigen (HEA) Molecular BeadChip test predicted a partial e antigen and potential hrB-. Genotyping for RHD variants was performed and revealed RHD*01 (homozygous or hemizygous). Targeted genomic testing did not detect variants associated with common partial or weak RhD antigens. Based on this testing, the patient was not predicted to be at risk of allo-anti-D. An RHCE genotype for C, c, E and e variants including hrB and hrS showed RHCE*ce48C,733G/RHCE*ce733G with predicated phenotype of D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/-. Based on the RHCE testing, the patient was predicted to be at risk for allo-anti-c, -e, -f (ce) and possibly anti-hrB. Results (if a Case Study enter NA) NA Conclusion In this case, serologic testing alone did not reveal a precise diagnosis. Molecular testing aided in the diagnosis of WAIHA and will help guide future transfusions. Pathologists must consider molecular testing when faced with a challenging serologic diagnosis.

Long-Term Human Immune Reconstitution, T-Cell Development, and Immune Reactivity in Mice Lacking the Murine Major Histocompatibility Complex: Validation with Cellular and Gene Expression Profiles.

Humanized mice transplanted with CD34+ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity. We evaluated the long-term (20-week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen. Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4+ and CD8+ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses. Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference.

An epitope imprinted electrochemical sensor for highly sensitive and selective determination of prostate-specific antigen.

Asimple method forhighly selective and sensitive prostate-specific antigen (PSA) detection using a molecularly imprinted electrochemical sensor ispresented. The sensor was developed through an epitope imprinted strategy combined with electrochemical measurement techniques. An epitope molecularly imprinted polymer (EMIP) film was constructed on a AuNPs-coated gold electrode surface through electropolymerization, utilizing the C-terminus epitope of PSA (KWIKDTIVANP) as the template molecular and o-phenylenediamine as the functional monomer. The characteristics of EMIP film were investigated by using a scanning electron microscope and electrochemical test methods, including electrochemical impedance spectroscopy and cyclic voltammetry. Key parameters such as electropolymerization cycles, elution and rebinding times, and the molar ratio of template molecular to functional monomer were systematically optimized. The sensor demonstrated a detection limit (LOD) of 0.31 fg/mL and exhibited an excellent linear response towards PSA concentration ranging from 1.0 fg/mL to 0.1 µg/mL. Furthermore, the EMIP sensor showed excellent selectivity against other biological macromolecules, such as bovine serum albumin, human serum albumin, alpha-fetoprotein, and carcinoembryonic antigen. With recoveriesbetween 95.89 and 106.04% for PSA detection in human serums theEMIP/AuNPs/AuE electrochemical sensor showed great potential in real sample analysis.

Upregulated DNMT3a coupling with inhibiting p62-dependent autophagy contributes to NNK tumorigenicity in human bronchial epithelial cells

NNK, formally known as 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanoe, is a potent chemical carcinogen prevalent in cigarette smoke and is a key contributor to the development of human lung adenocarcinomas. On the other hand, autophagy plays a complex role in cancer development, acting as a "double-edged sword" whose impact varies depending on the cancer type and stage. Despite this, the relationship between autophagy and NNK-induced lung carcinogenesis remains largely unexplored. Our current study uncovers a marked reduction in p62 protein expression in both lung adenocarcinomas and lung tissues of mice exposed to cigarette smoke. Interestingly, this reduction appears to be contingent upon the activity of extrahepatic cytochrome P450 (CYP450), revealing that NNK metabolic activation by CYP450 enzyme escalates its potential to induce p62 downregulation. Further mechanistic investigations reveal that NNK suppresses autophagy by accelerating the degradation of p62 mRNA, thereby promoting the malignant transformation of human bronchial epithelial cells. This degradation process is facilitated by the hypermethylation of the Human antigen R (HuR) promoter, resulting in the transcriptional repression of HuR - a key regulator responsible for stabilizing p62 mRNA through direct binding. This hypermethylation is triggered by the activation of ribosomal protein S6, which is influenced by NNK exposure and subsequently amplifies the translation of DNA methyltransferase 3 alpha (DNMT3a). These findings provide crucial insights into the nature of p62 in both the development and potential treatment of tobacco-related lung cancer.

A novel approach to simultaneous genotyping of human platelet antigen systems and human leucocyte antigen class I loci using PacBio long-read sequencing.

Accurate human leucocyte antigen (HLA) and human platelet antigen (HPA) typing is essential for establishing a blood platelet donor bank to deal with refractoriness in patients undergoing multiple platelet transfusions. Current methods, such as Sanger and next-generation sequencing, encounter difficulties in haplotyping. Herein, the aim of this study was to establish a method for HLA and HPA typing based on the long read sequencing. The HPA and HLA class I genotypes of 268 platelet donors from the Taiyuan Blood Center, China were identified using long-read sequencing on the PacBio platform. Allele frequencies for HPA systems and HLA class I genes were calculated, and genetic variability within HPA system genes was analysed. Polymorphisms were identified in 8 of the 35 HPA systems (HPA-1 to HPA-6w, HPA-15 and HPA-21w), with the frequencies of the 'b' allele at 0.0187, 0.0709, 0.4086, 0.0075, 0.0149, 0.0317, 0.4310 and 0.0019, respectively. The alleles with the highest frequencies at the HLA-A, HLA-B and HLA-C loci are HLA-A02:01, B51:01, B46:01 and C06:02, respectively. Additionally, several genetic patterns in HPA systems were identified, including the c.166-1029C>T variant, which was found exclusively in samples carrying the HPA-1b allele. This study developed a targeted long-read sequencing method characterized by high throughput and simultaneity, capable of resolving allele ambiguities for effective HLA class I genotyping in establishing a platelet donor bank.

Brugia malayi filarial helminth-derived extracellular vesicles suppress antigen presenting cell function and antigen-specific CD4+ T cell responses.

Live microfilariae (mf) and mf-derived extracellular vesicles (EVs) have been shown to modulate human antigen presenting cell (APC) function, most notably by suppressing the induction of IL-12 (and other pro-inflammatory cytokines) following activation with LPS and interferon-y. To explore further how EVs alter human APC function, we studied the effect ofmf and EVs on human elutriated monocyte-derived dendritic cells (DC) following exposure to Mf, mf-derived excretory/secretory (E/S) products, E/S depleted of EVs through ultracentrifugation and purified EVs. After demonstrating that the measurable responses induced by live mf could be recapitulated by EVs and EV-containing E/S, we next performed RNAseq analysis of human DC following exposure to live mf, EVs, E/S, or EV-depleted E/S. In our analyses of the data for the DC, using a false discovery rate (FDR)<0.05, EV-exposed DC had induced the expression of 212 differentially expressed genes (DEGs) when compared to unexposed DC and 157 when compared to E/S-depleted EVs. These genes were enriched in GO biological processes associated with neutrophil degranulation and 15 DEGs associated with KEGG Lysosome pathways. IPA analysis point to immune dysregulation. We next aimed to understand the intracellular processes altered by EVs and the effect these have on effector T cells. When SARS CoV-2 Membrane-specific CD4+ TCLs were assessed following EV conditioning of autologous DC and activation with the SARS CoV-2-Membrane peptide pool, we found conditioning reduced the frequency of SARS CoV-2 Membrane-specific CD3+ CD4+ CD154+ cells (p=.015). Similarly, EV-conditioning of SARS CoV-2 Membrane-specific CD3+ CD4+ cells induced fewer cell capable of producing IFN-γ (p=.045). Taken together, our data suggest a modulatory role of EVs on APC function that likely leads to defects in T cell effector function.

A cross-sectional pilot study to estimate the frequency of minor blood group alleles and phenotypes in RhD-negative North Indian blood donors by DNA microarray analysis

Abstract: INTRODUCTION: There are scarce data on Indian blood donors with respect to blood group phenotypes using molecular diagnostic modalities. Hence, we planned to estimate frequencies of blood group alleles/phenotypes using DNA microarray analysis in the north Indian RhD-negative blood donor population. With this initial pilot study, we plan to expand it to our entire donor population. METHODOLOGY: The cross-sectional prospective study was conducted on 50 Indian blood donors (O RhD negative), to study the blood group genotype frequency. Genotyping for the most relevant red blood cell antigens (Rh, Kell, Duffy, Kidd, MNS, Lutheran, and Dombrock) was done using Bioarray Precise TypeHM Human Erythrocyte Antigen BeadChip kit containing probes directed to polymorphic sites. RESULTS: In the Rh system, the most common alleles were RHCE*e/RHCE*e (98%) and RHCE*c/RHCE*c (80%). Phenotype K-k+ (genotype- KEL*02/KEL*02) was seen in 98% of samples, Js(a-b+) (KEL*02.07/KEL*02.07) was detected in 98% (49/50) of the samples tested. Jk(a + b+) (JK*01/JK*02) was the most common phenotype (48%) in the Kidd blood group system. In MNSs system, M+N+ (GYPA*01/GYPA*02) 44% and S+s+U+ (GYPB*03/GYPB*04) 34% were the most common phenotypes detected. CONCLUSION: This pilot study shows the feasibility of genotyping a Northern Indian donor population. To the best of our knowledge, it is the first study on molecular blood grouping in Indian blood donors using the Bioarray platform.

B-032 Evaluation of Assay Performance for Atellica CI 1900 analyzer in Routine Chemistry, Division of Laboratory Medicine, UMMC

Abstract Background The Atellica® CI 1900 (Siemens Healthiness) is a stand-alone, integrated clinical chemistry and immunoassay analyzer. Method verification has been done on this analyzer for 14 parameters based on the guideline of the Clinical and Laboratory Standard Institute (CLSI). Methods The method evaluation protocol consisted of determination of imprecision based CLSI EP 15-A3; CLSI EP06-A2 protocol was used for verification of linearity and method comparison was carry out in between Atellica® CI 1900 with our current Atellica® Solution based on CLSI EP09-A2 protocol. Method comparison data was analyzed by using statistical software, MedCalc. The data were used to calculate bias for Sigma metrics determination. Results Total of 14 analytes including sodium, potassium, chloride, creatinine, urea, alkaline phosphatase, total bilirubin, triglyceride, uric acid, glucose, thyroid stimulating hormone, free-thyroxine, human chorionic gonadotropin and carcinoembryonic antigen were tested. The results showed the repeatability and total imprecision were within acceptable limit as per manufacturer claims for all analytes. It was demonstrated that the analytical system operates within the claimed linearity ranges as stated in the information for use (IFU). Passing - Bablok analysis show the bias for all analytes were within our laboratory pre-set quality specification. The intercept and slope with 95% CI for Passing-Bablok analysis as shown in table 1. Sigma metrics were determined for each assay using the coefficient variation determined from precision study and bias obtained from the method comparison study. It showed that the Sigma for 14 analytes ranging from 3 to more than 6 with more than 93% assay has sigma scores more than 4. Conclusions Atellica® CI 1900 shows acceptable precision and bias for all analytes that evaluated. The analyser is fully comparable with Atellica® Solution. We conclude that Atellica® CI 1900 demonstrates good performance capabilities, making this analyzer suitable for low-to-medium volume laboratory.