Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model developed using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and treated with TCZ, BM-MSC, or both TCZ and BM-MSC. We compared HLA.A2-specific IgG levels and subsets of T cells and B cells using flow cytometry among groups. HLA.A2-specific IgG level was decreased in all treated groups in comparison with that in the allo-sensitized control (Allo-CONT) group. Its decrease was the most significant in the TCZ + BM-MSC group. Regarding the B cell subset, combined use of TCZ and BM-MSC increased proportions of pre-pro B cells but decreased proportions of mature B cells in BM (p < 0.05 vs. control). In the spleen, an increase in transitional memory was observed with a significant decrease in marginal, follicular, and long-lived plasma B cells (p < 0.05 vs. control) in the TCZ + BM-MSC group. In T cell subsets, Th2 and Th17 cells were significantly decreased, but Treg cells were significantly increased in the TCZ+BM-MSC group compared to those in the Allo-CONT group in the spleen. Regarding RNA levels, IL-10 and Foxp3 showed increased expression, whereas IL-23 and IFN-γ showed decreased expression in the TCZ + BM-MSC group. In conclusion, combined use of TCZ and BM-MSC can inhibit B cell maturation and up-regulate Treg cells, finally resulting in the reduction of HLA.A2-specific IgG in a highly sensitized mouse model. This study suggests that the combined use of TCZ and BM-MSC can be proposed as a novel strategy in a desensitization protocol for highly sensitized patients.

Repeated Infusions of Bone-Marrow-Derived Mesenchymal Stem Cells over 8 Weeks for Steroid-Refractory Chronic Graft-versus-Host Disease: A Prospective, Phase I/II Clinical Study.

Chronic graft-versus-host disease (cGVHD) is a long-term complication of allogeneic hematopoietic stem cell transplantation associated with poor quality of life and increased morbidity and mortality. Currently, there are several approved treatments for patients who do not respond to steroids, such as ruxolitinib. Nevertheless, a significant proportion of patients fail second-line treatment, indicating the need for novel approaches. Mesenchymal stem cells (MSCs) have been considered a potential treatment approach for steroid-refractory cGVHD. To evaluate the safety and efficacy of repeated infusions of MSCs, we administered intravenous MSCs every two weeks to ten patients with severe steroid-refractory cGVHD in a prospective phase I clinical trial. Each patient received a total of four doses, with each dose containing 1 × 106 cells/kg body weight from the same donor and same passage. Patients were assessed for their response to treatment using the 2014 National Institutes of Health (NIH) response criteria during each visit. Ten patients with diverse organ involvement were enrolled, collectively undergoing 40 infusions as planned. Remarkably, the MSC infusions were well tolerated without severe adverse events. Eight weeks after the initial MSC infusion, all ten patients showed partial responses characterized by the amelioration of clinical symptoms and enhancement of their quality of life. The overall response rate was 60%, with a complete response rate of 20% and a partial response (PR) rate of 40% at the last follow-up. Overall survival was 80%, with a median follow-up of 381 days. Two patients died due to relapse of their primary disease. Immunological analyses revealed a reduction in inflammatory markers, including Suppression of Tumorigenicity 2 (ST2), C-X-C motif chemokine ligand (CXCL)10, and Secreted phosphoprotein 1(SPP1), following the MSC treatment. Repeated MSC infusions proved to be both feasible and safe, and they may be an effective salvage therapy in patients with steroid-refractory cGVHD. Further large-scale clinical studies with long-term follow-up are needed in the future to determine the role of MSCs in cGVHD.

Impact of Induction Immunosuppressants on T Lymphocyte Subsets after Kidney Transplantation: A Prospective Observational Study with Focus on Anti-Thymocyte Globulin and Basiliximab Induction Therapies.

Induction immunosuppressive therapy for kidney transplant recipients (KTRs) primarily includes interleukin-2 receptor antagonists, such as basiliximab (BXM) or lymphocyte-depleting agents, and anti-thymocyte globulin (ATG). This study aimed to investigate their effects on T cell dynamics during the early post-transplantation period. This prospective observational study included 157 KTRs. Peripheral blood samples were collected from each patient within 5 days before and 4 and 12 weeks after transplantation. Flow cytometric analysis was performed to assess various T cell subsets whose changes were then analyzed. In the ATG group, CD4+ T cell expression decreased significantly compared with that in the BXM group. However, CD4+CD161+ and CD4+CD25+CD127low T cell expression levels increased significantly. In the CD8+ T cell subset, a decrease in CD8+CD28nullCD57+ and CD8+CCR7+ T cell expression was observed in the ATG group. However, among patients diagnosed with biopsy-proven acute rejection, T cell subset expression did not significantly differ relative to non-rejection cases. In conclusion, ATG induction therapy resulted in more pronounced changes in T lymphocyte subsets than BXM induction, with increased CD4+CD161+ and CD4+CD25+CD127low T cells and an early decrease in CD8+CD28nullCD57+ and CD8+CCR7+ T cells, some of which are associated with acute rejection.

Intra-host diversity of drug-resistant cytomegalovirus: A case report of cytomegalovirus infection after allogeneic hematopoietic cell transplantation.

Cytomegalovirus (CMV) is a major infectious agent causing severe complications in allogeneic hematopoietic cell transplantation (HCT) recipients, thereby warranting the need for aggressive preemptive or targeted antiviral therapy. However, prolonged or repeated use of antiviral agents, such as ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), can result in drug-resistant CMV infection, posing challenges to successful outcomes. Here, we report a case of a patient with acute myeloid leukemia and drug-resistant CMV infection who presented with persistent CMV DNAemia, colitis, pneumonia, and encephalitis. An intra-host diversity of UL97 and UL54 mutations were detected through the genotypic resistance testing conducted on two blood samples (D+199 and D+224) and a cerebrospinal fluid (CSF) specimen (D+260) collected from the patient. UL97 L595W/L595F and L595W mutations were detected in the blood and CSF samples, respectively, that conferred GCV resistance. UL54 F412L mutation detected in all three samples conferred GCV/CDV resistance. However, the V787L mutation of UL54, conferring GCV/FOS resistance, was observed only in the D+224 blood sample. Despite combination therapy with FOS and high dose GCV and adjunctive therapy with leflunomide, the patient died from CMV infection and multiple organ failure on D+279. Further data on resistant mutations and intra-host diversity of CMV should be accumulated to elucidate the antiviral resistance and related outcomes.

Inter-laboratory comparison of semiquantitative allergen-specific Immunoglobulin E test: 7 years of experience in Korea.

INTRODUCTION: Multiple allergen simultaneous test (MAST) is widely used as a screening tool for allergic diseases and has the advantage of providing specific IgE (sIgE) results for various allergens in semiquantitative class. We have continuously conducted external quality assessment (EQA) since 2012 for clinical laboratories performing MAST using AdvanSure allergy screen test (LG CHEM, Korea). This study provides an account of the EQA experience. METHODS: Samples were prepared using pooled sera collected from patients with suspected allergic disease and sent to each laboratory twice a year. Each round included 4-6 serum samples with sIgE for 10-20 inhaled or food allergens. The acceptable class value was the most frequently reported MAST class ±1 titer that exceeded 80% of the total laboratory results. RESULTS: The average number of participating laboratories was 76 (49-90) and the average response rate was 97.3% during the entire survey period. The acceptable rates were consistently high at 97.7% ± 3.7%. Of the total 537 trials, 18 trials (3.4%) were regarded as nonconsensus results, in which acceptable answers did not exceed 80%. For unacceptable results, the false-negative rate (1.5% ± 2.8%) was higher than the false-positive rate (0.8% ± 2.7%) (p < 0.001). MAST class results were correlated with quantitative IgE results by ImmunoCAP (Spearman's correlation coefficient of 0.682 (p < 0.001) and gamma index of 0.777 (p < 0.001). CONCLUSION: Although EQA for MAST showed a high level of acceptable answer, some allergen assays require harmonization. Continuous performance of systematic EQA is needed to improve the accuracy of sIgE assays and quality control in clinical laboratories.

Combined Analysis of HLA Class II Eplet Mismatch and Tacrolimus Levels for the Prediction of De Novo Donor Specific Antibody Development in Kidney Transplant Recipients.

We investigated whether HLA class II eplet mismatch was related to dnDSA development and analyzed its combined impact with tacrolimus levels for kidney transplantation outcomes. A total of 347 kidney transplants were included. HLA Matchmaker was used for the single molecular eplet, total eplet, antibody (Ab)-verified eplet mismatch analyses, and Ab-verified single molecular analysis to identify HLA-DR/DQ molecular thresholds for the risk of dnDSA development. A time-weighted tacrolimus trough level (TAC-C0) of 5 ng/mL and a TAC-C0 time-weighted coefficient variability (TWCV) of 20% were applied to find the combined effects on dnDSA development. A high level of mismatch for single molecular eplet (DQ ≥ 10), total eplet (DQ ≥ 12), Ab-verified eplet (DQ ≥ 4), and Ab-verified single molecular eplet (DQ ≥ 4) significantly correlated with HLA class II dnDSA development. Class II dnDSA developed mostly in patients with low TAC-C0 and high eplet mismatch. In the multivariable analyses, low TAC-C0 and high eplet mismatch showed the highest hazard ratio for the development of dnDSA. No significant combined effect was observed in dnDSA development according to TWCV. In conclusion, the determination of HLA class II eplet mismatch may improve the risk stratification for dnDSA development, especially in conjunction with tacrolimus trough levels.

BAFF Inhibition Effectively Suppresses the Development of Anti-HLA.A2 Antibody in the Highly Sensitized Mouse Model.

B cell activating factor (BAFF) is a cytokine that plays a role in the survival, proliferation and differentiation of B cells. We proposed to observe the effects of BAFF inhibition on the humoral immune responses of an allosensitized mouse model using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and were treated with anti-BAFF monoclonal antibody (mAb) (named Sandy-2) or control IgG1 antibody. HLA.A2-specific IgG was reduced in BAFF-inhibited mice compared to the control group (Δ-13.62 vs. Δ27.07, p < 0.05). BAFF inhibition also resulted in increased pre-pro and immature B cell proportions and decreased mature B cells in the bone marrow (p < 0.05 vs. control). In the spleen, an increase in transitional B cells was observed with a significant decrease in marginal and follicular B cells (p < 0.05 vs. control). There was no significant difference in the proportions of long-lived plasma and memory B cells. Microarray analysis showed that 19 gene probes were significantly up- (>2-fold, p < 0.05) or down-regulated (≤2-fold, p < 0.05) in the BAFF-inhibited group. BAFF inhibition successfully reduced alloimmune responses through the reduction in alloantibody production and suppression of B cell differentiation and maturation. Our data suggest that BAFF suppression may serve as a useful target in desensitization therapy.

Delayed NK Cell Reconstitution and Reduced NK Activity Increased the Risks of CMV Disease in Allogeneic-Hematopoietic Stem Cell Transplantation.

Cytomegalovirus (CMV) infection has a significant impact in patients after allogeneic hematopoietic stem cell transplantation (HSCT). We investigated natural killer (NK) cell reconstitution and cytotoxic/cytokine production in controlling CMV infection, especially severe CMV disease in HSCT patients. Fifty-eight patients with acute myeloid leukemia (AML) who received allo-HSCT were included. We monitored NK reconstitution and NK function at baseline, 30, 60, 90, 120, 150, and 180 days after HSCT, and compared the results in recipients stratified on post-HSCT CMV reactivation (n = 23), non-reactivation (n = 24) versus CMV disease (n = 11) groups. The CMV disease group had a significantly delayed recovery of CD56dim NK cells and expansion of FcRγ-CD3ζ+NK cells started post-HSCT 150 days. Sequential results of NK cytotoxicity, NK cell-mediated antibody-dependent cellular cytotoxicity (NK-ADCC), and NK-Interferon-gamma (NK-IFNγ) production for 180 days demonstrated delayed recovery and decreased levels in the CMV disease group compared with the other groups. The results within 1 month after CMV viremia also showed a significant decrease in NK function in the CMV disease group compared to the CMV reactivation group. It suggests that NK cells' maturation and cytotoxic/IFNγ production contributes to CMV protection, thereby revealing the NK phenotype and functional NK monitoring as a biomarker for CMV risk prediction, especially CMV disease.

Natural Killer Cell Function Tests by Flowcytometry-Based Cytotoxicity and IFN-γ Production for the Diagnosis of Adult Hemophagocytic Lymphohistiocytosis.

Although natural killer (NK) cell function is a hallmark of hemophagocytic lymphohistiocytosis (HLH), there is no standard method or data on its diagnostic value in adults. Thus, we performed a single-center retrospective study of 119 adult patients with suspected HLH. NK cell function was determined using both flowcytometry-based NK-cytotoxicity test (NK-cytotoxicity) and NK cell activity test for interferon-gamma (NKA-IFNγ). NK cell phenotype and serum cytokine levels were also tested. Fifty (42.0%) HLH patients showed significantly reduced NK cell function compared to 69 non-HLH patients by both NK-cytotoxicity and NKA-IFNγ (p < 0.001 and p = 0.020, respectively). Agreement between NK-cytotoxicity and NKA-IFNγ was 88.0% in HLH patients and 58.0% in non-HLH patients. NK-cytotoxicity and NKA-IFNγ assays predicted HLH with sensitivities of 96.0% and 92.0%, respectively. The combination of NKA-IFNγ and ferritin (>10,000 µg/L) was helpful for ruling out HLH, with a specificity of 94.2%. Decreased NK-cytotoxicity was associated with increased soluble IL-2 receptor levels and decreased CD56dim NK cells. Decreased NKA-IFNγ was associated with decreased serum cytokine levels. We suggest that both NK-cytotoxicity and NKA-IFNγ could be used for diagnosis of HLH. Further studies are needed to validate the diagnostic and prognostic value of NK cell function tests.

Comparison of allergen-specific IgE levels between Immulite 2000 and ImmunoCAP systems against six inhalant allergens and ten food allergens.

In vitro allergen-specific immunoglobulin E (sIgE) measurement has been used as an important diagnostic tool for allergic diseases. Currently, quantitative sIgE levels are detected mainly by using ImmunoCAP and Immulite 2000 assay system. These two systems have the same calibration scale at 0-100 kUA/L, but they differ in used allergens, detection methods and automation systems. We compared 1600 paired sIgE results for 204 allergic patients, including 100 paired sIgE assay results for each of 16 allergens (Alternaria alternata, birch-alder mix, cat dander, D. farinae, D. pteronyssinus, dog dander, buckwheat, crab, egg white, mackerel, milk, peach, peanut, shrimp, soybean and wheat flour). Inter-method comparison was performed for qualitative data with a cutoff of 0.35 kUA/L and a detection limit of 0.1 kUA/L, semi-quantitative class results and quantitative data. In qualitative comparisons, the overall concordance rate ranged from 81.0% to 99.0% (k: 0.599-0.949) with the cutoff value of 0.35 kUA/L. It also ranged from 80.0% to 99.0% (k: 0.521-0.951) with the detection limit of 0.1 kUA/L. The class results from these two assays showed good agreements for all allergens. For quantitative sIgE results, these two assays showed moderate positive correlations for Dog dander (r = 0.683) and Mackerel (r = 0.573) but high to very high correlations for the other 14 allergens (r = 0.734-0.972). Immulite 2000 and ImmunoCAP assays demonstrated good concordance and correlation for 16 common allergens, but international standards against each specific allergen for calibration and harmonization of sIgE tests are still needed.

Analytical Evaluation of Portable and Simple FREND Fluorescent Immunoassay for Rapid Quantification of Thyroid-Stimulating Hormone and Free Thyroxine.

BACKGROUND: The FREND™ thyroid stimulating hormone (TSH) and free thyroxine (FT4) assays are newly developed rapid quantitative immunoassays utilizing antibody-conjugated fluorescent nanoparticles in a microfluidic flow system. The FREND system is a simple and portable fluorescence reader with rapid turnaround time (4 - 10 minutes). METHODS: The analytical sensitivity, precision, and linearity were evaluated. For the method comparison, FREND TSH and FT4 levels were compared with those from ADVIA Centaur XP and Abbott ARCHITECT i2000. RESULTS: The limit of detection was 0.047 mIU/L and 5.031 pmol/L in FREND TSH and FT4 assays. FREND system had acceptable linearity and precision of  10% across the assay range (0.05 - 25.0 mIU/L for TSH and 1.4 - 96.8 pmol/L for FT4). The functional sensitivity was 0.057 mIU/L for the TSH assay and 4.644 pmol/L for the FT4 assay. Passing-Bablok regression analysis of TSH data showed good correlation among the three assays. For the FT4 assay, FREND FT4 displayed good correlation with the ARCHITECT FT4 assay, but the intercepts and slopes significantly differed between FREND and Centaur results: [FREND FT4] = 4.799 + 0.600 [Centaur FT4]. CONCLUSIONS: The analytical performance of FREND TSH and FT4 assays was satisfactory for use in the clinical laboratory. The FREND FT4 assay was in concordance with ARCHITECT FT4, but it needs further investigation and harmonization.

Quantitative detection of target cells using unghosted cells (UGCs) of DxH 800 (Beckman Coulter).

BACKGROUND: In the Retic channel of DxH 800 (Beckman Coulter), the red blood cells (RBCs) resistant to hemoglobin clearing are counted as unghosted cells (UGCs). The aim of this study was to evaluate that the UGC is a surrogate marker for both the detection and counting of target cells. METHODS: In total, 1181 samples including 22 from iron deficiency anemia (IDA) patients, 95 from jaundice, 2 from sickle cell anemia, 3 from thalassemia, 1 cord blood, and 269 from normal controls were analyzed. Slides were prepared from all samples except normal controls and target cells were counted for correlation analysis of target cell counts to UGCs. RESULTS: The normal control samples showed 0.01% (0%-0.01%) UGCs, and the reference range was set at ≤0.02%. The IDA samples showed 0.015% (0.01%-0.03%) UGC count and 0.05% (0%-0.2%) target cell count. The jaundice samples showed 0.98% (0.1%-5.36%) UGC count, and 1.4% (0.1%-7.0%) target cell count. The two sickle cell anemia samples showed 0.41% and 3.74% UGC counts and 0.4% and 11.5% target cell counts. A cord blood sample showed 0.01% UGCs and 0% target cells. The three thalassemia samples showed 0.01%, 1.99%, and 7.82% UGC counts and 0%, 1.4%, and 15.5% target cell counts. The samples showing poikilocytosis other than target cells showed normal UGC count (≤0.02%). The positive predictive value of UGCs was 58.2% (124/213) and the negative predictive value was 96.8% (674/696). The UGC counts were well correlated to the manual target cell counts (r=0.944, p=0.000). CONCLUSIONS: This study demonstrates for the first time in the literature that a hematological parameter obtained automatically every time a reticulocyte counting is performed can be used to both screen for the presence of target cells and reliably quantify them.

Effects of ECM protein mimetics on adhesion and proliferation of chorion derived mesenchymal stem cells.

BACKGROUND: We evaluated the effects of fibronectin, collagen, cadherin, and laminin based extracellular matrix (ECM) protein mimetics coated with mussel derived adhesive protein (MAP) on adhesion and proliferation of chorionic mesenchymal stem cells (cMSCs). METHODS: Human placental chorionic tissues from term third-trimester pregnancies (n=3) were used. The cMSCs were cultured on rationally designed ECM protein mimetics coated with MAP on plastic surfaces with the addition of reduced fetal bovine serum (0.5%, 1% FBS). Adhesion capabilities were monitored by a real time cell analysis system (RTCA) utilizing an impedance method. Proliferation capabilities were monitored by RTCA and MTS assay. RESULTS: Of the ECM protein mimetics tested, GRGDSP(FN) coated surfaces exhibited the highest adhesion and proliferation capabilities on RTCA at FBS concentration of 0.5% and 1%. When 0.5% FBS was added to ECM protein mimetics during the MTS assay, GRGDSP(FN), REDV(FN), and collagen mimetics, GPKGAAGEPGKP(ColI) showed higher cMSCs proliferation compared with the control. When 1% FBS was added, GRGDSP(FN) and TAIPSCPEGTVPLYS(ColIV) showed significant cMSCs proliferation capacity. CONCLUSIONS: Fibronectin mimetics, GRGDSP(FN) amino acid sequence showed the highest adhesion and proliferation capabilities. In addition, results from RTCA assessment of cell viability correlated well with the tetrazolium-based MTS assay.

New proposed guidelines for early identification of successful myeloid and erythroid engraftment in hematopoietic stem cell transplantation.

BACKGROUND: After hematopoietic stem cell transplantation (HSCT), early detection of engraftment would be of critical value for clinicians. The aim of this study was to identify faster parameters for engraftment. METHODS: We evaluated blood cell parameters including complete blood count (CBC), differential counts, and various reticulocyte parameters in 115 patients who received HSCT (allogeneic, n = 93; autologous, n = 22) in the purpose of identifying possible improved laboratory guidelines for engraftment prediction. RESULTS AND CONCLUSION: Days to white blood cell (WBC) count over 100 cells/µl with more than two-fold increase from nadir after transplantation (proposed new WBC guideline) preceded absolute neutrophil count (ANC) >500 cells/µl by 1.7 days. Among erythroid parameters, the earliest marker for erythroid engraftment was high light scattering reticulocytes (HLR) >0.1 (proposed new red blood cell guideline), which preceded reticulocyte counts (RET) >1% and immature reticulocyte fraction >0.5 by 3.9 and 1.6 days, respectively. Among the clinical parameters compared, those with statistically significant influence on myeloid engraftment were donor type (P = 0.009) and conditioning intensity (P = 0.009). As for erythroid recovery, ABO incompatibility was the only significant factor. In conclusion, the new guidelines may ensure engraftment several days earlier than the conventional parameters, which may help clinicians for decision-making on rescue therapy earlier.

Limited Contribution of Creatine Kinase-Myocardial Band Alongside High-Sensitivity Cardiac Troponin in Diagnosing Acute Myocardial Infarction in an Emergency Department.

Cardiac biomarkers, especially high-sensitivity cardiac troponin C or I (hs-cTnC or hs-cTnI, respectively), are vital for diagnosing acute myocardial infarction (AMI). Despite the specificity of hs-cTn as a biomarker, the creatine kinase-myocardial band (CK-MB) is commonly used alongside hs-cTn in emergency departments (EDs). We analyzed 23,771 simultaneous hs-cTn (hs-cTnT or hs-cTnI) and CK-MB requests for 17,185 patients in tertiary hospital ED in 2022. The objective of this study was to assess their practical value in diagnosing AMI in real-world settings. Among all 17,185 patients tested, 98.0% underwent hs-cTnT and CK-MB tests, and substantially fewer underwent hs-cTnI testing. We observed concordance between the initial hs-cTn and CK-MB results in 71.3% of patients. Of 131 AMI cases, 57 were positive for both biomarkers, 63 for hs-cTn only, and none for CK-MB alone. CK-MB positivity was often found in the absence of AMI. Discrepancies between the hs-cTnT and hs-cTnI results occurred in 30.0% of patients. Indiscriminate CK-MB testing for diagnosing AMI in EDs should be reconsidered. Efficient use of CK-MB is important for reducing costs and ensuring optimal patient care.

Evaluation of Long-Term Adaptive Immune Responses Specific to SARS-CoV-2: Effect of Various Vaccination and Omicron Exposure.

The immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) becomes increasingly complex as individuals receive different combinations of vaccine doses and encounter breakthrough infections. Our study focused on the immunogenicity observed over a two-year period in healthy individuals who completed a two-dose series and then experienced booster and/or Omicron infection. In June 2023, we recruited 78 healthcare workers who had previously participated in clinical research initiated in March 2021 at a single medical center in South Korea. At 1, 5, 11, and 25 months after a second dose, we assessed SARS-CoV-2-specific humoral and cellular immune responses. Longitudinal monitoring revealed a significant decline in humoral immunity levels after the second vaccine dose, followed by a substantial increase post-third vaccination or breakthrough infection. In contrast, stable cellular immune responses were consistently observed, with peak humoral and cellular immune measures reached at 25 months after the second dose. Among infection-naïve participants, three-dose vaccinated individuals had decreased neutralizing activity against wild-type (WT) and negative activities against Omicron subvariants BA.2 and BA.4/5, whereas those who received a fourth dose of bivalent BNT had significantly increased neutralizing activity (p < 0.05). All immune metrics tended to increase as the number of vaccine doses increased. Among participants with 4-exposure, homologous vaccination (mRNA × 4) led to higher humoral immunity, whereas heterologous vaccination (ChAd × 2/mRNA × 2) induced stronger cellular responses against multiple SARS-CoV-2 variants by enzyme-linked immunospot assays (p < 0.05). Immune responses from bivalent vaccines or Omicron infection did not show statistically significant differences among exposure number-matched participants (p > 0.05). Omicron exposure significantly increased cross-neutralizing activity, but magnitude of cellular immunity was not significantly altered by Omicron exposure. Our longitudinal study highlights the evolving complexity of SARS-CoV-2 immune responses, showing enhanced immunity with multiple vaccine doses and robust cellular responses from heterologous vaccination. These findings emphasize the need for ongoing surveillance to optimize vaccination strategies against emerging variants.

Single-cell RNA sequencing revealed the role of the Th17 pathway in the development of anti- human leukocyte antigen antibodies in a highly sensitized mouse model.

Background: The aim of this study is to investigate the specific pathway involved in human leukocyte antigen (HLA) sensitization using single-cell RNA-sequencing analysis and an allo-sensitized mouse model developed with an HLA.A2 transgenic mouse. Methods: For sensitization, wild-type C57BL/6 mouse received two skin grafts from C57BL/6-Tg(HLA-A2.1)1Enge/J mouse (allogeneic mouse, ALLO). For syngeneic control (SYN), skin grafts were transferred from C57BL/6 to C57BL/6. We performed single-cell RNA-sequencing analysis on splenocytes isolated from ALLO and SYN and compared the gene expression between them. Results: We generated 9,190 and 8,890 single-cell transcriptomes from ALLO and SYN, respectively. Five major cell types (B cells, T cells, natural killer cells, macrophages, and neutrophils) and their transcriptome data were annotated according to the representative differentially expressed genes of each cell cluster. The percentage of B cells was higher in ALLO than it was in SYN. Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the highly expressed genes in the B cells from ALLO were mainly associated with antigen processing and presentation pathways, allograft rejection, and the Th17 cell differentiation pathway. Upregulated genes in the T cells of ALLO were involved in the interleukin (IL)-17 signaling pathway. The ratio of Th17 cluster and Treg cluster was increased in the ALLO. On flow cytometry, the percentage of Th17 (IL-17+/CD4+ T) cells was higher and regulatory T cells (FOXP3+/CD4+ T) was lower in the ALLO compared to those in the SYN. Conclusion: Our results indicate that not only the B cell lineage but also the Th17 cells and their cytokine (IL-17) are involved in the sensitization to HLA.

Dextrose-containing fluids causing false-positive serum galactomannan: a case-control study and interrupted time series analysis.

OBJECTIVES: This study aimed to identify the cause of false-positive serum Aspergillus antigen galactomannan (GM) results in our centre. METHODS: We performed a case-control study aiming to elucidate the factors associated with false-positive GM results. Independent risk factors for false-positive GM were evaluated through a multivariable regression analysis. An interrupted time series analysis was used to evaluate the effectiveness of an intervention removing the identified factors. RESULTS: Among 568 patients tested, GM was positive in 130 patients of whom 97 had false-positive GM (cases). These were compared with 427 patients with true-negative GM (controls). Administration of dextrose-containing fluids within 6 days before GM testing was an independent predictor for false-positive GM results (adjusted odds ratio [aOR], 18.60; 95% CI, 8.95-38.66. An analysis of GM presence in different dextrose-containing fluids revealed positivity in 34.8% (8 of 23) (manufacturer A) and 33.3% (5 of 15) (manufacturer B) of the samples. Investigation of the manufacturing process revealed that the saccharification process employed enzymes derived from Aspergillus niger. After identifying the root cause of false positivity, GM-containing dextrose fluid use was restricted. Interrupted time series analysis showed an immediate reduction of GM false-positivity (-6.5% per week, p = 0.045) and a declining trend (-0.33% per week, p = 0.005) postintervention. CONCLUSIONS: Administering dextrose-containing fluids was the primary factor causing false-positive serum Aspergillus antigen GM assay results. Our investigation led to a modification of the manufacturing process of the dextrose-containing fluids.

Current Status of Flow Cytometric Immunophenotyping of Hematolymphoid Neoplasms in Korea.

Background: Flow cytometric immunophenotyping of hematolymphoid neoplasms (FCI-HLN) is essential for diagnosis, classification, and minimal residual disease (MRD) monitoring. FCI-HLN is typically performed using in-house protocols, raising the need for standardization. Therefore, we surveyed the current status of FCI-HLN in Korea to obtain fundamental data for quality improvement and standardization. Methods: Eight university hospitals actively conducting FCI-HLN participated in our survey. We analyzed responses to a questionnaire that included inquiries regarding test items, reagent antibodies (RAs), fluorophores, sample amounts (SAs), reagent antibody amounts (RAAs), acquisition cell number (ACN), isotype control (IC) usage, positive/negative criteria, and reporting. Results: Most hospitals used acute HLN, chronic HLN, plasma cell neoplasm (PCN), and MRD panels. The numbers of RAs were heterogeneous, with a maximum of 32, 26, 12, 14, and 10 antibodies used for acute HLN, chronic HLN, PCN, ALL-MRD, and multiple myeloma-MRD, respectively. The number of fluorophores ranged from 4 to 10. RAs, SAs, RAAs, and ACN were diverse. Most hospitals used a positive criterion of 20%, whereas one used 10% for acute and chronic HLN panels. Five hospitals used ICs for the negative criterion. Positive/negative assignments, percentages, and general opinions were commonly reported. In MRD reporting, the limit of detection and lower limit of quantification were included. Conclusions: This is the first comprehensive study on the current status of FCI-HLN in Korea, confirming the high heterogeneity and complexity of FCI-HLN practices. Standardization of FCI-HLN is urgently needed. The findings provide a reference for establishing standard FCI-HLN guidelines.