Immunofluorescence Research Articles

GZMA suppressed GPX4-mediated ferroptosis to improve intestinal mucosal barrier function in inflammatory bowel disease

BackgroundOur previous study has demonstrated a decreased colonic CD8+CD39+ T cells, enrichment of granzyme A (GZMA), was found in pediatric-onset colitis and inflammatory bowel disease (IBD) characterized by impaired intestinal barrier function. However, the influence of GZMA on intestinal barrier function remains unknown.MethodsWestern blotting(WB), real-time PCR (qPCR), immunofluorescence (IF) and in vitro permeability assay combined with intestinal organoid culture were used to detect the effect of GZMA on intestinal epithelial barrier function in vivo and in vitro. Luciferase, immunoprecipitation (IP) and subcellular fractionation isolation were performed to identify the mechanism through which GZMA modulated intestinal epithelial barrier function.ResultsHerein, we, for the first time, demonstrated that CD8+CD39+ T cells promoted intestinal epithelial barrier function through GZMA, leading to induce Occludin(OCLN) and Zonula Occludens-1(ZO-1) expression, which was attributed to enhanced CDX2-mediated cell differentiation caused by increased glutathione peroxidase 4(GPX4)-induced ferroptosis inhibition in vivo and in vitro. Mechanically, GZMA inhibited intestinal epithelial cellular PDE4B activation to trigger cAMP/PKA/CREB cascade signaling to increase CREB nuclear translocation, initiating GPX4 transactivity. In addition, endogenous PKA interacted with CREB, and this interaction was enhanced in response to GZMA. Most importantly, administration of GZMA could alleviate DSS-induced colitis in vivo.ConclusionThese findings extended the novel insight of GZMA contributed to intestinal epithelial cell differentiation to improve barrier function, and enhacement of GZMA could be a promising strategy to patients with IBD.

Carbohydrate antigens Lewis a and Lewis b act as tumor markers cooperating with CA19.9 in the management of PDAC patients

BackgroundCA19.9 is the unique marker recommended for the preoperative staging and the follow-up of patients suffering from pancreatic ductal adenocarcinoma (PDAC) but up to 30% of PDAC patients maintain normal CA19.9 values and cannot be monitored in this way. Lewis a (Lea Galβ1,3[Fucα1,4]GlcNAc) and b (Leb, Fucα1,2Galβ1,3[Fucα1,4]GlcNAc) are antigens which are structurally similar to sialyl-Lewis a (Siaα2,3Galβ1,3[Fucα1,4]GlcNAc), the epitope of CA19.9. MethodsWe set an ELISA procedure determining the levels of Lea, Leb, and CA19.9 in the blood of healthy individuals or PDAC patients. Moreover, such antigens were also detected in cancer resections by immunofluorescence microscopy, and the levels of glycosyltransferase transcripts involved in Lewis antigen biosynthesis were determined by RT-qPCR. ResultsIn our cohort of 116 healthy individuals, the distribution of circulating Lea and Leb was similar to that of CA19.9, allowing us to set putative cutoff values for both antigens. In a cohort of 115 PDAC patients, the differential distribution with respect to the controls was statistically significant for both antigens (p < 0.001). Out of 37 patients presenting normal CA19.9 values, 15 patients presented Lea or Leb above the cutoffs. By immunofluorescence, Lea, Leb and CA19.9 were all detected in cancer resections and expression levels were heterogeneous among patients in terms of intensity, localization and diffusion. The levels of relevant glycosyltransferase transcripts were found to be heterogeneous between cancers of different patients and no association was detectable with the levels of any circulating antigen. ConclusionsThe concurrent quantification of Lea and Leb together with CA19.9 improves the management of PDAC patients.

High-confidence and high-throughput quantification of synapse engulfment by oligodendrocyte precursor cells.

Oligodendrocyte precursor cells (OPCs) sculpt neural circuits through the phagocytic engulfment of synapses during development and adulthood. However, existing techniques for analyzing synapse engulfment by OPCs have limited accuracy. Here we describe the quantification of synapse engulfment by OPCs via a two-pronged cell biological approach that combines high-confidence and high-throughput methodologies. Firstly, an adeno-associated virus encoding a pH-sensitive, fluorescently tagged synaptic marker is expressed in neurons in vivo to differentially label presynaptic inputs, depending upon whether they are outside of or within acidic phagolysosomal compartments. When paired with immunostaining for OPC markers in lightly fixed tissue, this approach quantifies the engulfment of synapses by around 30-50 OPCs in each experiment. The second method uses OPCs isolated from dissociated brain tissue that are then fixed, incubated with fluorescent antibodies against presynaptic proteins, and analyzed by flow cytometry, enabling the quantification of presynaptic material within tens of thousands of OPCs in <1 week. The integration of both methods extends the current imaging-based assays, originally designed to quantify synaptic phagocytosis by other brain cells such as microglia and astrocytes, by enabling the quantification of synaptic engulfment by OPCs at individual and populational levels. With minor modifications, these approaches can be adapted to study synaptic phagocytosis by numerous glial cell types in the brain. The protocol is suitable for users with expertise in both confocal microscopy and flow cytometry. The imaging-based and flow cytometry-based protocols require 5 weeks and 2 d to complete, respectively.

Luteolin alleviates airway remodeling in asthma by inhibiting the epithelial-mesenchymal transition via β-catenin regulation

BackgroundAsthma is a prevalent long-term inflammatory condition that causes airway inflammation and remodeling. Increasing evidence indicates that epithelial-mesenchymal transition (EMT) holds a prominent implication in airway reconstruction in patients with asthma. Flavonoids obtained from Chinese Materia Medica (CMM), such as Luteolin (Lut), exhibit various beneficial effects in various asthma models. Lut has been shown to mitigate various asthma symptoms, including airway inflammation, hyperresponsiveness, bronchoconstriction, excessive mucus production, pulmonary autophagy, and neutrophilic asthma. However, whether flavonoids can suppress EMT-associated airway remodeling in asthma and the fundamental mechanisms involved remain unclear, with no studies specifically addressing Lut in this context. PurposeTo evaluate the inhibition of airway remodeling in asthma by Lut and its potential mechanisms, while examining the significance of β-catenin in this process through cellular and animal studies. MethodsA BEAS-2B cell model stimulated by lipopolysaccharide (LPS) was established in vitro. Wound closure and Transwell assays were utilized to assess the cellular migratory ability. EMT- and fibrosis-related markers in LPS-stimulated cells were evaluated using RT-qPCR and western blotting. The status of the β-catenin/E-cadherin and β-catenin destruction complexes was evaluated using western blotting, immunofluorescence (IF) staining, and co-immunoprecipitation (Co-IP) analysis. The regulatory function of Lut in β-catenin-dependent EMT was further validated by β-catenin overexpression with adenovirus transduction and siRNA-mediated knockdown of β-catenin. Moreover, the counts of different types of bronchoalveolar lavage fluid (BALF) inflammatory cells from mice with asthma induced by ovalbumin (OVA) were evaluated in vivo using Congo red staining. Hematoxylin and eosin (H&E), Masson's trichrome, and periodic acid-Schiff (PAS) staining were used to evaluate collagen deposition, mucus production, and inflammation in murine lung tissues. Western blotting and immunohistochemistry (IHC) assays were used to assess EMT- and fibrosis-related markers in the lung tissues in vivo. ResultSix naturally derived flavonoids, including Lut, attenuated cell migration and prevented EMT in LPS-treated BEAS-2B cells. Moreover, Lut suppressed TGF-β1, MMP-9, fibronectin (FN), and α-smooth muscle actin (α-SMA) levels in LPS-stimulated BEAS-2B cells. Additionally, Lut downregulated the levels of β-catenin by modulating the β-catenin/E-cadherin and β-catenin destruction complexes, highlighting the pivotal role of β-catenin in EMT inhibition by Lut in LPS-stimulated BEAS-2B cells. Furthermore, Lut suppressed airway inflammation and attenuated EMT-associated airway remodeling through β-catenin blockade in OVA-induced asthmatic mice. The bronchial wall thickness notably reduced from 37.24 ± 4.00 μm in the asthmatic model group to 30.06 ± 4.40 μm in the Lut low-dose group and 24.69 ± 2.87 μm in the Lut high-dose group. ConclusionAccording to our current understanding, this research is the first to reveal that Lut diminishes airway remodeling in asthma by inhibiting EMT via β-catenin regulation, thereby filling a research gap concerning Lut and flavonoids. These results provide a theoretical basis for treating asthma with anti-asthmatic CMM, as well as a candidate and complementary therapeutic approach to treat asthma.

BL-918 Alleviates Early Brain Injury in Rats After Subarachnoid Hemorrhage by Reducing Oxidative Stress and Promoting Mitophagy Through the ULK1/PINK1/Parkin Pathway

Background and purposeOxidative stress plays a critical role in early brain injury (EBI) following subarachnoid hemorrhage (SAH). The small molecule ULK1 agonist, BL-918, demonstrated neuroprotective effects in other central nervous system diseases; however, its role in SAH has not yet been explored. This study aimed to evaluate whether BL-918 could provide neuroprotective effects in rats following SAH. MethodsAn SAH model was established in Sprague-Dawley rats using endovascular perforation. BL-918 was administered intraperitoneally after SAH, while the ULK1 inhibitor SBI was given intraperitoneally prior to SAH modeling. PINK1 siRNA was administered into the lateral ventricle before SAH induction. The neuroprotective effects and mechanisms of BL-918 were assessed through SAH grading, brain water content measurement, blood-brain barrier permeability, neurobehavioral tests, Western blot, immunofluorescence, TUNEL staining, DHE staining, and transmission electron microscopy (TEM). ResultsAfter SAH, the expression levels of p-ULK1, PINK1, Parkin, and LC3Ⅱ increased, peaking at 24 hours post-SAH. BL-918 treatment improved neurological function in rats, reduced brain water content and blood-brain barrier permeability, and exhibited anti-oxidative stress and anti-apoptotic effects. Western blot analysis revealed that BL-918 increased the expression of p-ULK1, PINK1, Parkin, LC3Ⅱ, Bcl-xl, and Bcl-2 while inhibiting the expression of Bax and Cleaved Caspase-3. Oxidative stress-related indicators showed that BL-918 alleviated oxidative stress. Immunofluorescence and TEM results demonstrated that BL-918 promoted mitophagy and preserved mitochondrial morphology. Furthermore, the positive effects of BL-918 were reversed by SBI and PINK1 siRNA, respectively. ConclusionBL-918 promoted mitophagy through the ULK1/PINK1/Parkin signaling pathway, reduced oxidative stress following SAH, and improved both short-term and long-term neurological impairments. Thus, BL-918 treatment may offer a novel therapeutic approach for patients with SAH.

Inhibition of BCAT1 expression improves recurrent miscarriage by regulating cellular dysfunction and inflammation of trophoblasts.

Sustained or chronic inflammation in the placenta can result in placental insufficiency, leading to adverse reproductive outcomes such as pregnancy loss. Branched-chain amino acid transaminase 1 (BCAT1) expresses in the placenta and is involved in the pathological inflammatory response, but its role in recurrent miscarriage (RM) has not been fully investigated. In the present study, we delved into the effects of BCAT1 on trophoblast inflammation induced by lipopolysaccharide (LPS) and a mouse model of pregnancy loss induced by LPS. In vitro, after the HTR-8/SVneo cells were treated with LPS and BCATc inhibitor 2 (a selective BCAT inhibitor), the cell apoptosis was verified by TUNEL assay, and the activity of caspase-3 and caspase-9 was detected. Real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) were used to determine the expression of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and inflammasomes (NLRP3 and ASC) in LPS-treated trophoblast cells. Western blot analysis was performed to verify the expression of phospho-IκBα (p-IκBα) in cells and NF-κB p65 in the nuclei. IF staining was used to detect the nuclear translocation of NF-κB p65. The DNA binding activity of NF-κB was detected by an electrophoretic mobility shift assay (EMSA). The results demonstrated that inhibition of BCAT1 reduced trophoblast apoptosis, suppressed the release of proinflammatory cytokines, and prevented NLRP3 inflammasome activation in response to LPS. Additionally, BCAT1 inhibition blocked the activation of the NF-κB pathway in trophoblasts. This study highlights the potential therapeutic role of targeting BCAT1 in preventing adverse reproductive outcomes associated with chronic placental inflammation.

B-316 Evaluation of an FDA-cleared Chromogranin A Assay on an Automated Analyzer

Abstract Background Chromogranin A (CgA) is a hydrophilic and acidic protein, present in the chromaffin granules of neuroendocrine cells. It is the preferred tumor marker for the monitoring of neuroendocrine tumors (NETS), which are often found in the lungs, appendix, pancreas, and gastrointestinal tract. Previous CgA assays were performed by manual ELISA testing, and were not FDA-approved, leading to a longer, more rigorous validation process and longer run times. The introduction of an automated, FDA-cleared CgA assay allows the laboratory to process patient samples more quickly and reliably, shortening a multiple hour process to minutes. The objective of this study was to evaluate the performance of an automated CgA assay recently cleared by the FDA. Methods We validated the the B·R·A·H·M·S™ Chromogranin A II (CGAII) assay on the automated B·R·A·H·M·S KRYPTOR Compact PLUS (Thermo Fisher Scientific Inc.). The CGAII assay is an immunofluorescent assay, which uses Time-Resolved Amplified Cryptate Emission (TRACE) to quantitate CGA in human serum. Assay performance characteristics were evaluated including measuring range (MR) and maximum dilution, precision, method comparison, stability, reference interval, stability, and carryover. Results The MR for CGAII was established with triplicate analysis of serum at 8 levels, ranging from 25.8 - 2,700 ng/mL. Linearity was demonstrated with a slope of 0.907, intercept of 1.948 and observed error of 2.54 ng/mL. Recovery at each point ranged from 83.3-110.3%. Carryover (&amp;lt;5.0 ng/mL) was not observed at a concentration approximately 2 times the upper limit of the MR. A 1:25 or 1:500 automatic dilution was determined to be acceptable (% bias &amp;lt;16%). Intraday precision coefficient of variation (CV) ranged from 4.1-1.7% (SDs from 2.32-6.64 ng/mL) at concentrations of 56.7 and 393.0 ng/mL. The interday CVs ranged from 6.0-7.1% (SDs from 3.43 - 28.84 ng/mL) at concentrations of 57.5 and 403.8 ng/mL. Twenty samples with concentrations ranging from 56.4-2,907 ng/mL were compared to a reference laboratory utilizing a similar methodology. The correlation coefficient was 0.9934, with a slope of 1.014, and intercept of -32.95. An additional twenty samples with concentrations ranging from 67.0-2,446 ng/mL were compared to a CgA ELISA kit (CGA-ELISA-NG, CISBIO) to determine the bias between the two methodologies. The correlation coefficient was 0.9902, with a slope of 0.845, and intercept of -15.31, and a bias of -19.9%. The serum samples were stable for 3 days when stored at 2-8°C. Recovered CgA concentrations fell outside of the 95% confidence interval after day 3 at 2-8°C. The manufacturer provided reference interval of &amp;lt;187 ng/mL was verified by testing 20 healthy adult samples, all of which fell within this range. Conclusions This assay has been validated and shown to be an accurate and precise method for monitoring CgA. Further, the method was validated on an automated analyzer to allow expeditious turnaround time in a core laboratory.

B-104 Correlation of Anti-Centromere Antibodies (ACA) Detection by Indirect Immunofluorescence (IFA) on HEp-2 Cells and by ELISA Demonstrates High Concordance but Highlights Advantage of IFA

Abstract Background Anti-centromere antibodies (ACA), a type of anti-nuclear antibody (ANA) first identified as a discrete speckled pattern on indirect immunofluorescence assay (IFA), are a hallmark of systemic sclerosis (SSc), especially in its limited cutaneous form (lcSSc) or CREST (calcinosis, Raynaud’s, esophageal dysmotility, sclerodactyly and telangiectasia). These mitosis-related autoantibodies mainly recognize three centromere proteins (CENP), CENP-A, CENP-B, and CENP-C, that comprise the kinetochore important to chromosome segregation, but more recently, at least three other CENP antigens have been identified (CENP-D, CENP-E and CENP-F). Anti-CENP-B is considered the main autoantibody, and many, if not most, ELISA utilize recombinant CENP-B such that antibodies to CENP antigens other than CENP-B are not detected. Here, an ELISA using both CENP-B and CENP-A is compared to the reference method, HEp-2 cell IFA, highlighting its high, but still less than 100%, sensitivity due to multiple centromere subcomponents. Methods Anti-centromere antibodies are determined in patient serum using the reference method, IFA on the HEp-2 cell substrate (Sprinter XL/EUROpattern®, Euroimmun, NJ) which is also the current method for standalone ACA testing as well as screening by selected ANA profiles designed for the rheumatology specialist. Here ACA IFA- positive and negative serum samples were further analyzed by enzyme immunosorbent assay (ELISA) utilizing recombinant CENP-A and CENP-B (QuantaLyzer® 3000, Werfen, CA). Results When compared, samples between ACA pattern by HEp-2 cell IFA and anti- centromere antibodies by ELISA yielded an agreement of 93.2%. McNemar’s Test Chi Square of p&amp;gt;0.999, with a relative sensitivity of 89.7% and a specificity of 100.0%. Conclusions This study demonstrated very high concordance between ELISA and accurate ACA pattern identification on HEp-2 IFA. The ELISA studied here identifies autoantibodies to both CENP-A and CENP-B where many other ELISA identify only anti-CENP-B. Although ACA are relatively specific for SSc, they have also been reported in systemic lupus erythematosus (SLE), primary biliary cholangitis (PBC), rheumatoid arthritis (RA), Sjogren Syndrome, Raynaud's phenomenon and in cancer in individuals with or without evidence of systemic autoimmune rheumatic disease (SARD). Of note, many other commercially available ELISA for anti-centromere antibodies identify only anti-CENP-B, but there at least four other known CENP antigen targets for autoantibodies causing centromere pattern on IFA. Furthermore, almost all laboratories use the ELISA for ACA on ANA panel (or other connective tissue disease) screening. Hence, it should be duly noted that these testing strategies will not detect autoantibodies to all anti-centromere antibodies. Only specialized ANA profiles designed for the difficult-to-diagnose autoimmune patient begin screening using the IFA method for anti-centromere and therefore, are capable of detecting autoantibodies to all CENP antigens. Laboratorians should advise clinicians of these diagnostic limitations especially as our knowledge about centromere subcomponents and their clinical associations increases.

Enhancing diagnostic accuracy: Direct immunofluorescence assay as the gold standard for detecting Giardia duodenalis and Cryptosporidium spp. in canine and feline fecal samples

The enteric protozoan parasites Giardia duodenalis and Cryptosporidium spp. are common cause of diarrhea in pet dogs and cats, affecting primarily young animals. This comparative study evaluates the diagnostic performance of conventional and molecular methods for the detection of G. duodenalis and Cryptosporidium spp. infection in dogs and cats.The compared diagnostic assays included merthiolate-iodine-formalin (MIF) method, lateral flow immunochromatography rapid test (ICT) and real-time PCR; using direct immunofluorescence assay (DFA) as golden standard. The study included the analysis of 328 fecal samples from different dog (n = 225) and cat (n = 103) populations.According to DFA, the overall prevalence of G. duodenalis was 24.4% (80/328, 95% CI: 19.8–29.4), varying from 11.6% (12/103, 95% CI: 6.2–19.5) in cats to 30.2% (68/225, 95% CI: 24.3–36.7) in dogs. The overall prevalence of Cryptosporidium spp. was 4.0% (13/328, 95% CI: 2.1–6.7), varying from 2.9% (3/103, 95% CI: 0.6–8.3) in cats to 4.4% (10/225, 95% CI: 2.1–8.0) in dogs. MIF was only used for the detection of G. duodenalis, which was identified by this method in 22.7% of dogs and 7.8% of cats, respectively. DFA was the most sensitive technique for detecting G. duodenalis in samples from dogs and cats (p-value: < 0.001), followed by real-time PCR. Identification of Cryptosporidium infections was most effectively accomplished by the combination of DFA and PCR technique (p-value: < 0.001). In addition, epidemiological (sex, age, origin) and clinical (fecal consistency) variables were collected to assess their potential associations with an increased likelihood of infection by G. duodenalis and/or Cryptosporidium spp. Breeder dogs were more likely to harbor G. duodenalis infection (p-value: 0.004), whereas female cats were significantly more infected with Cryptosporidium (p-value: 0.003).In conclusion, DFA (alone or in combination with PCR) has been identified as the most accurate and cost-effective method for detecting G. duodenalis and Cryptosporidium spp. in fecal samples from pet dogs and cats. This highlights their importance in both veterinary and clinical settings for enabling prompt treatment and preventing potential transmission to humans.

Generation and Characterization of hiPS Lines from Three Patients Affected by Different Forms of HPDL-Related Neurological Disorders

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene, leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear, though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here, we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations, using episomal reprogramming. The patients’ clinical characteristics are carefully listed. The hiPS lines were meticulously characterized, demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4, TRA1-60, NANOG, and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition, HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation, similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83, offering insights into its molecular mechanisms and potential for developing targeted therapies.

B-082 Clinical Investigation of Rods and Rings Pattern in Anti-Nuclear Antibody Test in Korean Patients

Abstract Background In the mid-2000s, a novel pattern in antinuclear antibody (ANA) testing was reported. Indirect immunofluorescence assays on HEp-2 cells, conducted with sera from patients with Hepatitis C virus (HCV) infection, revealed cytoplasmic structures in the form of rods and rings (RR). These structures typically displayed rod lengths of 3-10μm and ring diameters of 2-5μm. Inosine monophosphate dehydrogenase (IMPDH) was identified as the primary component of the RR structures. Research on the administration of interferon/ribavirin (IFN/RBV) showed that ribavirin, an IMPDH inhibitor, induced RR formation in over 95% of cells. Additionally, RR patterns were reported in patients receiving other IMPDH inhibitors such as mycophenolic acid and azathioprine. While cytidine triphosphate synthase 1 (CTPS1) was suggested to be associated with RR structures, there were no reported cases in patients. Interestingly, patients receiving methotrexate, which does not directly inhibit IMPDH but hinders GTP biosynthesis, also exhibited RR structures. To explore the clinical implications of specimens showing RR patterns, a study was conducted to investigate the actual clinical scenarios associated with these findings. Methods This is a retrospective study conducted at a single center, covering the period from January 2022 to December 2023, focusing on the results of ANA testing. The ANA tests were performed using HEp-2 cells (FLUORO HEPANA TEST, MBL, Nagoya, Japan) on slides manufactured with AP 16 IF Plus (das, Palombara Sabina, RM, Italy). ANA results were obtained through qualitative testing, ranging from 1+ to 4+, or quantitative testing based on dilutions ranging from 1:40 to 1:640, depending on clinical requirements. Results Out of the 7,648 ANA test results, the RR pattern was observed in 40 cases (0.5%). Among these, 2.5% had a history of HCV infection with IFN/RBV treatment, and an additional 2.5% had concurrent use of IMPDH inhibitors. There were no reported cases of medication history related to the synthetic pathway of nucleic acids, including CTPS1. Cases where RR patterns coexisted with autoimmune diseases accounted for 35%, infections for 25%, and other inflammatory conditions for 22.5%. RR patterns reported with an intensity of 3+ or at dilutions of 1:160 or higher constituted 27.5%. Among cases displaying strong fluorescence, 45% were associated with infections, 18% with autoimmune conditions, and another 18% with unexplained inflammatory causes. In the overall sample, elevated CRP or ESR was noted in 54.5% of cases with infections, autoimmune diseases, or other inflammatory conditions. Among samples with intense intensity, 55.5% showed elevated CRP or ESR when inflammatory conditions were present. Conclusions The majority of cases exhibiting the RR pattern showed no history of exposure to IMPDH inhibitors or drugs affecting the nucleic acid synthetic pathway. A high proportion of cases clinically diagnosed with inflammation showed a concurrent RR pattern, with only approximately half of them demonstrating elevated CRP or ESR levels. This study suggests the possibility of identifying inflammatory responses not captured by conventional acute inflammation markers through the RR pattern observed in ANA testing.

B-076 Detection of KLHL11 Autoantibodies Using a New Cell-based Indirect Immunofluorescence Assay

Abstract Background Autoantibodies against kelch-like protein 11 (KLHL11) were first described in 2019 as markers of paraneoplastic encephalitis. Early diagnosis and treatment are imperative to improve prognosis and outcome of affected patients. Rat brain immunohistochemistry was reported to be not useful in routine screening for KLHL11 antibodies. This study evaluates the performance of a new recombinant cell-based assay for the standardized detection of KLHL11-specific IgG and reports the characteristics of the examined patients. Methods Serum (n=9), CSF (n=5) and/or plasmapheresis (n=1) samples from 10 patients with clinical presentations compatible with anti-KLHL11 encephalitis as well as sera from 100 healthy blood donors were analyzed using a prototype indirect immunofluorescence assay (IFA; EUROIMMUN) based on recombinant HEK293 cells expressing human KLHL11. Results KLHL11 autoantibodies were detected at high titers in all samples (serum &amp;gt;1:1,000; CSF &amp;gt;1:320; plasma &amp;gt;1:100,000) from all (10/10) patients but in none (0/100) of the blood donors, indicating 100% sensitivity and specificity. Anti-KLHL11-positive patients had a median age of 52 years (range 27-71) and 80% were male. The most common reported clinical presentations were cerebellar syndrome (ataxia), brainstem diencephalic encephalitis, rhombencephalitis and limbic encephalitis. Testicular or ovarian tumors were found in 87.5% (7/8) of the cases with available results from malignancy screening. Conclusions The new cell-based IFA enables the sensitive and specific detection of KLHL11 autoantibodies, thus supporting the diagnosis of patients with predominantly paraneoplastic brainstem cerebellar syndrome. Future studies will evaluate assay performance in larger patient cohorts to address the reported association of KLHL11 autoimmunity with a wider spectrum of syndromes and tumors.

B-053 Four years summary of flow cytometry live cell based assay for detecting serum anti-aquaporin 4 IgG and anti-myelin oligodendrocyte glycoprotein IgG in a Korean reference laboratory

Abstract Background In the diagnosis of neuromyelitis optica spectrum disorder (NMOSD), it is important to sensitively detect anti-aquaporin 4 (AQP4) IgG and anti-myelin oligodenrocyte glycoprotein (MOG) IgG in patients' serum. NMOSD is a disease that shows 3-7 times more common prevalence in Far East Asia than in the West. According to the IPND diagnostic criteria, it is recommended to diagnose as a cell based assay using full length recombinant protein. In this study, we would like to review 4 years results of AQP4, MOG requested to Eone laboratories in Korea performed with flow cytometry live cell based assay (FC-CBA). Methods FC-CBA was performed using piRES2 cell transfected with AQP4, MOG and stained with DsRed2. Subcultured cells were counted with 10^6, and reacted with patients' serum for 1 hour in a refrigerated state. Thereafter, reaction with FITC anti-human IgG for 2nd antibody for 30 minutes, and 10,000 cells were read from Navius (Beckman Coulter). The mean fluorescence intensity (MFI) ratio , which is the ratio obtained by dividing the MFI value of both FITC and DsRed2 positive cells and DsRed2 positive cells to negative cells, was used to determine positive, borderline, and negative for the serum sample. AQP4 reviewed cases during the period from November 2018 to January 2024, and MOG reviewed cases during the period from December 2019 to January 2024. Indirect immunofluorescence assay using formalin fixed cells is commercially available and compared to the FC-CBA of this institution. Results During the study period, 4,733 cases of AQP4 were performed, and 23,540 cases of MOG were performed by FC-CBA. Among them, AQP4 was positive in 405 cases (8.5%) and borderline in 71 cases (1.5%), and all others were negative. MOG was 2,597 positive (11%), borderline in 1,144 cases (4.8%), and all others were negative. The positive and borderline cutoffs for MFI ratio were changed during the study period, and the positive rate was significantly changed. Conclusions Eone laboratories are the only institution in Korea that performs live cell based assay using flow cytometry for AQP4 and MOG IgG, and receives samples from patients suspected of NMOSD at university hospitals. When reading with MFI ratio, it is important to properly set the cutoff to determine the positivity rate.

B-105 Farr Radioimmunoprecipitation Method Enhances Serial Monitoring of Quantitative Anti-dsDNA (double-stranded) Antibody Measurements in Lupus

Abstract Background Anti-double stranded DNA (anti-dsDNA) antibodies are an important criterion in the diagnosis, classification, and management of systemic lupus erythematous (SLE). High avidity anti-dsDNA IgG may be more clinically relevant and specific for SLE. As such, radioimmunoassay according to the Farr technique (Farr-RIPA) is often considered more sensitive and quantitative than enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae immunofluorescence assay (CLIFT) methods. Methods Measurements of anti-dsDNA antibodies in 147,016 serum patient samples were obtained by lab-developed Farr-RIPA. Serum with endogenous antibodies binds to 125I-radiolabelled dsDNA and undergoes subsequent immunoprecipitation under high salt conditions which selects for highly avidity anti-dsDNA antibodies. The analytic measurement range (AMR) of the assay is 5.7 to 85 IU/mL, and dilutional linearity is validated to 68,000 IU/mL for results &amp;gt;85 (ULOQ). Inter-assay precision is &amp;lt;10% CV. Results Of 147,016 measured samples, 5.4% (7800) were positive for anti-ds DNA antibodies (mean 251.7) with numeric values, ranging from 8.0 to 55,399 IU/mL. The numeric distribution of all positive results was such that 76% (5859/7800) were between 8.0 - 60 IU/mL, and the median was 19.4 with an interquartile range (IQR) of 36.1. A small percentage (3.9%) had very high anti-dsDNA levels greater than 1000 IU/mL and 0.4% (35/7800) were greater than 10,000 IU/mL. Serial measurements comprised almost half (45.5%) of all positives, occurring in 1016 unique patients an average of 3.5 levels within a 2-year time frame. A fifth of those patients, 200, were more frequently monitored with at least 4 levels. Of patients with the very highest dsDNA (&amp;gt;10,000), we analyzed the results of 17 unique patients who were frequently monitored an average of 6.1 times. Peak and nadir values yielded a mean delta of 20,751 with a mean of 264 days elapsing between peak and nadir. All except one showed a marked decrease in Anti-dsDNA over time with an average decrease of 15,685 IU/mL. Conclusions Here, we performed 147,016 Anti-dsDNA antibody measurements by Farr-RIPA, a precise method with a broad analytic range as compared to other methods which have a limited upper limit of quantitation or are semi-quantitative. Our 7800 positive Farr-RIPA results exhibited quantitative results from 8.0 to 55,399 IU/mL. The median of 19.4 and IQR of 36.1 showed most results to be &amp;lt;55.5 IU/mL. Very high levels &amp;gt;300 and &amp;gt;1000 IU/mL were seen in 10% and 4% of patient samples, respectively. Serial monitoring was frequent. In patients with the very highest levels &amp;gt;10,000, an-eight fold decrease in peak Anti-dsDNA levels was achieved over a mean of 264 days with more than half (9/17) achieving levels &amp;lt;250. The ability to detect and meaningfully monitor these patients is enhanced by the Farr-RIPA method. In conclusion, in 7800 positive samples, we demonstrated a wide range of inter- and intra-patient numeric Anti-dsDNA concentrations that denote the clinical usefulness of serial monitoring by Farr-RIPA.

Pestivirus bovis is implicated in repeated abortions and subfertility of vaccinated cattle in Egypt

The study of Pestivirus bovis, formerly known as Bovine Viral Diarrhea Virus (BVDV), is of significant importance due to the economic challenges it poses in cattle herds in several countries. This virus leads to decreased productivity, reproductive failures, and increased susceptibility to secondary infections. The current study aimed to investigate the role of BVDV in abortion and infertility among cattle in Egypt using advanced diagnostic techniques. We employed a one-step multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) to detect BVDV, and the results showed an overall prevalence of 11.2% among 178 tested samples. Notably, 11.2% tested positive for BVDV-1, with higher detection rates in adult cows (12.1%) compared to calves (9.85%). Importantly, 13.8% of samples from dams with subfertility and repeated abortions were positive for BVDV-1. The results of rRT-PCR guided virus isolation using Madin–Darby bovine kidney (MDBK) cells. Identifying cytopathic effects (CPEs) in 15% of the samples, consistent with cytopathic-BVDV (CP-BVDV). An indirect fluorescent antibody assay (IFA) confirmed the presence of CP-BVDV in these samples. For non-cytopathic (NCP)-strains, the immunoperoxidase (IP) test using bovine turbinate (BT) cells was more effective, detecting NCP-BVDV in samples without CPE in MDBK cells. The presence of mixed infection was indicated by an isolate from a diarrheic calf, showing positive results for both CP-BVDV and NCP-BVDV. Plaque assays further confirmed CP-BVDV and NCP-BVDV isolation from mixed infections, highlighting the selective amplification of both biotypes. These findings underscore the significant role of BVDV-1 in reproductive disorders in cattle and the importance of employing comprehensive diagnostic methods for effective control and management strategies. Our study provides valuable insights that can guide future studies and improve herd health and productivity.

Comprehensive analysis of bulk, single-cell RNA sequencing, and spatial transcriptomics revealed IER3 for predicting malignant progression and immunotherapy efficacy in glioma

BackgroundAs part of stress-triggered molecules, immediate early response 3 (IER3) dysregulation has been reported to sustain pro-oncogenic pathways and precede malignant transformation. However, the role of IER3 in glioma pathology is ill-defined.MethodsImmunohistochemistry (IHC) assay and publicly available glioma datasets were used to calculate the IER3 expression level in glioma. Wound healing, invasion and cell counting kit-8 (CCK8) assays were applied to measure the cell viability and capacities of migration and invasion of glioma cells in vitro. The immunofluorescence (IF) assay was used to assess the expression associations of IER3 with CCL2 and TGFBI. Cox regression analysis and Kaplan-Meier (K-M) curve were introduced to compute the prognosis-predicting value of IER3. Variations in copy number (CNVs), single nucleotide (SNVs), and methylation profiles were analyzed to illustrate the epigenetic modifications of IER3. Gliomas were divided into two subgroups using the restricted cubic spline (RCS) method.Results IER3was overexpressed and hypomethylated in gliomas and significantly associated with the dismal prognosis of glioma samples. Samples in the high IER3 subgroup were characterized by increased infiltration of tumor-associated monocytes/macrophages (TAMMs), as well as the elevated sensitivity to Dabrafenib, an inhibitor of BRAF. In addition, this subgroup demonstrated a low mutation rate of IDH, high gain rates of BRAF, ELTD1, and PDGFA. Gliomas with relatively low IER3 expression demonstrated a less invasive subtype and were featured by favorable prognosis, increased response to immunotherapy, and adjuvant chemotherapy plus radiotherapy. The IF assay revealed that IER3 was co-localized and co-expressed with TGFBI. The glioma cells with small interfering RNA (siRNA)-silenced IER3 displayed lower migration, invasion, proliferation, and cell viability than the control group.ConclusionsIn this study, we identified IER3 upregulation as an essential biomarker that could assist in adjuvant therapy and prognosis prediction for gliomas.

Isoliquiritigenin mitigates intervertebral disc degeneration induced by oxidative stress and mitochondrial impairment through a PPARγ-dependent pathway

ObjectivesOxidative stress, mitochondrial dysfunction, and apoptosis play significant roles in the degradation of extracellular matrix (ECM) in nucleus pulposus cells (NPCs), ultimately contributing to intervertebral disc degeneration (IVDD). This study investigates the potential of isoliquiritigenin (ISL), a natural extract known for its antioxidant, anti-inflammatory, and anti-atherosclerotic properties, to alleviate IVDD. MethodsThe viability of NPCs treated with ISL and tert-butyl hydroperoxide (TBHP) was assessed using the CCK-8 assay. Various techniques, including Western blot, qRT-PCR, immunofluorescence (IF), and immunohistochemistry, were employed to measure the expression of ECM components, oxidative stress markers, and apoptosis-related proteins. Mitochondrial function was evaluated through Western blot and IF analyses. Network pharmacology predicted ISL targets, and the expression levels of PPARγ were assessed using the aforementioned methods. The role of PPARγ in the therapeutic effects of ISL on IVDD was examined through siRNA knockdown. The therapeutic impact of ISL on puncture-induced IVDD in rats was evaluated using X-ray, MRI, and histological staining techniques. ResultsIn vitro, ISL reduced oxidative stress in NPCs, restored mitochondrial function, inhibited apoptosis, and improved the ECM phenotype. In vivo, ISL slowed the progression of IVDD in a rat model. Further analysis revealed that ISL enhances PPARγ activity and promotes its expression by direct binding, contributing to the delay of IVDD progression. ConclusionThis study demonstrates that ISL effectively treats puncture-induced IVDD in rats by inhibiting oxidative stress, restoring mitochondrial function, and reducing NPC apoptosis through a PPARγ-dependent mechanism. By balancing ECM synthesis and degradation, ISL presents a novel therapeutic approach for IVDD and identifies a promising target for treatment.

A proteomic atlas of glypican‐3 interacting partners: Identification of alpha‐fetoprotein and other extracellular proteins as potential immunotherapy targets in liver cancer

AbstractAntibody and cell‐based therapeutics targeting cell surface receptors have emerged as a major class of immune therapeutics for treating cancer. However, the number of cell surface targets for cancer immunotherapy remains limited. Glypican‐3 (GPC3) is a cell surface proteoglycan and an oncofetal antigen. In this study, we report a large‐scale tumor‐associated GPC3 co‐immunoprecipitation‐proteomic study using liver cancer xenograft tumors in mice. We identified 153 GPC3‐associated proteins through mass spectrometry. To identify potential drug targets, we categorized GPC3‐associated proteins based on their subcellular locations using UniProt annotations, with a focus on extracellular proteins. Additionally, we annotated differentially expressed proteins in hepatocellular carcinoma versus nontumor liver samples based on the literature, analyzed expression levels in tumor versus normal tissues using TCGA and GTEx databases via GEPIA, and identified prognostic liver cancer markers according to GEPIA. Among GPC3‐associated proteins, immunoglobulin superfamily member 1, alpha‐fetoprotein (AFP), FAT atypical cadherin 1, formin 1, and guanylate cyclase 2 C, were identified as potential therapeutic targets. Furthermore, we validated the direct protein interaction between GPC3 and AFP through immunoprecipitation with purified proteins and through co‐localization imaging using immunofluorescence microscopy. This study provides large proteomic datasets related to GPC3‐associated proteins, enhancing our understanding of glypican biology in cancer cells and offering a new approach to identifying immunotherapy targets.

Bioreactor-produced iPSCs-derived dopaminergic neuron-containing neural microtissues innervate and normalize rotational bias in a dose-dependent manner in a Parkinson rat model

A breadth of preclinical studies now support the rationale of pluripotent stem cell-derived cell replacement therapies to alleviate motor symptoms in Parkinsonian patients. Replacement of the primary dysfunctional cell population in the disease, i.e. the A9 dopaminergic neurons, is the major focus of these therapies. To achieve this, most therapeutical approaches involve grafting single-cell suspensions of DA progenitors. However, most cells die during the transplantation process, as cells face anoïkis. One potential solution to address this challenge is to graft solid preparations, i.e. adopting a 3D format. Cryopreserving such a format remains a major hurdle and is not exempt from causing delays in the time to effect, as observed with cryopreserved single-cell DA progenitors. Here, we used a high-throughput cell-encapsulation technology coupled with bioreactors to provide a 3D culture environment enabling the directed differentiation of hiPSCs into neural microtissues. The proper patterning of these neural microtissues into a midbrain identity was confirmed using orthogonal methods, including qPCR, RNAseq, flow cytometry and immunofluorescent microscopy. The efficacy of the neural microtissues was demonstrated in a dose-dependent manner using a Parkinsonian rat model. The survival of the cells was confirmed by post-mortem histological analysis, characterised by the presence of human dopaminergic neurons projecting into the host striatum. The work reported here is the first bioproduction of a cell therapy for Parkinson's disease in a scalable bioreactor, leading to a full behavioural recovery 16 weeks after transplantation using cryopreserved 3D format.

Comprehensive Proteomic Analysis of Dysferlinopathy Unveiling Molecular Mechanisms and Biomarkers Linked to Pathological Progression.

Previous proteomics studies in dysferlinopathy muscle have been limited in scope, often utilizing 2D-electrophoresis and yielding only a small number of differential expression calls. To address this gap, this study aimed to employ high-resolution proteomics to explore the proteomic landscapes of dysferlinopathy and analyze the correlation between muscle pathological changes and alterations in protein expression in muscle biopsies. We conducted a comprehensive approach to investigate the proteomic profile and disease-associated changes in the muscle tissue proteome from 15 patients with dysferlinopathy, exhibiting varying degrees of dystrophic pathology, alongside age-matched controls. Our methodology encompasses tandem mass tag (TMT)-labeled liquid chromatography-mass spectrometry (LC-MS/MS)-based proteomics, protein-protein interaction (PPI) network analysis, weighted gene co-expression network analysis, and differential expression analysis. Subsequently, we examined the correlation between the expression of key proteins and the clinical characteristics of the patients to identify pathogenic targets associated with DYSF mutations in dysferlinopathy. A total of 1600 differentially expressed proteins were identified, with 1321 showing high expression levels and 279 expressed at lower levels. Our investigation yields a molecular profile delineating the altered protein networks in dysferlinopathy-afflicted skeletal muscle, uncovering dysregulation across numerous cellular pathways and molecular processes, including mRNA metabolic processes, regulated exocytosis, immune response, muscle system processes, energy metabolic processes, and calcium transmembrane transport. Moreover, we observe significant associations between the protein expression of ANXA1, ANXA2, ANXA4, ANXA5, LMNA, PYGM, and the extent of histopathologic changes in muscle biopsies from patients with dysferlinopathy, validated through immunoblotting and immunofluorescence assays. Through the aggregation of expression data from dysferlinopathy-impacted muscles exhibiting a range of pathological alterations, we identified multiple key proteins associated with the dystrophic pathology of patients with dysferlinopathy. These findings provide novel insights into the pathogenesis of dysferlinopathy and propose promising targets for future therapeutic endeavors.