Specific Antibodies Research Articles

Seroprevalence and risk factors for toxoplasmosis and toxoplasmosis/cysticercosis coinfection in patients with schizophrenia: a case-control study

The protozoan Toxoplasma gondii and the helminth Taenia solium have been associated with the development of psychiatric diseases, including schizophrenia. This study aimed to determine the seroprevalence of these infections alone and/or in coinfection in a group of patients with schizophrenia and their respective control groups, in addition to assessing the epidemiological profile of these populations and risk factors. Serum samples from 31 individuals with schizophrenia and 31 control individuals were investigated for the presence of specific antibodies against T. solium and/or T. gondii by the ELISA method. A questionnaire was administered to all participants to determine the risk factors for the infections assessed. Toxoplasmosis is present in 58.1% of individuals with schizophrenia and 48.4% of control individuals. Toxoplasmosis/cysticercosis coinfection was observed in 6.5% of individuals with schizophrenia. The act of washing hands before meals was associated with infection by T. gondii in individuals with schizophrenia. There is a high seroprevalence of toxoplasmosis in individuals with schizophrenia, where the lack of hygienic habits is an important risk factor for acquiring the disease. The identification of toxoplasmosis/cysticercosis coinfection exclusively in individuals with schizophrenia demonstrates that both infections may be prevalent in individuals with this disorder.

Core fucosylation within the Fc-FcγR degradation pathway promotes enhanced IgG levels via exogenous L-fucose

α1,6-Fucosyltransferase (Fut8) is the enzyme responsible for catalyzing core fucosylation. Exogenous L-fucose upregulates fucosylation levels through the GDP-fucose salvage pathway. This study investigated the relationship between core fucosylation and IgG amounts in serum utilizing wild-type (Fut8+/+), Fut8 heterozygous knockout (Fut8+/−), and Fut8 knockout (Fut8−/−) mice. The IgG levels in serum were lower in Fut8+/− and Fut8−/− mice compared with Fut8+/+ mice. Exogenous L-fucose increased IgG levels in Fut8+/− mice, while the ratios of core fucosylated IgG versus total IgG showed no significant difference among Fut8+/+, Fut8+/−, and Fut8+/− mice treated with L-fucose. These ratios were determined by Western blot, lectin blot, and mass spectrometry analysis. Real-time PCR results demonstrated that mRNA levels of IgG Fc and neonatal Fc receptor, responsible for protecting IgG turnover, were similar among Fut8+/+, Fut8+/−, and Fut8+/− mice treated with L-fucose. In contrast, the expression levels of Fc-gamma receptor Ⅳ (FcγRⅣ), mainly expressed on macrophages and neutrophils, were increased in Fut8+/− mice compared to Fut8+/+ mice. The effect was reversed by administrating L-fucose, suggesting that core fucosylation primarily regulates the IgG levels through the Fc-FcγRⅣ degradation pathway. Consistently, IgG internalization and transcytosis were suppressed in FcγRⅣ-knockout cells while enhanced in Fut8-knockout cells. Furthermore, we assessed the expression levels of specific antibodies against ovalbumin and found they were downregulated in Fut8+/− mice, with potential recovery observed with L-fucose administration. These findings confirm that core fucosylation plays a vital role in regulating IgG levels in serum, which may provide insights into a novel mechanism in adaptive immune regulation.

Inhibition of Notch4 using Novel Neutralizing Antibodies Reduces Tumor Growth in Murine Cancer Models by Targeting the Tumor Endothelium.

Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed on tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancer. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms.

The gap-free genome of Forsythia suspensa illuminates the intricate landscape of centromeres

Abstract Forsythia suspensa, commonly known as weeping forsythia, holds significance in traditional medicine and horticulture. Despite its ecological and cultural importance, the existing reference genome presents challenges with duplications and gaps, hindering in-depth genomic analyses. Here, we present a Telomere-to-Telomere (T2T) assembly of the F. suspensa genome, integrating Oxford Nanopore Technologies (ONT) ultra-long, Hi-C datasets, and high-fidelity (HiFi) sequencing data. The T2T reference genome (Fsus-CHAU) consists of 14 chromosomes, totaling 688.79 Mb, and encompasses 33,932 predicted protein-coding genes. Additionally, we characterize functional centromeres in the F. suspensa genome by developing a specific CENH3 antibody. We demonstrate that centromeric regions in F. suspensa exhibit a diverse array of satellites, showcasing distinctive types with unconventional lengths across various chromosomes. This discovery offers implications for the adaptability of CENH3 and the potential influence on centromere dynamics. Furthermore, after assessing the insertion time of full-length LTRs within centromeric regions, we found that they are older compared to those across the entire genome, contrasting with observations in other species where centromeric retrotransposons are typically young. We hypothesize that asexual reproduction may impact retrotransposon dynamics, influencing centromere evolution. In conclusion, our T2T assembly of the F. suspensa genome, accompanied by detailed genomic annotations and centromere analysis, significantly enhances F. suspensa potential as a subject of study in fields ranging from ecology and horticulture to traditional medicine.

Immune cells phenotype and bioenergetic measures in CD4+ T cells differ between high and low feed efficient dairy cows

Identifying sources of variance that contribute to residual feed intake (RFI) can aid in improving feed efficiency. The objectives of this study were to investigate immune cells phenotype and bioenergetic measures in CD4+ T cells in low feed efficient (LE) and high feed efficient (HE) dairy cows. Sixty-four Holstein cows were enrolled at 93 ± 22 days in milk (DIM) and monitored for 7 weeks to measure RFI. Cows with the highest RFI (LE; n = 14) or lowest RFI (HE; n = 14) were selected to determine immune cells phenotype using flow cytometry. Blood was sampled in the same LE and HE cows at 234 ± 22 DIM to isolate peripheral blood mononuclear cells, followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The metabolic function of isolated CD4+ T lymphocytes was evaluated under resting and activated states. An increased expression of CD62L+ cells within CD8+ T lymphocytes and CD21+ B lymphocytes was observed in HE cows compared to LE cows. CD4+ T lymphocytes of HE cows exhibited an increased mitochondrial and glycolytic activity in resting and activated states compared to LE cows. These data suggest that immune cells in HE cows exhibit an increased metabolic function, which might influence nutrient partitioning and utilization and serve as a source of variation in feed efficiency that warrants future investigation.

Inhibition of Notch4 using Novel Neutralizing Antibodies Reduces Tumor Growth in Murine Cancer Models by Targeting the Tumor Endothelium.

Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed on tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancer. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms.

Immune cells phenotype and bioenergetic measures in CD4+ T cells differ between high and low feed efficient dairy cows

Identifying sources of variance that contribute to residual feed intake (RFI) can aid in improving feed efficiency. The objectives of this study were to investigate immune cells phenotype and bioenergetic measures in CD4+ T cells in low feed efficient (LE) and high feed efficient (HE) dairy cows. Sixty-four Holstein cows were enrolled at 93 ± 22 days in milk (DIM) and monitored for 7 weeks to measure RFI. Cows with the highest RFI (LE; n = 14) or lowest RFI (HE; n = 14) were selected to determine immune cells phenotype using flow cytometry. Blood was sampled in the same LE and HE cows at 234 ± 22 DIM to isolate peripheral blood mononuclear cells, followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The metabolic function of isolated CD4+ T lymphocytes was evaluated under resting and activated states. An increased expression of CD62L+ cells within CD8+ T lymphocytes and CD21+ B lymphocytes was observed in HE cows compared to LE cows. CD4+ T lymphocytes of HE cows exhibited an increased mitochondrial and glycolytic activity in resting and activated states compared to LE cows. These data suggest that immune cells in HE cows exhibit an increased metabolic function, which might influence nutrient partitioning and utilization and serve as a source of variation in feed efficiency that warrants future investigation.

Donor-derived cell-free DNA predicted allograft rejection and severe microvascular inflammation in kidney transplant recipients

IntroductionThe aim of this study is to investigate the clinical validity of donor-derived cell-free DNA (dd-cfDNA) in comparison with that of donor specific anti-HLA antibody (DSA) for predicting biopsy-proven rejection (BPR)and severe microvascular inflammation (severe MVI) in kidney transplant recipients (KTRs).MethodsIn this prospective observational investigation, 64 KTRs who underwent the indicated biopsies were included. Blood samples collected prior to biopsy were tested for dd-cfDNA and DSA. Biopsy specimens were classified by a renal pathologist according to the Banff classification. The predictive performance of dd-cfDNA and DSA for histological allograft diagnosis was assessed.ResultsKTRs were categorized into the high and low dd-cfDNA groups based on a level of 0.4%. Eighteen patients (28.1%) had positive DSA at biopsy, exhibiting higher dd-cfDNA levels than the DSA-negative patients. BPR and severe MVI incidences were elevated in the high dd-cfDNA group (BPR: 42.9% vs. 3.4%, P <0.001; severe MVI: 37.1% vs. 3.4%, P = 0.001). Also, elevated glomerulitis and MVI scores were observed in the high dd-cfDNA group. DSA showed the highest predictive value for BPR (AUC = 0.880), whereas dd-cfDNA alone excelled in predicting severe MVI (AUC = 0.855). Combination of DSA and dd-cfDNA (>0.4%) yielded sensitivities of 80.0% and 50.0% with specificities of 90.7% and 88.0% for antibody-mediated rejection and severe MVI detection, respectively.ConclusionThe dd-cfDNA test is a predictive tool for BPR and severe MVI, and it can improve the performance, especially when combined with DSA for BPR.

Donor-derived cell-free DNA predicted allograft rejection and severe microvascular inflammation in kidney transplant recipients

IntroductionThe aim of this study is to investigate the clinical validity of donor-derived cell-free DNA (dd-cfDNA) in comparison with that of donor specific anti-HLA antibody (DSA) for predicting biopsy-proven rejection (BPR)and severe microvascular inflammation (severe MVI) in kidney transplant recipients (KTRs).MethodsIn this prospective observational investigation, 64 KTRs who underwent the indicated biopsies were included. Blood samples collected prior to biopsy were tested for dd-cfDNA and DSA. Biopsy specimens were classified by a renal pathologist according to the Banff classification. The predictive performance of dd-cfDNA and DSA for histological allograft diagnosis was assessed.ResultsKTRs were categorized into the high and low dd-cfDNA groups based on a level of 0.4%. Eighteen patients (28.1%) had positive DSA at biopsy, exhibiting higher dd-cfDNA levels than the DSA-negative patients. BPR and severe MVI incidences were elevated in the high dd-cfDNA group (BPR: 42.9% vs. 3.4%, P <0.001; severe MVI: 37.1% vs. 3.4%, P = 0.001). Also, elevated glomerulitis and MVI scores were observed in the high dd-cfDNA group. DSA showed the highest predictive value for BPR (AUC = 0.880), whereas dd-cfDNA alone excelled in predicting severe MVI (AUC = 0.855). Combination of DSA and dd-cfDNA (>0.4%) yielded sensitivities of 80.0% and 50.0% with specificities of 90.7% and 88.0% for antibody-mediated rejection and severe MVI detection, respectively.ConclusionThe dd-cfDNA test is a predictive tool for BPR and severe MVI, and it can improve the performance, especially when combined with DSA for BPR.

Immersion immunization with recombinant Saccharomyces cerevisiae displaying ORF25 induced protective immunity against cyprinid herpesvirus 2.

Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV-2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 107 CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL-1β, and IFN-1 in vaccinated head-kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV-2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV-2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae-displayed ORF25 could be served as a candidate vaccine to prevent CyHV-2 infection.

Immersion immunization with recombinant Saccharomyces cerevisiae displaying ORF25 induced protective immunity against cyprinid herpesvirus 2.

Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV-2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 107 CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL-1β, and IFN-1 in vaccinated head-kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV-2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV-2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae-displayed ORF25 could be served as a candidate vaccine to prevent CyHV-2 infection.

Molecular and Serological Surveillance for Mycobacterium leprae and Mycobacterium lepromatosis in Wild Red Squirrels (Sciurus vulgaris) from Scotland and Northern England

Leprosy is a poverty-associated infectious disease in humans caused by Mycobacterium leprae or M. lepromatosis, often resulting in skin and peripheral nerve damage, which remains a significant public health concern in isolated areas of low- and middle-income countries. Previous studies reported leprosy in red squirrels in the British Isles, despite the fact that autochthonous human cases have been absent for centuries in this region. To investigate the extent of M. leprae and M. lepromatosis presence in wild red squirrels in the northern UK, we analyzed 220 blood/body cavity fluid samples from opportunistically sampled red squirrels (2004–2023) for specific antibodies against phenolic glycolipid-I, a cell wall component specific for these leprosy bacilli. Additionally, we assessed bacillus-derived DNA by real-time PCR (qPCR) in 250 pinnae from the same cohort. M. lepromatosis and M. leprae DNA were detected by qPCR in 20.4% and 0.8% of the squirrels, respectively. No cases of co-detection were observed. Detectable levels of anti-PGL-I antibodies by UCP-LFA were observed in 52.9% of animals with the presence of M. lepromatosis determined by qPCR, and overall in 15.5% of all animals. In total, 22.6% (n = 296) of this UK cohort had at least some exposure to leprosy bacilli. Our study shows that leprosy bacilli persist in red squirrels in the northern UK, emphasizing the necessity for ongoing molecular and serological monitoring to study leprosy ecology in red squirrels, gain insight into potential zoonotic transmission, and to determine whether the disease has a conservation impact on this endangered species.

Immune reactivity of two biological models to vaccination with inactivated vaccine QazVac against coronavirus infection COVID-19.

Specific prevention of a number of infectious diseases has been introduced into the vaccination schedule. The production of immunoprophylactic drugs, in order to establish standard properties, including safety and specific effectiveness, requires strict adherence to manufacturing regulations, and the reliability of the results obtained requires monitoring of these parameters. The specific effectiveness of vaccine preparations is standardized according to the indicators of stimulation of specific antibody response formed in the body of vaccinated model biological objects. Determination of the immune reactivity of white mice to vaccination with the QazVac vaccine to establish the possibility of using them as a biological model in assessing the immunogenicity of the vaccine instead of Syrian hamsters. The immune reactivity of model animals was assessed by the seroconversion rate, dynamics of antibody titers to the SARS-CoV-2 virus formed in the body after vaccination with the test vaccine. In the case of seropositivity of animals before administration of vaccine or placebo, the level of immune reactivity was calculated by the difference in antibody titers between control and vaccinated animals or by the difference in antibody titers before and after immunization. Specific antibodies were detected and their titer was determined using a neutralization reaction. The research results showed that the tested biological models had approximately the same immune reactivity to the administration of the QazVac vaccine, confirmed by the level and dynamics of antibody titers. When analyzing the fold increase in antibody titers in comparison to those of control animals, Syrian hamsters were more reactive compared to mice. But SPF white mice were standardized in their lack of the immune reactivity to SARS-CoV-2 virus before the immunization. The data obtained indicate that the immune reactivity of white mice to the administration of the QazVac vaccine in terms of the rate and dynamics of the formation of virus-neutralizing antibodies is approximately equivalent to the immune reactivity of Syrian hamsters. Before immunization with the vaccine, SPF white mice, in contrast to Syrian hamsters, do not have humoral immunity specific to the SARS-CoV-2 virus. The immune reactivity equivalent to that observed of Syrian hamsters and the absence of antibodies to the SARS-CoV-2 virus at a baseline indicate the superiority of the use of white mice in assessing the immunogenicity of vaccines against COVID-19 and/or obtaining specific factors of humoral immunity.

Enzyme immunoassay system for serological diagnosis of hemorrhagic fever with renal syndrome based on inactivated purified Puumala virus (Hantaviridae: Orthohantavirus)

Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS. Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent. Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA. For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays. The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.

Chitosan-alginate/R8 ternary polyelectrolyte complex as an oral protein-based vaccine candidate induce effective mucosal immune responses

Vaccination is the most effective method for preventing infectious diseases. Oral vaccinations have attracted much attention due to the ability to boost intestinal and systemic immunity. The focus of this study was to develop a poly (lactide-co-glycolide) acid (PLGA)-based ternary polyelectrolyte complex (PEC) with chitosan, sodium alginate, and transmembrane peptides R8 for the delivery of antigen proteins. In this study, the antigen protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis (MAP) antigens HBHA, Ag85B, and Bfra, was combined with R8 to generate self-assembled conjugates. The results showed that PEC presented a cross-linked reticular structure to protect the encapsulated proteins in the simulated gastric fluid. Then, the nanocomposite separated into individual nanoparticles after entering the simulated intestinal fluid. The ternary PEC with R8 promoted the in vivo uptake of antigens by intestinal lymphoid tissue. Moreover, the ternary PEC administered orally to mice promoted the secretion of specific antibodies and intestinal mucosal IgA. In addition, in the mouse models of MAP infection, the ternary PEC enhanced splenic T cell responses, thus reducing bacterial load and liver pathology score. These results suggested that this ternary electrolyte complex could be a promising delivery platform for oral subunit vaccine candidates, not limited to MAP infection.

Detection of Serum IgG Specific for Brachyspira pilosicoli and “Brachyspira canis” in Dogs

Brachyspira pilosicoli (B. pilosicoli) is a pathogen in pigs, poultry, and humans causing colitis, diarrhea, and poor growth rates. Its role as a canine pathogen is controversial, and the seroprevalence of specific IgG antibodies against B. pilosicoli in dogs is unknown. A further, not yet officially recognized Brachyspira species in dogs is “Brachyspira canis” (“B. canis“), which is proposed to be apathogenic. This study evaluates enzyme-linked immunosorbent assays (ELISAs) measuring serum IgG antibodies specific for B. pilosicoli or “B. canis” and investigates levels of specific IgG antibodies against B. pilosicoli and “B. canis” in a cohort of clinical patients presented at an animal referral clinic. These ELISAs use detergent-extracted antigens from B. pilosicoli and “B. canis”. To increase analytic specificity, we precipitated the antigens with trichloroacetic acid (TCA) to isolate and concentrate the respective protein fraction. Our results indicate that a large number of serum IgG antibodies bind to shared epitopes of detergent-extracted antigens of the two spirochaetes. Our data also suggest that dogs might not only carry B. pilosicoli but also have “B. canis”-specific serum IgG antibodies.

O-254 The relationship between dysbiosis of vaginal microbiome and proinflammatory cytokine levels in vaginal fluid in recurrent implantation failure patients

Abstract Study question Is there any relation between vaginal fluid cytokine levels and the composition of the vaginal microbiome in recurrent implantation failure (RIF) patients? Summary answer IL-1β and IL-2 levels are higher in patients with greater alpha diversity in vaginal microbiome, which associates dysbiotic bacteria with cytokine imbalance towards inflammation. What is known already In recent years, the development of sequencing-based technologies has enabled the evaluation of the vaginal microbiome. Studies have shown that a vaginal microbiota with less than 90% Lactobacillus is predictive of a poor IVF outcome, suggesting that dysbiosis in the reproductive tract negatively affects reproductive outcomes compared to women with a Lactobacillus-dominated microbiome. Furthermore, it is known that pregnancy is characterized by an anti-inflammatory cytokine pattern. In contrast, an altered Th1/Th2 ratio, and altered NK cell and macrophage numbers are more prevalent in women with RIF. Also, interleukin-1 system and interleukin-2 are involved in human implantation and pregnancy. Study design, size, duration 35 patients with RIF were included in a prospective study between November 2022 and July 2023. 21 had normal microbiome and 14 had dysbiotic microbiome. In all of them, two vaginal swabs were obtained to analyze microbiome composition and the levels of cytokines IL-2, IL-4, IL- 6, IL-8, IL-10, VEGF, IFNγ, TNFα, IL-1α, IL-1β, MCP-1 and EGF, respectively. Patients with uterine malformations, uterine surgery or other known factors which may influence RIF were excluded. Participants/materials, setting, methods To measure cytokine levels, one vaginal swab was resuspended in 500 μl PBS and a sandwich immunoassay with specific antibodies was performed for the cytokines IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFNγ, TNFα, IL -1α, IL-1β, MCP-1 and EGF. Vaginal microbiome was analyzed by mass sequencing of the V3V4 region of 16S rRNA. Statistical analysis was performed with R Statistical Software and bioinformatic analysis of vaginal microbiome was performed using QIIME2, Phyloseq and MicrobiomeAnalyst packages. Main results and the role of chance For the cytokines measured in vaginal fluid, we observe that IL-1β levels (p = 0.040) are higher in patients with dysbiotic microbiome, which have an imbalance towards inflammation. Comparing levels of cytokines with respect to alpha diversity, this is greater in patients with higher levels of IL-1β (p = 0.005) and IL-2 (p = 0.032). Furthermore, the multivariate model that predicts alpha diversity does consider IL-1β and IL-2 levels (p = 0.036; AIC=45.123). Alpha diversity, moreover, is higher in vaginal samples from patients with dysbiotic microbiome (p < 0.001). Regarding beta diversity, it is also higher in patients with altered microbiome (p = 0.001). Considering the relative abundance of each bacteria, we can predict the levels of the aforementioned cytokines: for IL-2, the multivariate model reaches 88.1% of prediction including the relative abundance of Aerococcus, Burkholderia, Dialister, Escherichia, Lactobacillus, Megasphaera, Porphyromonas, Streptococcus and Ralstonia. For IL-1β, the multivariate model reaches 76.0% of prediction including Aerococcus, Dialister, Enterobacter, Lactobacillus, Megasphaera, Porphyromonas, Streptococcus, Veillonella and Ureaplasma. Some of these genera cause dysbiotic microbiome, which suggests that the relative abundance of some pathogenic bacteria is what alters the production of cytokines. Limitations, reasons for caution The dysbiotic microbiome group includes patients with various vaginal microbiome compositions, which can make it difficult to observe the specific effects of certain bacteria. Additionally, the sample size in the dysbiotic microbiome group is limited. Further studies, incorporating a metagenomic approach, might be able to identify functional groups of bacteria. Wider implications of the findings These results indicate that dysbiotic bacteria, such as Aerococcus or Streptococcus, induce an imbalance in cytokine levels, resulting in inflammation associated with lower implantation rates. When the vaginal microbiome is altered, the administration of antibiotics or probiotics may improve the immunologic environment, thus, avoiding the use of immunosuppressants. Trial registration number -

A lateral flow immunoassay for the rapid identification of Candida auris from isolates or directly from surveillance enrichment broths.

Candida auris is a recently discovered yeast with a multi-drug resistant profile associated with high mortality rates. The rapid identification of Candida auris in hospital settings is crucial to allow appropriate therapeutic and rapid implementation of infection management measures. The aim of this study was to develop a lateral flow immunoassay (LFIA) for the rapid identification of Candida auris. Highly specific monoclonal antibodies were obtained by immunizing mice with membrane proteins from Candida auris which were then used to develop a LFIA whose performance was assessed by testing 12 strains of Candida auris and 37 strains of other Candida species. Isolates were grown on either Sabouraud dextrose, CHROMagarTM Candida Plus or HardyCHROMTM Candida + auris agar plates. The strains were also cultured on salt sabouraud-dextrose with chloramphenicol or a commercially available Salt-Sabouraud Dulcitol Broth with chloramphenicol and gentamicin, and processed using a simple centrifugation protocol to recover a pellet. Finally, the colonies or yeast extract were transferred to the LFIA to determine the specificity and sensitivity of the assay. The LFIA reached 100% specificity and sensitivity from solid agar plates. For both enrichment broths, some Candida non-auris species were able to grow, but the LFIA remained 100% specific. The use of a dextrose-based sabouraud broth resulted in earlier identification with the LFIA, with most of the Candida auris strains detected at 24 h. The developed LFIA prototype represents a powerful tool to fight the emerging threat of Candida auris. Clinical validation represents the next step.

P-035 Catecholamine neuroreceptors are differentially located in functional regions of the human sperm

Abstract Study question How are the expression and distribution of Adrenergic and Dopamine receptors in human sperm cells? Summary answer Using ultra-high-resolution microscopy techniques, we identified that each type of catecholamine receptor is expressed in a different functional region of the human sperm. What is known already Human sperm present complex cell signalling complexes with several neuroreceptors in their membrane. The spermatozoa is a terminal differentiated cell showing specialized regions optimized for physiological processes like motility, acrosome reaction and membrane fusion during fertilization. Although indirect methods have determined the presence of catecholamine receptors in human sperm, there is no direct evidence nor description of the exact localisation of these receptors. Catecholamine receptors are the direct target of medications used to treat very frequent health problems like hypertension (beta blockers), asthma (bronchodilators) and mental health disorders (antipsychotics and antidepressants). Study design, size, duration We design the study by developing new techniques that allow us to use high-resolution microscopes (i.e. Laser Scanning Confocal Microscopy LSCM and Field Emission Scanning Electron Microscopy or FE-SEM) to identify the localisation and distribution of catecholamine receptors. We determined sperm functional regions and quantified the relative amount of the different receptors on each one of those regions to perform a statistical analysis. Participants/materials, setting, methods After informed consent and ethical approval, sperm from young, healthy donors (n = 20) were included in the study. We used the neuroblastoma cell line SH-SY5Y as a positive control. Immunolabelling techniques used specific primary antibodies for each receptor, followed by the gold-conjugated secondary antibodies for FE-SEM. Negative controls were performed by omitting the primary antibody. In FE-SEM images, we quantify the number of gold nanoparticles in the sperm cells and the background. Main results and the role of chance We confirm the differential localisation of the different receptors studied. Dopamine D2 receptors are mainly expressed on the equatorial band of the acrosome, while beta-adrenergic receptors are restricted to the terminal piece of the flagellum. In both cases, the mean number of gold particles showed statistical differences (p < 0,001) between the region of expression and the rest of the regions and the background. Limitations, reasons for caution The present study only shows the presence of the receptors on the sperm plasma membrane. The activity of those receptors should be investigated by developing bioassays based on the use of agonists and/or antagonists. A more reliable genetic overexpression or knockout approach was impossible in human sperm. Wider implications of the findings The presence of receptors on restricted regions of the sperm suggests their participation in critical physiological events, like regulation of sperm motility hyperactivation or acrosome reaction. It may also suggest a possible side effect of beta-blockers or bronchodilators with the male fertility potential that needs to be investigated. Trial registration number not applicable

P-115 GATAD2B is required for pre-implantation embryo development by delaying zygotic genome activation

Abstract Study question Major zygotic genome activation (ZGA) involving the activation of thousands of genes occurs at the late-2cell stage, but the regulatory mechanisms governing ZGA remain unclear. Summary answer Our results demonstrate that GATAD2B is essential for early embryonic development, in part through facilitating zygotic genome activation (ZGA). What is known already Acetylation modifications of histone tails promote transcriptional activation, and the maternal deletion of H4K16ac leads to failure in ZGA. GATAD2B is one of the core subunits of the Nucleosome remodeling and histone deacetylation complex(NuRD). Study design, size, duration Using mouse embryos as the research model, the study was conducted over a period of approximately two years to ensure a relatively small-scale investigation. Participants/materials, setting, methods We examined the dynamic expression of NURD complex components, including GATAD2B, during pre-implantation embryonic stages. GATAD2B expression was specifically reduced via microinjection of GATAD2B-siRNA during the zygote stage, revealing its impact on early embryonic development in mice. Zygotic genome activation (ZGA), morula compaction, and blastocyst lineage differentiation were investigated using immunofluorescence, western blot, and other techniques. Additionally, we employed RNA-seq, protein immunoprecipitation, and mass spectrometry to uncover underlying molecular mechanisms. Main results and the role of chance Our research has shown that GATAD2B exhibits specific nucleus localization and high protein expression from the late-2-cell to 8-cell. This intriguing phenomenon prompted us to investigate the relationship between GATAD2B and the ZGA. We discovered a distinctive pattern of GATAD2B, starting from the late 2-cell stage with nuclear localization. GATAD2B depletion resulted in defective embryonic development, including increased DNA damage at morula, decreased blastocyst formation rate and abnormal differentiation of ICM/TE lineages. Consistent with the delay during the cleavage stage, the transcriptome analysis of the 2-cell revealed inhibition of the cell cycle G2/M phase transition pathway. Furthermore, the GATAD2B proteomics data provided clear evidence of a certain association between GATAD2B and molecules involved in the cell cycle pathway. As hypothesized, GATAD2B-deficient 2-cell embryos exhibited abnormalities in ZGA during the maternal-to-embryonic transition, with lower expression of the major ZGA mark MERVL. Limitations, reasons for caution Given the limitations of antibody effectiveness and specificity required for the experiments, the exploration of molecular mechanisms is still ongoing. Wider implications of the findings Our findings have elucidated the impact of GATAD2B on early embryonic development through the regulation of zygotic genome activation (ZGA), providing a novel genetic explanation for clinical defects in early embryonic development. Trial registration number not applicable