N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Claims-Based vs Agency-Reported Patient Outcomes Among Home Health Agencies, 2013-2019.

Importance: Given the growth of home health agency (HHA) care, it is important to understand whether quality reporting programs, such as star ratings, are associated with improved patient outcomes. Objective: To assess the immediate and long-term association of the introduction of HHA star ratings with patient-level quality outcomes, comparing claims-based and agency-reported measures. Design, Setting, and Participants: This cross-sectional study used Medicare HHA claims and agency-reported assessments to identify sequential patient episodes (ie, spells) among US adults with traditional Medicare who received HHA care (2013-2019). An interrupted time series (ITS) model was used to measure changes in trends and levels before and after the introduction of star ratings. Statistical analysis was performed from November 2022 to September 2023. Exposure: The exposure was the introduction of HHA star ratings. The postexposure period was set as starting January 1, 2016, to account for the period when both star ratings (quality of patient care and patient satisfaction rating) were publicly reported. Main Outcomes and Measures: The main outcomes included claims-based hospitalization measures (both during the patient spell and 30 days after HHA discharge) and agency-reported functional measures, such as improvement in ambulation, bathing, and bed transferring. There was also a measure to capture timely initiation of care among post-acute care HHA users, defined as HHA care initiated within 2 days of inpatient discharge. Results: This study identified 22 958 847 patient spells to compare annual changes over time; 9 750 689 patient spells were included during the pre-star ratings period from January 1, 2013, to December 31, 2015 (6 067 113 [62.2%] female; 1 100 145 [11.3%] Black, 512 487 [5.3%] Hispanic, 7 845 197 [80.5%] White; 2 656 124 [27.2%] dual eligible; mean [SD] patient spell duration, 70.9 [124.9] days; mean [SD] age, 77.4 [12.0] years); 13 208 158 patient spells were included during the post-star ratings period from January 1, 2016, to December 31, 2019 (8 104 69 [61.4%] female; 1 385 180 [10.5%] Black, 675 536 [5.1%] Hispanic, 10 664 239 [80.7%] White; 3 318 113 [25.1%] dual eligible; mean [SD] patient spell duration, 65.3 [96.2] days; mean [SD] age, 77.7 [11.6] years). Results from the ITS models found that the introduction of star ratings was associated with an acceleration in the mean [SE] hospitalization rate during the spell (0.39% [0.05%] per year) alongside functional improvements in ambulation (2.40% [0.29%] per year), bed transferring (3.95% [0.48%] per year) and bathing (2.34% [0.19%] per year) (P < .001). This occurred alongside a 1.21% (0.12%) per year reduction in timely initiation of care (P < .001). Conclusions and Relevance: This cross-sectional study found an observed improvement in agency-reported functional measures, which contrasted with slower increases in more objective measures such as hospitalization rates and declines in timely initiation of care. These findings suggest a complex picture of HHA quality of care after the introduction of star ratings.

The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis across Murine Models.

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).

Hybrid Materials Obtained by Immobilization of Biosynthesized Ag Nanoparticles with Antioxidant and Antimicrobial Activity.

Ag nanoparticles (AgNPs) were biosynthesized using sage (Salvia officinalis L.) extract. The obtained nanoparticles were supported on SBA-15 mesoporous silica (S), before and after immobilization of 10% TiO2 (Degussa-P25, STp; commercial rutile, STr; and silica synthesized from Ti butoxide, STb). The formation of AgNPs was confirmed by X-ray diffraction. The plasmon resonance effect, evidenced by UV-Vis spectra, was preserved after immobilization only for the sample supported on STb. The immobilization and dispersion properties of AgNPs on supports were evidenced by TEM microscopy, energy-dispersive X-rays, dynamic light scattering, photoluminescence and FT-IR spectroscopy. The antioxidant activity of the supported samples significantly exceeded that of the sage extract or AgNPs. Antimicrobial tests were carried out, in conditions of darkness and white light, on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Higher antimicrobial activity was evident for SAg and STbAg samples. White light increased antibacterial activity in the case of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In the first case, antibacterial activity increased for both supported and unsupported AgNPs, while in the second one, the activity increased only for SAg and STbAg samples. The proposed antibacterial mechanism shows the effect of AgNPs and Ag+ ions on bacteria in dark and light conditions.

Probing the expression and adhesion of glycans involved in Helicobacter pylori infection.

Helicobacter pylori infects approximately half the human population and has an unusual infective niche of the human stomach. Helicobacter pylori is a major cause of gastritis and has been classified as a group 1 carcinogen by the WHO. Treatment involves triple or quadruple antibiotic therapy, but antibiotic resistance is becoming increasingly prevalent. Helicobacter pylori expresses certain blood group related antigens (Lewis system) as a part of its lipopolysaccharide (LPS), which is thought to assist in immune evasion. Additionally, H. pylori LPS participates in adhesion to host cells alongside several adhesion proteins. This study profiled the carbohydrates of H. pylori reference strains (SS1 and 26695) using monoclonal antibodies (mAbs) and lectins, identifying interactions between two carbohydrate-targeting mAbs and multiple lectins. Atomic force microscopy (AFM) scans were used to probe lectin and antibody interactions with the bacterial surfaces. The selected mAb and lectins displayed an increased adhesive force over the surface of the curved H. pylori rods. Furthermore, this study demonstrates the ability of anti-carbohydrate antibodies to reduce the adhesion of H. pylori 26695 to human gastric adenocarcinoma cells via AFM. Targeting bacterial carbohydrates to disrupt crucial adhesion and immune evasion mechanisms represents a promising strategy for combating H. pylori infection.

Molecular characterization of the whole genome of H9N2 avian influenza virus isolated from Egyptian poultry farms.

H9N2 avian influenza viruses (AIVs) affect both poultry and humans on a global level, and they are especially prevalent in Egypt. In this study, we sequenced the entire genome of AIV H9N2 isolated from chickens in Egypt in 2021, using next-generation sequencing (NGS) technology. Phylogenetic analysis of the resulting sequences showed that the studied strain was generally monophyletic and grouped within the G1 sublineage of the Eurasian lineage. Four segments (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acidic [PA], and non-structural [NS]) were related to Egyptian genotype II, while the nucleoprotein (NP), neuraminidase (NA), matrix (M), and haemagglutinin (HA) segments were related to Egyptian genotype I. Molecular analysis revealed that HA protein contained amino acid residues (191H and 234L) that suggested a predilection for attaching to human-like receptors. The antigenic sites of HA had two nonsynonymous mutations: V194I at antigenic site A and M40K at antigenic site B. Furthermore, the R403W and S372A mutations, which have been observed in H3N2 and H2N2 strains that caused human pandemics, were found in the NA protein of the detected strain. The internal proteins contained virulence markers: 504V in the PB2 protein, 622G, 436Y, 207K, and 677T in the PB1 protein, 127V, 550L, and 672L in PA protein, and 64F and 69P in the M protein. These results show that the detected strain had undergone intrasubtype reassortment. Furthermore, it contains changes in the viral proteins that make it more likely to be virulent, raising a question about the tendency of AIV H9N2 to become highly pathogenic in the future for both poultry and humans.

[Molecular biology analysis of 2 rare RhD variant individuals with RHD*DEL37].

The Rh blood grouping system is a critical standardized test in transfusion medicine, especially for the cases related to haemolytic transfusion reactions and neonatal haemolytic disease caused by clinical RhD blood group incompatibility. In the present case report, we presented two cases with the uncommon RHD gene variation RHD*DEL37. The blood samples of the two subjects were mistakenly identified as RhD-negative through conventional serological testing. Firstly, both blood samples were tested negative for the RhD antigen using traditional tube test and gel microcolumn methods. The phenotyping of RhCE were identified as ccEe and ccee for each sample, respectively. Secondly, genetic analysis was performed using polymerase chain reaction-sequence specific prime (PCR-SSP) which revealed that neither sample belonging to the several common RHD gene variants which was found in Asia. Moreover, they turned out to be positive for the RHD haplotype, which indicated that exons 1-10 on one of the RHD alleles were entirely absent. In addition, a T>C mutation was observed at bases 1154-31 in intron 8 of the other allele, which was located at the intron 8 breakpoint. This result was obtained after further Sanger sequencing of exons 1-10 of the RHD gene. The mutant allele was designated as RHD*DEL37 by the International Society of Blood Transfusion (ISBT) and was identified as D-elute(Del) by phenotype ana-lysis. Both samples were genotyped as RHD*DEL37 and showed positive results. In summary, the true genotype of the two blood samples, of which the screening results only using serological testing method was negative, were RHD*DEL37 /RHD-(RHD*01N.01). Notably, this kind of genotype was reported for the first time in Chinese population. Moreover, the two individuals did not have ties of consanguinity, indicating that some of the Chinese individuals could be carriers of the genetic mutation. Therefore, it might be necessary to further confirm the frequency of this mutation in the Chinese population and the possibility of homozygosity for this mutation. This report identifies infrequent RHD gene mutation samples by coupling molecular biology and serological methods to prevent misclassification of blood groups. Combining serological and molecular biology test results to determine blood group is critical in protecting patients during clinical transfusion procedures.

Functional insights of Tyr37 in framework region 2 directly contributing to the binding affinities and dissociation kinetics in single-domain VHH antibodies.

Single-domain VHH antibody is regarded as one of the promising antibody classes for therapeutic and diagnostic applications. VHH antibodies have amino acids in framework region 2 that are distinct from those in conventional antibodies, such as the Val37Phe/Tyr (V37F/Y) substitution. Correlations between the residue type at position 37 and the conformation of the CDR3 in VHH antigen recognition have been previously reported. However, few studies focused on the meaning of harboring two residue types in position 37 of VHH antibodies, and the concrete roles of Y37 have been little to be elucidated. Here, we investigated the functional states of position 37 in co-crystal structures and performed analyses of three model antibodies with either F or Y at position 37. Our analysis indicates that Y at position 37 enhances the dissociation rate, which is highly correlated with drug efficacy. Our findings help to explain the molecular mechanisms that distinguish VHH antibodies from conventional antibodies.

Association of different milk fat content with coronary artery disease and myocardial infarction risk: A Mendelian randomization study.

BACKGROUND: Numerous observational studies have investigated on the correlation of whole, semi-skimmed, and skimmed milk with coronary artery disease (CAD) and myocardial infarction (MI) risk; However, no consensus has been reached and evidence on any causal links between these exposures and outcomes remains unclear. This study aimed to conduct univariate and multivariate Mendelian randomization (MR) analyses, using publicly released genome-wide association study summary statistics (GWAS) from the IEU GWAS database, to ascertain the causal association of milk with various fat content with CAD and MI risk. METHODS: For the exposure data, 29, 15, and 30 single-nucleotide polymorphisms for whole milk, semi-skimmed milk, and skimmed milk, respectively, obtained from 360,806 Europeans, were used as instrumental variables. CAD and MI comprised 141,217 and 395,795 samples, respectively. We used inverse variance weighted (IVW), weighted median, MR-Egger regression, and MR Pleiotropy Residual Sum and Outlier analyses to determine whether pleiotropy and heterogeneity could skew the MR results. Sensitivity tests were conducted to verify the robustness of the results. RESULTS: After adjusting for false discovery rates (FDR), we discovered proof that skimmed milk intake is a genetically predicted risk factor for CAD (odds ratio [OR] = 5.302; 95% confidence interval [CI] 2.261-12.432; P < 0.001; FDR-corrected P < 0.001) and MI (OR = 2.287; 95% CI 1.218-4.300; P = 0.010; FDR-corrected P = 0.009). Most sensitivity assessments yielded valid results. Multivariable MR for CAD and MI produced results consistent with those obtained using the IVW method. There was no causal relationship between whole or semi-skimmed milk, and CAD or MI. CONCLUSION: Our findings indicate that the consumption of skimmed milk may increase the risk of CAD and MI. This evidence may help inform dietary recommendations for preventing cardiovascular disease. Further studies are required to elucidate the underlying mechanisms.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients enrolled at 100 health facilities throughout Tanzania: February to July 2021.

Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.

Genotypic Frequency of Rh Cw Antigen in Blood Donors of Northern Pakistan.

OBJECTIVE: To determine the genotypic frequency of Rh Cw antigen in blood donors of Northern Pakistan. STUDY DESIGN: Descriptive cross-sectional study. Place and Duration of the Study: Department of Molecular Haematology, Armed Forces Institute of Transfusion (AFIT), Rawalpindi, Pakistan, from August 2022 to January 2023. METHODOLOGY: Blood donors were randomly selected. Venous blood samples were taken in K3-EDTA anticoagulant tubes. ABO and Rh D grouping were performed conventionally. DNA for Rh Cw genotyping was extracted via Chelex TM, followed by PCR amplification using an ABI 2700 thermal cycler. Human growth hormone (HGH) acts as an internal control. Amplified products underwent Polyacrylamide gel Electrophoresis (PAGE). RESULTS: There were 400 randomly chosen donors whose ages ranged from 26-35 years, with a predominantly male population (94.8%) of Punjabi origin (67.8%). The majority (87.3%) was RhD positive. Blood group B was the most prevalent (35%) in the studied population, followed by O (34.75%). Only 1.5% had Rh Cw antigen. Rh Cw was more prevalent in ABO-positive participants (87.25%) compared to ABO-negative (12.75%). CONCLUSION: There was a 1.5% prevalence of Rh Cw antigen genotype in randomly selected Northern Pakistani blood donors. Rh Cw prevalence was higher in ABO-positive participants. Significant correlation (<0.05) existed between RhD and Cw antigens. Given the implications of anti-Cw antibody, including Cw antigen-positive cells in antibody screening is recommended. KEY WORDS: Alloimmunisation, Blood donors, HDFN, Phenotype, Rh antigens, Transfusion.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants.

BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

Molecular and Family Analyses of a Novel RHD1058G>C Allele in a Chinese RhD Population.

BACKGROUND: Rh(D) phenotype in a sample from a 19-year-old female patient showed weak positivity (1+). A follow-up sample was requested to further define the Rh(D) phenotype, her Rh(D) phenotype was tested by using another reagent, Rh(D) phenotype still showed weak reactivity (1+), RhCcEe phenotype was Ccee. METHODS: Seven samples from the family members of the proposita were received. The RhDCcEe phenotypes were typed by the microcolumn gel card and the unexpected antibodies were assayed by indirect anti-human globulin test (IAT). Genomic DNA was extracted from the blood sample and the novel RHD1058G>C allele was detected through an established sequence-specific primer PCR (PCR-SSP), RHD exons 1 - 10 were sequenced afterward by exon-specific amplification. The distribution of RHD1058G>C allele and RHD weak positive phenotype were investigated in the pedigrees. RESULTS: The unexpected antibodies all were negative in the family members. The novel RHD1058G>C allele was found in the proposita, her father, and grandfather. Five family members were detected serologically with the common Rh(D)-positive phenotypes either as homozygote of RHD/RHD or heterozygote of RHD/RHd. Two family members were detected as weak D phenotypes in accordance with the genotyping results by PCR-SSP, and both of them have a D1058Ce haplotype and a dce haplotype. One member, her father, was tested common Rh(D)-positive with D1058Ce haplotype and a Dce haplotype. CONCLUSIONS: These data allow us to describe the characteristics of the weak D phenotype with a novel c.RHD-1058G>C allele, which may be partial D and increase the risk of RHD alloantibody. The novel RHD1058G>C allele was inherited in three generations in a family rather than spontaneous mutation in an individual.

Notch2 controls developmental fate choices between germinal center and marginal zone B cells upon immunization.

Sustained Notch2 signals induce trans-differentiation of Follicular B (FoB) cells into Marginal Zone B (MZB) cells in mice, but the physiology underlying this differentiation pathway is still elusive. Here, we demonstrate that most B cells receive a basal Notch signal, which is intensified in pre-MZB and MZB cells. Ablation or constitutive activation of Notch2 upon T-cell-dependent immunization reveals an interplay between antigen-induced activation and Notch2 signaling, in which FoB cells that turn off Notch2 signaling enter germinal centers (GC), while high Notch2 signaling leads to generation of MZB cells or to initiation of plasmablast differentiation. Notch2 signaling is dispensable for GC dynamics but appears to be re-induced in some centrocytes to govern expansion of IgG1+ GCB cells. Mathematical modelling suggests that antigen-activated FoB cells make a Notch2 dependent binary fate-decision to differentiate into either GCB or MZB cells. This bifurcation might serve as a mechanism to archive antigen-specific clones into functionally and spatially diverse B cell states to generate robust antibody and memory responses.

SARS-CoV-2-Specific Immune Responses in Vaccination and Infection during the Pandemic in 2020-2022.

To gain insight into how immunity develops against SARS-CoV-2 from 2020 to 2022, we analyzed the immune response of a small group of university staff and students who were either infected or vaccinated. We investigated the levels of receptor-binding domain (RBD)-specific and nucleocapsid (N)-specific IgG and IgA antibodies in serum and saliva samples taken early (around 10 days after infection or vaccination) and later (around 1 month later), as well as N-specific T-cell responses. One patient who had been infected in 2020 developed serum RBD and N-specific IgG antibodies, but declined eight months later, then mRNA vaccination in 2021 produced a higher level of anti-RBD IgG than natural infection. In the vaccination of naïve individuals, vaccines induced anti-RBD IgG, but it declined after six months. A third vaccination boosted the IgG level again, albeit to a lower level than after the second. In 2022, when the Omicron variant became dominant, familial transmission occurred among vaccinated people. In infected individuals, the levels of serum anti-RBD IgG antibodies increased later, while anti-N IgG peaked earlier. The N-specific activated T cells expressing IFN γ or CD107a were detected only early. Although SARS-CoV-2-specific salivary IgA was undetectable, two individuals showed a temporary peak in RBD- and N-specific IgA antibodies in their saliva on the second day after infection. Our study, despite having a small sample size, revealed that SARS-CoV-2 infection triggers the expected immune responses against acute viral infections. Moreover, our findings suggest that the temporary mucosal immune responses induced early during infection may provide better protection than the currently available intramuscular vaccines.

Humoral and cellular response of two different vaccines against SARS-CoV-2 in a group of healthcare workers: An observational study.

On March 13, 2021, Tunisia started a widespread immunization program against SARS-CoV-2 utilizing different vaccinations that had been given emergency approval. Herein, we followed prospectively a cohort of participant who received COVID-19 vaccine (Pfizer BioNTech and Sputnik-Gameleya V). The goal of this follow-up was to define the humoral and cellular immunological profile after immunization by assessing neutralizing antibodies and IFN- γ release. 26 vaccinated health care workers by Pfizer BioNTech (n=12) and Sputnik-Gameleya V (n=14) were enrolled from June to December 2021 in Military hospital of Tunis. All consenting participants were sampled for peripheral blood after three weeks of vaccination. The humoral response was investigated by the titer of anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies to S1 protein. The CD4 and CD8 T cell responses were evaluated by the QuantiFERON® SARS-CoV-2 (Qiagen® Basel, Switzerland). Regardless the type of vaccine, the assessment of humoral and cellular response following vaccination showed a strong involvement of the later with expression of IFN-γ as compared to antibodies secretion. Moreover, we showed that people with past SARS-CoV-2 infection developed high levels of antibodies than those who are not previously infected. However, no significant difference was detected concerning interferon gamma (IFN-γ) expression by CD4 and CD8 T cells in health care worker (HCW) previously infection or not with COVID-19 infection. Analysis of immune response according to the type of vaccine, we found that Pfizer BioNTech induced high level of humoral response (91.66%) followed by Sputnik-Gameleya V (64.28%). However, adenovirus vaccine gave a better cellular response (57.14%) than mRNA vaccine (41.66%). Regarding the immune response following vaccine doses, we revealed a significant increase of neutralizing antibodies and IFN-γ release by T cells in patients fully vaccinated as compared to those who have received just one vaccine. Collectively, our data revealed a similar immune response between Pfizer BioNTech and Sputnik-Gameleya V vaccine with a slight increase of humoral response by mRNA vaccine and cellular response by adenovirus vaccine. It's evident that past SARS-CoV-2 infection was a factor that contributed to the vaccination's increased immunogenicity. However, the administration of full doses of vaccines (Pfizer BioNTech or Sputnik-Gameleya V) induces better humoral and cellular responses detectable even more than three months following vaccination.

Comparative evaluation of Borrelia burgdorferi antibody detection between the VetScan Flex4 and SNAP 4Dx Plus.

Two studies were developed to compare Borrelia burgdorferi antibody detection between the VetScan Flex4 and SNAP 4Dx Plus tests. The objective of the first study was to evaluate the diagnostic sensitivity (Se) and specificity (Sp) of VetScan Flex4 and SNAP 4Dx Plus B. burgdorferi results using field sourced samples compared to a Western Blot reference method. The sensitivity and specificity of VetScan Flex4 were 81.9 % (95 % CI: 71.9 %-89.5 %) and 89.3 % (95 % CI: 85.2 %-92.9 %) respectively, and SNAP 4Dx Plus's sensitivity and specificity were 80.7 % (95 % CI: 70.6 %-88.6 %) and 92.8 % (95 % CI: 89.1 %-95.5 %) respectively. When comparing VetScan Flex4 and Snap 4Dx Plus, the Simple Kappa Coefficient estimate was 0.76 (95 % CI: 0.69-0.84) indicating substantial agreement between the two methods. McNemar's Test revealed concordance between the two methods was not statistically significant (P = 0.05). The objective of the second study was to evaluate whether VetScan Flex4 differentiates between B. burgdorferi antibodies derived from infection versus vaccination with commonly used canine Lyme vaccines. The sensitivity and specificity of the VetScan Flex4 in differentiating canine Lyme vaccination from infection with Borrelia burgdorferi were 100 % (Se 95 % CI: 78.2 %-100 %; Sp 95 % CI: 91.2 %-100 %). In conclusion, the VetScan Flex4 is a reliably sensitive and specific point-of-care test that is similar to Snap 4Dx Plus, can differentiate between infection and Lyme vaccination, and can be utilized by veterinarians for Lyme disease diagnosis and surveillance of B. burgdorferi exposure.

[Analysis of NF1 gene variants in a Chinese pedigree and a sporadic patient with Neurofibromatosis type 1].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree and a sporadic case with Neurofibromatosis type 1 (NF1). METHODS: Clinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from peripheral venous blood samples and subjected to whole exome sequencing. Candidate variants were validated by Sanger sequencing and bioinformatic analysis. RESULTS: All patients from the pedigree were found to harbor a c.3251delC variant in exon 25 of the NF1 gene, whilst a c.4312_4314delGAA variant was found in exon 32 of the NF1 gene in the sporadic case. CONCLUSION: Variants of the NF1 gene may account for the occurrence of NF1 in this pedigree and sporadic case.