Quantitative High-Throughput and High-Sensitivity Glycoprofile Analysis of Therapeutic Glycoproteins Using HILIC-FLD and ESI-MS.

Protein glycosylation is recognized as a Critical Quality Attribute for the biological and therapeutic activity of many recombinant proteins. Therefore, glycosylation should be monitored rigorously to ensure the desired quality, safety, and potency of monoclonal antibodies and other therapeutic glycoproteins. However, glycans are highly heterogeneous structures in proteins, and this poses a challenge for glycoprofile analysis. Hydrophilic interaction liquid chromatography with fluorescence detection has been recognized as a standard method for separation and quantification of released N-glycans after conventional 2-aminobenzamide labeling, but the sample preparation procedure is time-consuming with low sensitivity and poor reproducibility. Here we present a streamlined 96-well plate format platform for high-throughput and high-sensitivity glycan profiling. By taking advantage of rapid glycoprotein denaturation, enzymatic deglycosylation, highly sensitive fluorescent derivation, and on-matrix glycan cleanup, the reaction time has been shortened significantly to approximately 10 min. A detailed workflow including two-dimensional chromatography fractionation, exoglycosidase sequential digestion, mass spectroscopy glycan mass confirmation, and data interpretation is described. Potential pitfalls and precautions to be taken to ensure data accuracy are also discussed in this chapter.

Myelin oligodendrocyte glycoprotein-associated transverse myelitis after SARS-CoV-2 infection: A case report.

BACKGROUND: Cases of myelin oligodendrocyte glycoprotein (MOG) antibody-related disease have a history of coronavirus disease 2019 infection or its vaccination before disease onset. Severe acute respiratory syndrome virus 2 (SARS-CoV-2) infection has been considered to be a trigger of central nervous system autoimmune diseases. CASE SUMMARY: Here we report a 20-year male with MOG-associated transverse myelitis after a SARS-CoV-2 infection. The patient received a near-complete recovery after standard immunological treatments. CONCLUSION: Attention should be paid to the evaluation of typical or atypical neurological symptoms that may be triggered by SARS-CoV-2 infection.

Interactions that define the arrangement of sugar-binding sites in BDCA-2 and dectin-2 dimers.

The sugar-binding receptors dectin-2 and blood dendritic cell antigen 2 (BDCA-2) bind oligosaccharide ligands through extracellular carbohydrate-recognition domains (CRDs) and initiate intracellular signaling through Fc receptor γ adapters (FcRγ). Dectin-2 stimulates macrophages in response to pathogen binding while BDCA-2 modulates cytokine production in plasmacytoid dendritic cells. The oligomeric states of these receptors and the orientations of their CRDs have been investigated by analysis of a naturally occurring disulfide-bonded variant of BDCA-2 and by replacement of transmembrane domains with N-terminal dimerization domains to create extracellular domain dimers of both dectin-2 and BDCA-2. Analysis of these constructs, as well as previously described crystal structures of the CRDs from these proteins and a novel structure of an extended version of the extracellular domain of dectin-2, showed that there is only limited interaction of the CRDs in the dimers, but interactions can be stabilized by the presence of the neck region. The resulting orientation of sugar-binding sites in the dimers would favor crosslinking of multiple dimers by oligosaccharide ligands, causing clustering of FcRγ to initiate signaling.

NLRP12 inhibits PRRSV-2 replication by promoting GP2a degradation via MARCH8.

NLRP12, a member of the NLR family, has been shown to exert a vital function in orchestrating immune responses. Here, using the immunosuppressive porcine reproductive and respiratory syndrome virus (PRRSV) as a model, the role of NLRP12 in virus infection was deciphered. We demonstrated that overexpression of NLRP12 significantly restrained PRRSV replication, while NLRP12 silencing resulted in increased viral titer. Mechanistically, NLRP12 interacts with glycoprotein 2a (GP2a) through its LRR domain and recruits the membrane-associated RING-CH E3 ubiquitin ligase 8 (MARCH8) via the PYD domain. NLRP12 facilitates the lysine-48 (K48)-linked polyubiquitination of GP2a at K128 and induces its lysosome degradation via the MARCH8-NDP52 (nuclear dot protein 52 kDa) pathway. To counteract this, PRRSV Nsp2 effectively prevented the polyubiquitination of GP2a induced by NLRP12 by its deubiquitinating activity. Meanwhile, the overexpression of Nsp4 decreased the mRNA of endogenous NLRP12 and cleaved NLRP12 in a 3C-like protease activity-dependent manner, which collaboratively counteracts the antiviral function of NLRP12. Collectively, this study revealed the mechanisms of the NLRP12-MARCH8-NDP52 axis in the host defense against PRRSV, which might be harnessed for the development of anti-PRRSV therapies.

A public, cross-reactive glycoprotein epitope confounds Ebola virus serology.

Ebola disease (EBOD) in humans is a severe disease caused by at least four related viruses in the genus Orthoebolavirus, most often by the eponymous Ebola virus. Due to human-to-human transmission and incomplete success in treating cases despite promising therapeutic development, EBOD is a high priority in public health research. Yet despite almost 50 years since EBOD was first described, the sources of these viruses remain undefined and much remains to be understood about the disease epidemiology and virus emergence and spread. One important approach to improve our understanding is detection of antibodies that can reveal past human infections. However, serosurveys routinely describe seroprevalences that imply infection rates much higher than those clinically observed. Proposed hypotheses to explain this difference include existence of common but less pathogenic strains or relatives of these viruses, misidentification of EBOD as something else, and a higher proportion of subclinical infections than currently appreciated. The work presented here maps B-cell epitopes in the spike protein of Ebola virus and describes a single epitope that is cross-reactive with an antigen seemingly unrelated to orthoebolaviruses. Antibodies against this epitope appear to explain most of the unexpected reactivity towards the spike, arguing against common but unidentified infections in the population. Importantly, antibodies of cross-reactive donors from within and outside the known EBOD geographic range bound the same epitope. In light of this finding, it is plausible that epitope mapping enables broadly applicable specificity improvements in the field of serology.

Absence of the dolichol synthesis gene DHRSX leads to N-glycosylation defects in Lec5 and Lec9 Chinese hamster ovary cells.

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

Associations of coagulation parameters and thrombin generation potential with the incidence of type 2 diabetes: mediating role of glycoprotein acetylation.

Hypercoagulability is characterized by abnormal elevations of coagulation factor levels and increased thrombin generation potential. Prior studies demonstrated links between impaired glucose metabolism, endothelial dysfunction, and hypercoagulability. However, the associations between hypercoagulability and incident type 2 diabetes as well as its underlying mechanism remain unclear. We aimed to assess the associations between coagulation parameters including coagulation factor (F) VIII, FIX, FXI, fibrinogen, thrombin generation potential (lag time, endogenous thrombin potential [ETP], peak, time-to-peak, velocity) and incident type 2 diabetes, and to study the underlying mechanism by examining the mediating role of glycoprotein acetylation (GlycA). In the Netherlands Epidemiology of Obesity study, we applied a Cox Proportional-Hazards Model in 5718 participants after adjustment for confounders. We further conducted a mediation analysis investigating the mediation effect of GlycA on the observed associations. During a median follow-up of 6.7 years, 281 incident type 2 diabetes diagnoses were reported. Compared with the lowest quartile, hazard ratio (95% confidence interval) of the highest quartile was 2.47 (1.48-4.14) for FIX, 1.37 (0.85-2.20) for FVIII, 1.11 (0.76-1.63) for FXI, 0.98 (0.65-1.48) for fibrinogen, 1.56 (1.07-2.28) for ETP, 1.84 (1.23-2.74) for peak, 1.59 (1.08-2.33) for velocity, 0.92 (0.62-1.38) for lag time, and 1.21 (0.86-1.70) for time-to-peak. GlycA mediated only a small proportion of all observed associations. In conclusion, elevated levels of coagulation factor and thrombin generation potential are associated with incident type 2 diabetes, suggesting the involvement of hypercoagulability in the pathogenesis of type 2 diabetes.

Expression and immobilization of novel N-glycan-binding protein for highly efficient purification and enrichment of N-glycans, N-glycopeptides, and N-glycoproteins.

Comprehensive and selective enrichment of N-glycans, N-glycopeptides, and N-glycoproteins prior to analysis is of great significance in N-glycomics research, reducing sample complexity, removing impurity interference, increasing sample abundance and enhancing signal intensity. However, only an Fbs1 (F-box protein that recognizes sugar chain 1) GYR variant (Fg) can enrich these N-glycomolecules solely due to its substantial binding affinity for the core pentasaccharide motif of N-glycans. Stationary phase separation is commonly used to enrich N-glycomolecules efficiently. Herein, DNA encoding the Fg was cloned into pGEX-4T-1, and the protein was expressed with a GST tag, which facilitates the convenient and efficient immobilization of recombinant GST-tagged Fg to GSH agarose resin. The yield of the GST-tagged Fg reached to 0.05 g/L after optimization of the induction condition, and the purified protein exhibited good identification ability and excellent stability for months. In particular, the immobilized GST-tagged Fg can enrich N-glycans released by PNGase F and capture derivatized N-glycans possessing an intact terminal N-acetyl glucosamine (GlcNAc). Validation of immobilized GST-tagged Fg with standard N-glycopeptides and N-glycoproteins revealed its high loading capacity, sensitivity, and selectivity. The novel immobilized GST-tagged Fg is a convenient and efficient enrichment material specific for N-glycans, N-glycopeptides, and N-glycoproteins, suggesting excellent performance and prospects for industrial application.

A practical assessment protocol for clinically relevant P-glycoprotein-mediated drug-drug interactions.

Background: Drug-drug interactions (DDIs) may influence the effectiveness and safety of medication treatment, which may require additional monitoring, dose adjustment or avoidance of certain drugs. DDIs involving P-glycoprotein (P-gp) affect many drugs, but current official product information is often insufficient to guide the management of these DDIs in clinical practice. The aim of this paper is to describe a protocol to assess DDIs involving P-gp and to develop and implement practice recommendations for clinically relevant P-gp-mediated DDIs that affect clinical outcomes through changes in systemic drug exposure. Methods: A combined literature review and expert opinion approach will be used according to the following seven steps: set up an expert panel (step 1), establish core concepts and definitions (step 2), select potential P-gp-modulators (i.e., P-gp-inducers and -inhibitors) and P-gp-substrates to be evaluated (step 3), select and extract evidence-based data, and present findings in standardized assessment reports (step 4), discuss and adopt classifications and practice recommendations with the expert panel (step 5), publish and integrate information and alerts in clinical decision support systems (CDSS) (step 6), (re)assessments of DDIs and potential new DDIs when new information is available or when initiated by healthcare providers (step 7). Anticipated results: The expert panel will classify potential P-gp-modulators and -substrates as clinically relevant P-gp-inducer, -inhibitor and/or -substrate and draw conclusions about which combinations of classified modulators and substrates will lead to clinically relevant DDIs. This may include the extrapolation of conclusions for DDIs where limited or no data are available, based on the pharmacological characteristics of these drugs. For (potential) DDIs that are considered to be clinically relevant, practice recommendations will be developed. Discussion: This protocol describes a standardized, evidence- and expert opinion-based assessment of P-gp-mediated DDIs that affect clinical outcomes. This approach will generate alerts with practice recommendations for clinically relevant DDIs and transparent rationales for DDIs that are considered to be irrelevant. These recommendations will improve individual patient care by supporting healthcare professionals to make consistent decisions on how to manage P-gp mediated DDIs.

Performance of an envelope glycoprotein-based multiplex immunoassay for Ebola virus antibody detection in a cohort of Ebola virus disease survivors.

Serological surveillance in animal and human hosts can be a cost-effective strategy for orthoebolavirus detection, but is challenged by accurate estimates of seroprevalence, potential pauci-symptomatic disease presentation, and antigenic cross-reactivity. Here, we describe the use of an envelope glycoprotein (GP)-based multiplex microsphere immunoassay, consisting of nine filovirus GP antigens, for the detection of anti-Ebola virus (EBOV) antibodies in a well-characterized cohort of Guinean Ebola virus disease (EVD) survivors and contacts from the 2013 - 2016 West African EVD outbreak. We examined sensitivity and specificity for the detection of anti-EBOV antibodies by GP expressed as recombinant trimeric ectodomains, yielding an assay performance of 95.96% sensitivity and 98.61% specificity. Additionally, agreement between the multiplex test and a whole virus ELISA and virus neutralization test showed strong correlations. The results demonstrate this filovirus multiplex test is a sensitive tool for high-throughput serosurveillance.

HERV-Derived Syncytin-1 and Syncytin-2 as Sources of Linear and Discontinuous Epitopes in Antiphospholipid Syndrome: A Pivotal Computational Study.

BACKGROUND: To date, no studies have investigated the potential reactivation of human endogenous retroviruses (HERVs) in the pathogenesis of antiphospholipid syndrome (APS). HERV-derived syncytin-1 and syncytin-2 are localized in the plasma membrane of cells and physiologically expressed during pregnancy. The current study aimed to determine whether the epitopes of syncytins can trigger an immune response leading to APS in genetically predisposed individuals. METHODS: The TepiTool, ABCpred, and DiscoTope servers were utilized to predict T-cell and B-cell epitopes by inputting the FASTA sequences and 3D structures of syncytin-1, syncytin-2, and ß2-glycoprotein I (ß2GPI), which served as a reference antigen for APS. T-cell epitopes were selected based on their binding to a panel of human leukocyte antigen (HLA) class II alleles associated with APS according to the literature. Epitope predictions for the different proteins were statistically compared using GraphPad Prism. RESULTS: For syncytin-1, we identified a total of 721 T-cell epitopes, 51 linear B-cell epitopes, and up to 40 conformational epitopes. For syncytin-2, we predicted 705 T-cell epitopes and 28 linear B-cell epitopes, but a lower number of conformational epitopes, which also exhibited lower B-cell receptor (BCR)-binding scores. The predicted T-cell and B-cell conformational epitopes of both syncytin-1 and syncytin-2 demonstrated significantly higher binding affinity to selected HLA alleles and BCR compared with ß2GPI. Furthermore, syncytin-1 exhibited significantly higher immunogenicity than syncytin-2. CONCLUSIONS: Both syncytin-1 and syncytin-2 are computationally endowed with potential epitopes that may activate either T cells or B cells in individuals genetically predisposed to APS. While these findings may illuminate the possible role of HERVs in the development of APS, they warrant validation in further laboratory studies.

Analysis of the antigenic and immunogenic properties of the native rabies virus glycoprotein purified by Lens culinaris lectin affinity chromatography.

Rabies virus glycoprotein (RABV-G) is responsible for the recognition of specific cell surface receptors and induces the production of neutralizing antibodies (VNA). Since RABV-G is a glycoprotein, this work aimed to evaluate Lens culinaris (LCA) chromatography as a simple and effective purification method. The purity and identification of the protein obtained were analyzed by SDS-PAGE, ELISA and lectin-binding assay. The antigenic properties of the purified RABV-G were evaluated by direct ELISA using human serum samples from individuals who had received rabies pre-exposure vaccination. For the immunogenicity study, Swiss Webster mice were immunized with purified RABV-G and the specific antibodies were measured by direct ELISA and RFFIT. As results, it was observed that the purified protein reveled a molecular mass of 55 kDa and the presence of carbohydrate; additionally, it was recognized by anti-rabies virus glycoprotein monoclonal antibody. Purified RABV-G induced high VNA titers (>50.0 IU/ml) in vivo, as detected by RFFIT, as well as RABV-G specific IgG1 (0.8 mean OD±SD) and IgG2a (0.3 mean OD±SD) antibodies, with a predominance of IgG1 (p< 0.001). In addition, it was observed that RABV-G was efficient in selectively detecting anti- RABV-G IgG in the sera of vaccinated individuals compared to the negative control. Therefore, LCA chromatography was efficient in preserving the native properties of RABV-G that are essential in inducing an adequate humoral immune response. In addition, the purified RABV-G presented analytical potential as an ELISA reagent.

Platelet Function, Platelet Size and Content of Reticulated Platelets: Interactions in Patients Receiving Dual Antiplatelet Therapy.

Increased platelet activity is a risk factor of thrombotic events in cardiovascular patients. We studied the relationship between platelet function, platelet size, and the content of reticulated platelets (RP) in patients with coronary heart disease (CHD, n = 55) and acute coronary syndrome (ACS, n = 95) receiving acetylsalicylic acid + clopidogrel or ticagrelor, respectively. The control group consisted of patients with risk factors for CHD, but with no CHD/ACS and free of antiplatelet drugs (n = 66). Platelet function was evaluated by the exposure of activated glycoprotein (GP) IIb-IIIa and P-selectin. In the control group, platelets were activated by TRAP (Thrombin Receptor Activating Peptide) 10 µM, and ADP 20, 5, 2.5 µM, and in the CHD/ACS groups, by TRAP 10 µM, and ADP 20 5 µM (±epinephrine 20 µM). Platelet size was assessed by the mean volume, % large forms, and forward scattering. RP were stained by thiazole orange. In the control group, activated GP IIb-IIIa and P-selectin correlated with platelet size and RP content after platelet activation by all agonists. Despite the decrease in platelet activity by antiplatelet drugs, most correlations (primarily for activated GP IIb-IIIa) were preserved in the CHD/ACS patients. In conclusion, increased platelet size and RP content are associated with increased platelet activity and the reduced efficacy of antiplatelet therapy.

Vesicle-Transported Multidrug Resistance as a Possible Therapeutic Target of Natural Compounds.

BACKGROUND/OBJECTIVES: A key role of extracellular vesicles (EVs) is mediating both cell-cell and cell-stroma communication in pathological/physiological conditions. EVs from resistant tumor cells can transport different molecules like P-glycoprotein (P-gp), acting as a shuttle between donor and recipient cells, resulting in a phenotypic change. The aim of our work was to isolate, characterize, and inhibit the release of EVs in two multidrug resistance (MDR) cancer models: MCF-7R (breast cancer cell line) and HL-60R (acute myeloid leukemia cell line). METHODS: The existence of P-gp in EVs from MDR cells was confirmed by Western blotting assays. The characterization of EVs was carried out by evaluating the size using NTA and the presence of specific markers such as CD63, Hsp70 and Syntenin. The ability of HL-60R and MCF-7R to perform horizontal transfer of P-gp via EVs to sensitive cells was assessed using three different methods. The acquisition of resistance and its inhibition in recipient cells was confirmed by MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: Our data showed that cell lines (MDR) release P-gp-loaded EVs, unlike sensitive cells. The acquisition of resistance determined by the incorporation of P-gp into the membrane of sensitive cells was confirmed by the reduced cytotoxic activity of doxorubicin. Natural compounds such as curcumin, lupeol, and heptacosane can block vesicular transfer and restore the sensitivity of HL-60 and MCF-7 cells. CONCLUSIONS: Our study demonstrates that natural inhibitors able to reverse this mechanism may represent a new therapeutic strategy to limit the propagation of the resistant phenotype.

Soloxolone N-3-(Dimethylamino)propylamide Restores Drug Sensitivity of Tumor Cells with Multidrug-Resistant Phenotype via Inhibition of P-Glycoprotein Efflux Function.

Multidrug resistance (MDR) remains a significant challenge in cancer therapy, primarily due to the overexpression of transmembrane drug transporters, with P-glycoprotein (P-gp) being a central focus. Consequently, the development of P-gp inhibitors has emerged as a promising strategy to combat MDR. Given the P-gp targeting potential of soloxolone amides previously predicted by us by an absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, the aim of the current study was to experimentally verify their P-gp inhibitory and MDR reversing activities in vitro. Screening of soloxolone amides as modulators of P-gp using molecular docking and cellular P-gp substrate efflux assays revealed the ability of compound 4 bearing a N-3-(dimethylamino)propylamide group to interact with the active site of P-gp and inhibit its transport function. Blind and site-specific molecular docking accompanied by a kinetic assay showed that 4 directly binds to the P-gp transmembrane domain with a binding energy similar to that of zosuquidar, a third-generation P-gp inhibitor (ΔG = -10.3 kcal/mol). In vitro assays confirmed that compound 4 enhanced the uptake of Rhodamine 123 (Rho123) and doxorubicin (DOX) by the P-gp-overexpressing human cervical carcinoma KB-8-5 (by 10.2- and 1.5-fold, respectively (p < 0.05, unpaired t-test)) and murine lymphosarcoma RLS40 (by 15.6- and 1.75-fold, respectively (p < 0.05, unpaired t-test)) cells at non-toxic concentrations. In these cell models, 4 showed comparable or slightly higher activity than the reference inhibitor verapamil (VPM), with the most pronounced effect of the hit compound in Rho123-loaded RLS40 cells, where 4 was 2-fold more effective than VPM. Moreover, 4 synergistically restored the sensitivity of KB-8-5 cells to the cytotoxic effect of DOX, demonstrating MDR reversal activity. Based on the data obtained, 4 can be considered as a drug candidate to combat the P-gp-mediated MDR of tumor cells and semisynthetic triterpenoids, with amide moieties in general representing a promising scaffold for the development of novel therapeutics for tumors with low susceptibility to antineoplastic agents.

From Organotypic Mouse Brain Slices to Human Alzheimer's Plasma Biomarkers: A Focus on Nerve Fiber Outgrowth.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss and progressive deterioration of cognitive functions. Being able to identify reliable biomarkers in easily available body fluids such as blood plasma is vital for the disease. To achieve this, we used a technique that applied human plasma to organotypic brain slice culture via microcontact printing. After a 2-week culture period, we performed immunolabeling for neurofilament and myelin oligodendrocyte glycoprotein (MOG) to visualize newly formed nerve fibers and oligodendrocytes. There was no significant change in the number of new nerve fibers in the AD plasma group compared to the healthy control group, while the length of the produced fibers significantly decreased. A significant increase in the number of MOG+ dots around these new fibers was detected in the patient group. According to our hypothesis, there are factors in the plasma of AD patients that affect the growth of new nerve fibers, which also affect the oligodendrocytes. Based on these findings, we selected the most promising plasma samples and conducted mass spectrometry using a differential approach and we identified three putative biomarkers: aldehyde-dehydrogenase 1A1, alpha-synuclein and protein S100-A4. Our method represents a novel and innovative approach for translating research findings from mouse models to human applications.

Aggressive Course of Myelin Oligodendrocyte Glycoprotein Antibody-Associated Disease (MOGAD): An Illustration of Two Cases and Review of Literature.

Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is a central nervous system demyelinating disease that has become a major source of morbidity among children and adults. In the first case, we present an 18-year-old Hispanic female with a recently resolved upper respiratory infection who presented with fever, headache, progressive quadriparesis, urinary retention, and encephalopathy. The hospital course involved autonomic dysfunction and prolonged intubation requiring tracheostomy and gastrostomy. Cerebrospinal fluid (CSF) showed pleocytosis and a positive MOG titer (1:40). Magnetic resonance imaging (MRI) showed longitudinally extensive cervicothoracic T2 hyperintensity and brain multifocal T2 hyperintensities. After high-dose intravenous methylprednisolone (IVMP) and intravenous immunoglobulin (IVIG), she had full neurological recovery by the last follow-up. The second case is of a 22-year-old Hispanic male who presented with progressive lower extremity paresthesia and weakness over six weeks. CSF demonstrated pleocytosis, elevated protein, oligoclonal bands, and MOG antibody. MRI revealed multiple subcortical T2-hyperintense lesions and enhancing midcervical and lower thoracic lesions. Treatment with IVMP led to minor improvement with discharge on steroid taper and azathioprine. The patient's disease progressed with a fluctuating course requiring two readmissions with upper extremity weakness, right optic neuritis, and urinary sphincteric dysfunction with neuroradiologic worsening. Treatment throughout multiple admissions included intravenous steroids, IVIG, plasmapheresis, mycophenolate mofetil, and rituximab with minimal improvement, symptom recurrence, and progression of multifocal lesions. The patient died four months after the symptom onset. These cases had markedly different treatment responses despite similar baseline characteristics. The difference in morbidity and disability burden highlights the importance of further investigation of this condition through clinical trials.

Hypoxia-Associated GPNMB+ Macrophages Promote Malignant Progression of Colorectal Cancer and Its Related Risk Signature Are Powerful Predictive Tool for the Treatment of Colorectal Cancer Patients.

Colorectal cancer (CRC) is a highly malignant tumor with hypoxia being a crucial feature during its progression. This study utilized multiple independent CRC cohorts for bioinformatics analysis and in vitro experiments to investigate the role of hypoxia-related subgroups in CRC. Machine learning was employed to construct risk features associated with this subgroup and further explore its therapeutic value in CRC. The study identified the GPNMB+ Macrophage (GPNMB+ Macr) subgroup as most relevant to hypoxia. GPNMB+ Macr showed significantly higher infiltration in tumor tissues compared to non-tumor tissues, increasing with CRC stage. High infiltration of GPNMB+ Macr was associated with poor prognosis in terms of overall and recurrence-free survival in CRC patients. GPNMB+ Macrophages exhibit M2-like characteristics and have the ability to promote 5-FU resistance, proliferation, and metastasis of CRC cells. The study developed the Hypoxia-Related Macrophage Risk Score (HMRS), which not only served as an independent prognostic factor for CRC patients but also demonstrated robust prognostic performance compared to 84 previously published prognostic features. Patients with low HMRS were sensitive to fluorouracil, oxaliplatin (FOLFOX), and anti-PD-1 immunotherapy, while those with high HMRS showed resistance. Additionally, HMRS was identified as an independent prognostic factor in other digestive tract tumors (hepatocellular carcinoma, pancreatic cancer, esophageal cancer, and gastric cancer), indicating potential extrapolation to other tumor types. In conclusion, GPNMB+ Macr promotes the malignant progression of CRC, and HMRS serves as a powerful predictive tool for prognosis, chemotherapy, and immunotherapy in CRC patients, aiding in improving the quality of survival.

Preclinical evaluation and pilot clinical study of [68Ga]Ga-NOTA-H006 for non-invasive PET imaging of 5T4 oncofetal antigen.

PURPOSE: Trophoblast glycoprotein, the so-called 5T4, is an oncofetal antigen expressed in many different cancers. However, no 5T4-specific radioligand is employed in the clinic for non-invasive diagnosis. Thus, the aim of the current study was to develop a PET radiotracer for imaging 5T4 expression in preclinical and clinical stages. METHODS: A VHH library was constructed by camel immunization. The specificity of the VHHs toward 5T4 antigen was screened through phage display biopanning and periplasmic extract enzyme-linked immunosorbent assay. 1,4,7-Triazacyclononane-1,4,7-triacetate acid (NOTA) derivative was conjugated to the selected VHH. After radiolabeling, microPET/CT and ex vivo biodistribution were conducted using BxPC-3 and MDA-MB-468 tumor-bearing mice. Cold VHH was co-injected with the tracer to challenge its binding in vivo. For the pilot clinical study, PET/CT images were acquired at 1 h after injection of tracer in patients with pathologically confirmed primary and metastatic tumors. RESULTS: A library with a capacity of 1.2 × 1012 colony-forming units was constructed after successful camel immunization. Nb1-40 with a median effect concentration of 0.43 nM was selected. After humanization, the resulting H006 maintained a high affinity towards 5T4. [68Ga]Ga-NOTA-H006 with the molar activities of 6.48-54.2 GBq/µmol was prepared with high radiochemical purity (> 98%). Using [68Ga]Ga-NOTA-H006, microPET/CT revealed a clear visualization of 5T4 expression in BxPC-3 tumor-bearing mice. Ex vivo biodistribution showed that the highest tumor-to-blood ratio (∼ 3-fold) and tumor-to-muscle ratio (∼ 5-fold) were achieved at 60 min post-injection. Co-injection of the cold H006 at a dose of 1.5 mg/kg significantly reduced the tumor uptake (p < 0.0001). In the pilot clinical study, [68Ga]Ga-NOTA-H006 demonstrated its capacity to map 5T4-positive lesions in humans and yielded a mean effective dose of 3.4 × 10- 2 mSv/MBq. CONCLUSIONS: [68Ga]Ga-NOTA-H006, which can visualize 5T4 expression in vivo, has been successfully developed. This opens up opportunities for non-invasively studying 5T4 expression through nuclear medicine. Further clinical investigations are warranted to explore its clinical value in disease progression and companion diagnosis.

Low-dose aspirin and heparin treatment improves pregnancy outcome in recurrent pregnancy loss women with anti-ß2-glycoprotein I/HLA-DR autoantibodies: a prospective, multicenter, observational study.

Introduction: Anti-ß2-glycoprotein I (ß2GPI)/human leukocyte antigen (HLA)-DR antibodies may be a risk factor for recurrent pregnancy loss (RPL). The therapeutic modality for women with RPL and anti-ß2GPI/HLA-DR antibody positivity has not been evaluated. This prospective, multicenter, observational study aimed to assess whether low-dose aspirin (LDA) and/or heparin therapies improve pregnancy outcomes in women with RPL who tested positive for anti-ß2GPI/HLA-DR antibodies. Methods: Between August 2019 and December 2021, 462 women with RPL underwent anti-ß2GPI/HLA-DR antibody measurements and risk assessments for RPL. Each attending physician decided the treatment modality for women with RPL who tested positive for anti-ß2GPI/HLA-DR antibodies, and their pregnancy outcomes were followed up until December 2023. Finally, 47 pregnancies in 47 women with RPL and anti-ß2GPI/HLA-DR antibody positivity were included in the analysis and were divided into two groups regarding whether they were treated with LDA and/or unfractionated heparin (UFH) (LDA/UFH group, n = 39) or with neither of them (non-LDA/non-UFH group, n = 8). The rates of live birth and pregnancy complications (i.e., preeclampsia and preterm delivery before 34 gestational weeks due to placental insufficiency) were compared between the two groups. Results: The live birth rate in the LDA/UFH group was higher than that in the non-LDA/non-UFH group (87.2% vs 50.0%, p = 0.03). The pregnancy complication rate in the LDA/UFH group was significantly lower than that in the non-LDA/non-UFH group (5.9% vs 50.0%, p = 0.048). Among 21 women who tested positive for anti-ß2GPI/HLA-DR antibodies and had no other risk factors for RPL, the live birth rate in the LDA/UFH group (n = 14) was much higher than that in the non-LDA/non-UFH group (n = 7) (92.9% vs 42.9%, p = 0.03). Discussion: This study, for the first time, demonstrated that LDA and/or UFH therapies are effective in improving pregnancy outcomes in women with RPL and aß2GPI/HLA-DR antibody positivity.