Gene expression analysis revealed downregulation of complement receptor 1 in clonal B cells in cold agglutinin disease.

Cold agglutinin disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to the I antigen. Underlying genetic changes have previously been reported, but their impact on gene expression profile has been unknown. Here, we define differentially expressed genes in CAD B cells. To unravel downstream alteration in cellular pathways, gene expression by RNA sequencing was undertaken. Clonal B-cell samples from 12 CAD patients and IgM-expressing memory B cells from 4 healthy individuals were analyzed. Differential expression analysis and filtering resulted in 93 genes with significant differential expression. Top upregulated genes included SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, and IGHV4-34. They were upregulated at least 8-fold, while complement receptor 1 (CR1/CD35) was downregulated 11-fold in clonal CAD B cells compared to control B cells. Flow cytometry analyses further confirmed reduced CR1 (CD35) protein expression by clonal CAD IgM+ B cells compared to IgM+ memory B cells in controls. CR1 (CD35) is an important negative regulator of B-cell activation and differentiation. Therefore, reduced CR1 (CD35) expression may increase activation, proliferation, and antibody production in CAD-associated clonal B cells.

Soybean Agglutinin Alters the Gut Microbiota and Promotes Inflammation in Lupus-Prone MRL/lpr Mice.

BACKGROUND: Certain foods can trigger flares in patients with systemic lupus erythematosus. Lectins in edible plants have been reported to increase inflammation. OBJECTIVE: This study aimed to determine the effects of 1-time intake of soybean agglutinin (SBA) on the gut microbiota and immune response in lupus-prone MRL/MpJ (MRL)/lpr mice. METHODS: MRL/MpJ-Faslpr/J (MRL/lpr) and MRL mice were randomly assigned into 4 groups (8 mice/group): MRL mice + phosphate-buffered saline (PBS) (CON), MRL mice + SBA (CS), MRL/lpr mice + PBS (LPR), and MRL/lpr + SBA (LS). PBS and SBA were orally administered at 16 wk of age, and all mice were killed 24 h after oral challenge. The disease phenotype, levels of proinflammatory cytokines, and composition of the intestinal microbiota were determined. RESULTS: Interferon-gamma (IFN-γ) in the serum was significantly higher, whereas the level of serum IL-10 was significantly lower in LS mice than in LPR mice [fold change (FC) = 1.31 and FC = 0.36, respectively]. The expression levels of IL-6 and TNF-α in the spleen of LS mice were significantly higher than those in LPR mice (FC = 1.66 and FC = 1.96, respectively). The expression levels of IL-6, TNF-α, and IL-1ß in the kidney were also significantly higher in LS mice than in LPR mice (FC = 2.89, FC = 3.78, and FC = 2.02, respectively). The relative abundances of Erysipelotrichaceae and Turicibacter in LS mice were significantly higher than those in LPR mice (FC = 1.73 and FC = 1.74, respectively). The percentage of Breg cells in the mesenteric lymph nodes was significantly lower in LS mice than in LPR mice (FC = 0.53) (P < 0.05). No change was found between SBA treatment or not in the control (MRL) mice. CONCLUSIONS: One-time intake of SBA can promote the secretion of proinflammatory cytokines, downregulate Breg cells, and alter the intestinal flora in MRL/lpr mice within 24 h of oral challenge, which may contribute to exacerbation of lupus.

Retrograde tracing of breast cancer-associated sensory neurons.

Breast cancer is one of the leading causes of mortality among women. The tumour microenvironment, consisting of host cells and extracellular matrix, has been increasingly studied for its interplay with cancer cells, and the resulting effect on tumour progression. While the breast is one of the most innervated organs in the body, the role of neurons, and specifically sensory neurons, has been understudied, mostly for technical reasons. One of the reasons is the anatomy of sensory neurons: sensory neuron somas are located in the spine, and their axons can extend longer than a meter across the body to provide innervation in the breast. Next, neurons are challenging to culture, and there are no cell lines adequately representing the diversity of sensory neurons. Finally, sensory neurons are responsible for transporting several different types of signals to the brain, and there are many different subtypes of sensory neurons. The subtypes of sensory neurons, which innervate and interact with breast tumours, are unknown. To establish the tools for labelling and subtyping neurons that interact with breast cancer cells, we utilised two retrograde tracer's standards in neuroscience, wheat-germ agglutinin (WGA) and cholera toxin subunit B (CTB). In vitro, we employed primary sensory neurons isolated from mouse dorsal root ganglia, cultured in a custom-built microfluidic device DACIT, that mimics the anatomical compartmentalisation of the sensory neuron's soma and axons. In vivo, we utilised both syngeneic and transgenic mouse models of mammary carcinoma. We show that CTB and WGA trace different but overlapping sensory neuronal subpopulations: while WGA is more efficient in labelling CGRP+ neurons, CTB is superior in labelling the NF200+ neurons. Surprisingly, both tracers are also taken up by a significant population of breast cancer cells, both in vitro and in vivo. In summary, we have established methodologies for retrograde tracing of sensory neurons interacting with breast cancer cells. Our tools will be useful for future studies of breast tumour innervation, and development of therapies targeting breast cancer-associated neuron subpopulations of sensory neurons. Lay description: Breast cancer is an aggressive disease that affects both women and men throughout the world. While it has been reported that the increasing size of nerves in breast cancer correlates to bad prognosis in patients, the role of nerves, especially sensory nerves, in breast cancer progression, has remained largely understudied. Sensory nerves are responsible for delivering signals such as pain, mechanical forces (pressure, tension, stretch, touch) and temperature to the brain. The human body is densely innervated, and nerves extending into peripheral organs can be as long as a few meters. Nerve classification and function can be very complex, as they contain bundles of extensions (axons) originating in different neuronal bodies (soma). Maintaining neurons and growing axons in cell culture conditions in order to mimic innervation is technically challenging, as it involves multiple organs of the human body. Here, we focus on tracing sensory axons from the breast tumours back to the neuronal soma, located in the dorsal root ganglia, inside the spine. To do so, we are using two different 'retrograde' tracers, WGA and CTB, which are proteins with a natural ability to enter axons and travel in a retrograde fashion, arriving at the soma, even if it means to travel distances longer than a meter. Both tracers are fluorescently labelled, making them visible using high-resolution fluorescent microscopy. We show that both WGA and CTB can label sensory neurons in tumours, or in cell culture conditions. The two tracers differ in efficiency of tracing different sensory neurons subpopulations: while WGA is more efficient in tracing small C-fibres (CGRP-positive), CTB is more efficient in tracing A-fibres (NF200+) of sensory neurons. In summary, we have successfully established retrograde tracing techniques for sensory neurons towards studying and targeting breast cancer innervation.

A fungal-binding agglutinin in the skin slime of Japanese flounder (Paralichthys olivaceus) is glyceraldehyde 3-phosphate dehydrogenase.

Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast-binding protein, which consists of an approximately 35-kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time-of-flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An anti-GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well-known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts.

Cold-antibody Autoimmune Hemolytic Anemia: its Association with Neoplastic Disease and Impact on Therapy.

PURPOSE OF REVIEW: Cold-antibody mediated autoimmune hemolytic anemia (cAIHA) is subclassified as cold agglutinin disease (CAD), secondary cold agglutinin syndrome (CAS), and paroxysmal cold hemoglobinuria (PCH). This review aims to address the occurrence of neoplastic disorders with these three entities and analyze the impact of such neoplasias on treatment for cAIHA. RECENT FINDINGS: "Primary" CAD is a distinct clonal B-cell lymphoproliferative disorder in probably all cases, although not classified as a malignant lymphoma. CAS is secondary to malignant lymphoma in a minority of cases. Recent findings allow a further clarification of these differential diagnoses and the therapeutic consequences of specific neoplastic entities. Appropriate diagnostic workup is critical for therapy in cAIHA. Patients with CAD should be treated if they have symptomatic anemia, significant fatigue, or bothersome circulatory symptoms. The distinction between CAD and CAS and the presence of any underlying malignancy in CAS have essential therapeutic implications.

Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Successful treatment of concurrent cold agglutinin disease and myelodysplastic syndrome.

Herein, we describe a case of severe anemia presenting with myelodysplastic syndrome with cold agglutinin disease that was successfully treated by a moderate dose of steroids followed by cyclosporine. In patients with myelodysplastic syndrome, autoimmunity in erythroid cells is occasionally demonstrated, and autoimmune hemolytic anemia is seen in some patients. However, hemolytic anemia with cold agglutinin in patients with myelodysplastic syndrome is less common, and the effect of corticosteroids for autoimmune hemolytic anemia caused by cold agglutinin is thought to be limited. Although the elevated levels of reticulocytes and LDH are usually caused by ineffective hematopoiesis in myelodysplastic syndrome, clinicians should be aware of latent cold agglutinin disease. In the present case, in addition to the improvement of erythroid dysplasia, the corticosteroid-sparing effect on cold agglutinin disease may have played a role in the mechanism underlying the effectiveness of cyclosporine.

Construction of Biosensing System for Glycated Albumin Using an Electron Transfer Peptide-Modified Protein Probe.

Glycated albumin (GA) is one of the proteins that replaces several sugar moieties and can be used as an indicator of diabetes mellitus. We developed a sensing system that uses GA in the early detection of diabetes mellitus. In this study, H6Y4C acetylated (Ac-) at the N-terminals of the peptide was combined with wheat germ agglutinin (WGA) to recognize glucose moieties. The Ac-H6Y4C-WGA was constructed as a GA-sensing probe. The tyrosine residues of Y4C exhibited an oxidation peak, and His-tag moieties were introduced to separate Ac-H6Y4C-WGA in the synthesis of the probe. The Ac-H6Y4C-WGA probe binds with the 1-2 molecules of Ac-H6Y4C per WGA using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-MS. Next, the functions of Ac-H6Y4C-WGA were evaluated using voltammetry. The number of electron-transfers was calculated based on the relationship between the peak potential and logarithm of scan rate and was 3.03. In the electrochemical measurements with mannose and bovine serum albumin, the peak currents were similar to that of GA alone. By contrast, a decrease in the peak current was suppressed when glucose was added to the solution containing the probe. As a result, Ac-H6Y4C-WGA was selectively bound to the glucose moieties of GA. The calibration curve via differential pulse voltammetry was proportional to the concentrations of GA and ranged from 1.0 × 10-12 to 2.0 × 10-11 M with a detection limit of 3.3 × 10-13 M.

Ultrasensitive colorimetric detection of Staphylococcus aureus using wheat germ agglutinin and IgY as a dual-recognition strategy.

A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.

Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

[Cold agglutinin disease: pathology, diagnosis, and treatment].

Cold agglutinin disease (CAD), an immune hemolytic disease mediated by the classical complement-dependent pathway, accounts for approximately 8% of autoimmune hemolytic anemia (AIHA) cases. Primary CAD is a clonal B-cell lymphoproliferative disease of the bone marrow that produces IgM type-M protein, while conventional secondary CAD is cold agglutinin syndrome (CAS). Clinical findings are broadly classified into chronic anemia due to hemolysis and symptoms associated with peripheral circulatory failure due to erythrocyte aggregation under cold exposure. Not all patients require drug therapy, but monoclonal antibody therapy against complement C1s is preferred for the former presentation and B-cell suppressors for the latter. As cold AIHA is treated differently than warm AIHA, misdiagnosis can significantly impact the outcome of treatment. The most important aspect of blood testing is temperature control of specimens. Cold agglutinin titer, IgM quantification, electrophoresis, and immunofixation methods may produce false-negative results if the serum is not temperature-controlled at 37-38°C until serum separation. Correct handling of specimens, along with knowledge of the various clinical features of CAD, will lead to correct diagnosis and appropriate treatment.

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy.

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

Anti-C1s humanized monoclonal antibody SAR445088: A classical pathway complement inhibitor specific for the active form of C1s.

The objective of this study was to characterize the complement-inhibiting activity of SAR445088, a novel monoclonal antibody specific for the active form of C1s. Wieslab® and hemolytic assays were used to demonstrate that SAR445088 is a potent, selective inhibitor of the classical pathway of complement. Specificity for the active form of C1s was confirmed in a ligand binding assay. Finally, TNT010 (a precursor to SAR445088) was assessed in vitro for its ability to inhibit complement activation associated with cold agglutinin disease (CAD). TNT010 inhibited C3b/iC3b deposition on human red blood cells incubated with CAD patient serum and decreased their subsequent phagocytosis by THP-1 cells. In summary, this study identifies SAR445088 as a potential therapeutic for the treatment of classical pathway-driven diseases and supports its continued assessment in clinical trials.

The role of AtPP2-A3 and AtPP2-A8 genes encoding Nictaba-related lectin domains in the defense response of Arabidopsis thaliana to Heterodera schachtii.

MAIN CONCLUSION: Expression levels of AtPP2-A3 and AtPP2-A8 are reduced in syncytia induced by Heterodera schachtii and decline of their expression levels decreases host susceptibility, whereas their overexpression promotes susceptibility to parasite. Plant-parasitic nematodes cause huge crop losses worldwide. Heterodera schachtii is a sedentary cyst-forming nematode that induces a feeding site called a syncytium via the delivery of secreted chemical substances (effectors) to host cells, which modulate host genes expression and phytohormone regulation patterns. Genes encoding the Nictaba-related lectin domain have been found among the plant genes with downregulated expression during the development of syncytia induced by H. schachtii in Arabidopsis thaliana roots. To investigate the role of two selected Nictaba-related genes in the plant response to beet cyst nematode parasitism, mutants and plants overexpressing AtPP2-A3 or AtPP2-A8 were infected, and promoter activity and protein localization were analyzed. In wild-type plants, AtPP2-A3 and AtPP2-A8 were expressed only in roots, especially in the cortex and rhizodermis. After nematode infection, their expression was switched off in regions surrounding a developing syncytium. Astonishingly, plants overexpressing AtPP2-A3 or AtPP2-A8 were more susceptible to nematode infection than wild-type plants, whereas mutants were less susceptible. Based on these results and changes in AtPP2-A3 and AtPP2-A8 expression patterns after treatments with different stress phytohormones, we postulate that the AtPP2-A3 and AtPP2-A8 genes play important roles in the defense response to beet cyst nematode infection.

Candida parapsilosis cell wall proteins-CPAR2_404800 and CPAR2_404780-Are adhesins that bind to human epithelial and endothelial cells and extracellular matrix proteins.

One of the initial steps necessary for the development of Candida infections is the adherence to the host tissues and cells. Recent transcriptomic studies suggest that, in Candida parapsilosis-a fungal infectious agent that causes systemic candidiasis in immunosuppressed individuals-the adhesion is mediated by pathogen cell-exposed proteins belonging to the agglutinin-like sequence (Als) family. However, to date, the actual interactions of individual members of this family with human cells and extracellular matrix (ECM) have not been characterized in detail. In the current study, we focused attention on two of these C. parapsilosis Als proteins-CPAR2_404800 and CPAR2_404780-that were proteomically identified in the fungal cell wall of yeasts grown in the media suitable for culturing human epithelial and endothelial cells. Both proteins were extracted from the cell wall and purified, and using a microplate binding assay and a fluorescence microscopic analysis were shown to adhere to human cells of A431 (epithelial) and HMEC-1 (endothelial) lines. The human extracellular matrix components that are also plasma proteins-fibronectin and vitronectin-enhanced these interactions, and also could directly bind to CPAR2_404800 and CPAR2_404780 proteins, with a high affinity (KD in a range of 10-7 to 10-8 M) as determined by surface plasmon resonance measurements. Our findings highlight the role of proteins CPAR2_404800 and CPAR2_404780 in adhesion to host cells and proteins, contributing to the knowledge of the mechanisms of host-pathogen interactions during C. parapsilosis-caused infections.

Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination.

BACKGROUND AIMS: Cold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3-4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. CASE REPORTS: Four cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. CONCLUSIONS: While uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

Recognition factors of Dolichos biflorus agglutinin (DBA) and their accommodation sites.

Dolichos biflorus agglutinin (DBA) is one of the well known plant lectins that are widely used in clinical serology to differentiate human blood group A1 and A2 erythrocytes and also applied to glycobiology. However, the knowledge of recognition factors of polyvalent (super) glycotopes in glycans and the roles of functional group and epimer in monosaccharide (sub-monosaccharide recognition factor) have not been well established. The size and shape of the recognition (combining) site of DBA has not been clearly defined. In this study, many importnat recognition factors of DBA-glycan binding were characterized by our established enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results of these assays showed that the intensity profile of the recognition factors for the major combining site of DBA was expressed by Mass relative potency (Mass R.P.) and shown by decreasing order of high density of polyvalent GalNAcα1 → (super glycotopes, 3.7 × 103) >> the corresponding ß anomers >> monomeric GalNAcα1 → related glycotopes (GalNAc as 1.0) >> their GalNAc ß-anomers >> Gal (absence of NHCH3CO at carbon-2 of GAlNAc) and GlcNAc (different epimer of Carbon-4 in GalNAc). From the all data available, it is proposed that the combining site of DBA should consist of a small cavity shape as major site and most complementary to monomeric GalNAcα → located at both terminal reducing end (Tn) and nonreducing end of glycan chains, and with a wide and broad area as subsite to accomodate from mono- to tetra-saccharides (GalNAcß, Galß1 → 3/4GlcNAc, lFuc1 → 2Galß1 → 3/4GlcNAc, GalNAcß1 → 3Galα1 → 4Galß1 → 4Glc) at the nonreducing side. In this study, it has provided the most (comprehensive) recognition knowledge of DBA-glycan interactions at the factors of glycotope, super glycotope/sub-monosaccharide levels. Thus, it should expand and upgrade the conventional concept of the combining (recognition) site of DBA since 1980s.

Plant lectins as versatile tools to fight coronavirus outbreaks.

The S protein forming the homotrimeric spikes of pathogenic beta-coronaviruses, such as MERS-CoV, SARS-CoV and SARS-CoV-2, is a highly glycosylated protein containing mainly N-glycans of the complex and high-mannose type, as well as O-glycans. Similarly, the host cell receptors DPP4 for MERS-CoV and ACE2 for SARS-CoV and SARS-CoV-2, also represent N- and O-glycosylated proteins. All these glycoproteins share common glycosylation patterns, suggesting that plant lectins with different carbohydrate-binding specificities could be used as carbohydrate-binding agents for the spikes and their receptors, to combat COVID19 pandemics. The binding of plant lectins to the spikes and their receptors could mask the non-glycosylated receptor binding domain of the virus and the corresponding region of the receptor, thus preventing a proper interaction of the spike proteins with their receptors. In this review, we analyze (1) the ability of plant lectins to interact with the N- and O-glycans present on the spike proteins and their receptors, (2) the in vitro and in vivo anti-COVID19 activity already reported for plant lectins and, (3) the possible ways for delivery of lectins to block the spikes and/or their receptors.

Development of the Cold Agglutinin Disease Symptoms and Impact Questionnaire (CAD-SIQ).

OBJECTIVES: Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia. This study aimed to identify disease-related symptoms and impacts important to patients with CAD, and to develop a novel CAD-specific patient-reported outcome measure. METHODS: Adults with CAD were randomly selected from a United States patient panel to participate in concept elicitation (CE) interviews to identify important symptoms and impacts or cognitive debriefing (CD) interviews to assess the comprehension and relevance of the draft item set. RESULTS: Overall, 37 adults were included (mean [range] age 67.2 [35-87] years). In CE interviews (n = 16), the most frequently reported CAD-related symptoms were reactions to cold environments and fatigue (both 93.8%). CAD had negative impacts on enjoyable activities (81.3%) and daily activities (75.0%). Following CE, standard survey methodological principles were used to develop a draft item pool of 14 concepts. Items were refined through three iterative rounds of CD interviews (n = 21), yielding 11 final items: fatigue; cold sensitivity; dyspnea; wearing extra clothing; limited physical, social, and enjoyable activities; difficulty with usual activities; mood; frustration; and anxiety/stress. CONCLUSIONS: The novel 11-item CAD-Symptoms and Impact Questionnaire provides a measure of the symptoms and impacts of CAD that are important to patients.

Sutimlimab provides clinically meaningful improvements in patient-reported outcomes in patients with cold agglutinin disease: Results from the randomised, placebo-controlled, Phase 3 CADENZA study.

Cold agglutinin disease (CAD) is a rare chronic autoimmune haemolytic anaemia, driven mainly by classical complement pathway activation, leading to profound fatigue and poor quality of life. In the Phase 3 CADENZA trial, sutimlimab-a C1s complement inhibitor-rapidly halted haemolysis, increased haemoglobin levels and improved fatigue versus placebo in patients with CAD without a recent history of transfusion. Patient-reported outcomes (PROs) included Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue), 12-Item Short Form Health Survey (SF-12), EuroQol visual analogue scale (EQ-VAS), Patient Global Impression of Change (PGIC) and Patient Global Impression of (fatigue) Severity (PGIS). Sutimlimab resulted in significant rapid and meaningful improvements versus placebo in PROs. From Week 1, the FACIT-Fatigue mean score increased >5 points above baseline (considered a clinically important change [CIC]). Least-squares (LS) mean change in FACIT-Fatigue score from baseline to treatment assessment timepoint was 10.8 vs. 1.9 points (sutimlimab vs. placebo; p < 0.001). Improvements in physical (PCS) and mental (MCS) component scores of the SF-12 were also considered CICs (LS mean changes from baseline to Week 26: PCS 5.54 vs. 1.57 [p = 0.064]; MCS 5.65 vs. -0.48 [p = 0.065]). These findings demonstrate that in addition to improving haematologic parameters, sutimlimab treatment demonstrates significant patient-reported benefits. Study registered at www.clinicaltrials.gov: NCT03347422.