Tocilizumab in chronic active antibody-mediated rejection: rationale and protocol of an in-progress randomized controlled open-label multi-center trial (INTERCEPT study).

BACKGROUND: Chronic active antibody-mediated rejection (caAMR) in kidney transplants is associated with irreversible tissue damage and a leading cause of graft loss in the long-term. However, the treatment for caAMR remains a challenge to date. Recently, tocilizumab, a recombinant humanized monoclonal antibody directed against the human interleukin-6 (IL-6) receptor, has shown promise in the treatment of caAMR. However, it has not been systematically investigated so far underscoring the need for randomized controlled studies in this area. METHODS: The INTERCEPT study is an investigator-driven randomized controlled open-label multi-center trial in kidney transplant recipients to assess the efficacy of tocilizumab in the treatment of biopsy-proven caAMR. A total of 50 recipients with biopsy-proven caAMR at least 12 months after transplantation will be randomized to receive either tocilizumab (n = 25) added to our standard of care (SOC) maintenance treatment or SOC alone (n = 25) for a period of 24 months. Patients will be followed for an additional 12 months after cessation of study medication. After the inclusion biopsies at baseline, protocol kidney graft biopsies will be performed at 12 and 24 months. The sample size calculation assumed a difference of 5 ml/year in slope of estimated glomerular filtration rate (eGFR) between the two groups for 80% power at an alpha of 0.05. The primary endpoint is the slope of eGFR at 24 months after start of treatment. The secondary endpoints include assessment of the following at 12, 24, and 36 months: composite risk score iBox, safety, evolution and characteristics of donor-specific antibodies (DSA), graft histology, proteinuria, kidney function assessed by measured GFR (mGFR), patient- and death-censored graft survival, and patient-reported outcomes that include transplant-specific well-being, adherence to immunosuppressive medications and perceived threat of the risk of graft rejection. DISCUSSION: No effective treatment exists for caAMR at present. Based on the hypothesis that inhibition of IL-6 receptor by tocilizumab will reduce antibody production and reduce antibody-mediated damage, our randomized trial has a potential to provide evidence for a novel treatment strategy for caAMR, therewith slowing the decline in graft function in the long-term. TRIAL REGISTRATION: ClinicalTrials.gov NCT04561986. Registered on September 24, 2020.

Including the liver in the visceral allograft: Impact on donor-specific anti-HLA antibodies and long-term outcomes.

Humoral immunity emerges as a risk factor for graft failure after visceral transplantation (VTx) and development of donor-specific anti-HLA antibodies (DSAs) has been linked with poor outcomes. In most cases, a simultaneous liver transplant can be safely performed in sensitized patients with DSA and appears protective against lymphocytotoxic antibodies. We investigated the incidence of acute (AR) and chronic rejection (CR) in 32 VTx without any B cell-depleting pre-treatment (6 isolated intestinal transplants (IT) and 26 liver-containing, multivisceral transplants (MVT) and assessed the presence of donor-specific antibodies (DSA) pre- and post-transplantation. Twenty-one patients (65 %) developed AR, 15 (57 %) of the MVT and 6 (100 %) of the IT (p = 0.05). CR occurred in 4 IT (60 %, p < 0.001). At one month, de novo DSA were present in 71 % of VTx (66 % MVT vs 100 % IT, p = 0.09). At the last available follow-up, 69 % of the MVT and 50 % of the IT patients were DSA-free. De novo DSA seemed more persistent (7/19, 37 %) than pre-Tx DSA (1/6, 17 %; p = n.s.), de novo DSA were more frequently specific for HLA class II than class I, 16/19 (84 %) vs. 7/19 (37 %; p = 0.003), and HLA-DQ was their most frequent target HLA. DQ mismatches appeared to be a risk factor for developing de novo DSA. In conclusion, liver-containing visceral allografts have superior short- and long-term outcomes compared with liver-free allografts. De novo DSA develop early and frequently after VTx performed without B cell-depleting induction therapy, but the exact role of DSA in the pathogenesis of rejection remains unclear.

Heart transplantation in patients bridged with mechanical circulatory support: outcome comparison with matched controls.

AIMS: Due to the shortage of heart donors, increasing numbers of heart transplantation (HTx) candidates are receiving long-term mechanical circulatory support (MCS) as bridge-to-transplantation. Treatment with MCS is associated with increased formation of anti-human leukocyte antigen antibodies (allosensitization), but whether this affects post-HTx outcomes is unclear. METHODS AND RESULTS: We included all adult patients who received long-term MCS as bridge-to-transplantation and underwent subsequent HTx at our centre between 2008 and 2018. We also enrolled medically treated HTx recipients without prior MCS as controls. These controls were matched by age, sex, diagnosis, and transplantation era. Outcome parameters were compared between the two study groups. A total of 126 patients (48 ± 15 years, 84% male) were included of whom 64 were bridged with MCS and 62 were matched controls. Pre-HTx allosensitization occurred more frequently in the MCS group than in the control group (27% vs. 11%, P = 0.03). At post-HTx year 10, the overall survival probability was 84% among patients treated with MCS and 90% among those medically managed (P = 0.32). At post-HTx year 1, freedom from treated rejections (≥ISHLT 2R) was 69% in the MCS group and 70% in the control group (P = 0.94); and freedom from any rejection was 8% and 5%, respectively (P = 0.98). There were no differences in renal function or cardiac allograft vasculopathy (grade ≥ 1) between groups at 1, 3, and 5 years post-HTx. CONCLUSIONS: Although patients treated with MCS had a higher frequency of pre-HTx allosensitization, there were no significant differences in post-HTx graft survival, biopsy-proven rejections, or renal function as compared with patients not bridged with MCS.

Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.

The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration.

Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1-182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.

Sulfation of O-glycans on Mucin-type Proteins From Serous Ovarian Epithelial Tumors.

Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression with defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MSn were identified as core 1 and core 2 O-glycans up to the size of decamers and with 1 to 4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or -2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared with tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 was differentially expressed in benign, borderline, or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology.

Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N- and O-glycomes of porcine, bovine and equine heart tissues.

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by ß-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Recombinant mucin-type proteins carrying LacdiNAc on different O-glycan core chains fail to support H. pylori binding.

The ß4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a ß1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment of Helicobacter pylori to the gastric mucosa via binding to the LDN determinant. The O-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried by O-glycan core 2, core 3 and extended core 1 precursors using transient transfection of CHO-K1 cells and a mucin-type immunoglobulin fusion protein as reporter protein. Binding of the LabA-positive H. pylori J99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on different O-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of 125I-LDN-BSA to various clinical H. pylori isolates was assessed in solution. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blotting confirmed the requirement of a terminal GlcNAc for B4GALNT3 activity. B4GALNT3 added a ß1,4-linked GalNAc to GlcNAc irrespective of whether the latter was carried by a core 2, core 3 or extended core 1 chain. No LDN-mediated adhesion of H. pylori strains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1 O-glycans were detected in a microtiter well-based adhesion assay and no binding of a 125I-labelled LDN-BSA neoglycoconjugate to clinical H. pylori isolates was identified.

The Structural Complexity and Animal Tissue Distribution of N-Glycolylneuraminic Acid (Neu5Gc)-Terminated Glycans. Implications for Their Immunogenicity in Clinical Xenografting.

N-Glycolylneuraminic acid (Neu5Gc)-terminated glycans are present in all animal cells/tissues that are already used in the clinic such as bioprosthetic heart valves (BHV) as well as in those that potentially will be xenografted in the future to overcome end stage cell/organ failure. Humans, as a species lack this antigen determinant and can react with an immune response after exposure to Neu5Gc present in these products/cells/tissues. Genetically engineered source animals lacking Neu5Gc has been generated and so has animals that in addition lack the major αGal xenoantigen. The use of cells/tissues/organs from such animals may improve the long-term performance of BHV and allow future xenografting. This review summarizes the present knowledge regarding structural complexity and tissue distribution of Neu5Gc on glycans of cells/tissue/organs already used in the clinic or intended for treatment of end stage organ failure by xenografting. In addition, we briefly discuss the role of anti-Neu5Gc antibodies in the xenorejection process and how knowledge about Neu5Gc structural complexity can be used to design novel diagnostics for anti-Neu5Gc antibody detection.

Glyco-engineered cell line and computational docking studies reveals enterotoxigenic Escherichia coli CFA/I fimbriae bind to Lewis a glycans.

We have previously reported clinical data to suggest that colonization factor I (CFA/I) fimbriae of enterotoxigenic Escherichia coli (ETEC) can bind to Lewis a (Lea), a glycan epitope ubiquitous in the small intestinal mucosa of young children (<2 years of age), and individuals with a genetic mutation of FUT2. To further elucidate the physiological binding properties of this interaction, we engineered Chinese Hamster Ovary (CHO-K1) cells to express Lea or Leb determinants on both N- and O-glycans. We used our glyco-engineered CHO-K1 cell lines to demonstrate that CfaB, the major subunit of ETEC CFA/I fimbriae, as well as four related ETEC fimbriae, bind more to our CHO-K1 cell-line expressing Lea, compared to cells carrying Leb or the CHO-K1 wild-type glycan phenotype. Furthermore, using in-silico docking analysis, we predict up to three amino acids (Glu25, Asn27, Thr29) found in the immunoglobulin (Ig)-like groove region of CfaB of CFA/I and related fimbriae, could be important for the preferential and higher affinity binding of CFA/I fimbriae to the potentially structurally flexible Lea glycan. These findings may lead to a better molecular understanding of ETEC pathogenesis, aiding in the development of vaccines and/or anti-infection therapeutics.

The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells.

BACKGROUND: Cord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. STUDY DESIGN AND METHODS: Samples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. RESULTS: No nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r = 0. 72 vs. r = 0.66; p < 0.0001) with the results of the CFU assay. CONCLUSION: The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates better with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.

Concentration of the CDCP1 protein in human cord plasma may serve as a predictor of hematopoietic stem and progenitor cell content.

Successful hematopoietic stem and progenitor cell (HSPC) transplantation rests upon reliable methods for their enumeration in sources such as cord blood (CB). Methods used today are costly, time consuming and exhaust the limited number of cells needed for transplantation. The aim of this study was to analyze if surplus plasma from CB contains biomarkers that can predict HSPC content in CB. Frozen, surplus plasma from 95 CB units was divided into two groups based on CD34+ cell concentration. Birth weight, gestation age, gender, mode of delivery, collection volume, nucleated cell count and colony forming unit assay results were available. Samples were analyzed with a proximity ligation assay covering 92 different proteins. Two-group t-test with p-values adjusted for false discovery rate (FDR) identified 5 proteins that significantly differed between the two groups. CDCP1 was the most significant (FDR adjusted p-value 0.006). Correlation with CDCP1 concentration was most significant for CD34+ concentration and nucleated cell count. Multivariate analysis showed that CD34 and gender seemed to influence the level of CDCP1. In conclusion, CDCP1 was identified as a potential biomarker of HSPC content in CB. The finding also warrants further investigation for a possible role of CDCP1 in regulating HSPC presence in CB.

Influence of Glycosylation on Interfacial Properties of Recombinant Mucins: Adsorption, Surface Forces, and Friction.

Interfacial properties of two brush-with-anchor mucins, C-P55 and C-PSLex, have been investigated at the aqueous solution/poly(methyl methacrylate) (PMMA) interface. Both are recombinant mucin-type fusion proteins, produced by fusing the glycosylated mucin part of P-selectin glycoprotein ligand-1 (PSLG-1) to the Fc part of a mouse immunoglobulin in two different cells. They are mainly expressed as dimers upon production. Analysis of the O-glycans shows that the C-PSLex mucin has the longer and more branched side chains, but C-P55 has slightly higher sialic acid content. The adsorption of the mucins to PMMA surfaces was studied by quartz crystal microbalance with dissipation. The sensed mass, including the adsorbed mucin and water trapped in the layer, was found to be similar for these two mucin layers. Atomic force microscopy with colloidal probe was employed to study surface and friction forces between mucin-coated PMMA surfaces. Purely repulsive forces of steric origin were observed between mucin layers on compression, whereas a small adhesion was detected between both mucin layers on decompression. This was attributed to chain entanglement. The friction force between C-PSLex-coated PMMA is lower than that between C-P55-coated PMMA at low loads, but vice versa at high loads. We discuss our results in terms of the differences in the glycosylation composition of these two mucins.

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors.

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galß1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive ß-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galß1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.

High quality cord blood banking is feasible with delayed clamping practices. The eight-year experience and current status of the national Swedish Cord Blood Bank.

The National Swedish Cord Blood Bank (NS-CBB) is altruistic and publicly funded. Herein we describe the status of the bank and the impact of delayed versus early clamping on cell number and volume. Cord Blood Units (CBUs) were collected at two University Hospitals in Sweden. Collected volume and nucleated cell content (TNC) were investigated in 146 consecutive Cord Blood (CB) collections sampled during the first quarter of 2012 and in 162 consecutive CB collections done in the first quarter of 2013, before and after clamping practices were changed from immediate to late (60 s) clamping. NS-CBB now holds close to 5000 units whereof 30 % are from non-Caucasian or mixed origins. Delayed clamping had no major effect on collection efficiency. The volume collected was slightly reduced (mean difference, 8.1 ml; 95 % CI, 1.3-15.0 ml; p = 0.02), while cell recovery was not (p = 0.1). The proportion of CBUs that met initial total TNC banking criteria was 60 % using a TNC threshold of 12.5 × 10(8), and 47 % using a threshold of 15 × 10(8) for the early clamping group and 52 and 37 % in the late clamping group. Following implementation of delayed clamping practices at NS-CBB; close to 40 % of the collections in the late clamping group still met the high TNC banking threshold and were eligible for banking, implicating that that cord blood banking is feasible with delayed clamping practices.

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc) or type 2 (Galb4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs.

Glyco-engineering of host cells is used to increase efficacy, decrease immunogenicity and increase circulatory half-lives of protein biopharmaceuticals. The effect of transiently expressed O-glycan core chain glycosyltransferases on O-glycan biosynthesis pathways in CHO cells is reported. Liquid chromatography-mass spectrometry and Western blotting were used to map the O-glycome of a mucin-type fusion protein transiently co-transfected with ß1,3-N-acetylglucosaminyltransferase 3 (extended C1 ß3GnT3), core 2 ß1,6-N-acetylglucosaminyltransferase I (C2 ß3GnT1) or core 3 ß1,3-N-acetylglucosaminyltransferase 6 (C3 ß3GnT6) in CHO cells. Extended core 1 (GlcNAcß1,3Galß1,3GalNAc) and core 3 (GlcNAcß1,3GalNAc), and increased expression of core 2 [Galß1,3(GlcNAcß1,6)GalNAc], O-glycans were generated on P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL1/mIgG2b). Endogenous poly-N-acetyllactosamine (poly-LacNAc) synthase elongated extended core 1 and core 3 generating O-glycans with up to five LacNAc repeats. Low amounts of core 3 O-glycans appeared upon extended C1 ß3GnT3 expression. The α2,6-sialylated type 2 chain was detected upon co-transfection with the ß-galactoside α2,6-sialyltransferase I. N-acetylglucosamine-6-O-sulfotransferase 2 transferred sulfate to carbon 6 of GlcNAc in poly-LacNAc sequences. CHO cells with its known O-glycan repertoire can be used to express recombinant mucin-type proteins together with selected glycosyltransferases in order to recreate carbohydrate determinants on defined O-glycan chains. They will become important tools for assessing the core chain-dependent binding activity of carbohydrate-binding proteins.

Immunotoxicological effects of streptozotocin and alloxan: in vitro and in vivo studies.

Streptozotocin (STZ) and alloxan (ALX), widely used to induce diabetes in experimental animals, have different structures and mechanisms of action. We investigated those effects of these drugs on the immune system that might influence engraftment efficiency and graft survival in transplantation models, and their cytotoxicity on hematopoietic cell lines. We used the minimum dose to induce diabetes in a mouse, i.e. 180 mg/kg i.v. STZ and 75 mg/kg i.v. ALX. Both groups exhibited significant decrease in body weight during 4 days post-treatment as compared to controls. We found that blood glucose in ALX-injected mice increased faster than in STZ-injected mice. The total number of recovered splenocytes was lower in STZ-injected animals than in ALX-injected animals. The survival periods of rat islet grafts in recipient mice were longer and more diverse in STZ-injected recipients (7-24 days) compared to ALX-injected recipients (6-7 days). The in vitro study showed that ALX was less cytotoxic in cell lines with IC50 values of 2809, 3679 and >4000 µg/ml for HL60, K562 and C1498 cells respectively. STZ was more toxic, especially in HL60 cells, with IC50 values of 11.7, 904 and 1024 µg/ml for HL60, K562 and C1498 cells respectively. Furthermore, in response to concanavalin A (Con-A), splenocytes from STZ-injected mice produced higher amounts of interferon-gamma (IFN-γ) than those from ALX-injected mice. In conclusion, STZ was more cytotoxic than ALX in vitro and in vivo. STZ caused lymphocytopenia, which may result in longer graft survival in STZ-treated animals than in ALX-treated animals.