The limiting factor in organ transplantation is the availability of organs. Ongoing work to impro... more The limiting factor in organ transplantation is the availability of organs. Ongoing work to improve donation rates both at the public and the organizational level in donating hospitals is essential. We also think that encouragement of live donation is important, and the possibility of ABO incompatible transplantation has increased the number of LD transplantations. The one-year graft survival rate is excellent and focus has shifted towards achieving long-term results to reduce the attrition rate. There is also an increasing interest in studying and working to reduce comorbidities on a long-term basis and thus, improve survival rates and recipient quality of life.
Successful hematopoietic stem and progenitor cell (HSPC) transplantation rests upon reliable meth... more 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.
Extracellular adherence protein (Eap) from Staphylococcus aureus inhibits the adherence of neutro... more Extracellular adherence protein (Eap) from Staphylococcus aureus inhibits the adherence of neutrophils to nonstimulated and tumor necrosis factor alpha-stimulated endothelial cells in both static adhesion assays and flow adhesion assays. Consequently, Eap also impaired their transendothelial migration. During an S. aureus infection, Eap may thus serve to reduce inflammation by inhibiting neutrophil adhesion and extravasation. Staphylococcus aureus is one of the most common agents causing bacterial infections in humans and animals. The clinical manifestations of S. aureus infections range from mild wound infections to more life-threatening infections, such as endocarditis, osteomyelitis, and septic shock (14). S. aureus produces a wide range of extracellular matrix binding proteins, which are proposed to contribute to successful colonization and persistence at various sites in the host. Extracellular adherence protein (Eap) is an extracellular protein of S. aureus. Eap binds to many plasma proteins, including fibrinogen, fibronectin, and prothrombin (16). It has also been shown that Eap enhances the binding and internalization of S. aureus into eukaryotic cells (9, 12). In addition, Eap plays a role as an immunomodulating protein (1, 13). Chavakis et al. found a strong interaction of Eap and the cell adhesion molecule ICAM-1 (intercellular adhesion molecule 1) in vitro (1). No interaction could be seen between Eap and its ligands, Mac-1 and LFA-1 (lymphocyte function-associated antigen-1), expressed on leukocytes (1). During the infectious process, inflammatory stimuli activate vascular endothelial cells to express adhesion molecules and chemokines that physically engage circulating leukocytes. A coordinated sequence of adhesion and locomotion steps, including (i) leukocyte rolling, (ii) cell activation, (iii) firm cell adhesion, and (iv) transendothelial migration, requires that adhesion receptors on leukocytes and endothelial cells are up-regulated and activated (20). The binding of Eap to ICAM-1 suggests that Eap may inhibit the binding of leukocytes to endothelial cells and thereby inhibit the extravasation of leukocytes from the bloodstream into the site of infection (2). In this study, we show that Eap from S. aureus inhibits neutrophil binding to, and migration across, the endothelium in vitro. In addition, the inhibiting effect exerted by Eap was dose dependent and of the same magnitude as the blocking effect elicited by antibodies against ICAM-1. To determine the effects of Eap on the adhesion of neutrophils to nonstimulated or tumor necrosis factor alpha (TNF-␣)-stimulated endothelial cells, static and flow adhesion assays were performed as described previously (5). Human aortic
Rapid Communications in Mass Spectrometry, Jun 1, 1993
In a previous paper, the presence in human kidney vein tissue of a novel blood group A heptaglyco... more In a previous paper, the presence in human kidney vein tissue of a novel blood group A heptaglycosylceramide based on the type-3 carbohydrate chain GalNAcal-3(Fucal-2)G~~l-3GalNAcal-3(Fucal-Z)Gal~l-4Glc~l-l Ceramide, was suggested based on thin-layer immunostaining and electron ionization mass spectrometry. Ions corresponding to a structure containing two deoxyhexoses, two hexosamines and three hexoses were identified, but no information was obtained from mass spectrometry concerning the carbohydrate sequence.' In the present paper, we report the identification of carbohydrate sequence ions corresponding to a type-3 chain A heptaglycosylceramide by electron ionization-tandem mass spectrometry of a permethylated-reduced glycosphingolipid mixture isolated from human kidney vein tissue. The use of a microchannel-plate-array detector increased the sensitivity for collision-induced dissociation spectra by a factor of at least ten over a conventional electron multiplier.
Background. Our program for ABO-incompatible renal transplantation includes antigen-specific immu... more Background. Our program for ABO-incompatible renal transplantation includes antigen-specific immunoadsorption (extracorporeal columns with the A or B trisaccharides), rituximab, and standard maintenance immunosuppression. Anti-A orB titers Յ8 in the indirect antiglobulin test (IAT) against panel A1 or B RBC are acceptable for transplantation. Case Report. A previously healthy, 15-month-old girl was diagnosed with Wilms' tumor and proteinuria. Denys-Drash syndrome was confirmed. Bilateral nephrectomy was performed. At 3.5 years of age she received an ABO-incompatible renal transplant from her father (A1 to O). The anti-A titers before transplantation were low. She was treated preoperatively with rituximab, immunoadsorption, immunoglobulin and mycophenolate mofetil (MMF). The maintenance immunosuppression protocol included basiliximab, tacrolimus, MMF, and prednisolone. The initial postoperative course was uncomplicated with rapid normalization of serum creatinine. The anti-A titers started to increase on postoperative day 5 (8 NaCl/16 IAT). Despite daily immunoadsorptions the titers rose to 1024 NaCl/1024 IAT on day 9. Renal function deteriorated and hemodialysis was started. A renal biopsy on day 9 showed acute severe antibody-mediated rejection. Additional treatment with bortezomib was given and after 2 doses the titers started to decline, renal allograft function improved and hemodialysis was stopped. On day 21 posttransplant the titers went down, creatinine was 28 mol/L, and no more immunoadsorptions were performed. Conclusion. By using bortezomib, we were able to successfully reverse a severe ABO antibody-mediated rejection.
Journal of the American Society for Mass Spectrometry, May 1, 1992
A permethylated-reduced hexaglycosylceramide in a complex glycolipid mixture isolated from a uniq... more A permethylated-reduced hexaglycosylceramide in a complex glycolipid mixture isolated from a unique human tissue has been identifLed by using tandem mass spectrometry (MSjMS). The mass spectrum of this glycolipid mixture, obtained by using in-beam electron ionization, is very complex, and fragment ions derived from the hexaglycosylceramide cannot be distinguished from other ions. Tandem mass spectrometry using a four-sector mass spectrometer gave the mass spectrum of the immonium ion of the permethylated-reduced hexaglycosylceramide (m / z 1645.8), which is characteristic of its structure. Comparison of this MS/MS spectrum with those of two similarly derivatized blood group hexaglycosylceramide isomers permitted identification of the unknown glycolipid structure.
Total non-acid glycosphingolipids were isolated from the kidneys of single pigs serologically typ... more Total non-acid glycosphingolipids were isolated from the kidneys of single pigs serologically typed on their red blood cells as blood groups O and A. Glycolipid species were purified by HPLC and structurally characterized by thin-layer chromatography, mass spectrometry, proton NMR spectroscopy, degradation analysis, and reactivity with various monoclonal antibodies, Gal alpha 1-4Gal-specific E. coli bacteria, and lectins. Glucosyl-, globotriaosyl-, and globotetraosylceramides were the predominant molecular species with lactosyl- and globopentaosylceramides (IV3GalGb4Cer) as abundant constituents too. Small amounts of galactosyl- and digalactosylceramides were also present. In the blood group O pig kidneys, blood group H antigens based on four different core saccharides (types 1, 2, 4, and lactosyl core) were identified and the major blood group structure was V2FucIV3Gal-Gb4Cer. In the kidneys from the blood group A pig the corresponding blood group A antigens were found and in addition, a type 3 chain blood group A antigen was indicated by mass spectrometry and by its reactivity with a monoclonal antibody. Trace amounts of the type 2 chain-based X and Y antigens were found while blood group B antigens and the type 1 chain based Lewis antigens could not be detected. The ceramide part of the glycolipids was mainly composed of dihydroxy 18:0 long chain bases and non-hydroxy 16:0-24:0 fatty cids.
ABSTRACT Immortalized liver cell lines capable of continuous proliferation, and expressing stable... more ABSTRACT Immortalized liver cell lines capable of continuous proliferation, and expressing stable liver-specific functions, would be invaluable for in vitro toxicity testing in the pharmaceutical industries. The lack of stable in vitro culture systems for such cells justifies the need for generation of human foetal hepatocytes (hFH) by means of immortalization. Here we report the establishment of immortalized human foetal liver progenitor cells by expression of the Simian virus 40 large T (SV40LT) antigen. Methods: SV40LT-transfected cells were studied with immunohistochemistry, reverse transcription-polymerase chain reactions (RT-PCR), and flow cytometric analysis. Cells in the 11th passage were transplanted into nude mice (n=7) with acute liver injury and tested for functional capacity in vivo. Results: The SV40LT antigen-immortalized foetal liver cells showed similar morphology to that of the primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers like albumin, CK 8, CK18, transcription factors HNF-4a and HNF-1b, and CYP3A/7, which is normally present in adult hepatocytes. The cells did not stain for any of the cancer-associated markers Ber EP4 and MOC-31. Albumin, HNF-4a and CYP3A7 expression was confirmed by RT-PCR. Flow cytometry showed expression of different stem cell markers like CD133, DLK-1 and EPCAM. Trans-splenic transplantation of the cells into nude mice with injured liver revealed liver-reconstituting activity. In the regenerated liver of recipient animals, the cells localized in small-sized clusters expressing CK8, CK18, CK19, C-met, AFP, hepatocytes antigen. Conclusions: SV40 LT transformed foetal liver cells maintained morphological and functional characteristics of the primary cells with extended in vitro life-span. This cell line is an ideal tool for applications in diagnostic and toxicological assays.
AimsDue to the shortage of heart donors, increasing numbers of heart transplantation (HTx) candid... more AimsDue 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 resultsWe 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.ConclusionsAlthough 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.
bioRxiv (Cold Spring Harbor Laboratory), Sep 10, 2022
The gut mucolytic specialist Akkermansia muciniphila is strongly associated with the integrity of... more The gut mucolytic specialist Akkermansia muciniphila is strongly associated with the integrity of the mucus layer. Mucin glycan utilization requires the removal of diverse 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 with double sulphated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the exclusive sialyl T-antigen specificity of a sialidase of a previously unknown family and catalytic apparatus. Key cell attached sialidases and fucosidases conferred mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, the sialic acid fucose did not contribute to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unique mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and the nutrient sharing between key mucus-associated bacteria.
The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-... more 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 mucusassociated bacteria. The human gut microbiota exerts a major impact on our immune and metabolic homeostasis 1,2. The host's first line of defense against microbial insult is the intestinal mucosal barrier that has increased thickness towards the colon 3,4. Mucins are the main structural and gelforming scaffolds of the mucosa, which is dominated by Mucin 2 (MUC2) in the colon 5. Similarly to other mucins, MUC2, is an O-glycoprotein that is secreted by intestinal goblet cells and consists of up to 80% (w/w) glycan chains 6 that exhibit large structural diversity (>100 structures reported) 7. The O-glycan epitopes in mucin exhibit longitudinal variations along the gastrointestinal tract (GIT) 8. The outer mucus surface offers a steady nutritional resource and adhesion sites for adapted microbiota groups, while the inner mucus layer in the colon is sterile. In humans, O-glycans in the small intestine and cecum regions are densely fucosylated, which decreases gradually toward the
Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but... more 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) andN-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 fas...
The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked ... more 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, 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 (CHO) cells wit...
The β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a β1,4-linkage to GlcNA... more The β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a β1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides.
BACKGROUNDCord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic s... more BACKGROUNDCord 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 METHODSSamples from 57 ...
Intracerebral transplantation of porcine embryonic dopamine-producing neurons has been suggested ... more Intracerebral transplantation of porcine embryonic dopamine-producing neurons has been suggested as a method to treat patients with Parkinson's disease. Even though the brain is an immunologically privileged site, neuronal xenografts are usually rejected within a few weeks. T cells are important for this process, but the exact cellular events leading to rejection are poorly characterized. Brain cells from ventral mesencephalon of 26–27-day-old pig embryos were used as target cells in flow cytometry-assessed cytotoxicity assays using non- and IL-2-activated CD3–CD16+CD56+ human natural killer (NK) cells as effector cells. The ability of human NK cells to kill pig embryonic brain cells by antibody-dependent cellular cytotoxicity (ADCC) in the presence of nondepleted and anti-Galα1,3Gal antibody-depleted human blood group AB serum (AB serum) was evaluated using the same assay. Both nondepleted and anti-Galα1,3Gal antibody-depleted AB serum could mediate ADCC of pig embryonic VM cel...
The limiting factor in organ transplantation is the availability of organs. Ongoing work to impro... more The limiting factor in organ transplantation is the availability of organs. Ongoing work to improve donation rates both at the public and the organizational level in donating hospitals is essential. We also think that encouragement of live donation is important, and the possibility of ABO incompatible transplantation has increased the number of LD transplantations. The one-year graft survival rate is excellent and focus has shifted towards achieving long-term results to reduce the attrition rate. There is also an increasing interest in studying and working to reduce comorbidities on a long-term basis and thus, improve survival rates and recipient quality of life.
Successful hematopoietic stem and progenitor cell (HSPC) transplantation rests upon reliable meth... more 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.
Extracellular adherence protein (Eap) from Staphylococcus aureus inhibits the adherence of neutro... more Extracellular adherence protein (Eap) from Staphylococcus aureus inhibits the adherence of neutrophils to nonstimulated and tumor necrosis factor alpha-stimulated endothelial cells in both static adhesion assays and flow adhesion assays. Consequently, Eap also impaired their transendothelial migration. During an S. aureus infection, Eap may thus serve to reduce inflammation by inhibiting neutrophil adhesion and extravasation. Staphylococcus aureus is one of the most common agents causing bacterial infections in humans and animals. The clinical manifestations of S. aureus infections range from mild wound infections to more life-threatening infections, such as endocarditis, osteomyelitis, and septic shock (14). S. aureus produces a wide range of extracellular matrix binding proteins, which are proposed to contribute to successful colonization and persistence at various sites in the host. Extracellular adherence protein (Eap) is an extracellular protein of S. aureus. Eap binds to many plasma proteins, including fibrinogen, fibronectin, and prothrombin (16). It has also been shown that Eap enhances the binding and internalization of S. aureus into eukaryotic cells (9, 12). In addition, Eap plays a role as an immunomodulating protein (1, 13). Chavakis et al. found a strong interaction of Eap and the cell adhesion molecule ICAM-1 (intercellular adhesion molecule 1) in vitro (1). No interaction could be seen between Eap and its ligands, Mac-1 and LFA-1 (lymphocyte function-associated antigen-1), expressed on leukocytes (1). During the infectious process, inflammatory stimuli activate vascular endothelial cells to express adhesion molecules and chemokines that physically engage circulating leukocytes. A coordinated sequence of adhesion and locomotion steps, including (i) leukocyte rolling, (ii) cell activation, (iii) firm cell adhesion, and (iv) transendothelial migration, requires that adhesion receptors on leukocytes and endothelial cells are up-regulated and activated (20). The binding of Eap to ICAM-1 suggests that Eap may inhibit the binding of leukocytes to endothelial cells and thereby inhibit the extravasation of leukocytes from the bloodstream into the site of infection (2). In this study, we show that Eap from S. aureus inhibits neutrophil binding to, and migration across, the endothelium in vitro. In addition, the inhibiting effect exerted by Eap was dose dependent and of the same magnitude as the blocking effect elicited by antibodies against ICAM-1. To determine the effects of Eap on the adhesion of neutrophils to nonstimulated or tumor necrosis factor alpha (TNF-␣)-stimulated endothelial cells, static and flow adhesion assays were performed as described previously (5). Human aortic
Rapid Communications in Mass Spectrometry, Jun 1, 1993
In a previous paper, the presence in human kidney vein tissue of a novel blood group A heptaglyco... more In a previous paper, the presence in human kidney vein tissue of a novel blood group A heptaglycosylceramide based on the type-3 carbohydrate chain GalNAcal-3(Fucal-2)G~~l-3GalNAcal-3(Fucal-Z)Gal~l-4Glc~l-l Ceramide, was suggested based on thin-layer immunostaining and electron ionization mass spectrometry. Ions corresponding to a structure containing two deoxyhexoses, two hexosamines and three hexoses were identified, but no information was obtained from mass spectrometry concerning the carbohydrate sequence.' In the present paper, we report the identification of carbohydrate sequence ions corresponding to a type-3 chain A heptaglycosylceramide by electron ionization-tandem mass spectrometry of a permethylated-reduced glycosphingolipid mixture isolated from human kidney vein tissue. The use of a microchannel-plate-array detector increased the sensitivity for collision-induced dissociation spectra by a factor of at least ten over a conventional electron multiplier.
Background. Our program for ABO-incompatible renal transplantation includes antigen-specific immu... more Background. Our program for ABO-incompatible renal transplantation includes antigen-specific immunoadsorption (extracorporeal columns with the A or B trisaccharides), rituximab, and standard maintenance immunosuppression. Anti-A orB titers Յ8 in the indirect antiglobulin test (IAT) against panel A1 or B RBC are acceptable for transplantation. Case Report. A previously healthy, 15-month-old girl was diagnosed with Wilms' tumor and proteinuria. Denys-Drash syndrome was confirmed. Bilateral nephrectomy was performed. At 3.5 years of age she received an ABO-incompatible renal transplant from her father (A1 to O). The anti-A titers before transplantation were low. She was treated preoperatively with rituximab, immunoadsorption, immunoglobulin and mycophenolate mofetil (MMF). The maintenance immunosuppression protocol included basiliximab, tacrolimus, MMF, and prednisolone. The initial postoperative course was uncomplicated with rapid normalization of serum creatinine. The anti-A titers started to increase on postoperative day 5 (8 NaCl/16 IAT). Despite daily immunoadsorptions the titers rose to 1024 NaCl/1024 IAT on day 9. Renal function deteriorated and hemodialysis was started. A renal biopsy on day 9 showed acute severe antibody-mediated rejection. Additional treatment with bortezomib was given and after 2 doses the titers started to decline, renal allograft function improved and hemodialysis was stopped. On day 21 posttransplant the titers went down, creatinine was 28 mol/L, and no more immunoadsorptions were performed. Conclusion. By using bortezomib, we were able to successfully reverse a severe ABO antibody-mediated rejection.
Journal of the American Society for Mass Spectrometry, May 1, 1992
A permethylated-reduced hexaglycosylceramide in a complex glycolipid mixture isolated from a uniq... more A permethylated-reduced hexaglycosylceramide in a complex glycolipid mixture isolated from a unique human tissue has been identifLed by using tandem mass spectrometry (MSjMS). The mass spectrum of this glycolipid mixture, obtained by using in-beam electron ionization, is very complex, and fragment ions derived from the hexaglycosylceramide cannot be distinguished from other ions. Tandem mass spectrometry using a four-sector mass spectrometer gave the mass spectrum of the immonium ion of the permethylated-reduced hexaglycosylceramide (m / z 1645.8), which is characteristic of its structure. Comparison of this MS/MS spectrum with those of two similarly derivatized blood group hexaglycosylceramide isomers permitted identification of the unknown glycolipid structure.
Total non-acid glycosphingolipids were isolated from the kidneys of single pigs serologically typ... more Total non-acid glycosphingolipids were isolated from the kidneys of single pigs serologically typed on their red blood cells as blood groups O and A. Glycolipid species were purified by HPLC and structurally characterized by thin-layer chromatography, mass spectrometry, proton NMR spectroscopy, degradation analysis, and reactivity with various monoclonal antibodies, Gal alpha 1-4Gal-specific E. coli bacteria, and lectins. Glucosyl-, globotriaosyl-, and globotetraosylceramides were the predominant molecular species with lactosyl- and globopentaosylceramides (IV3GalGb4Cer) as abundant constituents too. Small amounts of galactosyl- and digalactosylceramides were also present. In the blood group O pig kidneys, blood group H antigens based on four different core saccharides (types 1, 2, 4, and lactosyl core) were identified and the major blood group structure was V2FucIV3Gal-Gb4Cer. In the kidneys from the blood group A pig the corresponding blood group A antigens were found and in addition, a type 3 chain blood group A antigen was indicated by mass spectrometry and by its reactivity with a monoclonal antibody. Trace amounts of the type 2 chain-based X and Y antigens were found while blood group B antigens and the type 1 chain based Lewis antigens could not be detected. The ceramide part of the glycolipids was mainly composed of dihydroxy 18:0 long chain bases and non-hydroxy 16:0-24:0 fatty cids.
ABSTRACT Immortalized liver cell lines capable of continuous proliferation, and expressing stable... more ABSTRACT Immortalized liver cell lines capable of continuous proliferation, and expressing stable liver-specific functions, would be invaluable for in vitro toxicity testing in the pharmaceutical industries. The lack of stable in vitro culture systems for such cells justifies the need for generation of human foetal hepatocytes (hFH) by means of immortalization. Here we report the establishment of immortalized human foetal liver progenitor cells by expression of the Simian virus 40 large T (SV40LT) antigen. Methods: SV40LT-transfected cells were studied with immunohistochemistry, reverse transcription-polymerase chain reactions (RT-PCR), and flow cytometric analysis. Cells in the 11th passage were transplanted into nude mice (n=7) with acute liver injury and tested for functional capacity in vivo. Results: The SV40LT antigen-immortalized foetal liver cells showed similar morphology to that of the primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers like albumin, CK 8, CK18, transcription factors HNF-4a and HNF-1b, and CYP3A/7, which is normally present in adult hepatocytes. The cells did not stain for any of the cancer-associated markers Ber EP4 and MOC-31. Albumin, HNF-4a and CYP3A7 expression was confirmed by RT-PCR. Flow cytometry showed expression of different stem cell markers like CD133, DLK-1 and EPCAM. Trans-splenic transplantation of the cells into nude mice with injured liver revealed liver-reconstituting activity. In the regenerated liver of recipient animals, the cells localized in small-sized clusters expressing CK8, CK18, CK19, C-met, AFP, hepatocytes antigen. Conclusions: SV40 LT transformed foetal liver cells maintained morphological and functional characteristics of the primary cells with extended in vitro life-span. This cell line is an ideal tool for applications in diagnostic and toxicological assays.
AimsDue to the shortage of heart donors, increasing numbers of heart transplantation (HTx) candid... more AimsDue 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 resultsWe 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.ConclusionsAlthough 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.
bioRxiv (Cold Spring Harbor Laboratory), Sep 10, 2022
The gut mucolytic specialist Akkermansia muciniphila is strongly associated with the integrity of... more The gut mucolytic specialist Akkermansia muciniphila is strongly associated with the integrity of the mucus layer. Mucin glycan utilization requires the removal of diverse 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 with double sulphated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the exclusive sialyl T-antigen specificity of a sialidase of a previously unknown family and catalytic apparatus. Key cell attached sialidases and fucosidases conferred mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, the sialic acid fucose did not contribute to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unique mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and the nutrient sharing between key mucus-associated bacteria.
The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-... more 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 mucusassociated bacteria. The human gut microbiota exerts a major impact on our immune and metabolic homeostasis 1,2. The host's first line of defense against microbial insult is the intestinal mucosal barrier that has increased thickness towards the colon 3,4. Mucins are the main structural and gelforming scaffolds of the mucosa, which is dominated by Mucin 2 (MUC2) in the colon 5. Similarly to other mucins, MUC2, is an O-glycoprotein that is secreted by intestinal goblet cells and consists of up to 80% (w/w) glycan chains 6 that exhibit large structural diversity (>100 structures reported) 7. The O-glycan epitopes in mucin exhibit longitudinal variations along the gastrointestinal tract (GIT) 8. The outer mucus surface offers a steady nutritional resource and adhesion sites for adapted microbiota groups, while the inner mucus layer in the colon is sterile. In humans, O-glycans in the small intestine and cecum regions are densely fucosylated, which decreases gradually toward the
Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but... more 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) andN-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 fas...
The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked ... more 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, 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 (CHO) cells wit...
The β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a β1,4-linkage to GlcNA... more The β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a β1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides.
BACKGROUNDCord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic s... more BACKGROUNDCord 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 METHODSSamples from 57 ...
Intracerebral transplantation of porcine embryonic dopamine-producing neurons has been suggested ... more Intracerebral transplantation of porcine embryonic dopamine-producing neurons has been suggested as a method to treat patients with Parkinson's disease. Even though the brain is an immunologically privileged site, neuronal xenografts are usually rejected within a few weeks. T cells are important for this process, but the exact cellular events leading to rejection are poorly characterized. Brain cells from ventral mesencephalon of 26–27-day-old pig embryos were used as target cells in flow cytometry-assessed cytotoxicity assays using non- and IL-2-activated CD3–CD16+CD56+ human natural killer (NK) cells as effector cells. The ability of human NK cells to kill pig embryonic brain cells by antibody-dependent cellular cytotoxicity (ADCC) in the presence of nondepleted and anti-Galα1,3Gal antibody-depleted human blood group AB serum (AB serum) was evaluated using the same assay. Both nondepleted and anti-Galα1,3Gal antibody-depleted AB serum could mediate ADCC of pig embryonic VM cel...
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Papers by Jan Holgersson