Collagen ß(1-O) galactosyltransferase 2 deficiency contributes to lipodystrophy and aggravates NAFLD related to HMW adiponectin in mice.

AIM: Our previous results showed that Colgalt1 knock-out resulted in fetal death on day E11.5, and collagen secretion was retarded. This study aimed to elucidate the role of Collagen ß(1-O) galactosyltransferase 2 (Colgalt2) in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). METHODS: Colgalt2-/- mice were fed a high-fat diet (HFD) or methionine-and choline-deficient diet (MCD). Nanopore long-read RNA-Seq analysis of liver tissues was used to profile genomic variation. In vitro, hepatocyte steatosis and differentiation of primary pre-adipocytes were induced. RESULTS: Colgalt2-/- mice exhibited lipodystrophy, increased body weight, and hepatic lipid accumulation at 6 weeks of age. Colgalt2 deficiency aggravated hepatic steatosis in mice fed an HFD or a standard laboratory chow diet. Colgalt2 deficiency promotes steatohepatitis in MCD-fed mice. In HFD mice, Colgalt2 deficiency caused lipodystrophy and decreased plasma HMW, total adiponectin, and leptin levels. Colgalt2 deficiency also reduced circulating HMW/Total adiponectin in mice fed a HFD diet without differences of adiponectin mRNA and protein level in WT and Colgalt2-/- mice. The nanopore long-read RNA-Seq analysis results revealed transcriptional changes in the adiponectin receptor downstream signaling pathway and lipogenic genes, including the AMPK signaling pathway, adipocytokine signaling pathway, and lipid metabolism (Cidea, Cidec, CD36, and PPARγ). Colgalt2 deficiency did not promote lipid accumulation in OA-induced HepG2 cells or primary hepatocytes. However, Colgalt2 deficiency inhibited adipogenesis and reduced PPARγ, adipogenesis-related transcription factors, and expression during adipocyte differentiation. CONCLUSIONS: In mice, Colgalt2 deficiency contributes to lipodystrophy and promotes NAFLD related to HMW adiponectin. These results suggest that Colgalt2 could be a novel and promising therapeutic strategy for the treatment of NAFLD.

Integrated bioinformatic analysis identifies UBE2Q1 as a potential prognostic marker for high grade serous ovarian cancer.

BACKGROUND: High grade serous ovarian cancer (HGSOC) accounts for nearly 60% of total cases of epithelial ovarian cancer. It is the most aggressive subtype, which shows poor prognosis and low patient survival. For better management of HGSOC patients, new prognostic biomarkers are required to facilitate improved treatment strategies and ensure suitable healthcare decisions. METHODS: We performed genome wide expression analysis of HGSOC patient samples to identify differentially expressed genes (DEGs) using R based Limma package, Clust and other statistical tools. The identified DEGs were subjected to weighted gene co-expression network analysis (WGCNA) to identify co-expression patterns of relevant genes. Module trait and gene ontology analyses were performed to establish important gene co-expression networks and their biological functions. Overlapping the most relevant DEG cluster 4 with prominent WGCNA cyan module identified strongest correlation of UBE2Q1 with ovarian cancer and its prognostic significance on survival probability of ovarian cancer patients was investigated. The predictive value of UBE2Q1 as a potential biomarker was analysed by correlating its expression with 12-months relapse free survival of patients in response to platin/taxane, the standard first-line chemotherapy for ovarian cancer, and analysing area under the ROC curve. RESULTS: An integrated gene expression analysis and WGCNA, identified UBE2Q1 as a potential prognostic marker associated with poor relapse-free survival and response outcome to platin/taxane treatment of patients with high grade serous ovarian cancer. CONCLUSIONS: Our study identifies a potential UBE2Q1 - B4GALT3 functional axis in ovarian cancer, where only the E2 conjugating enzyme showed a poor prognostic impact on the disease.

Abiotic stress regulates expression of galactinol synthase genes post-transcriptionally through intron retention in rice.

MAIN CONCLUSION: Expression of the Galactinol synthase genes in rice is regulated through post-transcriptional intron retention in response to abiotic stress and may be linked to Raffinose Family Oligosaccharide synthesis in osmotic perturbation. Galactinol synthase (GolS) is the first committed enzyme in raffinose family oligosaccharide (RFO) synthesis pathway and synthesizes galactinol from UDP-galactose and inositol. Expression of GolS genes has long been implicated in abiotic stress, especially drought and salinity. A non-canonical regulation mechanism controlling the splicing and maturation of rice GolS genes was identified in rice photosynthetic tissue. We found that the two isoforms of Oryza sativa GolS (OsGolS) gene, located in chromosomes 3(OsGolS1) and 7(OsGolS2) are interspersed by conserved introns harboring characteristic premature termination codons (PTC). During abiotic stress, the premature and mature transcripts of both isoforms were found to accumulate in a rhythmic manner for very small time-windows interrupted by phases of complete absence. Reporter gene assay using GolS promoters under abiotic stress does not reflect this accumulation profile, suggesting that this regulation occurs post-transcriptionally. We suggest that this may be due to a surveillance mechanism triggering the degradation of the premature transcript preventing its accumulation in the cell. The suggested mechanism fits the paradigm of PTC-induced Nonsense-Mediated Decay (NMD). In support of our hypothesis, when we pharmacologically blocked NMD, the full-length pre-mRNAs were increasingly accumulated in cell. To this end, our work suggests that a combined transcriptional and post transcriptional control exists in rice to regulate GolS expression under stress. Concurrent detection and processing of prematurely terminating transcripts coupled to repressed splicing can be described as a form of Regulated Unproductive Splicing and Translation (RUST) and may be linked to the stress adaptation of the plant, which is an interesting future research possibility.

Gal epitope expression and immunological properties in iGb3S deficient mice.

The Gal antigen is synthesized by glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) or (and) isoglobotrihexosylceramide 3 synthase (iGb3S). However, whether iGb3S deletion changes Gal epitope expression and immunological properties in animals is still not clear. The objective of this study was to develop iGb3S deficient mice, and characterize their Gal epitope expression and Gal epitope-related immunological properties. iGb3S gene knockout mice were generated on the C57BL/6 background using the bacterial artificial chromosome homology region recombination technique. Gal epitope expression in the iGb3S deficient mice was determined by using a monoclonal anti-Gal antibody. Immunological properties were analyzed by enzyme linked immune sorbent assay. It was found that Gal epitope expression was decreased from 5.19% to 21.74% in the main organs of iGb3S deficient mice, compared with that of C57BL/6 wild type mice, suggesting that the iGb3S gene participated to Gal epitope expression. However, iGb3S deletion alone did not cause significant changes in the immunological properties of iGb3S deficient mice with or without exogenous Gal antigen (Rabbit Red Blood Cell) stimulation. The data from this study suggest that the iGb3S gene likely contributes to Gal epitope expression, but may have a very weak effect on immunological properties of the iGb3S deficient mice.

Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice.

It is uncertain which ß4-galactosyltransferase (ß4GalT; gene name, B4galt), ß4GalT-5 and/or ß4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. METHODS: We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. RESULTS: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. CONCLUSIONS: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

T-Synthase Deficiency Enhances Oncogenic Features in Human Colorectal Cancer Cells via Activation of Epithelial-Mesenchymal Transition.

BACKGROUND: Immature truncated O-glycans such as Tn antigen are frequently detected in human colorectal cancer (CRC); however, the precise pathological consequences of Tn antigen expression on CRC are unknown. T-synthase is the key enzyme required for biosynthesis of mature O-glycans. Here we investigated the functional roles of Tn antigen expression mediated by T-synthase deficiency in CRC cells. METHODS: To knock out T-synthase, we used CRISPR-Cas9 technology to target C1GALT1, the gene encoding T-synthase, in a CRC cell line (HCT116). Deletion of T-synthase was confirmed by western blotting, and expression of Tn antigen was determined by flow cytometry in HCT116 cells. We then assessed the biological effects of T-synthase deficiency on oncogenic behaviors in HCT116 cells. Furthermore, we analyzed the mechanistic role of T-synthase deficiency in cancer cells by determining the epithelial-mesenchymal transition (EMT) pathway. RESULTS: We showed that forced knockout of T-synthase in HCT116 cells significantly induced Tn antigen expression, which represented the occurrence of aberrant O-glycosylation. Loss of T-synthase significantly enhanced cell proliferation and adhesion, as well as migration and invasiveness in culture. More importantly, we demonstrated that T-synthase deficiency directly induced classical EMT characteristics in cancer cells. E-cadherin, a typical epithelial cell marker, was markedly decreased in T-synthase knockout HCT 116 cells, accompanied by an enhanced expression of mesenchymal markers including snail and fibronectin (FN). CONCLUSIONS: These findings indicate that T-synthase deficiency in CRC cells not only is responsible for aberrant O-glycosylation, but also triggers the molecular process of EMT pathway, which may translate to increased invasiveness and metastasis in cancers.

Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila.

The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., ß-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. Although deviations from normal protein glycosylation have been documented in AD, their role in disease pathology has been barely explored. Here our analysis of available expression data sets indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled us to predict the occurrence of AD with remarkable accuracy in a test cohort and identify a set of key genes whose expression determines this classification. We then studied in vivo the effect of reducing expression of homologs of 6 of these genes in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. Our results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology.

DGE-seq analysis of MUR3-related Arabidopsis mutants provides insight into how dysfunctional xyloglucan affects cell elongation.

Our previous study of the Arabidopsis mur3-3 mutant and mutant plants in which the mur3-3 phenotypes are suppressed (xxt2mur3-3, xxt5mur3-3, xxt1xxt2mur3-3 and 35Spro:XLT2:mur3-3) showed that hypocotyl cell elongation is decreased in plants that synthesize galactose-deficient xyloglucan. To obtain genome-wide insight into the transcriptome changes and regulatory networks that may be involved in this decreased elongation, we performed digital gene expression analyses of the etiolated hypocotyls of wild type (WT), mur3-3 and the four suppressor lines. Numerous differentially expressed genes (DEGs) were detected in comparisons between WT and mur3-3 (1423), xxt2mur3-3 and mur3-3 (675), xxt5mur3-3 and mur3-3 (1272), xxt1xxt2mur3-3 and mur3-3 (1197) and 35Spro:XLT2:mur3-3 vs mur3-3 (121). 550 overlapped DEGs were detected among WT vs mur3-3, xxt2mur3-3 vs mur3-3, xxt5mur3-3 vs mur3-3, and xxt1xxt2mur3-3 vs mur3-3 comparisons. These DEGs include 46 cell wall-related genes, 24 transcription factors, 6 hormone-related genes, 9 protein kinase genes and 9 aquaporin genes. The expression of all of the 550 overlapped genes is restored to near wild-type levels in the four mur3-3 suppressor lines. qRT-PCR of fifteen of these 550 genes showed that their expression levels are consistent with the digital gene expression data. Overexpression of some of these genes (XTH4, XTH30, PME3, EXPA11, MYB88, ROT3, AT5G37790, WAG2 and TIP2;3) that are down-regulated in mur3-3 partially rescued the short hypocotyl phenotype but not the aerial phenotype of mur3-3, indicating that different mechanisms exist between hypocotyl cell elongation and leaf cell elongation.

ZFX modulates the growth of human leukemic cells via B4GALT1.

Zinc finger protein X-linked (ZFX) is a key regulator of both embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs), which is required for both Notch intracellular domain (NotchIC)-induced acute T-cell leukemia and MLL-AF9-induced myeloid leukemia in mouse models. However, the role of ZFX and its underlying mechanism in human leukemic cells remain unclear yet, though accumulating data have demonstrated that ZFX is aberrantly expressed in various human tumors and plays an important role. Herein, we found that ZFX was aberrantly expressed in various human leukemic cell lines and primary cells from leukemia patients compared with control cells. The silence of ZFX led to the growth suppression through either the deregulated cell cycle or the induction of apoptosis in various cells including K562, Jurkat, Namalwa, and THP-1 cells. The gene expression analysis revealed that UDP-Gal:ßGlcNAc ß 1,4-galactosyltransferase, polypeptide 1 (B4GALT1) was significantly down-regulated upon ZFX silencing, which is implicated in the response of K562 cells to the treatment of imatinib mesylate (IM). In addition, lectin blot assay showed that the galactosylation of glycoproteins in K562 cells was suppressed upon ZFX silencing. Interestingly, overexpression of B4GALT1 restored the growth and conferred drug resistance to ZFX-silenced cells. Taken together, we have demonstrated that ZFX is aberrantly expressed in multiple human leukemic cells and it modulates the growth and drug response of leukemic cells partially via B4GALT1, which suggests that ZFX is a new regulator of leukemic cells and warrants intensive investigations on this 'stemness' regulator in these deadly diseases.

Dysregulation of follicle development in a mouse model of premature ovarian insufficiency.

Premature ovarian insufficiency (POI) occurs in 1% of reproductive-age women. The ovarian manifestation ranges from the presence of a variable population of follicles (follicular) to the absence of follicles (afollicular), and in the majority of cases the cause is unknown. A transgenic mouse model of follicular POI, the Double Mutant (DM), arises from oocyte-specific deletion of Mgat1 and C1galt1 required for the generation of O- and N-glycans. DM females are subfertile at 6 weeks, infertile by 9 weeks and exhibit POI by 12 weeks of age. In this study we investigate the cause of the reduced fertility at 6 weeks and infertility at 9 weeks of DM females. Ovary sections were used to analyse follicle and corpora lutea (CL) numbers, apoptosis, and levels of laminin and 3ß-hydroxysteroid dehydrogenase using immunohistochemistry. After POI, DM females unexpectedly remained sexually receptive. At both 6 and 9 weeks, DM ovaries contained more primary follicles, however, at 9 weeks DM follicles were proportionally healthier, revealed by TUNEL analysis compared with Controls. In 9 week DM ovaries (collected post-mating), secondary follicles had theca and basal lamina structure abnormalities, whilst preovulatory follicles failed to ovulate resulting in the presence of numerous luteinised unruptured follicles, indicative of ovulation failure. Finally, DM ovaries contained more regressing CL with decreased luteal cell apoptosis indicative of a defect in CL regression. Identifying these follicular modifications have provided insight into the aetiology of a model of POI and highlight targets to investigate with the hope of developing new fertility treatments.

Metastatic Progression of Prostate Cancer Is Mediated by Autonomous Binding of Galectin-4-O-Glycan to Cancer Cells.

Metastatic prostate cancer continues to pose a difficult therapeutic challenge. Prostate cancer progression is associated with aberrant O-glycosylation of cancer cell surface receptors, but the functional impact of such events is uncertain. Here we report spontaneous metastasis of human prostate cancer xenografts that express high levels of galectin-4 along with genetic signatures of EGFR-HER2 signaling and O-glycosylation. Galectin-4 expression in clinical specimens of prostate cancer correlated with poor patient survival. Galectin-4 binding to multiple receptor tyrosine kinases stimulated their autophosphorylation, activated expression of pERK, pAkt, fibronectin, and Twist1, and lowered expression of E-cadherin, thereby facilitating epithelial-mesenchymal transition, invasion, and metastasis. In vivo investigations established that galectin-4 expression enabled prostate cancer cells to repopulate tumors in orthotopic and heterotopic tissues. Notably, these effects of galectin-4 relied upon O-glycosylation mediated by C1GALT1, a galactosyltransferase implicated in other cancers. Parallel changes in galectin-4 and O-glycosylation triggered aberrant receptor signaling and more aggressive invasive character in prostate cancer cells, which through better survival in the circulation also contributed to the bulk cell progeny of distal tumors. Our findings establish galectin-4 and C1GALT1-mediated glycosylation in a signaling axis that is activated during prostate cancer progression, with implications for therapeutic targeting of advanced metastatic disease. Cancer Res; 76(19); 5756-67. ©2016 AACR.

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

BACKGROUND: A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (α-gal) activity, that could conceivably prevent effective use. Use of lungs from α-gal knock out (α-gal KO) pigs presents a potential alternative and thus comparative de- and recellularization of wild-type and α-gal KO pig lungs was assessed. METHODS: Decellularized lungs were compared by histologic, immunohistochemical, and mass spectrometric techniques. Recellularization was assessed following compartmental inoculation of human lung bronchial epithelial cells, human lung fibroblasts, human bone marrow-derived mesenchymal stromal cells (all via airway inoculation), and human pulmonary vascular endothelial cells (CBF) (vascular inoculation). RESULTS: No obvious differences in histologic structure was observed but an approximate 25% difference in retention of residual proteins was determined between decellularized wild-type and α-gal KO pig lungs, including retention of α-galactosylated epitopes in acellular wild-type pig lungs. However, robust initial recellularization and subsequent growth and proliferation was observed for all cell types with no obvious differences between cells seeded into wild-type versus α-gal KO lungs. CONCLUSION: These proof of concept studies demonstrate that decellularized wild-type and α-gal KO pig lungs can be comparably decellularized and comparably support initial growth of human lung cells, despite some differences in retained proteins. α-Gal KO pig lungs are a suitable platform for further studies of xenogeneic lung regeneration.

Molecular analysis of the cumulus matrix: insights from mice with O-glycan-deficient oocytes.

During follicle development, oocytes secrete factors that influence the development of granulosa and cumulus cells (CCs). In response to oocyte and somatic cell signals, CCs produce extracellular matrix (ECM) molecules resulting in cumulus expansion, which is essential for ovulation, fertilisation, and is predictive of oocyte quality. The cumulus ECM is largely made up of hyaluronan (HA), TNF-stimulated gene-6 (TSG-6, also known as TNFAIP6), pentraxin-3 (PTX3), and the heavy chains (HCs) of serum-derived inter-α-inhibitor proteins. In contrast to other in vivo models where modified expansion impairs fertility, the cumulus mass of C1galt1 Mutants, which have oocyte-specific deletion of core 1-derived O-glycans, is modified without impairing fertility. In this report, we used C1galt1 Mutant (C1galt1(FF):ZP3Cre) and Control (C1galt1(FF)) mice to investigate how cumulus expansion is affected by oocyte-specific deletion of core 1-derived O-glycans without adversely affecting oocyte quality. Mutant cumulus-oocyte complexes (COCs) are smaller than Controls, with fewer CCs. Interestingly, the CCs in Mutant mice are functionally normal as each cell produced normal levels of the ECM molecules HA, TSG-6, and PTX3. However, HC levels were elevated in Mutant COCs. These data reveal that oocyte glycoproteins carrying core 1-derived O-glycans have a regulatory role in COC development. In addition, our study of Controls indicates that a functional COC can form provided all essential components are present above a minimum threshold level, and thus some variation in ECM composition does not adversely affect oocyte development, ovulation or fertilisation. These data have important implications for IVF and the use of cumulus expansion as a criterion for oocyte assessment.

Identification of a glycosylphosphatidylinositol anchor-modifying ß1-3 galactosyltransferase in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease nagana.  Trypanosoma brucei is dependent on glycoproteins for its survival and infectivity throughout its life cycle. Here we report the functional characterization of TbGT3, a glycosyltransferase expressed in the bloodstream and procyclic form of the parasite. Bloodstream and procyclic form TbGT3 conditional null mutants were created and both exhibited normal growth under permissive and nonpermissive conditions. Under nonpermissive conditions, the normal glycosylation of the major glycoprotein of bloodstream form T. brucei, the variant surface glycoprotein and the absence of major alterations in lectin binding to other glycoproteins suggested that the major function of TbGT3 occurs in the procyclic form of the parasite. Consistent with this, the major surface glycoprotein of the procyclic form, procyclin, exhibited a marked reduction in molecular weight due to changes in glycosylphosphatidylinositol (GPI) anchor side chains. Structural analysis of the mutant procyclin GPI anchors indicated that TbGT3 encodes a UDP-Gal: ß-GlcNAc-GPI ß1-3 Gal transferase. Despite the alterations in GPI anchor side chains, TbGT3 conditional null mutants remained infectious to tsetse flies under nonpermissive conditions.

Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model.

BACKGROUND: Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine cytomegalovirus (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be because of the inadvertent introduction of pCMV into our α1,3-galactosyltransferase gene knockout swine herd. METHODS: Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time polymerase chain reaction. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by cesarean section (C-section) and raised in isolation were likewise analyzed. RESULTS: Kidney grafts, from which 8 of the 18 archived samples were derived were found to be pCMV-negative, showed a mean graft survival of 48.3 days and were from transplants performed before 2008. None showed signs of disseminated intravascular coagulopathy and were lost because of proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008, and demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days. CONCLUSIONS: Decreased survivals of α1,3-galactosyltransferase gene knockout renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.

Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout Swine.

BACKGROUND: Various durations of survival have been observed in the xenotransplantation of life-supporting α-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates. Although others have demonstrated loss of GalT-KO-transplanted kidneys within 2 weeks, we have reported an average survival of 51 days with the cotransplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. To determine the factors responsible for this difference in survival time, we performed xenogeneic kidney transplantations into cynomolgus monkeys with an anti-CD40L-based regimen using two different strains of GalT-KO swine, one derived from MGH miniature swine and the other obtained from Meji University. MATERIALS AND METHODS: Eight cynomolgus moneys received GalT-KO kidneys. Three kidney grafts were from Massachusetts General Hospital (MGH)-Nippon Institute for Biological Science (NIBS) GalT-KO pigs and five GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. RESULTS: Recipients of kidneys from the MGH GalT-KO kidneys swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, whereas recipients of MEIJI GalT-KO kidneys swine survived an average of 9.2 days. Among the differences between these two groups, one potentially revealing disparity was that the MEIJI swine were positive for porcine cytomegalovirus, whereas the MGH-derived swine were negative. CONCLUSION: This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into nonhuman primates at a single center. The results demonstrate that porcine cytomegalovirus may be responsible for early loss of GalT-KO swine kidney xenografts.

Expression of human CD46 modulates inflammation associated with GalTKO lung xenograft injury.

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120' 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120' 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60' 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

Rituximab treatment prevents the early development of proteinuria following pig-to-baboon xeno-kidney transplantation.

We previously reported life-supporting α1,3-galactosyltransferase knockout (GalTKO) thymokidney xenograft survival of >2 months in baboons. However, despite otherwise normal renal function, recipients developed proteinuria with morphologic changes (podocyte effacement), a condition that presents a major obstacle to long-term studies in this model. A recent clinical study showed that rituximab therapy after allogeneic transplant prevented proteinuria possibly associated with loss of sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b). Here, we demonstrate that rituximab prevents the disruption of pig podocytes in an SMPDL-3b-dependent manner in vitro and the early development of proteinuria after xenogeneic kidney transplantation in baboons. Immunofluorescence showed SMPDL-3b expression in pig glomerular epithelium; immunoprecipitation demonstrated rituximab binding to SMPDL-3b in glomeruli. Culture of isolated pig podocytes with naive baboon sera, which has preformed antipig natural antibodies, reduced SMPDL-3b expression, disrupted podocyte morphology, and decreased podocyte proliferation, whereas pretreatment with rituximab prevented these effects. Six baboons received rituximab before transplantation to deplete B cells and again in the peri-transplant period; 18 baboons treated only before transplantation served as historical controls. The onset of post-transplant proteinuria was significantly delayed in a B cell-independent manner in the animals that received peri-transplant rituximab treatment. Although further optimization of this protocol is required, these data provide intriguing clues to the mechanisms of post-transplant proteinuria in xenogeneic kidney transplantation and a potential strategy for its prevention.

B4GALT family mediates the multidrug resistance of human leukemia cells by regulating the hedgehog pathway and the expression of p-glycoprotein and multidrug resistance-associated protein 1.

ß-1, 4-Galactosyltransferase gene (B4GALT) family consists of seven members, which encode corresponding enzymes known as type II membrane-bound glycoproteins. These enzymes catalyze the biosynthesis of different glycoconjugates and saccharide structures, and have been recognized to be involved in various diseases. In this study, we sought to determine the expressional profiles of B4GALT family in four pairs of parental and chemoresistant human leukemia cell lines and in bone marrow mononuclear cells (BMMC) of leukemia patients with multidrug resistance (MDR). The results revealed that B4GALT1 and B4GALT5 were highly expressed in four MDR cells and patients, altered levels of B4GALT1 and B4GALT5 were responsible for changed drug-resistant phenotype of HL60 and HL60/adriamycin-resistant cells. Further data showed that manipulation of these two gene expression led to increased or decreased activity of hedgehog (Hh) signaling and proportionally mutative expression of p-glycoprotein (P-gp) and MDR-associated protein 1 (MRP1) that are both known to be related to MDR. Thus, we propose that B4GALT1 and B4GALT5, two members of B4GALT gene family, are involved in the development of MDR of human leukemia cells, probably by regulating the activity of Hh signaling and the expression of P-gp and MRP1.