| 1. |
- Alsén, Samuel, et al.
(författare)
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Antigen-Presenting B Cells Program the Efferent Lymph T Helper Cell Response
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- B cells interact with T follicular helper (Tfh) cells in germinal centers (GCs) to generate high-affinity antibodies. Much less is known about how cognate T-B-cell interactions influence Th cells that enter circulation and peripheral tissues. Therefore, we generated mice lacking MHC-II expressing B cells and, by thoracic duct cannulation, analyzed Th cells in the efferent lymph at defined intervals post-immunization. Focusing on gut-draining mesenteric lymph nodes (MLNs), we show that antigen-specific alpha(4)beta(+)(7) gut-homing effector Th cells enter the circulation prior to CXCR5(+)PD-1(+) Tfh-like cells. B cells appear to have no or limited impact on the early generation and egress of gut-homing Th cells but are critical for the subsequent appearance of Tfh-like cells that peak in the lymph before GCs have developed. At this stage, antigen-presenting B cells also reduce the proportion of alpha(4)beta(+)(7) Th cells in the MLN and efferent lymph. Furthermore, cognate B-cell interaction drives a broad transcriptional program in Th cells, including IL-4 that is confined to the Tfh cell lineage. The IL-4-producing Tfh-like cells originate from Bcl6(+) precursors in the LNs and have gut-homing capacity. Hence, B cells program the efferent lymph Th cell response within a limited window of time after antigenic challenge.
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| 2. |
- Casselbrant, Anna, 1970, et al.
(författare)
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Intestinal Ketogenesis and Permeability
- 2024
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Ingår i: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - 1661-6596 .- 1422-0067. ; 25:12
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Tidskriftsartikel (refereegranskat)abstract
- Consumption of a high-fat diet (HFD) has been suggested as a contributing factor behind increased intestinal permeability in obesity, leading to increased plasma levels of microbial endotoxins and, thereby, increased systemic inflammation. We and others have shown that HFD can induce jejunal expression of the ketogenic rate-limiting enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS). HMGCS is activated via the free fatty acid binding nuclear receptor PPAR-alpha, and it is a key enzyme in ketone body synthesis that was earlier believed to be expressed exclusively in the liver. The function of intestinal ketogenesis is unknown but has been described in suckling rats and mice pups, possibly in order to allow large molecules, such as immunoglobulins, to pass over the intestinal barrier. Therefore, we hypothesized that ketone bodies could regulate intestinal barrier function, e.g., via regulation of tight junction proteins. The primary aim was to compare the effects of HFD that can induce intestinal ketogenesis to an equicaloric carbohydrate diet on inflammatory responses, nutrition sensing, and intestinal permeability in human jejunal mucosa. Fifteen healthy volunteers receiving a 2-week HFD diet compared to a high-carbohydrate diet were compared. Blood samples and mixed meal tests were performed at the end of each dietary period to examine inflammation markers and postprandial endotoxemia. Jejunal biopsies were assessed for protein expression using Western blotting, immunohistochemistry, and morphometric characteristics of tight junctions by electron microscopy. Functional analyses of permeability and ketogenesis were performed in Caco-2 cells, mice, and human enteroids. Ussing chambers were used to analyze permeability. CRP and ALP values were within normal ranges and postprandial endotoxemia levels were low and did not differ between the two diets. The PPAR alpha receptor was ketone body-dependently reduced after HFD. None of the tight junction proteins studied, nor the basal electrical parameters, were different between the two diets. However, the ketone body inhibitor hymeglusin increased resistance in mucosal biopsies. In addition, the tight junction protein claudin-3 was increased by ketone inhibition in human enteroids. The ketone body beta-Hydroxybutyrate (beta HB) did not, however, change the mucosal transition of the large-size molecular FD4-probe or LPS in Caco-2 and mouse experiments. We found that PPAR alpha expression was inhibited by the ketone body beta HB. As PPAR alpha regulates HMGCS expression, the ketone bodies thus exert negative feedback signaling on their own production. Furthermore, ketone bodies were involved in the regulation of permeability on intestinal mucosal cells in vitro and ex vivo. We were not, however, able to reproduce these effects on intestinal permeability in vivo in humans when comparing two weeks of high-fat with high-carbohydrate diet in healthy volunteers. Further, neither the expression of inflammation markers nor the aggregate tight junction proteins were changed. Thus, it seems that not only HFD but also other factors are needed to permit increased intestinal permeability in vivo. This indicates that the healthy gut can adapt to extremes of macro-nutrients and increased levels of intestinally produced ketone bodies, at least during a shorter dietary challenge.
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| 3. |
- Cervin, Jakob
(författare)
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Evaluation of fucosylated receptors for Cholera toxin in the human small intestine
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cholera toxin (CT) produced by Vibrio cholerae is the causative agent for the diarrheal disease cholera. Cholera is yearly afflicting millions and is estimated to kill over 100 000 people every year. In this thesis I aimed to better understand the role of noncanonical CT receptors, e.g. receptors other than the glycolipid GM1. Epidemiological studies have found a link between cholera severity and blood group indicating that histo-blood group antigens (HBGAs) could play a role as receptors for CT. The work presented in this thesis shows that CT readily binds to the HBGA Lewis X on cells and on some cells CTB binding correlates with the level of Lewis X. Furthermore, we show that other fucosylated glycans such as Lewis Y, A/BLewis Y and 2´-fucosyllactose (found in human breast milk) readily inhibit CT binding to cell lines and primary cells from human small intestine. In contrast, sialylated or non-fucosylated glycans did not show any inhibitory effect on CT binding to human cell lines indicating a fucose-dependent binding. This was further confirmed in blocking studies using long synthetic polymers displaying glucose, fucose, galactose or a mix of the latter two. Functional evaluation identified that the fucose-binding lectin AAL completely blocked the effect of CT, but so could the galactose-binding lectin PNA. The galactose-fucose polymers yielded a partial inhibition of CT intoxication of human small intestinal enteroids whereas GM1 glycan completely blocked the effect of CT. Hence, fucosylated glycans are involved in attachment of CT to the intestinal wall. However, if this binding assists or counteracts subsequent internalization by other receptors carrying terminal galactoses remains to be determined. Importantly, these receptors can be other glycans than GM1 as this thesis show GM1-independent CT-mediated intoxication.
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| 4. |
- Cervin, Jakob, et al.
(författare)
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Fucose-Galactose Polymers Inhibit Cholera Toxin Binding to Fucosylated Structures and Galactose-Dependent Intoxication of Human Enteroids.
- 2020
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Ingår i: ACS infectious diseases. - : American Chemical Society (ACS). - 2373-8227. ; 6:5, s. 1192-1203
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Tidskriftsartikel (refereegranskat)abstract
- A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. However, to date the efficacies of most of these blockers have been evaluated in noncellular systems that lack ligands other than GM1. Importantly, the CT B subunit (CTB) has a noncanonical site that binds fucosylated structures, which in contrast to GM1 are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to immobilized protein-linked glycan structures and also to primary human and murine small intestine epithelial cells (SI ECs). We show that the binding of CTB to human SI ECs is largely dependent on the noncanonical binding site, and interference with the canonical site has a limited effect while the opposite is observed with murine SI ECs. The galactose-fucose polymer blocks binding to fucosylated glycans but not to GM1. However, the preincubation of CT with the galactose-fucose polymer only partially blocks toxic effects on cultured human enteroid cells, while preincubation with GM1 completely blocks CT-mediated secretion. Our results support a model whereby the binding of fucose to the noncanonical site places CT in close proximity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication. Our finding also highlights the importance of complementing CTB binding studies with functional intoxication studies when assessing the efficacy inhibitors of CT.
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| 5. |
- Cervin, Jakob, et al.
(författare)
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GM1 ganglioside-independent intoxication by Cholera toxin
- 2018
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 14:2
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Tidskriftsartikel (refereegranskat)abstract
- Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.
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| 6. |
- Komban, Rathan, et al.
(författare)
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Activated Peyer's patch B cells sample antigen directly from M cells in the subepithelial dome.
- 2019
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The germinal center (GC) reaction in Peyer's patches (PP) requires continuousaccess to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines.
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| 7. |
- Olsson Hau, Sofie, et al.
(författare)
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PRR11 unveiled as a top candidate biomarker within the RBM3-regulated transcriptome in pancreatic cancer
- 2022
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Ingår i: Journal of Pathology: Clinical Research. - : Wiley. - 2056-4538. ; 8:1, s. 65-77
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Tidskriftsartikel (refereegranskat)abstract
- The outlook for patients with pancreatic cancer remains dismal. Treatment options are limited and chemotherapy remains standard of care, leading to only modest survival benefits. Hence, there is a great need to further explore the mechanistic basis for the intrinsic therapeutic resistance of this disease, and to identify novel predictive biomarkers. RNA-binding motif protein 3 (RBM3) has emerged as a promising biomarker of disease severity and chemotherapy response in several types of cancer, including pancreatic cancer. The aim of this study was to unearth RBM3-regulated genes and proteins in pancreatic cancer cells in vitro, and to examine their expression and prognostic significance in human tumours. Next-generation RNA sequencing was applied to compare transcriptomes of MIAPaCa-2 cells with and without RBM3 knockdown. The prognostic value of differentially expressed genes (DEGs) was examined in The Cancer Genome Atlas (TCGA). Top deregulated genes were selected for further studies in vitro and for immunohistochemical analysis of corresponding protein expression in tumours from a clinically well-annotated consecutive cohort of 46 patients with resected pancreatic cancer. In total, 19 DEGs (p < 0.01) were revealed, among which some with functions in cell cycle and cell division stood out; PDS5A (PDS cohesin associated factor A) as the top downregulated gene, CCND3 (cyclin D3) as the top upregulated gene, and PRR11 (proline rich 11) as being highly prognostic in TCGA. Silencing of RBM3 in MiaPaCa-2 cells led to congruent alterations of PDS5A, cyclin D3, and PRR11 levels. High protein expression of PRR11 was associated with adverse clinicopathological features and shorter overall survival. Neither PDS5A nor cyclin D3 protein expression was prognostic. This study unveils several RBM3-regulated genes with potential clinical relevance in pancreatic cancer, among which PRR11 shows the most consistent association with disease severity, at both transcriptome and protein levels.
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| 8. |
- Wands, Amberlyn M., et al.
(författare)
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Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells
- 2018
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Ingår i: ACS Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 4:5, s. 758-770
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Tidskriftsartikel (refereegranskat)abstract
- Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium.
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| 9. |
- Wands, Amberlyn M, et al.
(författare)
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Fucosylation and protein glycosylation create functional receptors for cholera toxin.
- 2015
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Ingår i: eLife. - 2050-084X. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera.
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| 10. |
- Youn, G., et al.
(författare)
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Targeting Multiple Binding Sites on Cholera Toxin B with Glycomimetic Polymers Promotes the Formation of Protein-Polymer Aggregates
- 2020
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 21:12, s. 4878-4887
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Tidskriftsartikel (refereegranskat)abstract
- The canonical binding site on the B subunit of cholera toxin (CTB) binds to GM1 gangliosides on host cells. However, the recently discovered noncanonical binding site on CTB with affinity for fucosylated molecules has raised the possibility that both sites can be involved in initiating intoxication. Previously, we showed that blocking CTB binding to human and murine small intestine epithelial cells can be increased by simultaneously targeting both binding sites with multivalent norbornene-based glycopolymers [ACS Infect. Dis.2020,6, 5, 1192-1203]. However, the mechanistic origin of the increased blocking efficacy was unclear. Herein, we observed that mixing CTB pentamers and glycopolymers that display fucose and galactose sugars results in the formation of large aggregates, which further inhibits binding of CTB to human granulocytes. Dynamic light scattering analysis, small-angle X-ray scattering analysis, transmission electron microscopy, and turbidimetric assays revealed that the facial directionality of CTB promotes interchain cross-linking, which in turn leads to self-assembly of proteinpolymer networks. This cross-linking-induced self-assembly occurs only when the glycopolymer system contains both galactose and fucose. In an assay of the glycopolymers ability to block CTB binding to human granulocytes, we observed a direct correlation between IC50 and self-assembly size. The aggregation mechanism of inhibition proposed herein has potential utility for the development of low-cost macromolecular clinical therapeutics for cholera that do not have exotic architectures and do not require complex synthetic sequences.
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