| 1. |
- Chai, Qian, et al.
(author)
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Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence
- 2014
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:16, s. 11342-11352
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Journal article (peer-reviewed)abstract
- Background: We studied ribosome and nucleoid distribution in Escherichia coli under growth and quiescence. Results: Spatially segregated ribosomes and nucleoids show drastically altered distribution in stationary phase or when treated with drugs affecting translation, transcription, nucleoid-topology, or cytoskeleton. Ribosome inheritance in daughter cells is frequently unequal. Conclusion: Cellular growth processes modulate ribosome and nucleoid distribution. Significance: This provides insight into subcellular organization of molecular machines. We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.
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| 2. |
- de la Rosa, Andrés
(author)
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Design, expression, and analysis of antibody-based blood-brain barrier shuttles
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Antibody therapeutics, with their strong and highly selective target binding, are now used to treat various diseases. However, to enable their use to treat brain disorders, they must be delivered across the blood-brain barrier (BBB), as without active transport, only around 0.01% of intravenously injected doses reach the brain. Brain delivery can be done by BBB shuttles capable of binding receptors that naturally transport proteins, e.g., the Transferrin receptor (TfR). This thesis has studied strategies for designing TfR-binding shuttles and how to enhance the protein expression of antibody therapeutics. In Paper I, we shared our updated transient gene expression (TGE) protocol and developed a small-scale version to surmount the cost limitations of testing many conditions. Large variations of protein expression were observed for both protocols, prompting future studies investigating its cause(s). In paper II, we investigated if binding to the glycosaminoglycan heparan sulfate (HS) present at the BBB could improve brain delivery. Our results indicate that the BBB shuttle scFv8D3 is not dependent on the HS-binding sites identified, and adding new HS-binding sites did not enhance delivery. However, further studies are required due to HS's complexity and heterogeneity. Decreasing the TfR affinity of BBB shuttles has been shown to boost the delivery of therapeutic doses of high affinity anti-TfR antibodies, e.g., bivalent 8D3 antibodies. In Paper III, we applied the strategy to a monovalent single-chain fragment variable (scFv) of 8D3 (scFv8D3) based BBB shuttle. Our affinity mutants exhibited lowered TfR affinity, longer blood half-life, and higher brain concentration. Using our In-Cell BBB Trans assay, we concluded that the increased brain concentration is likely due to extended blood half-life. In paper IV, we fused the TfR ligand holo-transferrin to the TfR binding arms of the partly bivalent RmAb158-scFv8D3 antibody. Our results indicate that the TfR binding shifted from partly to fully bivalent, resulting in markedly decreased in vitro transcytosis. The potential transcytosis-promoting effect of the fused holoTf was absent and/or counteracted by the bivalent binding of the design. However, the strategy may still prove useful for monovalent TfR binders. In conclusion, monovalent and low-to-moderate affinity are likely beneficial binding properties for TfR-mediated brain delivery at therapeutic doses. However, whether it is possible to enhance brain delivery with HS-binding or holoTf-fusion requires further study.
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| 3. |
- de la Rosa, Andres, et al.
(author)
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Introducing or removing heparan sulfate binding sites does not alter brain uptake of the blood-brain barrier shuttle scFv8D3
- 2022
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Journal article (peer-reviewed)abstract
- The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.
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| 4. |
- Gustavsson, Tobias, et al.
(author)
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Long-term effects of immunotherapy with a brain penetrating Aβ antibody in a mouse model of Alzheimer's disease
- 2023
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In: Alzheimer's Research & Therapy. - : BioMed Central (BMC). - 1758-9193. ; 15:1
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Journal article (peer-reviewed)abstract
- BackgroundBrain-directed immunotherapy is a promising strategy to target amyloid-β (Aβ) deposits in Alzheimer’s disease (AD). In the present study, we compared the therapeutic efficacy of the Aβ protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis.MethodsAppNL−G−F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL−G−F mice, with evaluation after 3 days. Second, to assess the antibodies’ ability to halt the progression of Aβ pathology, 3 months old AppNL−G−F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL−G−F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aβ aggregates and total Aβ42 were quantified with ELISA and immunostaining.ResultsNeither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aβ protofibrils or insoluble Aβ1-42 after a single injection treatment. After three successive injections, Aβ1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aβ aggregates, but a reduction in total Aβ42 was seen in the cortex of mice treated with both antibodies.ConclusionsBoth RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aβ immunotherapy.
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| 5. |
- Mattis, Katia K, et al.
(author)
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Loss of RREB1 in pancreatic beta cells reduces cellular insulin content and affects endocrine cell gene expression
- 2023
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In: Diabetologia. - : Springer Nature. - 0012-186X .- 1432-0428. ; 66:4, s. 674-694
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Journal article (peer-reviewed)abstract
- AIMS/HYPOTHESIS: Genome-wide studies have uncovered multiple independent signals at the RREB1 locus associated with altered type 2 diabetes risk and related glycaemic traits. However, little is known about the function of the zinc finger transcription factor Ras-responsive element binding protein 1 (RREB1) in glucose homeostasis or how changes in its expression and/or function influence diabetes risk.METHODS: A zebrafish model lacking rreb1a and rreb1b was used to study the effect of RREB1 loss in vivo. Using transcriptomic and cellular phenotyping of a human beta cell model (EndoC-βH1) and human induced pluripotent stem cell (hiPSC)-derived beta-like cells, we investigated how loss of RREB1 expression and activity affects pancreatic endocrine cell development and function. Ex vivo measurements of human islet function were performed in donor islets from carriers of RREB1 type 2 diabetes risk alleles.RESULTS: CRISPR/Cas9-mediated loss of rreb1a and rreb1b function in zebrafish supports an in vivo role for the transcription factor in beta cell mass, beta cell insulin expression and glucose levels. Loss of RREB1 also reduced insulin gene expression and cellular insulin content in EndoC-βH1 cells and impaired insulin secretion under prolonged stimulation. Transcriptomic analysis of RREB1 knockdown and knockout EndoC-βH1 cells supports RREB1 as a novel regulator of genes involved in insulin secretion. In vitro differentiation of RREB1KO/KO hiPSCs revealed dysregulation of pro-endocrine cell genes, including RFX family members, suggesting that RREB1 also regulates genes involved in endocrine cell development. Human donor islets from carriers of type 2 diabetes risk alleles in RREB1 have altered glucose-stimulated insulin secretion ex vivo, consistent with a role for RREB1 in regulating islet cell function.CONCLUSIONS/INTERPRETATION: Together, our results indicate that RREB1 regulates beta cell function by transcriptionally regulating the expression of genes involved in beta cell development and function.
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| 6. |
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| 7. |
- Metzendorf, Nicole G., 1979-, et al.
(author)
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Destination and Specific Impact of Different Bile Acids in the Intestinal Pathogen Clostridioides difficile
- 2022
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In: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 13
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Journal article (peer-reviewed)abstract
- The anaerobic bacterium Clostridioides difficile represents one of the most problematic pathogens, especially in hospitals. Dysbiosis has been proven to largely reduce colonization resistance against this intestinal pathogen. The beneficial effect of the microbiota is closely associated with the metabolic activity of intestinal microbes such as the ability to transform primary bile acids into secondary ones. However, the basis and the molecular action of bile acids (BAs) on the pathogen are not well understood. We stressed the pathogen with the four most abundant human bile acids: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA). Thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM), and electron microscopy (EM) were employed to track the enrichment and destination of bile acids in the bacterial cell. TLC not only revealed a strong accumulation of LCA in C. difficile, but also indicated changes in the composition of membrane lipids in BA-treated cells. Furthermore, morphological changes induced by BAs were determined, most pronounced in the virtually complete loss of flagella in LCA-stressed cells and a flagella reduction after DCA and CDCA challenge. Quantification of both, protein and RNA of the main flagella component FliC proved the decrease in flagella to originate from a change in gene expression on transcriptional level. Notably, the loss of flagella provoked by LCA did not reduce adhesion ability of C. difficile to Caco-2 cells. Most remarkably, extracellular toxin A levels in the presence of BAs showed a similar pattern as flagella expression. That is, CA did not affect toxin expression, whereas lower secretion of toxin A was determined in cells stressed with LCA, DCA or CDCA. In summary, the various BAs were shown to differentially modify virulence determinants, such as flagella expression, host cell adhesion and toxin synthesis. Our results indicate differences of BAs in cellular localization and impact on membrane composition, which could be a reason of their diverse effects. This study is a starting point in the elucidation of the molecular mechanisms underlying the differences in BA action, which in turn can be vital regarding the outcome of a C. difficile infection.
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| 8. |
- Morrison, Jamie I., et al.
(author)
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A single-chain fragment constant design enables easy production of a monovalent blood-brain barrier transporter and provides an improved brain uptake at elevated doses
- 2023
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In: Journal of Neurochemistry. - : John Wiley & Sons. - 0022-3042 .- 1471-4159. ; 165:3, s. 413-425
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Journal article (peer-reviewed)abstract
- The interest for developing antibody-driven therapeutic interventions has exponentially grown over the last few decades. Even though there have been promising leaps in the development of efficacious antibody therapies, problems revolving around production and site-directed delivery of these large macromolecules persist. This is especially pertinent when it comes to designing and producing antibodies to penetrate the blood-brain barrier (BBB) to tackle neurodegenerative diseases. One of the most effective approaches to alleviating this problem is to employ a "Trojan Horse " approach, using receptor-mediated transcytosis, such as those governed by the transferrin receptor (TfR)-mediated pathways, to deliver large protein payloads into the brain. Even though this method is effective, ideal limiting factors, related to how the antibody binds to the TfR, need to be elucidated to improve BBB penetrance. With this said, we have designed and produced a single-chain Fc antibody, conjugated to an scFv8D3 TfR binding motif, creating a single-chain monovalent BBB transporter (scFc-scFv8D3). This recombinant protein is easy to produce and purify, demonstrates monovalent binding to the TfR and is structurally stable at physiologically relevant temperatures. Using an in vitro BBB model system, we show a positive correlation between the concentration of administered antibody and transcytosis efficacy, with scFc-scFv8D3 demonstrating significantly higher transcytosis levels compared with scFv8D3-conjugated bivalent antibodies at elevated administered concentrations. Furthermore, in vivo studies recapitulate the in vitro results, with the scFc-scFv8D3 demonstrating an elevated brain uptake at higher therapeutic doses in wild-type mice, comparable with that of the scFv8D3-conjugated bivalent antibody control. In addition, the half-life of the single-chain monovalent BBB transporter is comparable with that of standard IgG antibodies, indicating that the scFc format does not exacerbate physiological degradation. Our results lead us to the conclusion that valency and affinity are important variables to consider when discerning optimal transport across the BBB using TfR-mediated transcytosis pathways. In addition, we believe the single-chain Fc antibody we have described, which can easily be manipulated to accommodate a bispecific target tactic, provides a simple and efficacious approach for delivering therapeutic payloads to the brain milieu.
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| 9. |
- Morrison, Jamie, et al.
(author)
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Standardized Preclinical In Vitro Blood-Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways
- 2023
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In: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 20:3, s. 1564-1576
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Journal article (peer-reviewed)abstract
- The presence of the blood-brain barrier (BBB) creates a nigh-on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a "Trojan Horse" strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB-penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that help delineate the ability of modified large bivalent IgG antibodies conjugated to the transferrin receptor binder scFv8D3 to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administration of bivalent antibodies into the endothelial monolayer, a highly sensitive enzyme-linked immunosorbent assay (ELISA) is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis, respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of receptors and proteins that are likely involved in the transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed that transcytosis of the transferrin-receptor-targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB-penetrating capabilities of transferrin-receptor-targeting antibodies. We believe that the In-Cell BBB-Trans assay can be used as a powerful, preclinical screening platform for therapeutic neurological pathologies.
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| 10. |
- Rofo, Fadi, et al.
(author)
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A Brain-Targeting Bispecific-Multivalent Antibody Clears Soluble Amyloid-Beta Aggregates in Alzheimer's Disease Mice
- 2022
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In: NEUROTHERAPEUTICS. - : Springer Nature. - 1933-7213 .- 1878-7479. ; 19:5, s. 1588-1602
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Journal article (peer-reviewed)abstract
- Amyloid-beta (A beta) oligomers and protofibrils are suggested to be the most neurotoxic A beta species in Alzheimer's disease (AD). Hence, antibodies with strong and selective binding to these soluble A beta aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional A beta-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of A beta protofibril selective antibody (RmAb158). Due to the additional binding sites and the short distance between them, HexaRmAb158 displayed a slow dissociation from protofibrils and strong binding to oligomers in vitro. In the current study, we aimed at investigating the therapeutic potential of this antibody format in vivo using mouse models of AD. To enhance BBB delivery, the transferrin receptor (TfR) binding moiety (scFv8D3) was added, forming the Bispecific-multivalent antibody (HexaRmAb158-scFv8D3). The new antibody displayed a weaker TfR binding compared to the previously developed RmAb158-scFv8D3 and was less efficiently transcytosed in a cell-based BBB model. HexaRmAb158 detected soluble A beta aggregates derived from brains of tg-ArcSwe and App(NL-G-F) mice more efficiently compared to RmAb158. When intravenously injected, HexaRmAb158-scFv8D3 was actively transported over the BBB into the brain in vivo. Brain uptake was marginally lower than that of RmAb158-scFv8D3, but significantly higher than observed for conventional IgG antibodies. Both antibody formats displayed similar brain retention (72 h post injection) and equal capacity in clearing soluble A beta aggregates in tg-ArcSwe mice. In conclusion, we demonstrate a Bispecific-multivalent antibody format capable of passing the BBB and targeting a wide-range of sizes of soluble A beta aggregates.
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