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  • 1.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mohammadamin, Sayran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Fc Receptors for Immunoglobulins and Their Appearance during Vertebrate Evolution2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 5, p. e96903-Article in journal (Refereed)
    Abstract [en]

    Receptors interacting with the constant domain of immunoglobulins (Igs) have a number of important functions in vertebrates. They facilitate phagocytosis by opsonization, are key components in antibody-dependent cellular cytotoxicity as well as activating cells to release granules. In mammals, four major types of classical Fc receptors (FcRs) for IgG have been identified, one high-affinity receptor for IgE, one for both IgM and IgA, one for IgM and one for IgA. All of these receptors are related in structure and all of them, except the IgA receptor, are found in primates on chromosome 1, indicating that they originate from a common ancestor by successive gene duplications. The number of Ig isotypes has increased gradually during vertebrate evolution and this increase has likely been accompanied by a similar increase in isotype-specific receptors. To test this hypothesis we have performed a detailed bioinformatics analysis of a panel of vertebrate genomes. The first components to appear are the poly-Ig receptors (PIGRs), receptors similar to the classic FcRs in mammals, so called FcRL receptors, and the FcR gamma chain. These molecules are not found in cartilagous fish and may first appear within bony fishes, indicating a major step in Fc receptor evolution at the appearance of bony fish. In contrast, the receptor for IgA is only found in placental mammals, indicating a relatively late appearance. The IgM and IgA/M receptors are first observed in the monotremes, exemplified by the platypus, indicating an appearance during early mammalian evolution. Clearly identifiable classical receptors for IgG and IgE are found only in marsupials and placental mammals, but closely related receptors are found in the platypus, indicating a second major step in Fc receptor evolution during early mammalian evolution, involving the appearance of classical IgG and IgE receptors from FcRL molecules and IgM and IgA/M receptors from PIGR.

  • 2.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Boinapally, Vamsi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11, article id e0143091Article in journal (Refereed)
    Abstract [en]

    Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

  • 3.
    Andersson, Mattias K.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 10, p. 2255-2267Article in journal (Refereed)
    Abstract [en]

    Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.

  • 4.
    Carnrot, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Prokopec, Kajsa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Råsbo, Kristina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Karlsson, Mikael
    Karolinska institutet, Clinical Allergy Research, Solna.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Marginal zone B cells are naturally reactive to collagen type II and are involved in the initiation of the immune response in collagen-induced arthritis2011In: Cellular & Molecular Immunology, ISSN 1672-7681, Vol. 8, no 4, p. 296-304Article in journal (Refereed)
    Abstract [en]

    Antibodies against type II collagen (CII) are essential for development of collagen-induced arthritis (CIA), but how and where the B-cell response to CII is initiated is not fully known. We show here that naive DBA/1 mice display naturally reactive IgM and IgG anti-CII producing B cells prior to immunization. The CII-reactive B cells were observed in the spleen and recognized as marginal zone (MZ) B cells. After CII immunization, CII-specific B cells expanded rapidly in the spleen, in contrast to the lymph nodes, with the initial response derived from MZ B cells and later by follicular (FO) B cells. This was evident despite that the MZ B cells were subject to stringent tolerance mechanisms by having a greater Fc gamma receptor IIb expression than the FO B cells. Further, the MZ B cells migrated to the FO areas upon immunization, possibly providing antigen and activating FO T cells and subsequently FO B cells. Thus, around CIA onset increased numbers of IgG anti-CII producing FO B cells was seen in the spleen, which was dominated by IgG2a- and IgG2b-positive cells. These data demonstrate that CII-reactive MZ B cells are present before and expand after CII immunization, suggesting an initiating role of MZ B cells in the development of CIA.

  • 5.
    Chahal, Gurdeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 6, article id e0129511Article in journal (Refereed)
    Abstract [en]

    Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.

  • 6.
    Das, Sarbashis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Pettersson, B M Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Behra, Phani Rama Krishna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Ramesh, Malavika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Bhattacharya, Alok
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Characterization of three Mycobacterium spp. with potential use in bioremediation by genome sequencing and comparative genomics2015In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 7, no 7, p. 1871-1886Article in journal (Refereed)
    Abstract [en]

    We provide the genome sequences of the type strains of the polychlorophenol-degrading Mycobacterium chlorophenolicum (DSM43826), the degrader of chlorinated aliphatics Mycobacterium chubuense (DSM44219) and Mycobacterium obuense (DSM44075) that has been tested for use in cancer immunotherapy. The genome sizes of M. chlorophenolicum, M. chubuense and M. obuense are 6.93, 5.95 and 5.58 Mbps with GC-contents of 68.4, 69.2 and 67.9%, respectively. Comparative genomic analysis revealed that 3254 genes are common and we predicted approximately 250 genes acquired through horizontal gene transfer from different sources including proteobacteria. The data also showed that the biodegrading Mycobacterium spp. NBB4, also referred to as M. chubuense NBB4, is distantly related to the M. chubuense type strain and should be considered as a separate species, we suggest it to be named M. ethylenense NBB4. Among different categories we identified genes with potential roles in: biodegradation of aromatic compounds, and copper homeostasis. These are the first non-pathogenic Mycobacterium spp. found harboring genes involved in copper homeostasis. These findings would therefore provide insight into the role of this group of Mycobacterium spp. in bioremediation as well as the evolution of copper homeostasis within the Mycobacterium genus.

  • 7.
    Das, Sarbashis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Pettersson, B. M. Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Behra, Phani Rama Krishna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Ramesh, Malavika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Bhattacharya, Alok
    Jawaharlal Nehru Univ, Sch Computat & Integrat Sci, New Delhi 110067, India.;Jawaharlal Nehru Univ, Sch Life Sci, New Delhi 110067, India..
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    The Mycobacterium phlei Genome: Expectations and Surprises2016In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 8, no 4, p. 975-985Article in journal (Refereed)
    Abstract [en]

    Mycobacterium phlei, a nontuberculosis mycobacterial species, was first described in 1898-1899. We present the complete genome sequence for the IV, phlei CCUG21000(T) type strain and the draft genomes for four additional strains. The genome size for all five is 5.3 Mb with 69.4% Guanine-Cytosine content. This is approximate to 0.35 Mbp smaller than the previously reported M. phlei RIVM draft genome. The size difference is attributed partly to large bacteriophage sequence fragments in the M. phlei RIVM genome. Comparative analysis revealed the following: 1) A CRISPR system similar to Type 1E (cas3) in M. phiei RIVM; 2) genes involved in polyamine metabolism and transport (potAD, potT) that are absent in other mycobacteria, and 3) strain specific variations in the number of sigma-factor genes. Moreover, M. phlei has as many as 82 mce (mammalian cell entry) homologs and many of the horizontally acquired genes in M. phlei are present in other environmental bacteria including mycobacteria that share similar habitat. Phylogenetic analysis based on 693 Mycobacterium core genes present in all complete mycobacterial genomes suggested that its closest neighbor is Mycobacterium smegmatis JS623 and Mycobacterium rhodesiae NBB3, while it is more distant to M. smegmatis mc2 155.

  • 8.
    Dhanraj, Santhosh
    et al.
    Univ Toronto, Inst Med Sci, Toronto, ON, Canada.;Hosp Sick Children, Genet & Genome Biol Program, Toronto, ON, Canada..
    Gunja, Sethu Madhava Rao
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Deveau, Adam P.
    IWK Hlth Ctr, Dept Pediat, Halifax, NS, Canada..
    Nissbeck, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Boonyawat, Boonchai
    Hosp Sick Children, Genet & Genome Biol Program, Toronto, ON, Canada..
    Coombs, Andrew J.
    IWK Hlth Ctr, Dept Pediat, Halifax, NS, Canada..
    Renieri, Alessandra
    Univ Siena, Med Genet, I-53100 Siena, Italy..
    Mucciolo, Mafalda
    Univ Siena, Med Genet, I-53100 Siena, Italy..
    Marozza, Annabella
    Univ Siena, Med Genet, I-53100 Siena, Italy..
    Buoni, Sabrina
    Univ Siena, Pediat Neurol, Siena, Italy..
    Turner, Lesley
    Mem Univ Newfoundland, Discipline Genet, St John, NF, Canada..
    Li, Hongbing
    Hosp Sick Children, Genet & Genome Biol Program, Toronto, ON, Canada..
    Jarrar, Ameer
    IWK Hlth Ctr, Dept Pediat, Halifax, NS, Canada..
    Sabanayagam, Mathura
    Hosp Sick Children, Genet & Genome Biol Program, Toronto, ON, Canada..
    Kirby, Melanie
    Hosp Sick Children, Dept Pediat, Div Hematol Oncol, Toronto, ON M5G 1X8, Canada..
    Shago, Mary
    Hosp Sick Children, Dept Paediat, Lab Med, Cytogenet Lab, Toronto, ON, Canada..
    Pinto, Dalila
    Mt Sinai Sch Med, Mindich Child Hlth & Dev Inst, Seaver Autism Ctr, Dept Psychiat, New York, NY USA.;Mt Sinai Sch Med, Mindich Child Hlth & Dev Inst, Seaver Autism Ctr, Dept Genet & Genom Sci, New York, NY USA..
    Berman, Jason N.
    Dalhousie Univ, IWK Hlth Ctr, Pediat, Microbiol & Immunol, Halifax, NS, Canada..
    Scherer, Stephen W.
    Hosp Sick Children, Genet & Genome Biol Program, Toronto, ON, Canada..
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Dror, Yigal
    Univ Toronto, Inst Med Sci, Toronto, ON, Canada.;Hosp Sick Children, Genet & Genome Biol Program, Toronto, ON, Canada.;Hosp Sick Children, Dept Pediat, Div Hematol Oncol, Marrow Failure & Myelodysplasia Program, Toronto, ON M5G 1X8, Canada..
    Bone Marrow Failure and Developmental Delay Caused By Mutations in Poly(A)-Specific Ribonuclease2015In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 126, no 23Article in journal (Other academic)
  • 9.
    Dhanraj, Santhosh
    et al.
    Hosp Sick Children, Res Inst, Genet & Genome Biol Program, Toronto, ON M5G 1X8, Canada.;Univ Toronto, Inst Med Sci, Toronto, ON, Canada..
    Gunja, Sethu Madhava Rao
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Deveau, Adam P.
    Dalhousie Univ, Dept Microbiol & Immunol, Halifax, NS, Canada..
    Nissbeck, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Boonyawat, Boonchai
    Hosp Sick Children, Res Inst, Genet & Genome Biol Program, Toronto, ON M5G 1X8, Canada.;Hosp Sick Children, Dept Paediat, Toronto, ON M5G 1X8, Canada..
    Coombs, Andrew J.
    IWK Hlth Ctr, Dept Pediat, Halifax, NS, Canada.;Dalhousie Univ, Halifax, NS, Canada..
    Renieri, Alessandra
    Univ Siena, Dept Med Genet, I-53100 Siena, Italy..
    Mucciolo, Mafalda
    Azienda Osped Univ Senese, Genet Med, Siena, Italy..
    Marozza, Annabella
    Azienda Osped Univ Senese, Genet Med, Siena, Italy..
    Buoni, Sabrina
    Univ Senese, Azienda Osped, Neuropsichiat Infantile, Siena, Italy..
    Turner, Lesley
    Mem Univ Newfoundland, Dept Discipline Genet, St John, NF, Canada..
    Li, Hongbing
    Hosp Sick Children, Res Inst, Genet & Genome Biol Program, Toronto, ON M5G 1X8, Canada..
    Jarrar, Ameer
    IWK Hlth Ctr, Dept Pediat, Halifax, NS, Canada.;Dalhousie Univ, Halifax, NS, Canada..
    Sabanayagam, Mathura
    Hosp Sick Children, Res Inst, Genet & Genome Biol Program, Toronto, ON M5G 1X8, Canada..
    Kirby, Melanie
    Shago, Mary
    Hosp Sick Children, Dept Paediat Lab Med, Toronto, ON M5G 1X8, Canada..
    Pinto, Dalila
    Hosp Sick Children, Res Inst, Genet & Genome Biol Program, Toronto, ON M5G 1X8, Canada.;Mt Sinai Sch Med, Mindich Child Hlth & Dev Inst, Seaver Autism Ctr, Dept Psychiat & Genet, New York, NY USA.;Mt Sinai Sch Med, Mindich Child Hlth & Dev Inst, Seaver Autism Ctr, Dept Genom Sci, New York, NY USA..
    Berman, Jason N.
    IWK Hlth Ctr, Dept Pediat, Halifax, NS, Canada.;Dalhousie Univ, Halifax, NS, Canada..
    Scherer, Stephen W.
    Hosp Sick Children, Res Inst, Genet & Genome Biol Program, Toronto, ON M5G 1X8, Canada.;Univ Toronto, Dept Mol Genet, Toronto, ON, Canada..
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Dror, Yigal
    Hosp Sick Children, Res Inst, Genet & Genome Biol Program, Toronto, ON M5G 1X8, Canada.;Univ Toronto, Inst Med Sci, Toronto, ON, Canada.;Hosp Sick Children, Dept Paediat, Toronto, ON M5G 1X8, Canada..
    Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ribonuclease (PARN)2015In: Journal of Medical Genetics, ISSN 0022-2593, E-ISSN 1468-6244, Vol. 52, no 11, p. 738-748Article in journal (Refereed)
    Abstract [en]

    Background Deadenylation regulates RNA function and fate. Poly(A)-specific ribonuclease (PARN) is a deadenylase that processes mRNAs and non-coding RNA. Little is known about the biological significance of germline mutations in PARN. Methods We identified mutations in PARN in patients with haematological and neurological manifestations. Genomic, biochemical and knockdown experiments in human marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease. Results We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination and bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and severely reduced PARN protein and deadenylation activity. Cells from this patient had impaired oligoadenylation of specific H/ACA box small nucleolar RNAs. Importantly, PARN-deficient patient cells manifested short telomeres and an aberrant ribosome profile similar to those described in some variants of dyskeratosis congenita. Knocking down PARN in human marrow cells and zebrafish impaired haematopoiesis, providing further evidence for a causal link with the human disease. Conclusions Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.

  • 10.
    Dixit, Shailesh S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Upadhayaya, Ram Shankar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    New parasite inhibitors encompassing novel conformationally-locked 5 '-acyl sulfamoyl adenosines2012In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 10, no 30, p. 6121-6129Article in journal (Refereed)
    Abstract [en]

    We describe the design, synthesis and biological evaluation of conformationally-locked 5'-acyl sulfamoyl adenosine derivatives as new parasitic inhibitors against Trypanosoma and Leishmania. The conformationally-locked (3'-endo, North-type) nucleosides have been synthesized by covalently attaching a 4'-CH2-O-2' bridge (Fig. 2) across C2'-C4' of adenosine in order to reduce the conformational flexibility of the pentose ring. This is designed to decrease the entropic penalty for complex formation with the target protein, which may improve free-energy of stabilization of the complex leading to improved potency. Conformationally-locked 5'-acyl sulfamoyl adenosine derivatives (16-22) were tested against parasitic protozoans for the first time in this work, and showed potent inhibition of Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma rhodesiense and Leishmania infantum with IC50 = 0.25-0.51 mu M. In particular, the potent 5'-pentanyl acyl sulfamoyl adenosine derivative 17 (IC50 = 0.25 mu M) against intracellular L. infantum amastigotes and Trypanosoma subspecies is interesting in view of its almost insignificant cytotoxicity in murine macrophage host cells (CC50 >4 mu M) and in diploid human fibroblasts MRC-5 cell lines (CC50 4 mu M). This work also suggests that variable alkyl chain length of the acyl group on the acylsulfamoyl side chain at 5' can modulate the toxicity of 5'-O-sulfamoylnucleoside analogues. This conformationally-locked sulfamoyl adenosine scaffold presents some interesting possibilities for further drug design and lead optimization.

  • 11. Dutta, Suman
    et al.
    Bhaduri, Nipa
    Rastogi, Neha
    Chandel, Sunita G.
    Vandavasi, Jaya Kishore
    Upadhayaya, Ram Shankar
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Carba-LNA modified siRNAs targeting HIV-1 TAR region downregulate HIV-1 replication successfully with enhanced potency2011In: MedChemComm, ISSN 2040-2503, Vol. 2, no 3, p. 206-216Article in journal (Refereed)
    Abstract [en]

    The conformationally-locked carbocyclic nucleosides carbaLNA ("jcLNA") (Gagnon et al., Biochemistry, 2010, 49, 10166; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362; Xu et al., J. Org. Chem., 2009, 74, 6534; Zhou and Chattopadhyaya, J. Org. Chem., 2010, 75, 2341; Zhou et al., J. Org. Chem., 2009, 74, 118) are chemically engineered by fusing a carbocyclic ring at the C2' to C4' chiral centres in a stereospecific manner at the alpha-face of the pentose-sugar of the native nucleosides. The benefit of the chemically-modified oligonucleotides with the jcLNA scaffold has been shown to be their uniquely enhanced nuclease resistance in the blood serum as well as their improved RNase H recruitment capability to cleave the target RNA in the hybrid antisense-RNA duplex when used as an antisense agent, compared to those of locked nucleic acid (LNA) modified counterparts. Herein we report the relative inhibition efficiency of HIV-1 by jcLNA modified siRNAs targeting TAR region compared to those of the LNA counterparts, in that the former were found to exhibit improved silencing efficiency and displayed enhanced stability in human serum with negligible cytotoxicity compared to those of the latter. A single jcLNA substitution as the 3'-overhang of the guide strand displayed near native-like IC50 value (of 4.01 +/- 0.87 nM compared to the nearly two-fold higher IC50 value of 7.15 +/- 1.57 nM for LNA modified counterparts, and of the native siRNA of 1.84 +/- 0.16 nM) and significantly higher hp value for the stability in serum (11.9 h for jcLNA, 6.8 h for LNA and 3.0 h for native), thereby showing that the efficiency of jcLNA-modified-siRNAs is supported by stability without compromising the native-like efficiency and target RNA recognition and subsequent down-regulation. Amongst all the modified siRNAs so far used to target HIV-1 TAR region, the best IC50 value was obtained for the doubly-modified siRNA in which jcLNA substitution was introduced both at position 1 and 20 of the antisense strand (T-1 + T-20, i.e. jcLNA11 which showed IC50 value of 0.54 +/- 0.14 nM). The IC50 of this doubly-modified siRNA was more than three-fold lower than that of the native and two-fold lower than that of LNA modified counterpart, i.e. LNA12: IC50: 1.13 +/- 0.27 nM. Hence the strategy to chemically modify the native siRNAs by substitution with the jcLNA can be considered as a significant development, leading to both enhanced siRNA efficiency and serum stability over that of the native.

  • 12.
    Femel, Julia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huijbers, Elisabeth J. M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Saupe, Falk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cedervall, Jessica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zhang, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Progression of metastatic breast cancer can be attenuated by therapeutic vaccination against the tumor vascular marker ED-A2014In: Angiogenesis, ISSN 0969-6970, E-ISSN 1573-7209, Vol. 17, no 3, p. 769-769Article in journal (Other academic)
  • 13.
    Femel, Julia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Huijbers, Elisabeth JM
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Saupe, Falk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Cedervall, Jessica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Zhang, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Roswall, Pernilla
    Larsson, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Olofsson, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Pietras, Kristian
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Therapeutic vaccination against fibronectin ED-A attenuates progression of metastatic breast cancer.2014In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 5, no 23, p. 12418-12427Article in journal (Refereed)
    Abstract [en]

    Therapeutic vaccination targeting self-molecules is an attractive alternative to monoclonal antibody-based therapies for cancer and various inflammatory diseases. However, development of cancer vaccines targeting self-molecules has proven difficult. One complicating factor is that tumor cells have developed strategies to escape recognition by the immune system. Antigens specifically expressed by the tumor vasculature can therefore provide alternative targets. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin are expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, these domains are re-expressed during tumor angiogenesis and matrix remodeling, which renders them highly interesting for targeted cancer therapies. Using the MMTV-PyMT transgenic model of metastatic mammary carcinoma, we show that tumor burden can be significantly decreased by immunization against ED-A in a therapeutic setting. Furthermore, we found that in mice carrying anti-ED-A antibodies the number of metastases was reduced. ED-A immunization increased infiltration of macrophages and compromised tumor blood vessel function. These findings implicate an attack of the tumor vasculature by the immune system, through a polyclonal antibody response. We conclude that tumor vascular antigens are promising candidates for development of therapeutic vaccines targeting growth of primary tumors as well as disseminated disease.

  • 14. Frankowiack, Marcel
    et al.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Zhao, Yaofeng
    Arnemo, Jon M.
    Lin, Miaoli
    Tengvall, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Moller, Torsten
    Lindblad-Toh, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hammarstrom, Lennart
    IgA deficiency in wolves2013In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 40, no 2, p. 180-184Article in journal (Refereed)
    Abstract [en]

    Low mean concentrations of serum immunoglobulin A (IgA) and an increased frequency of overt IgA deficiency (IgAD) in certain dog breeds raises the question whether it is a breeding-enriched phenomenon or a legacy from the dog's ancestor, the gray wolf (Canis lupus). The IgA concentration in 99 serum samples from 58 free-ranging and 13 captive Scandinavian wolves, was therefore measured by capture ELISA. The concentrations were markedly lower in the wolf serum samples than in the dog controls. Potential differences in the IgA molecule between dogs and wolves were addressed by sequencing the wolf IgA heavy chain constant region encoding gene (IGHA). Complete amino acid sequence homology was found. Detection of wolf and dog IgA was ascertained by showing identity using double immunodiffusion. We suggest that the vast majority of wolves, the ancestor of the dog, are IgA deficient.

  • 15. Frankowiack, Marcel
    et al.
    Olsson, Mia
    Cluff, H. Dean
    Evans, Alina L.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Mansson, Johan
    Arnemo, Jon M.
    Hammarstrom, Lennart
    IgA deficiency in wolves from Canada and Scandinavia2015In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 50, no 1, p. 26-28Article in journal (Refereed)
    Abstract [en]

    Immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in both humans and selected breeds of domestic dogs. In both species, IgAD is associated with recurrent infections and immune mediated diseases. Previous results imply that IgAD is also common in the wild ancestor of domestic dogs, the gray wolf. Here, we report that serum IgA concentrations are significantly different in Scandinavian and Canadian wolves (p =3.252e-15) with an increased prevalence for IgAD in Scandinavian wolves (60%), which is as high as those found in high-risk dog breeds. 

  • 16.
    Fu, Zhirong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Akula, Srinivas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 5, article id e0154886Article in journal (Refereed)
    Abstract [en]

    Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus.

  • 17.
    Fu, Zhirong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    rMCP-2, the Major Rat Mucosal Mast Cell Protease, an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 6, article id e0131720Article in journal (Refereed)
    Abstract [en]

    Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2, -3 and 4. rMCP-2, the most abundant of these enzymes, has been shown to increase the permeability of the intestinal epithelium, most likely by cleavage of cell adhesion and junction proteins and thereby play a role in intestinal parasite clearance. However, no target for this effect has yet been identified. To address this question we here present its extended cleavage specificity. Phage display analysis showed that it is a chymase with a specificity similar to the corresponding enzyme in mice, mMCP-1, with a preference for Phe or Tyr in the P1 position, and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junction molecules. To see if these proteins were also susceptible to cleavage in their native conformation we cleaved 5 different recombinant cell adhesion and cell junction proteins. Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.

  • 18. Gößringer, M.
    et al.
    Helmecke, D.
    Köhler, K.
    Schön, A.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Bindereif, A.
    Hartmann, R. K.
    Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase2014In: Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition / [ed] Roland K. Hartmann, Albrecht Bindereif, Astrid Schön, Eric Westhof, Weinheim: Wiley-Blackwell, 2014, 2, Vol. 1-2, p. 1-28Chapter in book (Other academic)
  • 19. Hagglund, Sara
    et al.
    Hu, Kefei
    Blodorn, Krister
    Makabi-Panzu, Boby
    Gaillard, Anne-Laure
    Ellencrona, Karin
    Chevret, Didier
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Bengtsson, Karin Lovgren
    Riffault, Sabine
    Taylor, Geraldine
    Valarcher, Jean Francois
    Eleouet, Jean-Francois
    Characterization of an Experimental Vaccine for Bovine Respiratory Syncytial Virus2014In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 21, no 7, p. 997-1004Article in journal (Refereed)
    Abstract [en]

    Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins alpha(V)beta(1), alpha(V)beta(3), and alpha(3)beta(1). The quantity of the major protein F was 4- to 5-fold greater than that of N (similar to 77 mu g versus similar to 17 mu g/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.

  • 20.
    Hellman, Lars
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Granule proteases of hematopoietic cells, a family of versatile inflammatory mediators - an update on their cleavage specificity, in vivo substrates, and evolution2014In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 395, no 1, p. 15-49Article, review/survey (Refereed)
    Abstract [en]

    Cells from several of the hematopoietic cell lineages including mast cells, basophils, neutrophils, cytotoxic T cells, and natural killer (NK) cells store proteases at very high levels within their cytoplasmic granules. In mast cells, these proteases can account for up to 35% of the total cellular protein, and the absolute majority of these belong to the chymotrypsin-related serine protease family. A number of very diverse functions have been identified for these proteases, including apoptosis induction, blood pressure regulation, inactivation of insect and snake toxins, intestinal parasite expulsion, killing of bacteria and fungi, induction, mobilization, or degradation of cytokines, and the degradation of connective tissue components. A very broad spectrum of primary cleavage specificities has also been observed, including chymase, tryptase, asp-ase, elastase, and met-ase specificities, which highlights the large flexibility in the active site of these proteases. Mast cells primarily express chymases and tryptases with chymotryptic or tryptic primary cleavage specificities, respectively. Neutrophils have several enzymes with chymase, elastase, and tryptase specificities. T cells and NK cells express between 5 and 14 different granzymes, depending on the species, and these enzymes have tryptase, asp-ase, chymase, and met-ase specificities. This review focuses on the appearance of these proteases during vertebrate evolution, their primary and extended cleavage specificities, and their potential in vivo substrates. The in vivo substrates and functions are a particular challenging issue because several of these enzymes have a relatively broad specificity and may therefore cleave a wide range of different substrates.

  • 21. Hu, Jiaxin
    et al.
    Gagnon, Keith T.
    Liu, Jing
    Watts, Jonathan K.
    Syeda-Nawaz, Jeja
    Bennett, C. Frank
    Swayze, Eric E.
    Randolph, John
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Corey, David R.
    Allele-selective inhibition of ataxin-3 (ATX3) expression by antisense oligomers and duplex RNAs2011In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 392, no 4, p. 315-325Article in journal (Refereed)
    Abstract [en]

    Spinocerebellar ataxia-3 (also known as Machado-Joseph disease) is an incurable neurodegenerative disorder caused by expression of a mutant variant of ataxin-3 (ATX3) protein. Inhibiting expression of ATX3 would provide a therapeutic strategy, but indiscriminant inhibition of both wild-type and mutant ATX3 might lead to undesirable side effects. An ideal silencing agent would block expression of mutant ATX3 while leaving expression of wild-type ATX3 intact. We have previously observed that peptide nucleic acid (PNA) conjugates targeting the expanded CAG repeat within ATX3 mRNA block expression of both alleles. We have now identified additional PNAs capable of inhibiting ATX3 expression that vary in length and in the nature of the conjugated cation chain. We can also achieve potent and selective inhibition using duplex RNAs containing one or more mismatches relative to the CAG repeat. Anti-CAG antisense bridged nucleic acid oligonucleotides that lack a cationic domain are potent inhibitors but are not allele-selective. Allele-selective inhibitors of ATX3 expression provide insights into the mechanism of selectivity and promising lead compounds for further development and in vivo investigation.

  • 22.
    Karimi, Mansoureh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Erfan, Sayeh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Földesi, András
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Steric Effects in the Tuning of the Diastereoselectivity of the Intramolecular Free-Radical Cyclization to an Olefin As Exemplified through the Synthesis of a Carba-Pentofuranose Scaffold2012In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 77, no 16, p. 6855-6872Article in journal (Refereed)
    Abstract [en]

    Two free-radical cyclization reactions with the radical at the chiral C4 of the pentose sugar and the intramolecularly C1-tethered olefin (on radical precursors 8 and 17) gave a new diastereospecific C4-C8 bond in dimethylbicyclo[2.2.1]heptane 9, whereas the new C4-C7 bond in 7-methyl-2-oxabicyclo[2.2.1]heptanes 18a/18b gave trans and cis diastereomers, in which the chirality of the C4 center is fully retained as that of the starting material. It has been shown how the chemical nature of the fused carba-pentofuranose scaffolds, dimethylbicyclo[2.2.1]heptane 9 vis-a-vis 7-methyl-2-oxabicyclo[2.2.1]heptanes 18a/18b (C7-Me in the former versus 2-O- in the latter), dictates the stereochemical outcome both at the Grignard reaction step as well as in the free-radical ring-closure reaction. The formation of pure 1,8-trans-bicyclo[2.2.1]heptane 9 from 8 suggests that the boat-like transition state is favored due to the absence of steric clash of the bulky 1(S)-O-p-methoxybenzyl (PMB) and 7(R)-Me substituents (both in the alpha-face) with that of the 8(R)-CH2 center dot radical in the beta-face. The conversion of 17 -> 18a-7(S) and 18b-7(R) in 6:4 ratio shows that the participation of both the chair- and the boat-like transition states is likely.

  • 23.
    Karimiahmadabadi, Mansoureh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Novel Pentofuranose Chemistry to Modulate RNA Function2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Chemical modifications of oligonucleotides provide an important tool to understand how the natural substrate works as well as how to improve their biochemical and biological properties as potential therapeutics and diagnostics. Our carba-LNA (2',4'-carba-bridged Locked Nucleic Acid) modified oligo-DNA or -RNA have been found to be useful to modulate oligo-RNA and -DNA activity. This thesis is based on four papers: Paper I (J. Org. Chem. 2010, 75, 7112-7128) deals with the synthesis of 2',4'-propylene-bridged (Carba-ENA) thymidine and its analogues. These carba-ENA nucleosides have been subsequently incorporated into 15mer antisense oligodeoxynucleotides (AON), and their affinity toward complementary mRNA and DNA, as well as their nuclease resistance and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. Paper II (J. Org. Chem. 2012, 77, 6855–6872) illustrates the synthesis of dimethylbicyclo[2.2.1]heptane and a diastereomeric mixture of oxabicyclo[2.2.1]heptanes by the free-radical ring-closure reaction approach. The role of steric factors for different chair- and the boat-like transition states was evaluated involving the 5-exo radical ring closure reaction to a tethered olefin. Paper III (J. Org. Chem. 2012, 77, 9747-9755) shows an unusual strain releasing reaction of 1-mesyloxy-8,7-dimethylbicyclo[2.2.1]heptane by a base-promoted substitution at the chiral C3 followed by spontaneous concerted ring opening involving the most strained C2-C3-C4 bonds (with bond angle 94°) and the C2 bridgehead leading to anti-endo elimination of the C1-mesyloxy group by the conjugate base of adenine or thymine to give two diastereomeric C3'(S) and C3'(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene, and a mechanistic rational has been formulated. Paper IV (J. Org. Chem. 2014, 79, 7266−7276) focuses on the diastereospecific synthesis of E/Z bicyclo[2.2.1]heptane-7- and oxabicyclo[2.2.1]heptane-8-oximes and their corresponding C-nitroso derivatives. The comparative kinetic and thermodynamic studies of the conversions of the C-nitroso side products to the required oximes have been delineated leading to the synthesis of desmethyl sugar derivatives.

    List of papers
    1. Synthesis of 2',4' -Propylene-Bridged (Carba-ENA) Thymidine and Its Analogues: The Engineering of Electrostatic and Steric Effects at the Bottom of the Minor Groove for Nuclease and Thermodynamic Stabilities and Elicitation of RNase H
    Open this publication in new window or tab >>Synthesis of 2',4' -Propylene-Bridged (Carba-ENA) Thymidine and Its Analogues: The Engineering of Electrostatic and Steric Effects at the Bottom of the Minor Groove for Nuclease and Thermodynamic Stabilities and Elicitation of RNase H
    Show others...
    2010 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 75, no 21, p. 7112-7128Article in journal (Refereed) Published
    Abstract [en]

    2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH2/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C=N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON: RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance hut 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH2 substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase HI with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-134109 (URN)10.1021/jo101207d (DOI)000283531100009 ()
    Available from: 2010-12-03 Created: 2010-11-22 Last updated: 2017-12-12Bibliographically approved
    2. Steric Effects in the Tuning of the Diastereoselectivity of the Intramolecular Free-Radical Cyclization to an Olefin As Exemplified through the Synthesis of a Carba-Pentofuranose Scaffold
    Open this publication in new window or tab >>Steric Effects in the Tuning of the Diastereoselectivity of the Intramolecular Free-Radical Cyclization to an Olefin As Exemplified through the Synthesis of a Carba-Pentofuranose Scaffold
    2012 (English)In: J. Org. Chem., Vol. 77, p. 6855-6872Article in journal (Refereed) Published
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-233861 (URN)
    Available from: 2014-10-11 Created: 2014-10-11 Last updated: 2014-11-06
    3. Unusual strain-releasing nucleophilic rearrangement of a bicyclo[2.2.1]heptane system to a cyclohexenyl derivative
    Open this publication in new window or tab >>Unusual strain-releasing nucleophilic rearrangement of a bicyclo[2.2.1]heptane system to a cyclohexenyl derivative
    2012 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 77, no 21, p. 9747-9755Article in journal (Refereed) Published
    Abstract [en]

    We report an unusual strain-releasing reaction of 1-mesyloxy-8,7- dimethylbicyclo[2.2.1]heptane (3) by a base-promoted substitution at the chiral C3 followed by spontaneous concerted ring opening involving the most strained C2-C3-C4 bonds (with bond angle 94°) and the C2 bridgehead leading to anti-endo elimination of the C1-mesyloxy group by the conjugate base of adenine or thymine to give two diastereomeric C3′(S) and C3′(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene: 3 → T-4a + T-4b and 3 → A-5a + A-5b. These products have been unambiguously characterized by detailed 1D and 2D NMR (J-coupling constants and nOe analysis), mass, and UV spectroscopy. Evidence has been presented suggesting that the origin of these diastereomeric C3′(S) and C3′(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene from 3 is most probably a rearrangement mechanism of a trigonal bipyramidal intermediate formed in the S N2 displacement-ring-opening reaction.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-184927 (URN)10.1021/jo301871d (DOI)000311190200034 ()
    Available from: 2012-11-19 Created: 2012-11-15 Last updated: 2017-12-07Bibliographically approved
    4. Distal Two-Bond versus Three-Bond Electronegative Oxo-Substituent Effect Controls the Kinetics and Thermodynamics of the Conversion of a C-Nitroso Function to the Corresponding Oxime in the Conformationally Locked Pentofuranose (Bicyclo[2.2.1]heptane) System
    Open this publication in new window or tab >>Distal Two-Bond versus Three-Bond Electronegative Oxo-Substituent Effect Controls the Kinetics and Thermodynamics of the Conversion of a C-Nitroso Function to the Corresponding Oxime in the Conformationally Locked Pentofuranose (Bicyclo[2.2.1]heptane) System
    2014 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 79, no 16, p. 7266-7276Article in journal (Refereed) Published
    Abstract [en]

    We report the high-yielding and scalable diastereospecific synthesis of isomeric bicyclo [2.2.1]heptane-7- and -8-oximes and their corresponding C-nitroso derivatives, which are the key intermediates for the synthesis of carbanucleosides. Neither the (C7-R)-nitroso- nor (C8-S)-nitrosobicycloheptane system requires any external base in DMSO-d(6) to afford the corresponding oxime, and no reverse isomerization from the oxime to the C-nitroso compound was observed. The conversion of the (C8-S)-nitroso compound to the E/Z-oximes was similar to 8 times faster (at 40 degrees C) than that of the (C7-R)-nitroso derivative. The mechanism involves first-order reaction kinetics for the conversion of either the (C7-R)- or (C8-S)-nitroso derivative to the corresponding E/Z-oximes. The lower rate of conversion of the (C7-R)-nitroso compound to the corresponding crimes compared with that of the (C8-S)-nitroso derivative is attributed to the fact that the acidic H8 ionizing center is two bonds away from the OPMB group on C1 in the latter whereas H7 is three bonds away from the C1 OMe group in the former, making the effect of the electron-withdrawing group on C1 stronger in the latter.

    National Category
    Organic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-231998 (URN)10.1021/jo500266k (DOI)000340517600002 ()
    Available from: 2014-09-15 Created: 2014-09-12 Last updated: 2017-12-05Bibliographically approved
  • 24.
    Karimiahmadabadi, Mansoureh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Erfan, Sayeh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Földesi, András
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Distal Two-Bond versus Three-Bond Electronegative Oxo-Substituent Effect Controls the Kinetics and Thermodynamics of the Conversion of a C-Nitroso Function to the Corresponding Oxime in the Conformationally Locked Pentofuranose (Bicyclo[2.2.1]heptane) System2014In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 79, no 16, p. 7266-7276Article in journal (Refereed)
    Abstract [en]

    We report the high-yielding and scalable diastereospecific synthesis of isomeric bicyclo [2.2.1]heptane-7- and -8-oximes and their corresponding C-nitroso derivatives, which are the key intermediates for the synthesis of carbanucleosides. Neither the (C7-R)-nitroso- nor (C8-S)-nitrosobicycloheptane system requires any external base in DMSO-d(6) to afford the corresponding oxime, and no reverse isomerization from the oxime to the C-nitroso compound was observed. The conversion of the (C8-S)-nitroso compound to the E/Z-oximes was similar to 8 times faster (at 40 degrees C) than that of the (C7-R)-nitroso derivative. The mechanism involves first-order reaction kinetics for the conversion of either the (C7-R)- or (C8-S)-nitroso derivative to the corresponding E/Z-oximes. The lower rate of conversion of the (C7-R)-nitroso compound to the corresponding crimes compared with that of the (C8-S)-nitroso derivative is attributed to the fact that the acidic H8 ionizing center is two bonds away from the OPMB group on C1 in the latter whereas H7 is three bonds away from the C1 OMe group in the former, making the effect of the electron-withdrawing group on C1 stronger in the latter.

  • 25.
    Karimiahmadabadi, Mansoureh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Földesi, András
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Unusual strain-releasing nucleophilic rearrangement of a bicyclo[2.2.1]heptane system to a cyclohexenyl derivative2012In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 77, no 21, p. 9747-9755Article in journal (Refereed)
    Abstract [en]

    We report an unusual strain-releasing reaction of 1-mesyloxy-8,7- dimethylbicyclo[2.2.1]heptane (3) by a base-promoted substitution at the chiral C3 followed by spontaneous concerted ring opening involving the most strained C2-C3-C4 bonds (with bond angle 94°) and the C2 bridgehead leading to anti-endo elimination of the C1-mesyloxy group by the conjugate base of adenine or thymine to give two diastereomeric C3′(S) and C3′(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene: 3 → T-4a + T-4b and 3 → A-5a + A-5b. These products have been unambiguously characterized by detailed 1D and 2D NMR (J-coupling constants and nOe analysis), mass, and UV spectroscopy. Evidence has been presented suggesting that the origin of these diastereomeric C3′(S) and C3′(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene from 3 is most probably a rearrangement mechanism of a trigonal bipyramidal intermediate formed in the S N2 displacement-ring-opening reaction.

  • 26.
    Kikovska, Ema
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Wu, Shiying
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Mao, Guanzhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Cleavage mediated by the P15 domain of bacterial RNase P RNA2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 5, p. 2224-2233Article in journal (Refereed)
    Abstract [en]

    Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg2+ at the cleavage site. Here we show that small model-RNA molecules (similar to 30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for 'RNA-mediated' cleavage of RNA.

  • 27.
    Kirsebom, Leif A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Ciesiolka, Jerzy
    Polish Academy of Sciences, Institute of Bioorganic Chemistry, Laboratory of RNA Biochemistry, Poznan, Poland.
    Pb2+-Induced Cleavage of RNA2014In: Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition / [ed] Roland K. Hartmann, Albrecht Bindereif, Astrid Schön, Eric Westhof, Wiley-Blackwell, 2014, 2, Vol. 1-2, p. 269-284Chapter in book (Refereed)
  • 28.
    Kirsebom, Leif A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Pettersson, Fredrik M. Brännvall
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Pleiomorphism in Mycobacterium2012In: Advances in Applied Microbiology, Vol 80, ELSEVIER ACADEMIC PRESS INC , 2012, p. 81-112Chapter in book (Refereed)
    Abstract [en]

    Morphological variants in mycobacterial cultures under different growth conditions, including aging of the culture, have been shown to include fibrous aggregates, biofilms, coccoids, and spores. Here we discuss the diversity in shape and size changes demonstrated by bacterial cells with special reference to pleiomorphism observed in Mycobacterium spp. in response to nutritional and other environmental stresses. Inherent asymmetry in cell division and compartmentalization of cell interior under different growth conditions might contribute toward the observed pleiomorphism in mycobacteria. The regulatory genes comprising the bacterial signaling pathway responsible for initiating morphogenesis are speculated upon from bioinformatic identifications of genes for known sensors, kinases, and phosphatases existing in mycobacterial genomes as well as on the basis of what is known in other bacteria.

  • 29. Lei, Ying
    et al.
    Boinapally, Vamsi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Zoltowska, Anna
    Adner, Mikael
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Nilsson, Gunnar
    Vaccination against IL-33 Inhibits Airway Hyperresponsiveness and Inflammation in a House Dust Mite Model of Asthma2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0133774Article in journal (Refereed)
    Abstract [en]

    In several clinical and experimental studies IL-33 and its receptor have been found to play important roles in the development of asthma and allergic airway inflammation. We evaluated the effects of vaccination against IL-33 in a mouse model of airway inflammation induced by house dust mite (HDM) allergen. Balb/c mice received the IL-33 vaccine subcutaneously, followed by intranasal administration of HDM for up to six weeks. Vaccination against IL-33 induced high titers of specific anti-IL-33 IgG antibodies that inhibited HDM-induced airway hyperresponsiveness (AHR) in the conducting airways and tissue damping. The vaccination also attenuated the HDM-induced elevation in the numbers of eosinophils in bronchoalveolar lavage fluid (BALF) and suppressed the accumulation of inflammatory cells in the airways. Furthermore, the levels of IL-17A, IL-25, IL-33 and TSLP in lung tissue homogenates were reduced by vaccination against IL-33. These observations demonstrate that vaccination against IL-33 inhibits HDM-induced development of AHR, airway inflammation and production of inflammatory cytokines. The results also indicate an important role of IL-33 in the regulation of AHR of the distal lung compartments. Thus, administration of such a vaccine is potentially an effective therapeutic tool for treating allergic asthma.

  • 30.
    Li, Qing
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Conformationally Constrained Oligonucleotides for RNA Targeting2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems.

    In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application.

    AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate.

    Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

    List of papers
    1. Carba-LNA-5MeC/A/G/T Modified Oligos Show Nucleobase-Specific Modulation of 3′-Exonuclease Activity, Thermodynamic Stability, RNA Selectivity, and RNase H Elicitation: Synthesis and Biochemistry
    Open this publication in new window or tab >>Carba-LNA-5MeC/A/G/T Modified Oligos Show Nucleobase-Specific Modulation of 3′-Exonuclease Activity, Thermodynamic Stability, RNA Selectivity, and RNase H Elicitation: Synthesis and Biochemistry
    Show others...
    2011 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 76, no 11, p. 4408-4431Article in journal (Refereed) Published
    Abstract [en]

    Using the intramolecular 5-exo-5-hexenyl radical as a key cyclization step, we previously reported an unambiguous synthesis of carba-LNA thymine (cLNA-T), which we subsequently incorporated in antisense oligonudeotides (AON) and investigated their biochemical properties [J. Am. Chem. Soc. 2007, 129 (26), 8362-8379]. These cLNA-T incorporated oligos showed specific RNA affinity of +3.5-5 degrees C/modification for AON:RNA heteroduplexes, which is comparable to what is found for those of LNAs (Locked Nucleic Acids). These modified oligos however showed significantly enhanced nuclease stability (ca. 100 times more) in the blood serum compared to those of the LNA modified counterparts without compromising any RNase H recruitment capability. We herein report the synthesis of 5-methylcytosine-1-yl (C-Me), 9-adeninyl (A), and 9-guaninyl (G) derivatives of cLNA and their oligonucleotides and report their biochemical properties as potential RNA-directed inhibitors. In a series of isosequential carba-LNA modified AONs, we herein show that all the cLNA modified AONs are found to be RNA-selective, but the magnitude of RNA-selectivity of 7'-R-Me-cLNA-G (cLNA-G) (Delta T-m = 2.9 degrees C/modification) and intractable isomeric mixtures of 7'-(S/R)-Me-cLNA-T (cLNA-T, Delta T-m = 2.2 degrees C/modification) was found to be better than diastereomeric mixtures of 7'-(S/R)-Me-cLNA-C-Me with trace of cENA-C-Me (cLNA-C-Me, Delta T-m = 1.8 degrees C/modification) and 7'-R-Me-cLNA-A (cLNA-A, Delta T-m = 0.9 degrees C/modification). cLNA-C-Me modified AONs however exhibited the best nuclease stability, which is 4-, 7-, and 20-fold better, respectively, than cLNA-T, cLNA-A, and cLNA-G modified counterparts, which in turn was more than 100 times stable than that of the native. When the modification sites are appropriately chosen in the AONs, the cLNA-A, -G, and -C-Me modified sites in the AON:RNA hybrids can be easily recognized by RNase H, and the RNA strand of the hybrid is degraded in a specific manner, which is important for the design of oligos for therapeutic purposes. The cLNA-C-Me modified AON/RNA, however, has been found to be degraded 4 times faster than cLNA-A and G modified counterparts. By appropriately choosing the carba-LNA modification sites in AON strands, the digestion of AON:RNA can be either totally repressed or be limited to cleavage at specific sites or at a single site only (similar to that of catalytic RNAzyme or DNAzyme). Considering all physico- and biochemical aspects of cLNA modified oligos, the work suggests that the cLNA modified antisense oligos have the potential of being a promising therapeutic candidate due to their (i) higher nucleobase-specific RNA affinity and RNA selectivity, (ii) greatly improved nuclease stability, and (iii) efficient RNase H recruitment capability, which can induce target RNA cleavage in a very specific manner at multiple or at a single site, in a designed manner.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-155235 (URN)10.1021/jo200073q (DOI)000291128300009 ()
    Available from: 2011-06-21 Created: 2011-06-20 Last updated: 2017-12-11Bibliographically approved
    2. Free-Radical Ring Closure to Conformationally Locked α-l-Carba-LNAs and Synthesis of Their Oligos:: Nuclease Stability, Target RNA Specificity, and Elicitation of RNase H
    Open this publication in new window or tab >>Free-Radical Ring Closure to Conformationally Locked α-l-Carba-LNAs and Synthesis of Their Oligos:: Nuclease Stability, Target RNA Specificity, and Elicitation of RNase H
    Show others...
    2010 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 75, no 18, p. 6122-6140Article in journal (Refereed) Published
    Abstract [en]

    A new class of conformationally constrained nucleosides, α-L-ribo-carbocyclic LNA thymidine (α-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" α-L-ribo-configured tetracyclic thymidine (6,7'-methylene-bridged-α-L-carba-LNA-T) in which both the sugar puckering and glycosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These α-L-carba-LNA analogues have been subsequently transformed to corresponding phosphoramidites and incorporated into isosequential antisense oligonucleotides (AONs), which have then been examined for the thermal denaturation of their duplexes, nuclease stability, and RNase H recruitment capabilities. Introduction of a single 6',7'-substituted α-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T(m) reduction by 2-3 °C as compared to the native heteroduplex, whereas the parent 2'-oxa-α-L-LNA-T modification at the identical position in the AON strand has been found to lead to an increase in the T(m) by 3-5 °C. This suggests that the 6' and 7' substitutions lead to much reduced thermal stability for the modified heteroduplex, especially the hydrophobic 7'-methyl on α-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted α-L-carba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-α-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted α-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the β-D-LNA-T or β-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNase H recruitment capabilities elevate the α-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.

    Place, publisher, year, edition, pages
    U. S. A: American Chemical Society (ACS), 2010
    Keyword
    conformationally constrained nucleoside, free-radical intramolecular cyclization, antisense oligonucleotide, RNA target therapeutic
    National Category
    Organic Chemistry
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:uu:diva-176562 (URN)10.1021/jo100900v (DOI)
    Available from: 2012-06-20 Created: 2012-06-20 Last updated: 2017-12-07Bibliographically approved
    3. The Physicochemical Properties of DNA-RNA Duplexes Containing Pure 7′R-Me- or 7′S-Me-Carba-LNA Derivatives of A, G, 5-MeC or T in the DNA Strand: Diastereomer Specific Comparison of The 3′-Exonuclease Stability and RNase H Elicitation
    Open this publication in new window or tab >>The Physicochemical Properties of DNA-RNA Duplexes Containing Pure 7′R-Me- or 7′S-Me-Carba-LNA Derivatives of A, G, 5-MeC or T in the DNA Strand: Diastereomer Specific Comparison of The 3′-Exonuclease Stability and RNase H Elicitation
    Show others...
    (English)In: Article in journal (Refereed) Submitted
    Abstract [en]

       Recently, the intramolecular 5-exo-5-hexenyl free-radical cyclization gave access to 2′, 4′-locked carba-LNAs with different nucleobase moieties, i.e. 7′S- and 7′R-Me-cLNA-A, -G, -MeC and -T nucleosides (J. Am. Chem. Soc. 2007, 129, 8362-8379; J. Org. Chem. 2011, 76, 4408-4431). In these studies, diastereomeric mixtures of 7′S/R-Me-cLNA-MeC and -T and diastereomerically pure 7′R-Me-cLNA-A and -G have been incorporated into antisense oligonucleotides (AONs) for biological evaluations. These cLNA modified oligos have shown to have comparable RNA affinity and highly improved nuclease and blood serum stabilities relative to that of their LNA modified counterparts. In order to fully understand the spatial effect of diastereomeric orientation of 7′-methyl group in cLNA-A/G/MeC/T on the RNA affinity, nuclease stability and RNase H elicitation efficiency, we have synthesized and preparatively HPLC separated and tested each of the 7′S- (minor) and 7′R- (major) pure diastereomer of 7′S/R-Me-cLNA-A/G/MeC/T nucleosides. Incorporation into oligos of each pure diastereomer of cLNA led to higher RNA affinity (1-4°C/mod).  Tm increase was found to be dependent both on the modification site in the AON as well as whether it is 7′S or 7′R modified cLNA is incorporated. RNA selectivity (DDTm) was found to be in the range of 3.1-6.7°C compared to DDTm of 2.7°C for the native counterpart. The Tm variations modulated by 7′S- and 7′R-Me-cLNAs in the AON have been found to be sequence and position-dependent. Molecular dynamics (MD) simulations of DNA-RNA duplexes with AON2 and AON5, with pure diastereomer incorporated at the 7th position of the AON strand from 3′-end, revealed that both 7′S- and 7′R-Me-cLNA-A modifications have only small local effect on stacking and hydrogen bonding within the duplexes, with Watson-Crick base-pairing remained intact during 98-100% duration of the MD simulations. It has been however found that the Tm of each of the modified heteroduplex is dictated by the individual solvation energy (CPCM) of the 7′S- or 7′R-Me-cLNA diastereomer of A, G, MeC or T nucleobase. This demonstrates that the major factor behind variation in the thermal stabilities of the 7′R- or 7′S-Me-cLNA modified AON-RNA duplexes lies in the intrinsic hydrophobicity, hence its relative solvation energy, inherent in the 7′R- vis-a-vis 7′S-Me-cLNA modified monomer blocks, compared to those of the native and LNA counterparts. We have also found that AONs containing 7′S- and 7′R-Me-cLNA-MeC modifications exhibited unprecedented nuclease stabilities: the most stable AON is the one that contains 7′S-Me-cLNA-MeC, which is ~40 times more stable towards 3′-exonuclease (SVPDE) than 7′S- and 7′R-Me-cLNA-T modified AONs, which was in turn much more stable than 7′S- and 7′R-Me-cLNA-A and G modified counterparts. It is noteworthy that 7′S-methyl group of cLNAs endows the AON strand with more nuclease stability than that of 7′R configured counterpart when compared within the same nucleobase. Thus the carba-LNA modified AONs show nucleobase-dependent activity in the following order: MeC > T > A > G, regardless of 7′S- or 7′R-configurations in the carba-LNA. All of the cLNA and LNA modified AON/RNA hybrids can elicit RNase H activity with similar or even more enhanced rates of digestion by E. coli RNase H1 compared to that of the native AON/RNA. The cleavage rates and patterns of modified AON/RNA hybrids by E. coli RNase H1 are only correlated with the modification site in AON sequence of AON/RNA hybrids, but irrelevant to the structural features of incorporated modifications.

    National Category
    Organic Chemistry
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:uu:diva-179066 (URN)
    Available from: 2012-08-07 Created: 2012-08-07 Last updated: 2015-05-19Bibliographically approved
  • 31.
    Li, Qing
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Upadhayaya, RamShankar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Deshpande, Sachin Gangadhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Földesi, Andras
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    The Physicochemical Properties of DNA-RNA Duplexes Containing Pure 7′R-Me- or 7′S-Me-Carba-LNA Derivatives of A, G, 5-MeC or T in the DNA Strand: Diastereomer Specific Comparison of The 3′-Exonuclease Stability and RNase H ElicitationIn: Article in journal (Refereed)
    Abstract [en]

       Recently, the intramolecular 5-exo-5-hexenyl free-radical cyclization gave access to 2′, 4′-locked carba-LNAs with different nucleobase moieties, i.e. 7′S- and 7′R-Me-cLNA-A, -G, -MeC and -T nucleosides (J. Am. Chem. Soc. 2007, 129, 8362-8379; J. Org. Chem. 2011, 76, 4408-4431). In these studies, diastereomeric mixtures of 7′S/R-Me-cLNA-MeC and -T and diastereomerically pure 7′R-Me-cLNA-A and -G have been incorporated into antisense oligonucleotides (AONs) for biological evaluations. These cLNA modified oligos have shown to have comparable RNA affinity and highly improved nuclease and blood serum stabilities relative to that of their LNA modified counterparts. In order to fully understand the spatial effect of diastereomeric orientation of 7′-methyl group in cLNA-A/G/MeC/T on the RNA affinity, nuclease stability and RNase H elicitation efficiency, we have synthesized and preparatively HPLC separated and tested each of the 7′S- (minor) and 7′R- (major) pure diastereomer of 7′S/R-Me-cLNA-A/G/MeC/T nucleosides. Incorporation into oligos of each pure diastereomer of cLNA led to higher RNA affinity (1-4°C/mod).  Tm increase was found to be dependent both on the modification site in the AON as well as whether it is 7′S or 7′R modified cLNA is incorporated. RNA selectivity (DDTm) was found to be in the range of 3.1-6.7°C compared to DDTm of 2.7°C for the native counterpart. The Tm variations modulated by 7′S- and 7′R-Me-cLNAs in the AON have been found to be sequence and position-dependent. Molecular dynamics (MD) simulations of DNA-RNA duplexes with AON2 and AON5, with pure diastereomer incorporated at the 7th position of the AON strand from 3′-end, revealed that both 7′S- and 7′R-Me-cLNA-A modifications have only small local effect on stacking and hydrogen bonding within the duplexes, with Watson-Crick base-pairing remained intact during 98-100% duration of the MD simulations. It has been however found that the Tm of each of the modified heteroduplex is dictated by the individual solvation energy (CPCM) of the 7′S- or 7′R-Me-cLNA diastereomer of A, G, MeC or T nucleobase. This demonstrates that the major factor behind variation in the thermal stabilities of the 7′R- or 7′S-Me-cLNA modified AON-RNA duplexes lies in the intrinsic hydrophobicity, hence its relative solvation energy, inherent in the 7′R- vis-a-vis 7′S-Me-cLNA modified monomer blocks, compared to those of the native and LNA counterparts. We have also found that AONs containing 7′S- and 7′R-Me-cLNA-MeC modifications exhibited unprecedented nuclease stabilities: the most stable AON is the one that contains 7′S-Me-cLNA-MeC, which is ~40 times more stable towards 3′-exonuclease (SVPDE) than 7′S- and 7′R-Me-cLNA-T modified AONs, which was in turn much more stable than 7′S- and 7′R-Me-cLNA-A and G modified counterparts. It is noteworthy that 7′S-methyl group of cLNAs endows the AON strand with more nuclease stability than that of 7′R configured counterpart when compared within the same nucleobase. Thus the carba-LNA modified AONs show nucleobase-dependent activity in the following order: MeC > T > A > G, regardless of 7′S- or 7′R-configurations in the carba-LNA. All of the cLNA and LNA modified AON/RNA hybrids can elicit RNase H activity with similar or even more enhanced rates of digestion by E. coli RNase H1 compared to that of the native AON/RNA. The cleavage rates and patterns of modified AON/RNA hybrids by E. coli RNase H1 are only correlated with the modification site in AON sequence of AON/RNA hybrids, but irrelevant to the structural features of incorporated modifications.

  • 32.
    Magnusson, Sofia E
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wennerberg, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Matt, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology.
    Lindqvist, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Dysregulated Fc receptor function in active rheumatoid arthritis2014In: Immunology Letters, ISSN 0165-2478, E-ISSN 1879-0542, Vol. 162, no 1 Pt A, p. 200-206Article in journal (Refereed)
    Abstract [en]

    Given the critical role of Fc gamma receptors (FcgammaR) as primary targets for autoantibody-mediated effects an important issue is how the FcgammaR pathway is affected in autoimmune disorders. Here we investigated the FcgammaR function in monocytes from rheumatoid arthritis (RA) patients in relation to immunoglobulin levels and disease activity. Peripheral blood was obtained from 30 RA patients with clinical acute joint synovitis (active RA), 28 RA patients with no clinical signs of acute joint synovitis (non-active RA) and 34 healthy controls. Prior the functional studies the monocytes were characterized of their FcgammaRI (CD64), II (CD32), IIb (CD32b) and III (CD16) expression as well as their cell surface bound IgG. The monocytic FcgammaR function was assessed by binding of human IgG1 and IgG3 immune complexes (IC) and TNF secretion in vitro. IgG anti-citrullinated peptide antibodies (ACPA) were analyzed in the plasma. We found that monocytes from active RA patients had increased levels of FcgammaRI, II and cell surface IgG concurrently with impaired FcgammaR function. This was evident by reduced IgG1-IC binding and decreased TNF secretion in response to IgG3-IC. In contrast, monocytes from non-active RA patients displayed a normal FcgammaR function and had increased FcgammaRIIb expression together with elevated FcgammaRI, II and cell surface IgG. The ACPA levels did not differ in active and non-active RA patients but correlated with the monocytic FcgammaRIII expression in the patients. In conclusion, active RA patients display a dysregulated FcgammaR function that may represent a novel phenotypic and likely pathogenetic marker for active RA. A disease and FcgammaR function controlling effect is suggested by the increased inhibitory FcgammaRIIb in non-active RA.

  • 33.
    Mao, Guanzhong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chen, Tien-Hao
    Ohio State Univ, Dept Chem & Biochem, Ctr RNA Biol, Columbus, OH 43210 USA..
    Srivastava, Abhishek S.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology. AstraZeneca R&D, Discovery Sci, Cambridge Sci Pk, Cambridge CB4 0WG, England..
    Kosek, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Biswas, Pradip K.
    Ohio State Univ, Dept Chem & Biochem, Ctr RNA Biol, Columbus, OH 43210 USA..
    Gopalan, Venkat
    Ohio State Univ, Dept Chem & Biochem, Ctr RNA Biol, Columbus, OH 43210 USA..
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 8, article id e0160246Article in journal (Refereed)
    Abstract [en]

    Two broad classes of RNase P trim the 5' leader of precursor tRNAs (pre-tRNAs): ribonucleoprotein (RNP)- and proteinaceous (PRORP)-variants. These two RNase P types, which use different scaffolds for catalysis, reflect independent evolutionary paths. While the catalytic RNA-based RNP form is present in all three domains of life, the PRORP family is restricted to eukaryotes. To obtain insights on substrate recognition by PRORPs, we examined the 5' processing ability of recombinant Arabidopsis thaliana PRORP1 (AtPRORP1) using a panel of pre-tRNA(Ser) variants and model hairpin-loop derivatives (pATSer type) that consist of the acceptor-T-stem stack and the T-/D-loop. Our data indicate the importance of the identity of N-1 (the residue immediately 5' to the cleavage site) and the N-1: N+73 base pair for cleavage rate and site selection of pre-tRNA(Ser) and pATSer. The nucleobase preferences that we observed mirror the frequency of occurrence in the complete suite of organellar pre-tRNAs in eight algae/plants that we analyzed. The importance of the T-/D-loop in pre-tRNA(Ser) for tight binding to AtPRORP1 is indicated by the 200-fold weaker binding of pATSer compared to pre-tRNA(Ser), while the essentiality of the T-loop for cleavage is reflected by the near-complete loss of activity when a GAAA-tetraloop replaced the T-loop in pATSer. Substituting the 2'-OH at N-1 with 2'-H also resulted in no detectable cleavage, hinting at the possible role of this 2'-OH in coordinating Mg2+ ions critical for catalysis. Collectively, our results indicate similarities but also key differences in substrate recognition by the bacterial RNase P RNP and AtPRORP1: while both forms exploit the acceptor-T-stem stack and the elbow region in the pre-tRNA, the RNP form appears to require more recognition determinants for cleavage-site selection.

  • 34. Martinez-Montero, Saul
    et al.
    Fernandez, Susana
    Sanghvi, Yogesh S.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Ganesan, Muthupandian
    Ramesh, Namakkal G.
    Gotor, Vicente
    Ferrero, Miguel
    Design and Divergent Synthesis of Aza Nucleosides from a Chiral Imino Sugar2012In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 77, no 10, p. 4671-4678Article in journal (Refereed)
    Abstract [en]

    Several novel nucleoside analogues as potential inhibitors of glycosidases and purine nucleoside phosphorylase (PNP) have been synthesized via selective coupling of an appropriate nucleobase at different positions of an orthogonally protected imino sugar as a common precursor. This synthetic strategy offers a straightforward protocol for the assembly of imino sugar containing nucleosides, establishing a new repertoire of molecules as potential therapeutics.

  • 35.
    Matt, Peter
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Lindqvist, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology.
    FC Gamma Receptors in Active Psoriatic Arthritis (PSA)2012In: Clinical and Experimental Rheumatology, ISSN 0392-856X, E-ISSN 1593-098X, Vol. 30, no 4, p. 634-634Article in journal (Other academic)
  • 36.
    Matt, Peter
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Lindqvist, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Elevated Membrane and Soluble CD64: A Novel Marker Reflecting Altered Fc gamma R Function and Disease in Early Rheumatoid Arthritis That Can Be Regulated by Anti-Rheumatic Treatment2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 9, article id e0137474Article in journal (Refereed)
    Abstract [en]

    Objectives Fc receptors (FcR) interacting with immune complexes (ICs) is a central event in the immune pathogenesis of rheumatoid arthritis (RA). Here we asked if a specific FcR is linked to RA pathogenesis and if FcR activities relate to disease and treatment outcome in early RA. Material and Methods Twenty autoantibody-positive RA patients and 33 HC were included. The patients were evaluated before and after treatment with methotrexate and prednisolone. At follow-up, the EULAR response criteria were applied to determine the individual treatment outcomes. Serum immunoglobulin levels were measured and the expression of FcR for IgG (Fc gamma R) and IgA (Fc alpha R) on peripheral blood monocytes were determined by flow cytometry. The monocytic Fc gamma R function was evaluated by human IgG1 and IgG3 IC-binding and TNF alpha stimulated release. Plasma levels of soluble FcRs (sFcRs) were determined with ELISA. Results The IgG1 and IgG3 levels were elevated in the RA sera. The RA monocytes expressed more CD64 and cell surface-bound IgG than HC monocytes, and showed an impaired Fc gamma R function as reflected by changes in IC-binding and decreased IC-stimulated TNF alpha secretion. These findings correlated significantly with different disease activity markers. Furthermore, sFcRs were elevated in the patient plasma, and sCD64 was specific for RA (compared with a reference group of patients with active psoriatic arthritis). Following treatment, immunoglobulins and sFcR levels were reduced, whereas membrane CD64 was only decreased in patients with good response to treatment. Conclusions Early RA patients display increased membrane and soluble CD64 and an impaired Fc gamma R function correlating with joint disease activity. Beneficial responses of anti-rheumatic treatment in patients reduce CD64. These data suggest sCD64 as an important objective biomarker in RA.

  • 37.
    Matt, Peter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology.
    Lindqvist, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Up-regulation of CD64-expressing monocytes with impaired Fc gamma R function reflects disease activity in polyarticular psoriatic arthritis2015In: Scandinavian Journal of Rheumatology, ISSN 0300-9742, E-ISSN 1502-7732, Vol. 44, no 6, p. 464-473Article in journal (Refereed)
    Abstract [en]

    Objectives: The aim of this study was to assess monocyte Fc receptor (FcR) status and function in patients with active psoriatic arthritis (PsA) in relation to healthy controls (HC) and to disease activity.Method:The study population comprised 23 patients with active polyarticular PsA and 33 age- and gender-matched HC. Immunoglobulin (Ig) levels, inflammatory laboratory parameters, patient-reported outcomes of joint disease activity, skin scoring (Psoriasis Area and Severity Index, PASI), and joint status were determined in the patients. Monocytes were analysed for the expression of FcRs for IgG (FcR) class I (CD64), IIa (CD32a), IIb (CD32b), and III (CD16), the FcR for IgA (FcR) (CD89), and surface-bound IgG. The monocytic FcR function was assessed by evaluating IgG immune complex (IC) binding and tumour necrosis factor (TNF)- production following IgG-IC stimulation. The monocytes were further subdivided and analysed according to their CD14 and CD16 expression.Results:The PsA patients presented elevated serum levels of IgG1, 2, and 3 and increased numbers of CD64(+) monocytes. Furthermore, the PsA monocytes exhibited increased cell-bound IgG, and the FcR function was affected in terms of reduced IgG-IC-mediated TNF- release. These findings correlated significantly with different markers of joint disease activity. PsA was also accompanied by an increase in the CD16 low-expressing monocyte subset.Conclusions:An intensified humoral immune response affects monocytes and their FcR status in active polyarticular PsA. The up-regulated CD64(+) monocytes seem to be have an important role in psoriatic joint inflammation. These cells may prove to be a useful target in future PsA therapeutic interventions.

  • 38. Moriou, Celine
    et al.
    Denhez, Clement
    Plashkeyych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Coantic-Castex, Stephanie
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Guillaume, Dominique
    Clivio, Pascale
    A Minute Amount of S-Puckered Sugars Is Sufficient for (6-4) Photoproduct Formation at the Dinucleotide Level2015In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 80, no 1, p. 615-619Article in journal (Refereed)
    Abstract [en]

    The di-2'-alpha-fluoro analogue of thymidylyl(3',5')thymidine, synthesized to probe the effect of a minimum amount of S conformer on the photoreactivity of dinucleotides, is endowed with only 3% and 8% of S sugar conformation at its 5'- and 3'-end, respectively. This analogue gives rise to the (6-4) photoproduct as efficiently as the dithymine dinucleotide (74% and 66% at the 5'- and 3'-end, respectively) under 254 nm. Our results suggest that the 5'-N, 3'-S conformer gives rise to the (6-4) photoproduct.

  • 39. Olaru, Florina
    et al.
    Wang, Xu-Ping
    Luo, Wentian
    Ge, Linna
    Miner, Jeffrey H.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Geiger, Xochiquetzal J.
    Wasiluk, Andrew
    Heidet, Laurence
    Kitching, A. Richard
    Borza, Dorin-Bogdan
    Proteolysis Breaks Tolerance toward Intact alpha 345(IV) Collagen, Eliciting Novel Anti-Glomerular Basement Membrane Autoantibodies Specific for alpha 345NC1 Hexamers2013In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 190, no 4, p. 1424-1432Article in journal (Refereed)
    Abstract [en]

    Goodpasture disease is an autoimmune kidney disease mediated by autoantibodies against noncollagenous domain 1 (NC1) monomers of alpha 3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis (GN). We identified a novel type of human IgG4-restricted anti-GBM autoantibodies associated with mild nonprogressive GN, which specifically targeted alpha 345NC1 hexamers but not alpha 3NC1 monomers. The mechanisms eliciting these anti-GBM autoantibodies were investigated in mouse models recapitulating this phenotype. Wild-type and Fc gamma RIIB-/- mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoantibodies specific for alpha 345NC1 hexamers, which bound to the GBM in vivo but did not cause GN. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3(-/-) Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG Abs specific for alpha 345NC1 hexamers, which were not subclass restricted. As heterologous Ag in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a, and IgG2b autoantibodies specific for alpha 345NC1 hexamers and induced anti-GBM Ab GN. These findings indicate that tolerance toward autologous intact alpha 345(IV) collagen is established in hosts expressing this Ag, even though autoreactive B cells specific for alpha 345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic alpha 345NC1 hexamers. This provides a mechanism eliciting autoantibodies specific for alpha 345NC1 hexamers, which are restricted to noninflammatory IgG subclasses and are nonnephritogenic. In Alport syndrome, lack of tolerance toward alpha 345(IV) collagen promotes production of alloantibodies to alpha 345NC1 hexamers, including proinflammatory IgG subclasses that mediate posttransplant anti-GBM nephritis.

  • 40.
    Palm, Anna-Karin E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Function and Regulation of B-cell Subsets in Experimental Autoimmune Arthritis2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    B lymphocytes play a significant role in autoimmune arthritis, with their function stretching beyond autoantibody production to cytokine secretion and presentation of autoantigen. However, the involvement and activation of different B-cell subset in the autoimmune response is not fully clear. The main focus of this thesis has been to understand the contribution of marginal zone (MZ) B cells in the induction of collagen-induced arthritis (CIA), a mouse model for rheumatoid arthritis (RA).

    We show that MZ B cells in the spleen of naïve mice display a natural self-reactivity to collagen type II (CII), the autoantigen used for immunization of CIA. The CII-reactive MZ B cells expand rapidly following immunization with CII, and produce IgM and IgG antibodies to CII. They also very efficiently present CII to cognate T cells in vitro and in vivo. Moreover, absence of regulatory receptors such as CR1/2 or FcγRIIb on the MZ B cells increases their proliferation and cytokine production in response to toll-like receptor, but not B-cell receptor, activation. Further, FcγRIIb-deficient MZ B cells present CII to T cells more efficiently than wild-type MZ B cells. We additionally demonstrate for the first time the existence of a small population of nodal MZ B cells in mouse lymph nodes. Similar to splenic MZ B cells, the nodal MZ B cells expand after CIA induction, secrete IgM anti-CII antibodies and can present CII to cognate T cells. Finally, we show that mast cells, associated with ectopic B cell follicles in inflamed RA joints, in coculture with B cells promote their expansion, production of IgM and IgG antibodies as well as upregulation of CD19 and L-selectin. Coculture with mast cells further causes the B cells to upregulate costimulators and class II MHC, important molecules for antigen-presenting function.

    In summary, my findings suggest that splenic and nodal self-reactive MZ B cells participate in breaking T-cell tolerance to CII in CIA. B-cell intrinsic regulation is needed to keep such autoreactive B cells quiescent. Mast cells can potentiate B-cell responses locally in the arthritic joint, thus feeding the autoimmune reaction.

    List of papers
    1. Function and regulation of self-reactive marginal zone B cells in autoimmune arthritis
    Open this publication in new window or tab >>Function and regulation of self-reactive marginal zone B cells in autoimmune arthritis
    Show others...
    2015 (English)In: Cellular & Molecular Immunology, ISSN 1672-7681, E-ISSN 2042-0226, Vol. 12, no 4, p. 493-504Article in journal (Refereed) Published
    Abstract [en]

    Polyreactive innate-type B cells account for many B cells expressing self-reactivity in the periphery. Improper regulation of these B cells may be an important factor that underlies autoimmune disease. Here we have explored the influence of self-reactive innate B cells in the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis. We show that splenic marginal zone (MZ), but not B-1 B cells exhibit spontaneous IgM reactivity to autologous collagen II in naive mice. Upon immunization with heterologous collagen II in complete Freund's adjuvant the collagen-reactiveMZ B cells expanded rapidly, while the B-1 B cells showed a modest anti-collagen response. The MZ B cells were easily activated by toll-like receptor (TLR) 4 and 9-ligands in vitro, inducing proliferation and cytokine secretion, implying that dual engagement of the B-cell receptor and TLRs may promote the immune response to self-antigen. Furthermore, collagen-primed MZ B cells showed significant antigen-presenting capacity as reflected by cognate T-cell proliferation in vitro and induction of IgG anti-collagen antibodies in vivo. MZ B cells that were deficient in complement receptors 1 and 2 demonstrated increased proliferation and cytokine production, while Fc gamma receptor IIb deficiency of the cells lead to increased cytokine production and antigen presentation. In conclusion, our data highlight self-reactive MZ B cells as initiators of the autoimmune response in CIA, where complement and Fc receptors are relevant in controlling the self-reactivity in the cells.

    Keyword
    arthritis, complement receptors, Fc receptors, marginal zone B cells, mice
    National Category
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-264328 (URN)10.1038/cmi.2015.37 (DOI)000360939000012 ()25958842 (PubMedID)
    Funder
    Swedish Research CouncilSwedish Rheumatism Association
    Available from: 2015-10-15 Created: 2015-10-09 Last updated: 2017-12-01Bibliographically approved
    2. Nodal marginal zone B cells in mice: a novel subset with dormant self-reactivity
    Open this publication in new window or tab >>Nodal marginal zone B cells in mice: a novel subset with dormant self-reactivity
    2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 27687Article in journal (Refereed) Published
    Abstract [en]

    Marginal zone (MZ) B cells, representing a distinct subset of innate-like B cells, mount rapid T-independent responses to blood-borne antigens. They express low-affinity polyreactive antigen receptors that recognize both foreign and self-structures. The spleen is considered the exclusive site for murine MZ B cells. However, we have here identified B cells with a MZ B-cell phenotype in the subcapsular sinuses of mouse lymph nodes. The nodal MZ (nMZ) B cells display high levels of IgM, costimulators and TLRs, and are represented by naive and memory cells. The frequency of nMZ B cells is about 1-6% of nodal B cells depending on mouse strain, with higher numbers in older mice and a trend of increased numbers in females. There is a significant expansion of nMZ B cells following immunization with an autoantigen, but not after likewise immunization with a control protein or with the adjuvant alone. The nMZ B cells secrete autoantibodies upon activation and can efficiently present autoantigen to cognate T cells in vitro, inducing T-cell proliferation. The existence of self-reactive MZ B cells in lymph nodes may be a source of autoantigen-presenting cells that in an unfortunate environment may activate T cells leading to autoimmunity.

    National Category
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-265022 (URN)10.1038/srep27687 (DOI)000377368700001 ()27277419 (PubMedID)
    Funder
    Swedish Rheumatism Association
    Available from: 2015-10-20 Created: 2015-10-20 Last updated: 2017-12-01Bibliographically approved
    3. Activated mast cells promote a pro-inflammatory phenotype of B cells
    Open this publication in new window or tab >>Activated mast cells promote a pro-inflammatory phenotype of B cells
    Show others...
    (English)Article in journal (Refereed) Submitted
    National Category
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-265023 (URN)
    Available from: 2015-10-20 Created: 2015-10-20 Last updated: 2016-01-13
  • 41.
    Palm, Anna-Karin E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Friedrich, H. C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Identification of self-reactive marginal-zone B cell like cells in lymph nodes of mice2016In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 46, p. 931-931Article in journal (Other academic)
  • 42.
    Palm, Anna-Karin E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Friedrich, Heike C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Nodal marginal zone B cells in mice: a novel subset with dormant self-reactivity2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 27687Article in journal (Refereed)
    Abstract [en]

    Marginal zone (MZ) B cells, representing a distinct subset of innate-like B cells, mount rapid T-independent responses to blood-borne antigens. They express low-affinity polyreactive antigen receptors that recognize both foreign and self-structures. The spleen is considered the exclusive site for murine MZ B cells. However, we have here identified B cells with a MZ B-cell phenotype in the subcapsular sinuses of mouse lymph nodes. The nodal MZ (nMZ) B cells display high levels of IgM, costimulators and TLRs, and are represented by naive and memory cells. The frequency of nMZ B cells is about 1-6% of nodal B cells depending on mouse strain, with higher numbers in older mice and a trend of increased numbers in females. There is a significant expansion of nMZ B cells following immunization with an autoantigen, but not after likewise immunization with a control protein or with the adjuvant alone. The nMZ B cells secrete autoantibodies upon activation and can efficiently present autoantigen to cognate T cells in vitro, inducing T-cell proliferation. The existence of self-reactive MZ B cells in lymph nodes may be a source of autoantigen-presenting cells that in an unfortunate environment may activate T cells leading to autoimmunity.

  • 43.
    Palm, Anna-Karin E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Friedrich, Heike C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Mezger, Anja
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Myers, Linda K.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Identification of a Population of Lymph Node Marginal Zone B Cells that Expand in Mice Developing Autoimmune Arthritis2014In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 80, no 3, p. 230-230Article in journal (Other academic)
  • 44.
    Palm, Anna-Karin E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Friedrich, Heike C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mezger, Anja
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Salomonsson, Maya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Myers, Linda K.
    Univ Tennessee, Hlth Sci Ctr, Dept Pediat, Memphis, TN USA..
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Function and regulation of self-reactive marginal zone B cells in autoimmune arthritis2015In: Cellular & Molecular Immunology, ISSN 1672-7681, E-ISSN 2042-0226, Vol. 12, no 4, p. 493-504Article in journal (Refereed)
    Abstract [en]

    Polyreactive innate-type B cells account for many B cells expressing self-reactivity in the periphery. Improper regulation of these B cells may be an important factor that underlies autoimmune disease. Here we have explored the influence of self-reactive innate B cells in the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis. We show that splenic marginal zone (MZ), but not B-1 B cells exhibit spontaneous IgM reactivity to autologous collagen II in naive mice. Upon immunization with heterologous collagen II in complete Freund's adjuvant the collagen-reactiveMZ B cells expanded rapidly, while the B-1 B cells showed a modest anti-collagen response. The MZ B cells were easily activated by toll-like receptor (TLR) 4 and 9-ligands in vitro, inducing proliferation and cytokine secretion, implying that dual engagement of the B-cell receptor and TLRs may promote the immune response to self-antigen. Furthermore, collagen-primed MZ B cells showed significant antigen-presenting capacity as reflected by cognate T-cell proliferation in vitro and induction of IgG anti-collagen antibodies in vivo. MZ B cells that were deficient in complement receptors 1 and 2 demonstrated increased proliferation and cytokine production, while Fc gamma receptor IIb deficiency of the cells lead to increased cytokine production and antigen presentation. In conclusion, our data highlight self-reactive MZ B cells as initiators of the autoimmune response in CIA, where complement and Fc receptors are relevant in controlling the self-reactivity in the cells.

  • 45.
    Palm, Anna-Karin E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Garcia Faroldi, Gianni
    Sveriges lantbruksuniversitet, Institutionen för anatomi, fysiologi och biokemi.
    Lundberg, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Sveriges lantbruksuniversitet, Institutionen för anatomi, fysiologi och biokemi.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Activated mast cells promote a pro-inflammatory phenotype of B cellsArticle in journal (Refereed)
  • 46.
    Palm, Anna-Karin E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Garcia-Faroldi, Gianni
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden..
    Lundberg, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden..
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Activated mast cells promote differentiation of B cells into effector cells2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 20531Article in journal (Refereed)
    Abstract [en]

    Based on the known accumulation of mast cells (MCs) in B cell-dependent inflammatory diseases, including rheumatoid arthritis, we hypothesized that MCs directly modulate B cells. We show here that degranulated, and to a lesser extent naive or IgE-sensitized, MCs activate both naive and B cell receptor-activated B cells. This was shown by increased proliferation, blast formation, and expression of CD19, MHC class II and CD86 in the B cells. Further, MCs stimulated the secretion of IgM and IgG in IgM(+) B cells, indicating that MCs can induce class-switch recombination in B cells. We also show that coculture of MCs with B cells promotes surface expression of L-selectin, a homing receptor, on the B cells. The effects of MCs on B cells were partly dependent on cell-cell contact and both follicular and marginal zone B cells could be activated by MCs. Our findings suggest that degranulated MCs support optimal activation of B cells, a finding that is in line with in vivo studies showing that MCs frequently degranulate in the context of B-cell driven pathologies such as arthritis. Together, our findings show that MCs have the capacity to differentiate B cells to effector cells.

  • 47.
    Palm, Anna-Karin E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Marginal zone B cells in autoimmune arthritis2012In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 137, p. 336-336Article in journal (Other academic)
  • 48.
    Palm, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Friedrich, Heike
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Myers, Linda
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Evidence of Marginal Zone B Cells in the Lymph Nodes Presenting Autoantigen to T Cells in a Model of Autoimmune Arthritis2014In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 79, no 6, p. 455-455Article in journal (Other academic)
  • 49.
    Palm, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Expansion of a marginal zone B cell-like population in lymph nodes of mice developing collagen-induced arthritis2013In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 190, p. P4042-Article in journal (Other academic)
  • 50. Park, Jeoung-Eun
    et al.
    Cullins, David
    Zalduondo, Lillian
    Barnett, Stacey L.
    Yi, Ae-Kyung
    Kleinau, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Stuart, John M.
    Kang, Andrew H.
    Myers, Linda K.
    Molecular Basis for T Cell Response Induced by Altered Peptide Ligand of Type II Collagen2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 23, p. 19765-19774Article in journal (Refereed)
    Abstract [en]

    Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9.I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-zeta and signal via FcR gamma and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcR gamma, or CD3-zeta, we demonstrate that A9.I-A(q) preferentially activates FcR gamma/Syk but not CD3-zeta/Zap-70. Moreover, a genetic absence of Syk or FcR gamma significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcR gamma and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.

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