Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease

Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.     

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

We previously reported that c-kit⁺ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3^? IL-2Rβ⁺) and extrathymic T cells (CD3^int IL-2Rβ⁺) before and after irradiation. To determine their function, post-irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8⁺ CD3^int IL-2Rβ⁺ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1⁺ and NK1.1^? subsets of CD3^int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1⁺ subset of CD3^int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1^? subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.     

Preparation of 67Ga-labeled antibodies using deferoxamine as a bifunctional chelate. An improved method

Radionuclides or anti-cancer drugs may be coupled to antibodies for specific transport to target tissues. We have previously reported that several proteins could be rapidly and efficiently labeled with gallium (67Ga) by using deferoxamine (DFO) as a bifunctional chelating agent. In the present paper, we have described the use of hetero-bifunctional agents for the conjugation of DFO with antibodies and investigated the effect of coupling agents on in vitro properties and biodistribution of 67Ga-labeled antibodies. 67Ga-labeled monoclonal antibodies retained antigen-binding activity when prepared under optimum conditions. The use of hetero-bifunctional reagents, such as succinimidyl 6-maleimido-hexanoate (EMCS) or N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), which link thioether bonds and disulfide bridges prevented the formation of polymerized antibodies. Although high non-specific uptake in the liver was observed with radiolabels prepared by the homo-bifunctional agent glutaraldehyde, uptake in the liver was low with conjugates linked by hetero-bifunctional agents. 67Ga-labeled antibodies with thioether bonds showed in vivo stability, but the clearance from the circulation was the fastest with the radiolabel holding disulfide bonds. The coupling reagents used to link DFO and antibodies greatly influenced both in vitro properties and in vivo distribution of labeled antibodies and 67Ga-labeled antibodies provide a good model for the study of coupling methods and biodistribution of antibody conjugates.     

Activation of hepatic NKT cells and subsequent liver injury following administration of alpha-galactosylceramide

It has been established that alpha-galactosylceramide (alpha-GalCer), a glycolipid, is recognized by natural killer T (NKT) cells together with the monomorphic MHC-like antigen, CD1d, in mice and humans. In this study, we examined how NKT cells are modulated by in vivo administration of alpha-GalCer in mice. When 2 microg (or more)/mouse of alpha(-GalCer was injected i.p., the majority of NKT cells disappeared in the liver and spleen, possibly undergoing apoptosis, on day 1. At this time, NKT cytotoxicity seen in liver lymphocytes also disappeared. In parallel with this numerical and functional change of NKT cells, there was always concomitant hepatocyte damage, as shown by histology and elevated levels of transaminases. Subsequently, the number and function of NKT cells continued to increase from day 3 to day 7. The response seen in hepatic (and splenic) NKT cells did not occur in thymic NKT cells. All these phenomena induced in the liver did not appear in NKT-deficient mice such as beta2-microglobulin(-/-) and CD1d(-/-) mice. These results shed further light on the in vivo interaction between NKT cells and alpha-GalCer in mice.     

Lipopolysaccharide Induces CD25-Positive, IL-10-Producing Lymphocytes Without Secretion of Proinflammatory Cytokines in the Human Colon: Low MD-2 mRNA Expression in Colonic Macrophages

Despite the huge number of colonized Gram-negative bacteria in the colon, the normal colon maintains its homeostasis without any excessive immune response. To investigate the potential mechanisms involved, human colonic lamina propria mononuclear cells (LPMCs) obtained from uninflamed mucosa were cultured with lipopolysaccharide (LPS) prepared from Bacteroides vulgatus (BV-LPS) or Bacteroides fragilis (BF-LPS), as representatives of indigenous flora, or pathogenic Salmonella minnesota (SM-LPS). Colonic LPMCs failed to produce inflammatory cytokines in response to any type of LPS. Colonic macrophages barely expressed mRNA for MD-2, an essential association molecule for LPS signaling via Toll-like receptor 4. Further, BV-LPS induced CD25 and Foxp3 expression in lymphocytes and CD4(+)CD25(+) cells expressed IL-10 mRNA. Thus, the low expression of functioning LPS receptor molecules and induction of IL-10-producing CD4(+)CD25(+) lymphocytes by indigenous LPS may play a central role in the maintenance of colonic immunological homeostasis.     

Metal ions induce bone-resorbing cytokine production through the redox pathway in synoviocytes and bone marrow macrophages

To evaluate the biological reactions to metal ions potentially released from prosthetic implants, we examined the ability of metal ions to produce bone-resorbing cytokines and the underlying mechanism using synoviocytes and bone marrow (BM) macrophages. The cells were incubated with NiCl(2), CoCl(2), CrCl(3) or Fe(2)(SO(4))(3) at optimal concentrations, which are detectable in joint fluid following total joint arthroplasty. The production of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha were enhanced by all metal ions tested as determined by enzyme-linked immunosorbent assay. From the results of electrophoresis mobility shift assay, all metal ions enhanced the DNA-binding activity of nuclear factor kappaB (NF-kappaB), and p50-p65 heterodimers and p50 homodimers were the major subunits. These effects of the metal ions were considerably blocked by pyrrolidine dithiocarbamate (PDTC) known as a radical scavenger. An electron spin resonance study clearly demonstrated the ability of metal ions to generate activated oxygen species (AOS), especially hydroxyl radicals (*OH), which accounts for PDTC-blockade of metal ion-induced NF-kappaB activation and subsequent cytokine production. Taken together, our data raised the possibility that small amounts of metal ions released from prosthetic implants activate synoviocytes and BM macrophages through the AOS-mediated process (i.e. the redox pathway), and contribute to the initiation of osteolysis at the bone-implant interface.     

Whole genome association study of rheumatoid arthritis using 27 039 microsatellites

A major goal of current human genome-wide studies is to identify the genetic basis of complex disorders. However, the availability of an unbiased, reliable, cost efficient and comprehensive methodology to analyze the entire genome for complex disease association is still largely lacking or problematic. Therefore, we have developed a practical and efficient strategy for whole genome association studies of complex diseases by charting the human genome at 100 kb intervals using a collection of 27,039 microsatellites and the DNA pooling method in three successive genomic screens of independent case-control populations. The final step in our methodology consists of fine mapping of the candidate susceptible DNA regions by single nucleotide polymorphisms (SNPs) analysis. This approach was validated upon application to rheumatoid arthritis, a destructive joint disease affecting up to 1% of the population. A total of 47 candidate regions were identified. The top seven loci, withstanding the most stringent statistical tests, were dissected down to individual genes and/or SNPs on four chromosomes, including the previously known 6p21.3-encoded Major Histocompatibility Complex gene, HLA-DRB1. Hence, microsatellite-based genome-wide association analysis complemented by end stage SNP typing provides a new tool for genetic dissection of multifactorial pathologies including common diseases.     

Antiphospholipid antibodies

The concept of antiphospholipid syndrome(APS) has been widely accepted. Antiphospholipid antibodies originally included anticardiolipin antibodies and lupus anticoagulants as serological marker of APS. However, recent advances have shown that most pathogenic antiphospholipid antibodies are directed to phospholipid binding proteins such as beta 2-glycoprotein I and prothrombin as well as phospholipids. The preliminary classification criteria for definite APS have been advocated as the "Sapporo criteria". Further prospective investigations are required to re-evaluate the clinical significance of so-called antiphospholipid antibodies.     

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-alpha), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.