Haplotype structure,LD blocks,and uneven recombination within the LRP5 gene

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.     

c-fos overexpression in splenic B cells augments development of marginal zone B cells

Marginal zone (MZ)-B cells participate in very early immune responses and play a pivotal role in the first-line of defense against blood-borne Ags including bacterial LPS. Since splenic B cells from c-fos transgenic (H2-c-fos) mice are hyper-sensitive to LPS stimulation, we examined LPS-sensitivity of MZ-B cells in the spleen of H2-c-fos mice. Here, we show that proliferation of MZ-B cells from H2-c-fos mice stimulated with LPS was larger than that from control mice. Proliferation and IgM production of the H2-c-fos MZ-B cells were also larger than those of splenic follicular (FO)-B cells from the H2-c-fos mice, suggesting that c-fos overexpression augments LPS-sensitivity of MZ-B cells more than that of FO-B cells. Furthermore, the number of MZ-B cells but not that of FO-B cells in the spleen of H2-c-fos mice was two- to three-fold larger than that in control littermates. The number of transitional type II (T2)-B cells in H2-c-fos mice was also larger than that of control littermates. However, the number of transitional type I (T1)-B cells in the spleen of H2-c-fos mice was not larger than that of control mice. Moreover, this c-fos effect on differentiation of MZ-B cells was intrinsic in B cells by the competitive repopulation assay with hematopoietic stem cells of H2-c-fos and control mice. These results suggest that c-fos overexpression in B cells augments differentiation and accumulation of MZ-B cells.     

Evidence for increased expression of eotaxin and monocyte chemotactic protein-4 in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with tissue eosinophilia and the activation of T lymphocytes. The novel eosinophil chemoattractants, eotaxin and monocyte chemotactic protein (MCP)-4, are up-regulated at sites of allergic inflammation, yet their contribution to the pathophysiologic mechanisms of AD remains to be determined. OBJECTIVE: We sought to investigate the expression of eotaxin and MCP-4 in acute and chronic lesions from patients with AD and to determine their relationship to the numbers of resident inflammatory cells. METHODS: With use of in situ hybridization, the expression of eotaxin and MCP-4 messenger RNA (mRNA) in skin biopsy specimens from patients with acute and chronic AD skin lesions was compared with that of uninvolved skin from these patients and skin from healthy volunteers. RESULTS: There was a constitutive expression of eotaxin and MCP-4 mRNA in skin biopsy specimens from healthy subjects. Positive signal for chemokine mRNA was observed both within the epidermis and inflammatory cells (macrophages, eosinophils, and T cells) of the subepidermis in AD skin lesions. Within the subepithelium acute and chronic skin lesions exhibited a significant increase in the numbers of eotaxin and MCP-4 mRNA-positive cells compared with uninvolved skin (P <.01), whereas the numbers of eotaxin and MCP-4 mRNA-positive cells were significantly higher in chronic AD compared with acute AD skin lesions (P <.005, P <.001, respectively). Correlations were observed between the expression of eotaxin and MCP-4 mRNA and the presence of eosinophils and macrophages, respectively, in AD lesions (r(2) = 0.84, r(2) = 0.94). CONCLUSION: There is an increased expression of eotaxin and MCP-4 in acute and chronic lesions, suggesting that these chemotactic factors play a major role in the pathophysiologic mechanisms of AD.     

Culture of chondrocytes in fibroin-hydrogel sponge

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.     

Effects of estrogen on susceptibility to oxidation of low-density and high-density lipoprotein in postmenopausal women

Objective: To investigate the effects of estrogen on the susceptibility to oxidation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in postmenopausal women. Methods: A total of 23 postmenopausal women were treated with 0.625 mg of conjugated equine estrogen daily for 3 months. Blood samples were obtained before and after therapy. Plasma levels of total cholesterol and triglyceride and the concentrations of cholesterol, triglyceride, phospholipid in LDL and HDL were determined enzymatically and the levels of apolipoprotein A-I, A-II in HDL and apolipoprotein B in LDL were measured by turbidimetric immunoassay. The isolated LDL and HDL were incubated at 37°C for 24 h with CuSO₄ 5 μmol/l and the lipid peroxide concentration of LDL and HDL was measured. Results: Estrogen significantly reduced the plasma level of total cholesterol and significantly increased the plasma level of triglyceride. The LDL concentrations of cholesterol, phospholipid and apolipoprotein B were significantly decreased following estrogen therapy. The triglyceride level of LDL did not change significantly. The HDL concentrations of cholesterol, triglyceride, phospholipid and apolipoprotein A-I and A-II were all significantly elevated after estrogen therapy. Estrogen significantly inhibited the peroxidation of LDL at 50–2000 μg of LDL protein (14.17±4.17–11.49±1.42 nmol/200 μg of LDL protein, P<0.001) and of HDL (4.49±1.74–3.37±1.24 nmol/200 μg of HDL protein, P<0.03) induced by their incubation in the presence of CuSO₄. Conclusions: Estrogen inhibited the susceptibility of LDL and HDL to oxidative modification and favorably affected lipid metabolism by reducing the number of LDL particles and increasing the number of HDL particles in plasma that were resistant to oxidation.     

Autoepitopes on autoantigen centromere protein-A (CENP-A) are restricted to the N-terminal region, which has no homology with histone H3

Anti-centromere autoantibodies (ACA) are commonly found in the serum of patients with a limited type of scleroderma and other systemic autoimmune diseases. CENP-A is one of the major antigens against ACA and a histone H3-like protein. To analyse the autoantigenic epitopes of CENP-A, a series of truncated peptides of human CENP-A were expressed in Escherichia coli and immunoblotting analysis was performed with 91 ACA+ sera. Eighty sera (88%) with the ACA reacted to the 52-amino acids N-terminal region which is not homologous to H3, while no sera reacted to the C-terminus which has a sequence similarity with H3. Moreover, ELISA was also employed in this study using two synthetic peptides corresponding to the amino acid sequences 3-17 (peptide A) and 25-38 (peptide B). Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively. Core antigens of hepatitis B virus (HBV) and hepatitis C virus (HCV) have similar sequences to peptide A and/or peptide B, but three sera containing HBV without ACA and five sera containing HCV without ACA were found to be reactive to neither peptide. Centromere localization of CENP-A is dependent on the H3-like C-terminal domain which is not autoantigenic, while the antigenic N-terminal domain, which might play unidentified functional roles, should be an important region for the induction of ACA.     

Spinocerebellar ataxia 27 with a novel nonsense variant (Lys177X) in FGF14

Spinocerebellar ataxia 27 (SCA27) is an autosomal dominant SCA caused by variants in the fibroblast growth factor 14 (FGF14) gene. We examined a Japanese SCA patient whose deceased father also suffered from SCA. The patient was a 63-year-old male. He graduated from junior high school but received no further education. The predominant complaint was slowly progressive dysarthria and gait disturbance, which appeared at age 47. He showed pathological saccadic dysmetria, saccadic intrusions into smooth pursuit eye movements, dysarthria, and limb and truncal ataxia. His gait was wide-based but he did not require a walking stick. Limb muscle strength was intact. Deep tendon reflexes were normal or slightly reduced. Pathological reflexes were absent. He demonstrated mildly impaired vibration sense in the lower limbs. There was no urinary dysfunction. Brain MRI showed cerebellar atrophy without brainstem involvement. We first confirmed the absence of repeat expansion in genes known to be responsible for SCAs 1–3, 6–8, 10, 12, 17, 36 and dentatorubral-pallidoluysian atrophy. By exome analysis, we identified a novel heterozygous variant (NM_004115, c.529A>T; Lys177X) in exon 4 of the FGF14 gene. This variant is expected to generate a truncated FGF14 protein lacking the heparin binding sites, those are likely to modify the activity of FGF14. We confirmed the absence of the variant in 502 healthy Japanese individuals by Sanger sequencing. There is no record of the variant in public databases. We conclude that the novel variation in FGF14 is causative for SCA27 in this patient.     

ICAM-3, the third LFA-1 counterreceptor,is a co-stimulatory molecule for both resting and activated T lymphocytes

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co-stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter-receptors on antigen-presenting cells. Soluble ICAM-3, anti-ICAM-3 and anti-CD3 mAb were utilized to address the role of the ICAM-3/LFA-1 pathway in TcR/CD3-dependent or -independent T cell activation. Immunoaffinity-purified ICAM-3 co-immobilized with suboptimal concentrations of anti-CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM-3 with a β2 integrin, likely to be LFA-1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti-ICAM-3 mAb were able to co-stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti-ICAM-1 mAb were only co-stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some γδ T cell clones bearing the Vδ1 segment were activated by direct mAb engagement of ICAM-3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti-ICAM-3 mAb also induced development of dentritic processes. In conclusion, our data suggest that ICAM-3 on the surface of both T cells and antigen-presenting cells plays an essential role in the initiation of the immune response.