Tetsuroh Okano

Autoantibodies to the Amino-Terminal Fragment of β-Fodrin Expressed in Glandular Epithelial Cells in Patients with Sjögren’s Syndrome

Sjögrens’s syndrome (SS) is an autoimmune disease characterized by destruction of lacrimal and salivary glands, but the mechanisms underlying the disease process are unclear. By immunoscreening a HepG2 cDNA library with serum from an SS patient we isolated a cDNA encoding amino-terminal 616 aa of β-fodrin, a membrane skeleton protein associated with ion channels and pumps. Serum Ab to the amino-terminal fragment of β-fodrin was frequently detected in SS patients compared with rheumatic disease patients without SS or healthy controls (70 vs 12 or 4%; p < 0.00001). All the anti-β-fodrin-positive sera recognized the amino-terminal fragment with no homology to α-fodrin. Anti-β-fodrin Abs in patients’ sera as well as mouse polyclonal sera raised against the amino-terminal β-fodrin fragment did not react with intact β-fodrin, but recognized the 65-kDa amino-terminal fragment generated through cleavage by caspase-3 or granzyme B. When expression of intact and fragmented β-fodrin in lacrimal glands was assessed by immunohistochemistry, the antigenic amino-terminal fragment was distributed diffusely in acinar epithelial cell cytoplasm, whereas the carboxyl-terminal fragment and/or intact β-fodrin were localized in peripheral cytoplasm, especially at the basal membrane, in SS patients. In contrast, intact β-fodrin was detected primarily at the apical membrane of epithelia, and the amino-terminal fragment was scarcely detected in control patients with chronic graft-vs-host disease. These findings suggest that cleavage and altered distribution of β-fodrin in glandular epithelial cells may induce impaired secretory function and perpetuate an autoimmune response to β-fodrin, leading to autoantibody production and glandular destruction in SS.

α2-HS glycoprotein is an essential component of cryoglobulin associated with chronic hepatitis C.

*Corresponding author: Toshio Okazaki, Yamazaki Gakuen University School of Animal Health Sciences, 4-7-2 Minami-osawa, Hachiouji, Tokyo 192-0364, Japan, E-mail: t_okazaki@yamazaki.ac.jp Toshio Okazaki, Tetumi Iwasaki, Tetsuroh Okano and Yoshifumi Kurosaki: Kitasato University School of Allied Health Sciences , Sagamihara, Kanagawa , Japan Kaoru Yamazaki: Yamazaki Gakuen University School of Animal Health Sciences , Hachiouji, Tokyo , Japan Kazuo Nakamura: Center for Natural Sciences , Kitasato University, Sagamihara, Kanagawa , Japan Takahiro Fujioka: Department of Internal Medicine , Shiki Community Hospital, Shiki, Saitama , Japan Hiroshi Yotsuyanagi: Department of Infectious Diseases , Internal Medicine, University of Tokyo, Tokyo , Japan

Quantitative scanning analysis of a cryoglobulin ring detected in sera of patients with hepatitis C using a cooling gel diffusion method

Figure 1 Cryoglobulin ring and its scanning pattern on cooling gel diffusion from serum of a patient with hepatitis C. (A) Cryoglobulin ring from serum of a patient with hepatitis C following cooling gel diffusion. (B) Scanning point of the cryoglobulin ring

Composition of cryoglobulin and cryoprecipitate

Figure 1 Comparison of cryoglobulin and cryoprecipitate. (A) Precipitation ring of cryoglobulin generated by the cooling gel diffusion method and the cryoprecipitate generated by the cooling precipitation method. a: cryoglobulin ring on a gel plate; b: gel plate control; c: cryoprecipitate from cooling precipitation method. (B) Relationship between the quantitaties of the cryoglobulin ring and cryoprecipitate. To quantify the precipitation ring obtained by the cooling gel diffusion method, we used ImageJ scanning software for scanning and calculated the area under the curve (AUC). In addition, the cryoprecipitate obtained by the cooling precipitation method was washed and redissolved at 378C, followed by measurement of the protein concentration using the biuret method. Toshio Okazaki*, Asami Nakahashi, Takaharu Uchiyama, Akemi Imoto, Kohei Morikawa, Tetsuroh Okano, Kazuo Nakamura, Shinichiro Takahashi and Takahiro Fujioka 1 Department of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan 2 Department of Hematology, Kitasato University School of Allied Health Sciences, Sagamihara, Japan 3 Department of Immunology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan 4 Division of Complementary Medicine, Kitasato University School of Allied Health Sciences, Sagamihara, Japan 5 Center for Natural Sciences, College of Liberal Arts and Sciences, Kitasato University, Sagamihara, Japan 6 Department of Internal Medicine, Self-Defense Forces Central Hospital, Tokyo, Japan

Effects of albumin-bound-fatty acids on the growth of the human T lymphoblastic cell line Jurkat

To culture a T lymphoblastic cell line, JURKAT, in RPMI 1640 medium, human albumin, instead of fetal bovine serum (FBS), was added to a final concentration of 1% to facilitate growth comparable with FBS. In addition, several kinds of animal albumin were added to the culture, resulting in markedly different growth rates (Fig. 1). In RPMI 1640 medium, amino acids and carbohydrates are included as nutrients, but no lipids. Most fatty acids are binding to serum albumin and alpha fetoprotein (Reed 1986; Copado et al. 1999), and albumin may be the only source of lipids for cultured cells in this study. On the other hand, it is known that fatty acids have various functions to cell growth; i.e., cytotoxicity (Lima et al. 2002; Martins de Lima et al. 2006), induction of apoptosis (Welters et al. 2004; Artwohl et al. 2008), and regulation of signaling (Pawar and Jump 2003). Here, we measured the concentrations of non-esterified fatty acids (NEFAs) in animal albumin (10% solution) using a measuring kit (Wako, Tokyo, Japan), and analyzed albumin-bound NEFAs employing gas chromatography. The amounts of bound NEFAs varied with albumins, being largest for rabbit albumin, followed by sheep, rat, human, and bovine albumins in this order. NEFAs in FBS showed values intermediate between rat and human albumins (Fig. 2A). Using gas chromatography, fatty acids bound to animal albumins were analyzed to be consisting of palmitic (C16:0), stearic (C18:0), and oleic (C18:1) or linoleic acids (C18:2) similar to NEFAs in FBS used for Jurkat cell culture (Fig. 2B). However, only rabbit albumin attached a large amount of caprylic acid (C8:0), which presumably caused contamination during the purification process, and sheep albumin attached more than twice as large amount of stearic acid (C18:0) as to rat or human albumin (Fig. 2C). In media supplemented with albumins derived from various T. Okazaki : S. Takahashi Department of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan

Cysteine analog breaks cryoprecipitate associated with chronic hepatitis C

*Corresponding author : Toshio Okazaki, Yamazaki Gakuen University, School of Animal Health Sciences, 4-7-2 Minami-osawa, Hachiouji, Tokyo 192-0364, Japan, Phone: + 81 42 6530901, Fax: + 81 42 6530902, E-mail: t_okazaki@yamazaki.ac.jp Kaoru Yamazaki: Yamazaki Gakuen University, School of Animal Health Sciences , Hachiouji, Tokyo , Japan Toshio Okazaki, Tetsumi Iwasaki, Yoshifumi Kurosaki and Tetsuroh Okano: Kitasato University, School of Allied Health Sciences , Sagamihara, Kanagawa , Japan Mikio Taniguchi: Department of Research and Development , Showa Yakuhin Kako Co., Ltd., Kawasaki, Kanagawa , Japan Takahiro Fujioka: Department of Internal Medicine , Shiki Community Hospital, Shiki, Saitama , Japan