Clinical and immunological studies for 105 Japanese seropositive patients of epidermolysis bullosa acquisita examined at Kurume University.

OBJECTIVES: Using our serological diagnostic criteria, we selected 105 Japanese patients with epidermolysis bullosa acquisita (EBA), an autoimmune bullous disease (AIBD) reacting with type VII collagen, from our cohort of 5063 AIBD patients. METHODS: We examined the patients clinically and immunologically. RESULTS: We found diversity of clinical manifestations in both cutaneous and oral mucosal lesions and a high rate of inflammatory-type EBA patients in Japan. Common treatments were systemic steroids, followed by immunosuppressives, DDS, tetracycline/minocycline and colchicine. Immunological studies revealed that indirect immunofluorescence of 1M-NaCl-split skin, immunoblotting of dermal extract, and type VII collagen ELISA were sensitive methods, with possible multiplicity of circulating autoantibodies against other basement membrane autoantigens. CONCLUSION: The present study analyzed the largest cohort of EBA patients, confirming the scarcity of EBA (only 105 of the 5063 AIBD patients), and showed that the three serological tests are useful for the diagnosis of EBA.

Integrin ß4 is a major target antigen in pure ocular mucous membrane pemphigoid.

Previous studies of ocular mucous membrane pemphigoid (OMMP) have identified several components of the basement membrane zone to be autoantigens, including integrin ß4. However, there are no extensive or definitive reported studies that address this, particularly in pure OMMP. To clarify the major autoantigens in pure OMMP. In this study, we examined sera from 43 pure OMMP patients for both IgG and IgA antibodies using newly developed immunoblotting analyses with a hemidesmosome-rich fraction and various recombinant proteins of integrin α6ß4, in addition to our routine immune-serological tests. Using a hemidesmosome-rich fraction, sera from patients with pure OMMP demonstrated reactivity of IgG and/or IgA antibodies to integrin ß4, BP180 and laminin-332. The reactivity of pure OMMP sera to integrin ß4 was further confirmed by immunoblotting using integrin ß4 recombinant proteins. Using concentrated supernatant of HaCaT cells, only one serum sample showed positive IgG and IgA reactivity to LAD-1, the ectodomain of BP180. None of the pure OMMP sera reacted with any autoantigens on immunoblotting using normal human epidermal or dermal extracts, or purified human laminin-332. Integrin ß4 was considered to be the major and specific autoantigen for pure OMMP. The new methods established in this study are useful for detection of various autoantigens, particularly integrin ß4.

[Analysis and identification of Semen Glycines Nigrae and Semen Pharbitidis by infrared spectroscopy].

Semen Glycines Nigrae and Semen Pharbitidis containing a large amount of fats and proteins are commonly used in Chinese herbal medicine. Tri-step infrared spectroscopy was applied to fast analyze and identify the two samples. In the conventional infrared spectroscopy, the samples both have obvious characteristic absorption peaks at 1,745 cm(-1) assigned to the stretching mode of C==O in esters. Furthermore, the two kinds of herbs have the peaks at 1,656 and 1,547 cm(-1) assigned to the amide I and II bands of protein. Obviously, the infrared spectra of herbs demonstrate that protein and fat is the major component in two kinds of herbs, and the relative intensity of the peaks assigned to fat and protein indicate their relative content is different. And the result is consistent with the reported. In the second derivative spectra, Semen Pharbitidis has a peak at 1,712 cm(-1) assigned to the organic acid, however, Semen Glycines Nigrae has not this absorption peak. In addition, in the second derivative spectra, appeared more differences between the two samples in shape and intensity of the peaks. In two-dimensional correlation infrared spectra, the two samples were visually distinguished due to their significant differences in auto-peak position and intensity. In the region of 1,500-1,700 cm(-1), Semen Glycines Nigrae has two autopeaks and Semen Pharbitidis has three autopeaks. In the region of 2,800-3,000 cm(-1), the samples both have two autopeaks, but the position of the strongest autopeak is different. It was demonstrated that the Tri-step infrared spectroscopy were successfully applied to fast analyze and identify the two kinds of samples containing the same major component, and made sure the foundation for future researches.

[The analyses and identification of Flos rhododendri mollis and Flos chrysanthemi indici via infrared spectroscopy].

In this study, major chemical components of Flos rhododendri mollis and Flos chrysanthemi indici were characterized using Fourier transform infrared spectroscopy (FTIR). For Flos rhododendri mollis, the bands at 1,648 and 1,543 cm(-1) were attributed to amide I and amide II , respectively, indicating that it contained proteins probably resulting in immunization. In case of Flos chrysanthemi indici, stretching vibration of C==O function group was responsible for the bands at 1,734 and 1,515 cm(-1), as a result of essential oils, lipids, etc. Since FTIR spectra of Flos rhododendri mollis and Flos chrysanthemi indici are almost identical and it is difficult to discriminate them, two-step identification was investigated via secondary derivative of the FTIR spectra. The bands at 1,656 and 1,515 cm(-1) corresponds to flavonoides in Flos rhododendri mollis and Flos chrysanthemi indici. In the secondary derivative of the FTIR spectrum of Flos chrysanthemi indici, characteristic bands of inulin were present at 1,163, 1,077, 1,026, 986 and 869 cm(-1), and therefore Flos chrysanthemi indici contained inulin as well. Tri-step identification was carried out for Flos rhododendri mollis and Flos chrysanthemi indici by means of comparing their 2D-IR correlation spectra in different wave number range. In the characteristic range of flavonoides (1,700-1,400 cm(-1)), Flos rhododendri mollis exhibited 3 obvious autopeaks, while 10 autopeaks were visualized in the 2D-IR correlation spectrum of Flos chrysanthemi indici Moreover, in the characteristic range of glucoside (1,250-900 cm(-1)), 10 and 9 autopeaks were present in the 2D-IR correlation spectra of Flos rhododendri mollis and Flos chrysanthemi indici, respectively. Therefore, the tri-step identification of FTIR is a time-saving; accurate, cost-saving and convenient method to effectively distinguish traditional Chinese medicines.

[Discrimination of eleven genera of Chinese herbs in Geraniaceae by FTIR spectroscopy and clustering analysis].

A fast identification method of eleven genera of Chinese herbs in Geraniaceae was developed by the combination of Fourier transform infrared spectroscopy with clustering analysis. FTIR spectroscopy was employed to identify and analyze eleven genera of Chinese herbs in Geraniaceae. On the basis of a principal component analysis (PCA) model, three genera of Chinese herbs were rapidly classified by using the method of SIMCA clustering analysis. These samples could be successfully classified by SIMCA. Recognition rate and rejection rate reached up to 98%. The accuracy of clustering reached up to 91% during blind sample testing. It is concluded that in combination with clustering analysis, FTIR method provides an effective way to rapidly evaluate Chinese herbs in Geraniaceae.

[Analysis and identification of Geranium by two-dimensional correlation infrared spectroscopy].

Tri-step infrared spectroscopy (Fourier transform infrared spectroscopy (FTIR) combined with second derivative spectra and two-dimensional correlation infrared spectroscopy (2D-COS)) was employed to identify and analyze the main components of Heilongjiang (HLJG), Jilin (JLG), Liaoning (LNG) genuine Herba Geranium. The emergence of several characteristic absorption peaks of tannins including 1 730 and 1 337 cm(-1) and peaks around 1 618 and 1 318 cm(-1) belonging to calcium oxalate suggested that Herba Geranii contained tannins and calcium oxalate. Differences near 1 370 and 1 230 cm(-1) were found among the three Herba Geranii. In light of second derivative spectra, four more peaks of tannin components around 1 509, 1 204, 764 and 763 cm(-1) and evident differences around C=O stretching bands (1 750-1 600 cm(-1)) were observed. By 2D-COS spectra with further improved resolution, the three genuine Geraniums were visually distinguished due to their significant differences in auto-peak profile. HLJG has 7 auto peaks with a strongest peak around 1 621 cm(-1), while JLG and LNG both have only 4 auto peaks with a strongest peak around 1 580 and 1 659 cm(-1), respectively. It was demonstrated that the Tri-step infrared spectroscopy was successfully applied to fast analyze and identify genuine Geraniums from different geographical regions and subsequently would be applicable to the study of Chinese medicinal resources and quality standards.

Prevention of fasting-mediated bone marrow atrophy by leptin administration.

Leptin is an adipokine that regulates body weight. In the current study, we demonstrate that continuous injection of leptin prevents the lymphocyte reduction observed in fasted mice, especially the immature B cell populations in the bone marrow. Although leptin administration reduced apoptotic cells in the bone marrow of fasted mice, it did not prevent glucocorticoid-mediated apoptosis in vitro. Bone marrow atrophy has also been shown in the leptin receptor-deficient db/db mice. In order to investigate the mechanisms underlying these processes, we transplanted bone marrow cells from db/db or control (+m/+m) mice into C.B-17/lcr-scid/scid mice. We found that the spleen and bone marrow B cell populations were completely reconstituted when db/db and +m/+m cells were transplanted into scid mice. Our findings suggest that direct interactions between leptin and bone marrow cells are not essential for the development of B cells in a metabologically normal environment.

High-dose methotrexate with R-CHOP therapy for the treatment of patients with primary central nervous system lymphoma.

We describe MR-CHOP therapy, a novel treatment regimen consisting of high-dose methotrexate and R-CHOP that provides systemic anti-tumor activity with penetration of the blood-brain barrier in patients with newly diagnosed primary central nervous system lymphoma. The MR-CHOP regimen was administered with 2 g/m(2) of methotrexate and 375 mg/m(2) of rituximab on day 1, 750 mg/m(2) of cyclophosphamide on day 3, 50 mg/m(2) of doxorubicin on day 3, 1.4 mg/m(2) of vincristine on day 3 and 100 mg of prednisolone on days 1-5 in this pilot study of seven patients. Six cycles of MR-CHOP therapy were administered every 3 weeks, followed by high-dose chemotherapy with stem cell rescue in young patients, or an additional two cycles of 4 g/m(2) methotrexate and rituximab in older patients. The overall response rate was 100%, with 85.7% complete remission (CR). One patient showed partial response, relapsed and subsequently died. Another relapsed following CR, and was rescued by further salvage therapy. The others survive without relapse at a median observation period of 24 months. Hematological toxicity included grade 4 leukocytopenia in 4/7 and neutropenia in 5/7, which were transient and tolerated well. Non-hematological toxicities were tolerated well. The efficacy of this novel regimen as remission induction therapy was found to be promising in this pilot study, although the number of patients was small and follow-up short.

Cholesterol-dependent induction of dendrite formation by ginsenoside Rh2 in cultured melanoma cells.

Herbal remedies containing root extracts of Panax ginseng are commonly used for complementary or alternative therapies. Ginsenosides, the major components of root extracts, are responsible for ginseng's pharmacological and biological effects; however, their mechanisms of action are unclear. We examined whether membrane cholesterol was involved in the mechanism of action of ginsenoside Rh2 in cultured cells. In B16 melanoma cells, Rh2 (18.5 µM) induced dendrite formation within 2 h. Depletion of cholesterol by pretreatment with 10 mM methyl-ß-cyclodextrin suppressed this effect of Rh2. Rh2 did not change the cellular cholesterol content and the immunofluorescence staining pattern of the lipid-raft-associated molecules, ganglioside GM3, Caveolin-1, Flotillin-1, and Flotillin-2, for up to 3 or 6 h. However, within 2 min of addition, Rh2 changed the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) but not of 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). DPH is more sensitive than TMA-DPH to changes in the physical properties of membrane lipid bilayers regulated by cholesterol. These results suggest that Rh2 affects the physical properties of cholesterol-regulated membrane lipid bilayers and could lead to changes in cellular functions.

Synergistic effects of topoisomerase I inhibitor, SN38, on Fas-mediated apoptosis.

Inhibitors of topoisomerase I, such as camptothecin, have proven to be among the most promising new classes of anti-neoplastic agents introduced into the clinical setting in recent years. Irinotecan (CPT-11) is one of the most widely used camptothecin analogs and is converted to form the active metabolite SN-38. The present study was designed to explore apoptosis induced by SN38 and anti-Fas antibody (CH11) in WR/Fas-SMS1 cells and its possible mechanisms. The results demonstrate that combination of SN38 and CH11 synergistically enhanced cell apoptosis in WR/Fas-SMS1 cells. Western blotting analysis showed that combination of SN38 and CH11 activated the ATM-Chk1-p53 pathway, increased protein expression of phospho-p53 and cleavaged caspase-3, but down-regulated expression of phospho-p21. Our data suggest that combination of SN38 and CH11 enhanced apoptosis through down-regulation of p21 phosphorylation. In conclusion, inhibition of p21 could be a new adjuvant approach in cancer therapy.

DIP2 disco-interacting protein 2 homolog A (Drosophila) is a candidate receptor for follistatin-related protein/follistatin-like 1--analysis of their binding with TGF-ß superfamily proteins.

Follistatin-related protein (FRP)/follistatin-like 1 (FSTL1) is a member of the follistatin protein family, all of which share a characteristic structure unit found in follistatin, called the FS domain. Developmental studies have suggested that FRP regulates organ tissue formation in embryos. Immunological studies showed that FRP modifies joint inflammation in arthritic disease, and modulates allograft tolerance. However, the principle physiological function of FRP is currently unknown. To address this issue, we cloned four FRP-associated proteins using a two-hybrid cloning method: disco-interacting protein 2 homolog A from Drosophila (DIP2A), CD14, glypican 1 and titin. Only DIP2A was expected to be a membrane receptor protein with intracellular regions. Over-expression of FLAG epitope-tagged DIP2A augmented the suppressive effect of FRP on FBJ murine osteosarcoma viral oncogene homolog (FOS) expression, and the Fab fragment of IgG to FLAG blocked this effect. Knockdown of Dip2a leaded to Fos gene up-regulation, and this was not affected by exogenous FRP. These in vitro experiments confirmed that DIP2A could be a cell-surface receptor protein and mediate a FOS down-regulation signal of FRP. Moreover, molecular interaction analyses using Biacore demonstrated that FRP bound to DIP2A and CD14, and also with proteins of the TGF-ß superfamily, i.e. activin, TGF-ß, bone morphogenetic protein 2/4 (BMP-2/4), their receptors and follistatin. FRP binding to DIP2A was blocked by CD14, follistatin, activin and BMP-2. FRP blocked the ligand-receptor binding of activin and BMP-2, but integrated itself with that of BMP-4. This multi-specific binding may reflect the broad physiological activity of FRP.

Cisplatin augments FAS-mediated apoptosis through lipid rafts.

Cisplatin is widely and effectively used for the treatment of various types of cancer. However, its biochemical mechanisms are still unelucidated. Previously, we reported that membrane sphingomyelin (SM) was important for FAS-mediated apoptosis through lipid raft function. In this study, we strikingly show that cisplatin combined with CH11 (anti-FAS antibody, IgM) was able to induce marked apoptosis in SM synthase-restored WR/Fas-SMS1 cells, but not in SM synthase-deficient WR/FAS-SM(-) cells. In addition, we demonstrated that membrane SM played an important role in cisplatin/CH11-induced apoptosis through the classical caspase-dependent pathway, mainly by enhancing the formation of FAS-associated signaling complexes.

[Discrimination of seven species of Agrimonia pilosa Ledeb and its extracts by FTIR and 2D-IR].

The objective of the present study is to discriminate seven species of Agrimonia pilosa Ledeb herbs and their total tannin extracts by Fourier transform infrared spectroscopy (FTIR), second derivative infrared spectroscopy, and two-dimensional correlation infrared spectroscopy (2D-IR) under thermal perturbation. The structural information of the samples indicated that Agrimonia pilosa Ledeb and their extract residues contain a large amount of starch and calcium oxalate, since some characteristic absorption peaks of the starch, such as 1 151, 1 101, 1 032 and 988 cm(-1) can be observed; and some characteristic absorption peaks of the calcium oxalate, such as 1 618, 1 318 and 780 cm(-1), can be observed. Further more, the characteristic absorption peaks of the sulfate which arouse at 1 711 and 1 447 cm(-1) in the IR spectra of Agrimonia pilosa Ledeb acetone extracts can be found. The macroscopical fingerprint characters of FTIR and 2D-IR spectra can not only provide the information of main chemical constituents in medicinal materials and their different extracts, but also compare the components differences among the similar samples. In conclusion, the multi-steps IR macro-fingerprint method is rapid, effective, visual and accurate for pharmaceutical research.

[Study on hydrolysable tannin constituents of seed of Juglans regia II].

OBJECTIVE: To study hydrolysable tannin constituents of the seed of Juglans regia. METHOD: The chemical constituents were isolated by Diaion HP-20, Toyopaerl HW-40 and MCI gel CHP-20P column chromatogramphy and identified by physicochemical identification and spectral data. RESULT: Six compounds obtained from the 70% ethanol extract were identified as 1, 2, 3, 4, 6-penta-O-galloyl-3-D-glucose (1), rugosin C (2), 1, 2, 3, 6-tetra-O-galloyl-3-D-glugose (3), tellimagrandin II (4), casuarictin (5), 1-degalloylrugosin F (6). CONCLUSION: All compouds were isolated from the seeds of J. regia for the first time.

Impaired TCR signaling through dysfunction of lipid rafts in sphingomyelin synthase 1 (SMS1)-knockdown T cells.

During T cell activation, TCRs cluster at the center of the T cell-antigen-presenting cell interface forming the central supramolecular activation cluster. Although it has been suggested that sphingolipid- and cholesterol-rich microdomains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals, an actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has not been reported. After cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1, we established a SM-knockdown cell line (Jurkat-SMS1/kd) by transfection of SMS1-short-interfering RNA into Jurkat T cells, which is deficient in membrane expression of SM. Upon CD3 stimulation, expression of CD69 (the earliest leukocyte activation antigen), activation-induced cell adhesion and proliferation as well as TCR clustering was severely impaired in Jurkat-SMS1/kd cells. CD3-induced tyrosine phosphorylation and association of linker for activation of T cell with ZAP-70 and Grb2 and phosphorylation of protein kinase C (PKC) were also severely impaired in Jurkat-SMS1/kd cells. Finally, translocation of TCR, ZAP-70 and PKC into lipid rafts was markedly decreased in Jurkat-SMS1/kd cells. These findings indicate that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.

[Analysis of Acanthopanax senticosus harms for different parts using Fourier transform infrared spectroscopy].

In the present paper, raw radix, stem and leaf and the total glycoside extract of them of Acanthopanax senticosus Harms were studied via the multi-steps IR macro-fingerprint method. The spectra of raw medicinal materials show that the spectra of radix and stem are similar and mainly show characteristic peaks of calcium oxalate and starch, whereas the characteristic peaks of calcium oxalate of leaf almost disappear and the shape of characteristic peak of starch also gets unconspicuous. The FT-IR spectra of total glycoside extract of radix, stem and leaf present characteristic peaks at 1602, 1514, 1452 cm(-1) (vibration of phenyl framework) and 1271 cm(-1) (==C--O), respectively, therefore, the authors speculated that their mutual component is the compound of phenolic glycoside. Through observing the second derivative IR spectra of the total glycoside extract of different parts of Acanthopanax senticosus Harms, the authors found that the characteristic peak of leaf is stronger than that of radix and stem at 1656 cm(-1) (flavone C==O), this proves that the quantity of component of flavone in the leaf is higher than that in the radix and stem. In the two-dimensional correlation spectra, the radix and stem both have five automatic peaks (vibration of phenyl framework) in 1350-1700 cm(-1), whereas the leaf still shows another automatic peak at 1656 cm(-1) (flavone C==O), and this further proves that the quantity of component of flavone in the leaf is higher than that in the radix and stem.

[Studies on hydrolysable tannin constituents in seed of Juglans regia(I)].

OBJECTIVE: To study hydrolysable tannin constituents of the seeds of Juglans regia. METHOD: The chemical constituents were isolated by Diaion HP-20 and Toyopaerl HW-40 MCI gel CHP-20P column chromatogramphy and identified by physicochemical identification and spectral data. RESULT: The compounds obtained from the 70% acetone extract were identified as gemin D (1), casuariin (2), pedunculagin (3), tellimagrandin I (4), rugosin F (5), heterophylliin D (6). CONCLUSION: All other compounds which were isolated from the seeds of J. regia for the first time.

Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen.

Serum titers of antibody to Epstein-Barr virus (EBV) viral capsid antigen (VCA) have been positively correlated with malignancies of lymphoid proliferation, such as Burkitt's lymphoma and Hodgkin's lymphoma. We have constructed a phage display combinatorial antibody Fab library from a patient with marginal zone B cell lymphoma associated with Sjögren's syndrome and carrying high serum anti-EBV-VCA IgG titer. Fab fragments were selected by panning against EBV-VCA protein coated onto ELISA plates, and selected Fab clones were characterized by ELISA, western blotting (WB), indirect immunofluorescence assay and immunohistochemistry. We have established two Fab clones, Fab-aVCA1 and Fab-aVCA21, which specifically recognize EBV-VCA by ELISA and WB. Inhibition ELISA competition showed that both clones could significantly reduce the binding of specific anti-EBV-VCA mAb to its relevant proteins. Furthermore, these two Fab clones could localize VCA protein in the EBV-positive P3HR1 and Daudi cell lines, as well as in tissue samples from patients with EBV-infected lymphoid malignancies. These results indicate that our two Fab clones are novel human mAbs specific for EBV-VCA protein and may have potential benefits for development of novel diagnostic and therapeutic approaches in EBV-related lymphoid malignancies.

[Identification of human salivary stem cells from cultured labial minor salivary cells].

Stem cell therapy is expected to be a promising approach for the compensation of lost organs. The various organization cells that compose an animal's body are always being renewed for the maintenance of homeostasis. The cells that become the source of new cells are a body's own stem cells. Cell therapy, using stem cells, has a few of bioethical problems but there is the advantage that it is not necessary to worry about the immunity rejection of the transplant because the body's stem cell is from it's own body. In our present study we identified side population cells (SP cells), which are highly enriched for stem cell activity in human salivary glands. Isolated SP cells expressed high level of DNp63 and PSCA (prostate stem cell antigen), but not nestin, Oct4 and CD34. Real time PCR analysis revealed that the expression of DNp63, detected in immature salivary epithelial cells, gradually decreased through cell differentiation. In contrast, PSCA can be distinguished among early differentiating and later transit-amplifying salivary epithelial cells in tissue culture. Our study suggested that these markers may mark the transition of human salivary epithelial cells.

Fractalkine in rheumatoid arthritis and allied conditions.

Leukocyte adhesion and trafficking at the endothelium requires both adhesion molecules and chemotactic factors. Fractalkine (CX3C) is a unique chemokine, and is expressed on tumor necrosis factor-alpha- and interleukin-1-activated endothelial cells (ECs). Fractalkine receptor, CX3CR1, is expressed on NK cells, monocytes, and some portion of CD4- and CD8-positive T cells. Interactions between fractalkine and CX3CR1 can mediate not only chemotaxis, but also cell adhesion in the absence of substrates for other adhesion molecules. Furthermore, fractalkine activates NK cells, leading to increased cytotoxicity and interferon-gamma production. Recently, accumulating evidence has shown that fractalkine is involved in the pathogenesis of rheumatoid arthritis and allied conditions. This review examines new concepts underlying fractalkine-mediated leukocyte migration and tissue damage, focusing primarily on the pathophysiological roles of fractalkine in rheumatic diseases.