A Splice Variant of ASC Regulates IL-1β Release and Aggregates Differently from Intact ASC

The apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) is involved in apoptosis and innate immunity and is a major adaptor molecule responsible for procaspase-1 activation. ASC mRNA is encoded by three exons: exons 1 and 3 encode a pyrin domain (PYD) and caspase recruit domain (CARD), respectively, and exon 2 encodes a proline and glycine-rich (PGR) domain. Here, we identified a variant ASC protein (vASC) lacking the PGR domain that was smaller than full length ASC (fASC) derived from fully transcribed mRNA and searched for differences in biochemical and biological nature. Both fASC and vASC were found to activate procaspase-1 to a similar degree, but the efficiency of IL-1β excretion was significantly higher for vASC. There was also a marked structural difference observed in the fibrous aggregates formed by fASC and vASC. These results suggest that although the PGR domain is dispensable for procaspase-1 activation, it plays an important role in the regulation of the molecular structure and activity of ASC

Localized laccase activity modulates distribution of lignin polymers in gymnosperm compression wood

The woody stems of coniferous gymnosperms produce specialised compression wood to adjust the stem growth orientation in response to gravitropic stimulation. During this process, tracheids develop a compression-wood-specific S2L cell wall layer with lignins highly enriched with p-hydroxyphenyl (H)-type units derived from H-type monolignol, whereas lignins produced in the cell walls of normal wood tracheids are exclusively composed of guaiacyl (G)-type units from G-type monolignol with a trace amount of H-type units. We show that laccases, a class of lignin polymerisation enzymes, play a crucial role in the spatially organised polymerisation of H-type and G-type monolignols during compression wood formation in Japanese cypress (Chamaecyparis obtusa). We performed a series of chemical-probe-aided imaging analysis on C. obtusa compression wood cell walls, together with gene expression, protein localisation and enzymatic assays of C. obtusa laccases. Our data indicated that CoLac1 and CoLac3 with differential oxidation activities towards H-type and G-type monolignols were precisely localised to distinct cell wall layers in which H-type and G-type lignin units were preferentially produced during the development of compression wood tracheids. We propose that, not only the spatial localisation of laccases, but also their biochemical characteristics dictate the spatial patterning of lignin polymerisation in gymnosperm compression wood

Decreased levels of regulatory B cells in patients with systemic sclerosis: Association with autoantibody production and disease activity

Objective. B cell abnormalities characterized by autoantibody production and polyclonal B cell activation play an important role in the pathogenesis of SSc. IL-10 producing regulatory B (Breg) cells also play an important role in the negative immune response. We identified a human Breg cell subset that was predominantly found within the CD24hiCD27+ B cell subpopulation. However, the role of Breg cells in SSc remains unknown. The aim of this study was to investigate the clinical association of Breg cells in SSc patients. Methods. Blood IL-10 producing Breg cell levels were determined by FACS in 35 SSc patients and 30 healthy subjects. In a follow-up study, we analysed six individual dcSSc patients before and after treatment. Results. The frequency of blood Breg cells was significantly lower in SSc patients than in healthy controls (P<0.0001). Similarly, the frequency of CD24hiCD27+ B cells was significantly lower in SSc patients than in healthy controls (P<0.0001). SSc patients with decreased Breg cell levels often had interstitial lung disease (P<0.05). Furthermore, Breg cell levels correlated negatively with the titre of anti-topo I antibody (Ab) and anticentromere Ab in SSc patients. For a follow-up study, Breg cell levels in dcSSc patients after treatment were found to be significantly increased compared with those before treatment (P<0.05), accompanied by decreased disease activity. Thus, Breg cell levels were inversely correlated with disease activity of SSc. Conclusion. These results suggest that decreased Breg cell levels may contribute to the development of SSc. © The Author 2015.Embargo Period 12 month

Microscopic distribution of alkaloids in freeze-fixed stems of Phellodendron amurense

IntroductionPhellodendron amurense Rupr. contains rich alkaloids, which have been extensively applied in clinical treatments for their various biological activities. However, detailed microscopic distribution and roles of such alkaloids in P. amurense stem still need to be clarified.MethodsIn this study, the distribution of eight alkaloids in the transverse surface of freeze-fixed P. amurense stems in fall and summer has been visualized by cryo-time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM), which was found in living tissues with relative contents of different alkaloids varying with the position. In addition, the contents of these alkaloids quantified by high-performance liquid chromatography (HPLC) analysis suggested the seasonal variation from fall to the following summer.Results and discussionDistribution of eight alkaloids in the freeze-fixed stems of P. amurense from fall and summer seasons has been visualized and assigned into specific living tissues, with relative contents varying in different positions with seasons, which suggested their possible roles in the physiological processes of the plant itself or plant responding to changes in the surrounding conditions.ConclusionThis study provided a significant basis for further discussion of the genes or enzymes involved in these processes, which will contribute to investigating biosynthetic pathways and specific in planta roles of alkaloids

BAFF antagonist attenuates the development of skin fibrosis in Tight-Skin Mice

The tight-skin (TSK/+) mouse, a genetic model for systemic sclerosis (SSc), develops cutaneous fibrosis and autoimmunity. Although immunological abnormalities have been demonstrated in TSK/+ mice, the roles of B-cell-activating factor belonging to the tumor necrosis factor family (BAFF), a potent B-cell survival factor, have not been investigated. Serum BAFF levels in TSK/+ mice were examined by ELISA. Newborn TSK/+ mice were treated with BAFF antagonist, and then skin fibrosis of 8-week-old mice was assessed. Serum BAFF levels were significantly elevated in TSK/+ mice. Remarkably, BAFF antagonist inhibited the development of skin fibrosis, hyper-γ-globulinemia, and the autoantibody production in TSK/+ mice. The skin from TSK/+ mice showed upregulated expressions of fibrogenic cytokines, such as IL-6 and IL-10, while BAFF antagonist significantly suppressed them. Reciprocally, BAFF antagonist augmented antifibrogenic cytokines, such as IFN-γ, in the skin of TSK/+ mice. Furthermore, TSK/+ B cells with BAFF stimulation had a significantly enhanced ability to produce IL-6. The results suggest that BAFF/BAFF receptor system is critical for the development of skin fibrosis in TSK/+ mice and could be a potent therapeutical target. © 2007 The Society for Investigative Dermatology

Elevated Serum BAFF Levels in Patients with Localized Scleroderma in contrast to other organ-specific autoimmune diseases

Serum levels of B-cell activating factor belonging to the tumor necrosis factor family (BAFF), a potent B-cell survival factor, are elevated in patients with systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis and systemic sclerosis (SSc). The objective of this study was to determine serum BAFF levels and relate the results to the clinical features in patients with organ-specific autoimmune diseases of the skin, such as localized scleroderma and autoimmune bullous diseases. Serum BAFF levels were examined by enzyme-linked immunosorbent assay in 44 patients with localized scleroderma, 20 with pemphigus vulgaris/pemphigus foliaceus, 20 with bullous pemphigoid and 30 healthy controls. Twenty patients with SSc and 20 with SLE were also examined as disease controls. Serum BAFF levels were elevated in localized scleroderma patients compared with healthy controls. Concerning localized scleroderma subgroups, patients with generalized morphea, the severest form of localized scleroderma, had higher serum BAFF levels than linear scleroderma or morphea patients. The BAFF levels of generalized morphea were comparable with those of SSc or SLE. Furthermore, serum BAFF levels correlated positively with antihistone antibody levels and the severity of skin lesion as well as the number of skin lesions. By contrast, serum BAFF levels were not significantly elevated in patients with pemphigus or pemphigoid. These results suggest that BAFF may be contributing to autoimmunity and disease development in localized scleroderma. © 2007 The Authors Journal compilation © 2007 Blackwell Munksgaard

Elevated Serum BAFF Levels in Patients with Systemic Sclerosis (SSc): Enhanced BAFF Signaling in SSc B Lymphocytes

Objective. To determine serum levels of BAFF, a potent B cell survival factor, in patients with systemic sclerosis (SSc) and relate the results to the clinical features of SSc. Methods. Serum BAFF levels in 83 patients with SSc were examined by enzyme-linked immunosorbent assay (ELISA). In a longitudinal study, 131 serum samples obtained from 21 patients with SSc were analyzed. The expression of BAFF messenger RNA (mRNA) in the skin was quantified by real-time reverse transcription-polymerase chain reaction. The expression of BAFF receptor (BAFFR) on CD19+ B cells was assessed by flow cytometry. The production of IgG and interleukin-6 (IL-6) by isolated B cells was examined by ELISA. Results. Serum BAFF levels were elevated in SSc patients compared with healthy controls and correlated positively with the extent of skin fibrosis. Among the 21 patients with SSc in the longitudinal study, 7 had decreased BAFF levels, 11 had levels that remained unchanged, and 3 patients had increased levels. Decreasing BAFF levels were accompanied by regression of skin sclerosis, whereas increasing levels of BAFF were associated with the new onset or worsening of organ involvement. BAFF mRNA expression was up-regulated in the affected skin of patients with early diffuse cutaneous SSc. BAFFR expression on B cells was increased in SSc patients relative to healthy controls. Furthermore, SSc B cells that were stimulated by BAFF exhibited an enhanced ability to produce IgG and IL-6. Conclusion. These results suggest that BAFF and its signaling in B cells contribute to B cell abnormalities and disease development in patients with SSc. © 2006, American College of Rheumatology

Detection of Disease-specific Fusion Genes of Soft Tissue Tumors Using Formalin-fixed Paraffin-embedded Tissues; Its Diagnostic Usefulness and Factors Affecting the Detection Rates

[Background] Recent rapid advances in molecular biology have led the discovery of disease-specific novel fusion genes in a variety of soft tissue tumors. In this study, we attempted to detect these fusion genes using formalin-fixed paraffin-embedded (FFPE) tumor tissues and investigated their clinical utility and factors that affect the results of examination. [Methods] Reverse transcription polymerase chain reaction for the detection of tumor-specific fusion genes was performed using 41 FFPE tumor samples obtained from 37 patients representing nine histological types of soft tissue tumors that were diagnosed from 2006 to 2017 in our laboratory. [Results] Fusion genes in 19 (51.3%) out of 37 cases were detected successfully. Relatively high detection rates were observed in synovial sarcomas (100%, 4/4) and alveolar rhabdomyosarcomas (75%, 3/4). The detection rates of fusion genes were inversely correlated with the storage period of FFPE blocks. Decalcification by Plank-Rychlo solution significantly affected detection rates of the internal control gene (P = 0.0038). In contrast, there was no significant difference in detection rates between primary and metastatic lesion, or biopsy and resection material, or presence and absence of treatment history. [Conclusion] In certain histological types, detection of disease-specific fusion genes of soft tissue tumors using FFPE tissues showed high sensitivity and thus had diagnostic utility. However, due to the diversity of fusion patterns and the low-quality of nucleic acid, the detection rate as a whole was sluggish and required further improvement. For factors affecting the detection results, our results suggested that it was impossible to detect fusion genes by decalcified FFPE tissues, but it may be not necessary to consider factors such as the type of specimen (biopsy or resection) and treatment history of the patients when selecting the FFPE tissues