Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.     

Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity

Defects in death receptor-mediated apoptosis have been linked to cancer and autoimmune disease in humans. The in vivo role of caspase 8, a component of this pathway, has eluded analysis in postnatal tissues because of the lack of an appropriate animal model. Targeted disruption of caspase 8 is lethal in utero. We generated mice with a targeted caspase 8 mutation that is restricted to the T-cell lineage. Despite normal thymocyte development in the absence of caspase 8, we observed a marked decrease in the number of peripheral T-cells and impaired T-cell response ex vivo to activation stimuli. caspase 8 ablation protected thymocytes and activated T-cells from CD95 ligand but not anti-CD3-induced apoptosis, or apoptosis activated by agents that are known to act through the mitochondria. caspase 8 mutant mice were unable to mount an immune response to viral infection, indicating that caspase 8 deletion in T-cells leads to immunodeficiency. These findings identify an essential, cell-stage-specific role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. This is consistent with the recent identification of caspase 8 mutations in human immunodeficiency.     

Enzymatic determination of a molar ratio of free branched-chain amino acids to tyrosine (BTR) and its clinical significance in plasma of patients with various liver diseases

A molar ratio of free branched-chain amino acids to tyrosine (BTR) was determined in the plasma of patients with liver diseases using a new enzymatic method. In addition, clinical significance of BTR was studied by comparing particularly with that of Fischer's ratio (a molar ratio of branched-chain amino acids to aromatic amino acids (tyrosine+phenylalanine], which was obtained by conventional HPLC (Amino acid autoanalyzer, Hitachi 835). Following results were obtained: 1) Enzymatically determined branched-chain amino acids and tyrosine showed significant correlations with respective results obtained by HPLC (r = 0.937, 0.972). 2) Significant correlation was also found between enzymatically determined BTR and Fischer's ratio obtained by HPLC. Changes of BTR in clinical courses were found to be in parallel with those of Fischer's ratio. 3) BTR as well as Fischer's ratio correlated significantly with ICG R15, KICG, prothrombin time (%) and serum albumin level. 4) BTRs in patients with decompensated liver cirrhosis or with fulminant hepatitis were significantly lower than those in patients with acute or chronic hepatitis. In conclusion, new enzymatic assay of branched-chain amino acids and tyrosine as described here is quite simple method, and is also considered to be very useful parameter of the clinical conditions of patients with liver diseases, particularly representing the severity of liver diseases and the protein nutritional status.     

Evaluation of endocrine disrupting activity of plasticizers in polyvinyl chloride tubes by estrogen receptor alpha binding assay

Polyvinyl chloride (PVC) tubing is an indispensable medical material for extracorporeal circulation therapy. However, di(2-ethylhexyl)phthalate (DEHP), a suspected endocrine disruptor, can be eluted from PVC, suggesting that an alternative material that does not contain DEHP is needed for clinical applications. First, we evaluated the endocrine disrupting risks of the plasticizers contained in PVC tubes by investigating their binding affinities for the human estrogen receptor alpha (ERα). Our results revealed that, while DEHP has some binding affinity for ERα, neither epoxidized soybean oil nor tris(2-ethylhexyl)trimellitate (an alternative to DEHP) has any affinity for ERα. Second, we evaluated the endocrine disrupting risks of a tube made of newly developed plasticizer-free (PF) materials. We confirmed the presence of DEHP and detected several unidentified substances in plasma stored within the PVC tube. This plasma's competitive binding affinity for ERα was significantly higher than that of control plasma (P < 0.01). In contrast, the profile of plasma stored in the PF tube was similar to that of the control, both in terms of high-performance liquid chromatography chromatograms and competitive binding capacity for ERα, suggesting that the PF tube is biocompatible and is useful for reducing the elution of substances capable of binding to ERα. Presented in part at the 42nd Congress of the Japanese Society for Artificial Organs, October 5–7, 2004, Tokyo, Japan     

Significance of an insular component in follicular thyroid carcinoma with distant metastasis at initial presentation

Risk factors for distant metastasis were studied in 82 patients with follicular thyroid carcinoma (FTC). Metastases to either the lung or bone existing at the time of presentation were confirmed by I-131 radio-iodine uptake in 10 patients. FTC with an insular component was found in eight patients. Univariate analysis of 14 possible risk factors showed 7 to be statistically significant: insular component, poorly differentiated carcinoma, trabecular component, serum thyroglobulin level before surgery, patient age at the time of presentation, solid component, and vascular invasion (ranked by p values). After further analysis of the interrelation of the factors and of the logistic regression curves, we concluded that presence of an insular component and patient age were the only independent risk factors. Distant metastasis was not detected in any of the 27 patients ≤49 yr old. Among the 55 older patients (≥50 yr old), 5 of the 49 (10%) without an insular component and 5 of the 6 (83%) with an insular component had distant metastasis. The remaining older patient with an insular component but without distant metastasis showed a gradual increase in thyroglobulin levels after total thyroidectomy.     

Interaction of poly(styrene sulfonic acid) with the alternative pathway of the serum complement system

Bioartificial pancreas, in which the islets of Langerhans are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement reaction. In this study, we examined the effects of poly(styrene sulfonic acid) (PSSa) on the alternative pathway of the serum complement system to identify the mechanism(s) involved. PSSa was dissolved in pooled normal human serum (NHS), and the mixtures were incubated at 37 degrees C for 30 min. Complement activities in sera were determined by hemolytic assays. Amounts of complement activation products released were determined by ELISA. Interactions of PSSa with complement components and fragments were examined with electrophoresis and immunoblotting. From these examinations, it appeared that the manner of PSSa effects on the alternative pathway (AP) highly depends on its concentration. PSSa seemingly acted as an activator when its concentration was 0.005 g/dl to 0.05 g/dl, while it acted as an inhibitor when its concentration was more than 0.1 g/dl. In terms of activation or inhibition of the AP, forming complex of PSSa with factor H induced activation, and that with factor D induced inhibition.     

IL-6-STAT3 controls intracellular MHC class II alphabeta dimer level through cathepsin S activity in dendritic cells

We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alphabeta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alphabeta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alphabeta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alphabeta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alphabeta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alphabeta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.     

Histological studies on the ontogeny of bovine gut-associated lymphoid tissue: appearance of T cells and development of IgG+ and IgA+ cells in lymphoid follicles

The development and distribution of lymphocyte subsets in bovine gut-associated lymphoid tissues (ileal and jejunal Peyer's patches (PP)) were examined. Before birth, the composition of lymphocyte subsets in both PP follicles did not differ except for the dimensions of the interfollicular area and the dome region. Many IgM+ cells were observed in these follicles, but very few CD3+, IgG+, and IgA+ cells could be found. At neonatal period, the IgG+ cells, which did not produce IgG mRNA, were dominant within both PP follicles. From 1 month after birth, many CD3+ cells, IgG mRNA expression, and IgA mRNA expression were detected within the jejunal PP follicles, but very few were in the ileal PP follicles. These data suggest that the characteristics of the jejunal PP follicles metamorphose into secondary lymphoid tissue such as germinal centers at around 1 month after birth, whereas the characteristics of ileal PP follicles were distinct from those of germinal centers.     

Expression of tocopherol-associated protein in mast cells

Tocopherol-associated protein (TAP) was expressed in mouse mast cells. TAP was predominantly localized in the cytoplasm, and the subcellular localization was not changed by alpha-tocopherol. The results suggest that the physiological role of TAP in mast cells is not regulation of tocopherol function but an as-yet-unidentified activity.     

A consistent region of deletion on 1p36 in meningiomas: identification and relation to malignant progression

We analyzed the genetic aberrations on chromosome arms 1p, 10q, and 14q, which are thought to be loci that include putative tumor suppressor genes in meningiomas. We initially conducted molecular genetic testing on a total of 72 tumors including 15 atypical and 8 anaplastic meningiomas using double-target fluorescence in situ hybridization. An incidence of deletion of 1p was observed in 16.3% of histologically benign, 86.7% of atypical, and 87.5% of anaplastic meningiomas. Microsatellite analysis for loss of heterozygosity on 1p, 10q, and 14q was performed in 15 tumors (6 benign, 6 atypical, and 3 anaplastic meningiomas). We detected a limited deleted region on 1p36 in two tumors and suggest a new consistent region of deletion at 1p36.21p23 distal to D1S507 and proximal to D1S214, which spans 8.21 megabases. In addition, loss of 10q was detected in two of three secondary atypical meningiomas, and loss of 14q in two of three primary anaplastic meningiomas. We suggest that one of the putative suppressor genes is located at 1p36.21p23, and that 10q loss may contribute to the malignant progression from benign to atypical meningiomas.