Identification of the optimal structure required for a Shiga toxin neutralizer with oriented carbohydrates to function in the circulation

Shiga toxin (Stx) is a major virulence factor of Stx-producing Escherichia coli. Recently, we developed a therapeutic Stx neutralizer with 6 trisaccharides of globotriaosyl ceramide, a receptor for Stx, in its dendrimer structure (referred to as "SUPER TWIG [1]6") to function in the circulation. Here, we determined the optimal structure of SUPER TWIG for it to function in the circulation and identified a SUPER TWIG with 18 trisaccharides, SUPER TWIG (2)18, as another potent Stx neutralizer. SUPER TWIGs (1)6 and (2)18 shared a structural similarity, a dumbbell shape in which 2 clusters of trisaccharides were connected via a linkage with a hydrophobic chain. The dumbbell shape was found to be required for formation of a complex with Stx that enables efficient uptake and degradation of Stx by macrophages and, consequently, for potent Stx-neutralizing activity in the circulation. We also determined the binding site of the SUPER TWIGs on Stx.     

Sensitization against pancreatic antigen in Sj?gren's syndrome and chronic pancreatitis. Preliminary communication.

A fractionated pancreatic antigen was prepared using a monoclonal antibody, SP3-1, which reacts with the duct cells of various exocrine organs. The cell-mediated immune response to this antigen was studied by the leukocyte migration inhibition test (LMT) in patients with chronic pancreatitis (CP), Sj?gren's syndrome (SjS), and chronic sialoadenitis (CSA). The migration index (MI) for the LMT was 0.97 +/- 0.07 in normal controls (mean +/- SD, n = 11, range: 0.88-1.08). A positive LMT result (MI less than 0.82 = mean -2 SD in controls) was obtained in 7/8 (88%) SjS patients, 4/22 (18%) CP patients, 2/3 CP patients with SjS, and 0/3 CSA patients. These results indicated sensitization against the pancreatic antigen in most SjS patients and some CP patients, and may suggest that an immune response to a common antigenic determinant of the duct cells of exocrine glands plays a role in the pathophysiology of both diseases.     

Overexpression of src family gene for tyrosine-kinase p59fyn in CD4-CD8- T cells of mice with a lymphoproliferative disorder.

Overexpression of a src family gene, lck, has been associated with differentiation of the murine thymic lymphoma line LSTRA. Recent findings by several groups strongly suggest a functional role for the gene product p56lck protein-tyrosine kinase (PTK) in the activation of normal T cells. A single recessive gene, lpr or gld, induces a lymphoproliferative disorder concomitant with autoimmune disease in mice. In this study, a 10-fold elevated activity of PTK encoded by fyn, another src family gene, was demonstrated in CD4-CD8- T cells in mutant mice. The increased PTK activity was consistent with overexpression of fyn mRNA. The elevated fyn mRNA expression appeared to be a characteristic of CD4-CD8- T cells, since it was not observed in normal T cells at any stage of differentiation. The fact that fyn mRNA expression was markedly induced in normal T cells by mitogenic stimulation with anti-T3 epsilon antiserum supports the possibility that p59fyn PTK is a signal-generating molecule in T cells. Thus, our findings provide insight into the physiological role for a src gene family kinase in T-cell development and contribute to a better understanding of the molecular mechanisms of disease-inducing recessive genes.     

Mutations of the p53 and ras genes in childhood t(1;19)-acute lymphoblastic leukemia

We have investigated the alterations of p53 and ras genes including H-, K-, and N-ras genes in 22 acute lymphoblastic leukemia (ALL) cases and five cell lines carrying t(1;19) by use of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis and direct sequencing. The mutations of the p53 gene were found in 2 of 20 t(1;19)-ALL cases at diagnosis (10%), all of 4 cases at relapse (100%), and 4 of the 5 cell lines (80%). Four of the five patients who died had missense mutations at codons 49, 177, 179, and 248. In cases examined sequentially, one had the same point mutation at codon 179 at both diagnosis and relapse, and another had the same p53 gene mutation at codon 240 both in leukemic cells at relapse and in a cell line derived at that time. The other case had no mutation at diagnosis but had the mutation at codon 177 at relapse and cell lines derived from blast cells at diagnosis, suggesting that a small number of leukemic cells with the p53 gene mutation at diagnosis might have escaped PCR-SSCP analysis. In cell lines, SCMC-L9 had three point mutations in the p53 gene at codons 175, 248, and 358, whereas SCMC-L10 had frame shift at codons 209-211. One case had a rare polymorphism at codon 11. We found only one mutation of the N-ras gene that was a 2-bp substitution of GGT(Gly) to GTC(Val) at codon 13 among 22 t(1;19)-ALL cases and five cell lines. This case showed no mutation of the p53 gene and has had a good course. These results suggest that in t(1;19)-ALL, mutations of the p53 and ras genes are infrequent at diagnosis and that p53 gene alterations may be associated with relapse phase or progression of t(1;19)-ALL.     

Pharmacobezoar complicating treatment with sodium alginate

We encountered a gastric bezoar that had developed in a 9-year-old girl treated with sodium alginate (Alloid G) for acute gastritis associated with systemic lupus erythematosus. A hard mass palpated in the left upper abdomen proved, upon gastric endoscopy, to be an intragastric foreign body. Sodium alginate was detected in an analysis of a sample from this bezoar. In an in vitro simulation, sodium alginate solidified when mixed with the patients other medicines. The bezoar caused no complications, and disappeared spontaneously after discontinuation of the medications. This case indicates that this sodium alginate preparation, Alloid G, can be a cause of pharmacobezoar.     

Serum markers of liver fibrosis and histologic severity of fibrosis in resected liver

BACKGROUND/AIMS: Serum concentrations of the 7S fragment of type IV collagen (7S collagen), amino-terminal propeptide of type III procollagen (PIIIP), and hyaluronic acid (HA) have been reported to serve as serologic markers of liver fibrosis in hepatitis and cirrhosis. We investigated whether these fibrosis markers reliably reflect histologic changes in the livers of patients with hepatocellular carcinoma. METHODOLOGY: Subjects included 165 patients undergoing liver resection for hepatocellular carcinoma. Most were seropositive for chronic hepatitis B or C. Histopathologic changes in liver tissue resected with the tumor were scored according to Knodell's histologic activity index. Serum was sampled for assays shortly before surgery. RESULTS: Significant correlations were found between hepatitis activity score and 7S collagen, PIIIP, and HA. Concentrations of 7S collagen differed significantly between activity grades, but differences were not significant for PIIIP or HA. Significant correlations were found between fibrosis staging score and all these three markers. When patients were divided according to activity grade, 7S collagen showed stronger correlation with fibrosis staging score than did PIIIP or HA. CONCLUSIONS: The 7S collagen fragment correlated more strongly than PIIIP or HA with stage and activity grade in patients with hepatocellular carcinoma. However, overlapping of results between histologically defined groups appeared to limit clinical diagnostic usefulness of all markers in individual patients.     

A prospective clinical study of theophylline safety in 3810 elderly with asthma or COPD

A large-scale prospective study was conducted in 3810 Japanese elderly (> or =65 years old) patients with asthma or chronic obstructive pulmonary disease (COPD) who had been treated with sustained-release theophylline tablets (THEODUR) at a dose of 400 mg/day for 1-6 months, in principle. Among 3798 protocol-complying patients (mean age: 73.8 +/- 0.10 years, 1997 with COPD), 261 theophylline-related adverse events were observed in 179 (4.71%) patients. The 5 most frequently observed adverse events were "nausea" (40 episodes, 1.05%), "loss of appetite" (22 episodes, 0.56%), "hyperuricemia" (16 episodes, 0.42%), "palpitation" (15 episodes, 0.39%), and "increased alkaline phosphatase" (11 episodes, 0.28%). No convulsions were reported. Six patients had serious adverse events. The incidence of theophylline-related adverse events was higher in patients with hepatic disease (odds ratio: 1:1.81) and in patients with arrhythmia (odds ratio: 1:1.88). Blood drug concentration measurements in 736 patients indicated that the drug levels were < or =15 microg/ml in 641 patients (87.1%), and no correlation was noted between dose and theophylline-related adverse events. These results suggest that sustained-release theophylline can be used safely in elderly patients with asthma or COPD.