Purification and some properties of a Vero toxin from a human strain of Escherichia coli that is immunologically related to Shiga-like toxin II (VT2)

A cytotoxin to Vero cells (Vero toxin), which was immunologically related to Shiga-like toxin II (SLT-II) (or VT2), was purified from a stain of Escherichia coli isolated from a patient with hemolytic uremic syndrome. The toxin was active on Vero cells but much less active on HeLa cells, a property similar to that of the recently identified SLT-II variant from E. coli strains that caused edema disease of swine. Thus the toxin purified in this report was tentatively named Shiga-like toxin II variant (Vero toxin 2 variant). The purification procedures consisted of ammonium sulfate fractionation, DEAE-Sepharose CL-6B column chromatography, chromatofocusing column chromatography, and repeated high performance liquid chromatography (HPLC) on TSK-gel G-2000SW column and on TSK-gel DEAE-5PW columns. About 90 micrograms of purified toxin was obtained from 451 of the culture supernatant with a yield of about 16%. The purified toxin consisted of A and B subunits of molecular sizes similar to those of SLT-II (VT2). The isoelectric point of the purified toxin was 6.1, which was different from that of SLT-II (VT2) (pI = 4.1). In an Ouchterlony double gel diffusion test, purified toxin and SLT-II (VT2) formed precipitin lines with spur formation against anti-purified toxin and anti-SLT-II (anti-VT2), respectively. The purified toxin was cytotoxic to Vero cells, about 6 pg of the toxin killing 50% of the Vero cells, and showed lethal toxicity to mice when injected intraperitoneally, the LD50 being about 2.7 ng per mouse.     

Depolarization thresholds for hippocampal damage, ischemic preconditioning, and changes in gene expression after global ischemia in the rat

Induced ischemic tolerance in rat hippocampus was investigated in a forebrain ischemia model of repeated 4-vessel occlusion (4-VO). Ischemic insult variability was reduced by the use of dc potential measurements to determine the duration of ischemic depolarization in hippocampus. The results demonstrate a depolarization threshold for ischemic injury to CA1 neurons of 4-6 min and a window for optimal preconditioning of 2.5-3.5 min. Levels of induced mRNAs encoding hsp72 and several immediate-early genes were also shown to vary with depolarization interval. Immediate-early genes were maximally induced after depolarization periods inducing optimal preconditioning, while hsp72 expression increased with insult severity over the range leading to neuron loss. These results are similar to those obtained in gerbil studies indicating that preconditioning does not require large increases in hsp72 expression, and demonstrate the fundamental comparability of rodent global ischemia models when monitored by this approach.     

Immunohistochemical Heterogeneity Within Clinically Nonfunctioning Pituitary Adenomas

To investigate whether adenohypophysial hormone expression is heterogeneous within individual clinically nonfunctioning pituitary adenomas, immunohistochemical examinations were performed on tissues obtained by multiple sampling of 11 adenomas. Stained sections were assessed by morphometric image analysis as well as semiquantitative estimation under microscopy. All tumors except one were immunopositive for one or more gonadotropins. Results were divided into five grades based on the proportion of immunoreactive cells per section. Semiquantitative estimation showed only a one-grade difference among samples from the same tumor in four cases for FSHβ and in two cases for LHβ. These qualitative similarities between multiple samples were confirmed by morphometric image analysis. From the practical standpoint of making a diagnosis of nonfunctioning pituitary adenoma, it is not necessary to take into account immunohistochemical heterogeneity within an individual tumor, and immunohistochemical findings in a given sample obtained at surgery can be regarded as representative of the entire adenoma.     

Comparison of low attenuation areas on computed tomographic scans between inner and outer segments of the lung in patients with chronic obstructive pulmonary disease: incidence and contribution to lung function

BACKGROUND: The low attenuation areas on computed tomographic (CT) scans have been reported to represent emphysematous changes of the lung. However, the regional distribution of emphysema between the inner and outer segments of the lung has not been adequately studied. In this study the regional distribution of low attenuation areas has been compared by quantitative CT analysis and the contribution of the regional distribution to pulmonary function tests evaluated in patients with chronic obstructive pulmonary disease (COPD). METHODS: Chest CT images and the results of pulmonary function tests were obtained from 73 patients with COPD. The lung images were divided into inner and outer segments in the upper (cranial), middle, and lower (caudal) sections. The percentage ratio of low attenuation area to corresponding lung area (LAA%) was then calculated. The LAA% of each segment was also compared with the results of pulmonary function tests. RESULTS: The mean (SD) LAA% of the inner segment was 39.1 (18.5) compared with 28.1 (13.2) for the outer segment (p<0.0001). Linear and multiple regression analyses revealed that airflow limitation is closely correlated with the inner segment LAA% of the lower lung. In contrast, the carbon monoxide transfer factor is closely correlated with the inner segment LAA% of the upper lung. CONCLUSION: Low attenuation areas on CT scans are more often found in the inner segment of the lung than in the outer segment, and the contribution of the inner segment to pulmonary function tests may be greater than the outer segment.     

Suppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the high-affinity antibody response in C57BL/6 mice.

In the humoral immune response to an invasion of foreign antigens, B cells differentiate into low-affinity antibody-forming cells (AFCs) that mainly secrete IgM or, through germinal center (GC) formation, into high-affinity AFCs that secrete IgG-class antibodies with a higher affinity for the antigen. Previous studies have established the suppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on low-affinity antibody responses to antigens. However, whether and how TCDD affects the high-affinity antibody response to antigens has not yet been clarified. In this paper we investigate the effects of TCDD on GC formation, high-affinity AFC generation, and high-affinity antibody production in the primary humoral immune response. C57BL/6 mice were orally administered 0 or 20 microg/kg of TCDD and subsequently immunized with alum-precipitated ovalbumin (OVA) on day 0. Then the GC formation in the spleen and OVA-specific antibodies in the plasma, was evaluated until day 14 postimmunization. TCDD exposure reduced the production of OVA-specific IgG1 on days 10 and 14. GC formation in the spleen was also suppressed by TCDD exposure, and the suppression persisted from day 7 until day 14. In TCDD-administered mice, on day 7, cellular proliferation in the GCs was significantly suppressed, although apoptosis was not markedly affected. In order to measure high-affinity antibody and high-affinity AFCs, the mice were administered TCDD followed by immunization with alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl linked to chicken gamma-globulin (NP-CG). The frequency of high-affinity NP-specific AFCs that bind to low-haptenated antigen was clearly shown to be reduced in the spleen on days 10 and 14. Furthermore, the high-affinity anti-NP IgG1 levels on days 10 and 14 postimmunization were significantly reduced by TCDD exposure. Taken together, the results of this paper demonstrate that TCDD exposure inhibits the generation of high-affinity AFCs and high-affinity antibody production during the primary humoral immune response and suggest that these alterations were caused by the suppression of antigen-responding B-cell proliferation induced by TCDD during GC formation.     

Role of Sialylglycoconjugate(s) in the Initial Phase of Metastasis of Liver-metastatic RAW117 Lymphoma Cells

To elucidate the early events of blood-borne metastasis under actual blood flow, real-time trafficking of RAW117 large cell lymphoma cells, namely parental RAW117-P and liver-metastatic RAW117-H10 cells, was investigated using positron emission tomography (PET). Both types of cells accumulated in the liver immediately after injection via the portal vein, and were eliminated from the liver time-dependently. The elimination rate of RAW117-H10 cells, however, was slower than that of RAW117-P cells, suggesting that RAW117-H10 cells interact more strongly with hepatic sinusoidal endothelium than the parental cells. This result correlated with the metastatic potential of these cells: RAW117-H10 cells metastasized in the liver to a greater extent than RAW117-P cells after injection via this route. To investigate the role of sialylglycoconjugates in the interaction of RAW117-H10 cells with the hepatic endothelium after injection via the portal vein, the trafficking of RAW117-H10 cells was examined after the cells had been treated with sialidase. The elimination rate of RAW117-H10 cells from liver was observed to be greatly accelerated by sialidase treatment. To elucidate what kind of sialylglycoconjugates is related to this phenomenon, we analyzed the distribution of sialyl Lewis A and sialyl Lewis X antigens of both sublines of RAW117 by using flow cytometry. RAW117-H10 cells were found to express a much higher level of sialyl Lewis A than RAW117-P cells, whereas the amount of sialyl Lewis X did not differ significantly. These findings suggest that some sialylglycoconjugates, perhaps sialyl Lewis A in particular, play an important role in the initial interaction of RAW117-H10 cells with the hepatic endothelium, leading to metastasis.     

Relationship between brain zinc and transient learning impairment of adult rats fed zinc-deficient diet

Because of the reported presence of both CART peptide and NOS activity in the same hypothalamic nuclei, their colocalization was examined. Eighteen percent of the neurons in the supraoptic nuclei, and 16% of the neurons in the paraventricular nucleus contained both CART immunoreactivity and NOS activity. Many other neurons in these regions stained for only one marker although they were often close by. Thus, CART peptides and NO may interact in these regions.     

Case of follicular mucinosis showing brownish yellow and red dots via dermoscopy

We herein describe a 68‐year‐old man with follicular mucinosis. A dermoscopic examination showed multiple, round, brownish yellow dots with a whitish rim in the follicular ostium and red dots in the interfollicular area. This case report is the first to suggest that follicular mucinosis shows these dermoscopic findings.     

Relationship of coffee consumption with a decline in kidney function among patients with type 2 diabetes: The Fukuoka Diabetes Registry

Aims/IntroductionThe evidence regarding the effects of coffee consumption on incident chronic kidney disease is inconclusive, and no studies have investigated the relationship in patients with diabetes. We aimed to prospectively investigate the relationship between coffee consumption and the decline in estimated glomerular function rate (eGFR) in patients with type 2 diabetes.Materials and MethodsA total of 3,805 patients (2,112 men, 1,693 women) with type 2 diabetes (mean age 64.2 years) and eGFR ≥60 mL/min/1.73 m² were followed (completion of follow up, 97.6%; median 5.3 years). Coffee consumption was assessed at baseline. The end‐point was a decline in eGFR to <60 mL/min/1.73 m² during the follow‐up period.ResultsDuring follow up, 840 participants experienced a decline in eGFR to <60 mL/min/1.73 m². Higher coffee consumption reduced the risk of decline in eGFR. Compared with no coffee consumption, the multivariate‐adjusted hazard ratios (95% confidence intervals) were 0.77 (0.63–0.93) for less than one cup per day, 0.77 (0.62–0.95) for one cup per day and 0.75 (0.62–0.91) for two or more cups per day (P for trend 0.01). This trend was unaffected by further adjustment for baseline eGFR and albuminuria. The mean eGFR change per year was −2.16 mL/min/1.73 m² with no coffee consumption, −1.89 mL/min/1.73 m² with less than one cup per day, −1.80 mL/min/1.73 m² with one cup per day and −1.78 mL/min/1.73 m² with two or more cups per day (P for trend 0.03).ConclusionsCoffee consumption is significantly associated with a lower risk of decline in eGFR in patients with type 2 diabetes.