Evaluation of norovirus removal performance in a coagulation-ceramic microfiltration process by using recombinant norovirus virus-like particles.

Norovirus (NV) is a prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis worldwide. Because of the lack of a cell culture system or an animal model for this virus, studies on drinking water treatment such as separation and disinfection processes are still hampered. In the present study, we investigated NV removal performance as particles during a coagulation-ceramic microfiltration (MF) process by using recombinant NV virus-like particles (rNV-VLPs), which are morphologically and antigenically similar to native NV. We also experimentally investigated the behaviors of two widely accepted surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, for comparison with the behavior of rNV-VLPs. More than 4-log removal was observed for rNV-VLPs with a 1.08 mg-Al/L dose of polyaluminium chloride in the coagulation-ceramic MF process. This high removal ratio of rNV-VLPs satisfies the U.S. Environmental Protection Agency requirement of 4-log removal or inactivation. In addition, the removal ratios of Qbeta and MS2 were approximately 2-log and 1-log, smaller than the ratio of rNV-VLPs. Accordingly, both bacteriophages have the potential to become appropriate surrogates for native NV in the coagulation-ceramic MF process, and, of the two, Qbeta is the more conservative surrogate.

Comparison of behaviors of two surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, during the aluminum coagulation process.

Differences in the behaviors of two surrogates for pathogenic waterborne viruses, F-specific RNA bacteriophages Qbeta and MS2, were investigated during the coagulation process by using river water spiked with these bacteriophages. The particle size and electrophoretic mobility of Qbeta and MS2 were similar, but the removal performances of infectious Qbeta and MS2, as measured by a plaque forming unit (PFU) method, differed markedly during the coagulation process. The removal ratio of the infectious Qbeta concentration was approximately 2log higher than that of the infectious MS2 concentration at all coagulant doses tested. The total Qbeta and MS2 bacteriophage concentrations, which were measured by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method and represented the total number of bacteriophages regardless of their infectivity, were similar after the coagulation process, suggesting that the behaviors of Qbeta and MS2 as particles were similar during the coagulation process. The difference between total concentration and infectious concentration indicated that some of the bacteriophages were probably inactivated during the coagulation process. This difference was larger for Qbeta than MS2, meaning that Qbeta was more sensitive to the virucidal activity of the aluminum coagulant. Analysis of the PFU and real-time RT-PCR findings together suggested that the difference in removal performances of Qbeta and MS2 during the coagulation process was probably caused by differences not in the extent of bacteriophage entrapment in the aluminum floc particles but in the sensitivity to virucidal activity of the aluminum coagulant.

Pivotal role of 5-lipoxygenase in the activation of human eosinophils: platelet-activating factor and interleukin-5 induce CD69 on eosinophils through the 5-lipoxygenase pathway.

CD69 is an activation-related cell surface molecule on human eosinophils. It has been reported that interleukin (IL)-5, but not platelet-activating factor (PAF), can induce CD69 on human eosinophils in vitro. In this study, PAF induced CD69 intensely on eosinophils from patients with hypereosinophilic syndrome (HES), while only weakly on those from normal donors. Because HES eosinophils contain abundant cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), we examined the roles of several enzymes involved in the metabolism of arachidonic acid in the PAF- or IL-5-induced CD69 expression on eosinophils. The CD69 expression induced by PAF and IL-5 on HES eosinophils and that by IL-5 on normal eosinophils were both inhibited by AA861 and MK-886, inhibitors of 5-LO activity. In addition, AACOCF3, a selective cPLA2 inhibitor, inhibited IL-5-induced CD69 expression on normal eosinophils, although it hardly affected either IL-5- or PAF-induced CD69 expression on HES eosinophils. Moreover, PAF alone induced CD69 only weakly on normal eosinophils, but exogenous arachidonic acid remarkably enhanced PAF-induced CD69 expression on them. These findings suggest that IL-5 activates both cPLA2 and 5-LO but PAF activates only 5-LO. It is suggested that 5-LO plays a critical role in the induction of CD69 on eosinophils.

Induction of the activation-related antigen CD69 on human eosinophils by type IIA phospholipase A2.

OBJECTIVE: High levels of human type IIA phospholipase A2 (PLA2-IIA) have been found in the eosinophil-mediated inflammation sites, although the pathophysiological role of PLA2-IIA in the eosinophil activation has remained poorly understood. We investigated the effects of PLA2-IIA on eosinophil activation. METHODS: Eosinophils were incubated with recombinant human PLA2-IIA or other stimuli, and then eosinophil peroxidase (EPO) (by colorimetric assay) and leukotriene C4 (by enzyme immunoassay) released in the incubation buffer were measured. Expression of CD11b and CD69 on the cell surface was also measured by flow cytometry (by mean fluorescence intensity (MFI)). EPO, LTC4, and CD11b are thought to be markers for early phase activation (occurred in an hour after stimulation), and CD69 is to be a marker for late phase activation (occurred after several hours). RESULTS: While PLA2-IIA (5 microg/ml) did not induce any early phase activation, it induced significant expression of an activation-related antigen, CD69, on human blood eosinophils. The PLA2-IIA, when enzymatically inactivated by either p-bromophenacyl bromide or EDTA, lost its effect on the CD69 induction. Similarly to PLA2-IIA, several lysophospholipids (1 microg/ml) also induced CD69 on eosinophils significantly (control, 0.71 +/- 0.11; PLA2-IIA, 3.29 +/- 0.37*; lysophosphatidic acid, 2.57 +/- 0.43*; specific MFI +/- S.E.M., n = 4, * indicate p < 0.05 vs. control). CONCLUSIONS: PLA2-IIA induces CD69 expression on the eosinophils through its catalytic activity at least partly via the enzymatic products such as several lysophospholipids from the eosinophil membrane phospholipids. PLA2-IIA may contribute to the eosinophilic inflammation synergistically with other factors.

Reevaluation of discograms not classified into usual classifications.

Discograms of images that were eccentrically dyed because of insufficient infiltration of contrast medium are difficult to classify into the usual past discogram patterns. In this study, these types of images were detected in 40 discs of 36 patients with lumbar disc disease. We classified these images into the following three types, and analyzed the dye mechanisms in each case by computed tomography discographic findings: (1) type A (image of the annulus fibrosus only). Nine discs in nine cases. A part of the marginal annulus fibrosus was dyed. (2) type B (image of the right or left half of the nucleus pulposus). Eighteen discs in 15 cases. Unilateral dyeing was considered nucleus pulposus existing in the central region of the disc. (3) type C (partial image of the superior or inferior half of the nucleus pulposus). Thirteen discs in 12 cases. Only the superior or inferior half showing a cotton-ball pattern was dyed.

Cytosolic phospholipase A2, increased and activated in the eosinophils of patients with hypereosinophilic syndrome in vivo, is involved in the augmented release of leukotriene C4.

OBJECTIVE: Eosinophils from patients with hypereosinophilic syndrome (HES) showed augmented release of leukotriene C4 (LTC4) by stimulation with A23187. In the present study, we investigated the involvement of cytosolic phospholipase A2 (cPLA2) in this phenomenon. METHODS: Eosinophils from normal and HES donors (2.5 x 10(6) cells/ml) were incubated with A23187 (0.03-3 microM) for 60 min in the presence or absence of a cPLA2 inhibitor, AACOCF3. The LTC4 released from eosinophils was measured by enzyme immunoassay. Distribution of cPLA2 and 5-lipoxygenase (5-LO) proteins within the eosinophils were detected by Western immunoblotting. RESULTS: The level of LTC4 released from the HES eosinophils by stimulation with A23187 was higher than that from normal eosinophils. The A23187-induced LTC4 release was inhibited by AACOCF3 in a dose-dependent manner. The amounts of cPLA2 seemed to be increased in the non-stimulated HES eosinophils by an analysis of immunoblotting. To be noticed was that cPLA2 was detected as a phosphorylated and membrane-bound form in the HES eosinophils, but not in the normal eosinophils. In contrast, localization of 5-LO within the eosinophils under A23187 stimulation was not different between normal and HES donors, while the amounts of 5-LO also seemed to be increased in the HES eosinophils. CONCLUSIONS: These results suggest that cPLA2, increased and activated (phosphorylated and membrane-translocated) in vivo, is involved in the augmented release of LTC4 from the HES eosinophils.

Consistency of lumbar discograms of the same disc obtained twice at a 2-week interval: influence of needle tip position.

: Although numerous papers have emphasized the importance of accurate needle positioning in lumbar discography, no concrete evidence is available to support this contention, and no study has evaluated the image consistency of discography as influenced by this factor. By observing the consistency of two images in relation to needle tip position we aimed to clarify the importance of needle positioning in discography. One hundred and ninety-two patients (324 discs) receiving steroid intradiscal therapy in whom discography of the same disc was performed twice at a 2-week interval and in whom the needle tip position was within the acceptable range (as defined by us) were studied. The patients were divided into two groups: in group G, in whom the needle tip was within a limited range on both discograms, and group P, in whom the needle tip was in this range on only one discogram. Image consistency was compared roentgenographically in the two groups. The consistent image rate for the total number of discs was 48.5%, being significantly higher in group G (53.2%) than in group P (39.0%). The rates were lower in the nucleus pulposus and the posterior portion of the disc than in the other disc areas, but were significantly higher in group G (85. 4% and 75.0%, respectively, for these two areas). The necessity for accurate needle tip positioning was proved roentgenographically.

Effects of long-term ursodeoxycholate administration on expression levels of secretory low-molecular-weight phospholipases A2 and mucin genes in gallbladders and biliary composition in patients with multiple cholesterol stones.

Group IIA phospholipase A2 (PLA2), a secretory low-molecular-weight PLA2, may play a critical role in the process of gallbladder mucosal inflammation in multiple cholesterol stones, which in turn may produce biliary pronucleating proteins as well as mucin. On the other hand, ursodeoxycholate (UDC) decreases biliary levels of various pronucleating proteins, possibly because of its membrane-protective effects on the inflamed gallbladder mucosa. To elucidate that beneficial effect of UDC, the expression levels of low-molecular-weight PLA2s, group IIA PLA2 (PLA2-IIA), and group V PLA2 (PLA2-V), and mucin core polypeptide genes in the gallbladders were studied for UDC-treated patients and untreated patients with multiple cholesterol stones. Furthermore, the results were correlated with alterations in biliary composition. With long-term administration of UDC, the PLA2-IIA protein mass (2.7 +/- 0.5 vs. 5.0 +/- 0.4 ng/mg x protein [mean +/- SEM]; P < .01) and steady-state mRNA level, as well as the PLA2-V mRNA level, were significantly decreased in the gallbladders, where the prostaglandin E2 (PGE2) level was concomitantly decreased (190.7 +/- 27.9 vs. 393.6 +/- 55.3 pg/mg x protein; P < .01). In the gallbladder bile, the immunoradiometrically determined PLA2-IIA levels were significantly decreased in the UDC-treated patients (43 +/- 4 ng/dL; P < .01) in comparison with untreated patients (78 +/- 6 ng/dL). Significant decreases were similarly found for total protein, mucin, and free arachidonate concentrations, as well as nucleation activity in the bile. The degree of the changes was found to be rather small in solitary stones. In contrast to the decreased mucin concentration, however, there were no significant changes in the expression levels of mucin core polypeptide genes (MUC1-MUC6) between the UDC-treated and untreated patients. Long-term UDC administration was observed to lower the increased PLA2-IIA protein mass and mRNA level, as well as the PLA2-V mRNA level, in the gallbladders of patients with multiple cholesterol stones, which in turn may be of therapeutic importance in improving the gallbladder mucosal inflammation. Effects of UDC on secretory low-molecular-weight PLA2s as inflammatory mediators may relate to the reported efficacy of UDC treatment in cholesterol gallstone disease.

Surrogate thrombopoietin.

The extracellular domain of human c-Mpl, the receptor for thrombopoietin (TPO), was expressed as a chimeric protein with the interleukin-2 receptor alpha chain on the surface of murine B cell-line B300-19. BALB/c mice were immunized with cells expressing the chimeric protein. The IgG purified from the resulting immune serum immunoprecipitated human c-Mpl. The immune IgG supported proliferation of both stable transfectant Ba/F3 cells expressing whole c-Mpl molecules (c-Mpl-Ba/F3 No. 9) and UT7/TPO cells bearing naturally occurring c-Mpl, whereas it did not support the growth of the untransfected parental Ba/F3 cells. Cell growth was induced using 3 to 100 microg/ml of immune IgG in a dose-dependent manner, but this induction was decreased at doses higher than 100 microg/ml. Non-immune IgG did not affect cell growth of c-Mpl-Ba/F3 No. 9 cells. Although the Fab fragment of immune IgG also immunoprecipitated c-Mpl, it did not support cell growth at concentrations as high as 180 microg/ml, implying that the bivalent binding of receptors by antibodies is essential for cell proliferation. These results suggest that antibodies against human c-Mpl stimulate the proliferation and differentiation of megakaryocytes by their bivalent binding to receptors like TPO.

Antibodies against type II phospholipase A2 prevent renal injury due to ischemia and reperfusion in rats.

This study was performed to determine the involvement of type II phospholipase A2 (PLA2-II) in renal injury caused by ischemia and reperfusion. Ischemia and reperfusion significantly elevated levels of blood urea nitrogen and serum creatinine in rats. These increases were significantly reduced by i.v. administration of rabbit IgG F(ab')2 fragments against rat PLA2-II. Increased levels of acid-stable PLA2 activity in the kidney were caused by ischemia and reperfusion, and were suppressed by administration of anti-PLA2-II F(ab')2. Increased levels of myeloperoxidase activity, a marker of neutrophil infiltration, in the kidney were also reduced after anti-PLA2-II F(ab')2 treatment. These results suggest that PLA2-II plays a pivotal role in pathogenesis of ischemia and reperfusion injury through induction of neutrophil infiltration.