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The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.

Background: A chronic osseous Bankart lesion has traditionally been treated with soft-tissue repair and/or open bone-grafting for a large glenoid defect. We developed an arthroscopic method of osseous reconstruction of the glenoid without bone-grafting. The purpose of this study was to evaluate the postoperative outcomes of our technique for chronic recurrent traumatic anterior glenohumeral instability., Methods: A consecutive series of forty-two shoulders in forty-one patients with chronic recurrent traumatic glenohumeral instability underwent an arthroscopic osseous Bankart repair. All shoulders were evaluated preoperatively with three-dimensionally reconstructed computed tomography, which confirmed an osseous fragment at the anteroinferior portion of the glenoid. The average bone loss in the glenoid was 24.8% (range, 11.4% to 38.6%), and the average fragment size was 9.2% (range, 2.1% to 20.9%) of the glenoid fossa. In all shoulders, a displaced osseous fragment, firmly attached to the labroligamentous complex, was separated from the glenoid neck before reduction and fixation in the optimal position with use of suture anchors. All patients were assessed with use of the scoring systems of Rowe et al. and the University of California at Los Angeles preoperatively and at the final evaluation., Results: The mean duration of follow-up was thirty-four months. At that time, thirty-nine of the forty-two shoulders were rated as having a good or excellent result. The mean Rowe score improved from 33.6 points preoperatively to 94.3 points postoperatively (p < 0.01). The mean score on the University of California at Los Angeles system improved from 20.5 points preoperatively to 33.6 points at the final evaluation (p < 0.01). The average passive external rotation was 75 degrees with the arm at the side and 93 degrees with the arm at 90 degrees of abduction. Two patients had a reinjury. Eventually, thirty-five of thirty-seven patients who were active participants in sports returned to the sport they had played before the injury., Conclusions: Arthroscopic osseous Bankart repair with use of suture anchors yields a successful outcome even in shoulders with a chronic large glenoid defect.

Purpose: The purpose of this study was to compare the functional as well as the structural outcomes of single-row and dual-row fixation after arthroscopic full-thickness rotator cuff repair., Type of Study: Retrospective cohort study., Methods: A consecutive series of 80 shoulders in 78 patients with full-thickness rotator cuff tears was evaluated using the rating scale of the University of California Los Angeles (UCLA) and the shoulder index of the American Shoulder and Elbow Surgeons (ASES) at an average of 35 months (range, 24 to 60 months) after arthroscopic rotator cuff repair. Thirty-nine shoulders were repaired using the single-row technique and 41 shoulders using the dual-row technique. Postoperative cuff integrity was determined through magnetic resonance imaging and was classified into 5 categories: type I, sufficient thickness with homogenously low intensity; type II, sufficient thickness with partial high intensity; type III, insufficient thickness without discontinuity; type IV, presence of a minor discontinuity; type V, presence of a major discontinuity., Results: The average UCLA score improved significantly to 32.4 in the single-row and to 33.1 in the dual-row group. The ASES shoulder index improved significantly to 93.0 in the single-row group and to 94.6 in the dual-row group. However, there was no statistical difference between the groups in the postoperative scores. Postoperative MRI revealed 11 type I, 6 type II, 12 type III, 4 type IV, and 6 type V in the single-row group, and 22 type I, 8 type II, 7 type III, 4 type IV, and no type V in the dual-row group. A statistical difference was observed between the groups (P < .01)., Conclusions: Arthroscopic rotator cuff repair yielded successful functional outcomes without significant difference between single and dual-row fixation techniques. However, dual-row repairs excelled in structural outcome over the single-row technique., Level of Evidence: Level III.

Background: A chronic osseous Bankart lesion has traditionally been treated with soft-tissue repair and/or open bone-grafting for a large glenoid defect. We developed an arthroscopic method of osseous reconstruction of the glenoid without bone-grafting. The purpose of this study was to evaluate the postoperative outcomes of our technique for chronic recurrent traumatic anterior glenohumeral instability., Methods: A consecutive series of forty-two shoulders in forty-one patients with chronic recurrent traumatic glenohumeral instability underwent an arthroscopic osseous Bankart repair. All shoulders were evaluated preoperatively with three-dimensionally reconstructed computed tomography, which confirmed an osseous fragment at the anteroinferior portion of the glenoid. The average bone loss in the glenoid was 24.8% (range, 11.4% to 38.6%), and the average fragment size was 9.2% (range, 2.1% to 20.9%) of the glenoid fossa. In all shoulders, a displaced osseous fragment, firmly attached to the labroligamentous complex, was separated from the glenoid neck before reduction and fixation in the optimal position with use of suture anchors. All patients were assessed with use of the scoring systems of Rowe et al. and the University of California at Los Angeles preoperatively and at the final evaluation., Results: The mean duration of follow-up was thirty-four months. At that time, thirty-nine of the forty-two shoulders were rated as having a good or excellent result. The mean Rowe score improved from 33.6 points preoperatively to 94.3 points postoperatively (p < 0.01). The mean score on the University of California at Los Angeles system improved from 20.5 points preoperatively to 33.6 points at the final evaluation (p < 0.01). The average passive external rotation was 75 degrees with the arm at the side and 93 degrees with the arm at 90 degrees of abduction. Two patients had a reinjury. Eventually, thirty-five of thirty-seven patients who were active participants in sports returned to the sport they had played before the injury., Conclusions: Arthroscopic osseous Bankart repair with use of suture anchors yields a successful outcome even in shoulders with a chronic large glenoid defect.

Background: The majority of gastro-oesophageal reflux disease (GERD) seems to be non-erosive reflux disease. Nonerosive reflux disease includes minimal change oesophagitis (whitish or reddish, oedematous change and erosion that is not regarded as mucosal break) and no endoscopic abnormalities., Aim: To investigate the accurate proportion of those with minimal change oesophagitis and to clarify its characteristics. In addition, we evaluated the effect of famotidine (40 mg/day) in those with minimal change., Methods: Prospective endoscopic assessment was performed for consecutive 606 out-patients. Of the 582 patients suitable for analysis, 347 were non-treated. The latter were divided into those with erosive GERD or minimal change, and their endoscopic findings and characteristics were compared., Results: Among 347 non-treated patients, 88 (25%) had erosive GERD and 249 (72%) had minimal change. Compared with patients who have erosive GERD and those with minimal change, the latter were less likely to have hiatal hernia or bile reflux, but more likely to have gastric atrophy. Symptomatic patients (n = 55) with minimal change oesophagitis were more likely to have hiatal hernia than those who were asymptomatic (n= 194). Most patients preferred taking famotidine on-demand, during a 4-week follow-up period., Conclusions: Most non-erosive reflux disease can be classified as minimal change oesophagitis, and that have different characteristics from erosive GERD. On-demand famotidine may be a suitable alternative treatment for patients with minimal change disease.

Background: In patients with recurrent anterior dislocations of the shoulder, it is well known that the glenoid rim is often deficient (8%-95%). However, little is known regarding the precise location of the bony defect of the glenoid., Hypothesis: The bony defect is anterior rather than anteroinferior to the glenoid., Study Design: Cohort study (symptom prevalence); Level of evidence, 2., Methods: The authors studied 3-dimensional images of the glenoid reconstructed from computed tomography to determine the location of the glenoid defect in 123 shoulders of 123 patients with recurrent anterior dislocations of the shoulder. They measured the location, extent, and orientation of the defect based on the clock face of the glenoid., Results: The defects were located between 12:08 and 6:32, with the range between 2:30 and 4:20 being the most frequent. The extent of the glenoid defect was 106.7 degrees +/- 27.1 degrees (mean +/- standard deviation). The mean orientation of the defect was pointing toward 3:01 on the clock face of the glenoid, at a mean angle of 90.5 degrees +/- 10.4 degrees from the 12-o'clock direction., Conclusion: The glenoid defect is located almost anterior to the glenoid.

Anterior glenoid fractures associated with glenohumeral instability are normally treated with open fixation of the fragment. However, recent technical development enables this procedure to be performed under arthroscopic control using screws or even suture anchors if the fragment is small enough. We developed a unique method of stabilizing a larger fragment with suture anchors and the use of a clear imaging method of three-dimensional computed tomography (3D-CT) with the humeral head eliminated, preoperatively and postoperatively. In the present article, we report a detailed method of arthroscopic stabilization for a consecutive series of 8 patients, with an average of 27% bone loss (fragment size) against the lower part of the circular glenoid portion, using suture anchors.

We describe 3 cases of an all-arthroscopic technique for repair of a humeral avulsion of the glenohumeral ligament (HAGL) lesion and the postoperative clinical outcomes. From a technical perspective, the most critical part of the surgeries was the anchor insertion at an optimal position on the humerus in order to achieve proper tension of the glenohumeral ligament. The arm-free beach-chair position, which facilitates maximum internal rotation, use of a 70 degrees angled arthroscope, and an anterior-inferior trans-subscapularis tendon portal were considered key factors to accomplish this procedure.

In shoulder arthroscopy, cannulas with large diameters (7-9 mm) are commonly used both in the lateral decubitus and the beachchair position because they ensure the easy introduction of instruments into the intra-articular space and provide a clear arthroscopic view through the application of high intra-articular pressure. On the other hand, fluid extravasation and the resulting inconvenience due to the narrowing of the intra-articular working space are also obvious issues. Consequently, this makes arthroscopic stabilization surgery all the more challenging. To avoid these shortcomings of this procedure, we believe cannulas should be used only when necessary such as for knot tying and for transtendon portals. In this article, we report a unique and original method of arthroscopic Bankart repair in the beachchair position with limited use of cannulas of smaller diameter (5 mm) and its advantages over ordinary arthroscopic techniques using large-diameter cannulas in terms of preventing fluid extravasation and keeping a wide working space, which facilitates beneficial intra-articular techniques.

Aim: To investigate if esophageal varices can be evaluated using external contrast-enhanced ultrasonography with Levovist and coded harmonic angio (CHA)., Methods: Subjects were six healthy adult volunteers and 23 patients with liver cirrhosis. After identification of the lower esophagus under B-mode scanning, 300 mg/mL of Levovist was intravenously injected into the cubital vein at a rate of 1 mL/s under observation by CHA-mode scanning. Approximately 30 s after intravenous administration, interval-delay scanning was performed every second to visualize the area around the lower esophageal lumen. The degree of ultrasonographic enhancement was assessed as either no enhancement (negative); linear enhancement along the esophageal wall (weak) or full enhancement of the esophageal lumen (strong). Endoscopic evaluation of esophageal varices was also performed., Results: The CHA enhancement around the lower esophageal lumen was identified in 21 of the 23 patients. Of these 21 patients, endoscopic assessments of varices were as follows: F0 in four patients, F1 in seven patients, F2 in three patients, and F3 in seven patients. Nine patients were red color sign (RCS)-positive. Regarding the relationship between ultrasonographic enhancement and endoscopic assessment, enhancement was identified as negative in all four F0 patients, negative in three and weak in three and strong in one of the seven F1 patients, weak in one and strong in two of the three F2 patients, and weak in two and strong in five of the seven F3 patients, respectively. Furthermore, of the nine RCS-positive patients, enhancement was recognized as strong in seven and weak in two patients. Ultrasonographic enhancement was identified as negative in all six healthy volunteers., Conclusions: By performing contrast-enhanced CHA ultrasonography using Levovist, ultrasonographic enhancement was detectable in all patients with varices categorized as F2 or above. Because the present method is easy to perform and causes less pain to patients compared to endoscopy, it is useful for following and assessing esophageal varices in patients with liver cirrhosis.

We revealed morphology and physicochemical behavior of a widely used powerful hydrophilizing agent, polyvinylpyrrolidone (PVP), present on polysulfone (PS)/PVP films by atomic force microscopy (AFM). This is the first time such clear PS/PVP phase-separated morphology was observed by nanoscopic technique. The film surfaces were observed by the identical observation mode, probe and scanning conditions to reveal the change of PVP morphology and behavior between dry and wet conditions. Morphology was related to biocompatibility by combining AFM data with results of surface element composition, contact angle, adhesion amount of rabbit platelet and relative amount of adsorbed fibrinogen. PVP nano-particles of one or several molecules were formed on the dry PS/PVP film surfaces. Amount of PVP present on the surfaces increased with the molecular weight of PVP. At a mixed amount of 1-5 wt%, PVP K90 formed crowded particles on the dry surface. When wet, they swelled, followed by their union to produce a smooth surface leading to improved biocompatibility. The highest biocompatibility with excellent mechanical strength is achieved by blending the highest molecular weight PVP K90 at 1-5 wt%.

MUC2 mucin is a secretory glycoprotein which is produced from the intestinal goblet cells and is a major component of the intestinal epithelial mucus. The biological function of MUC2 mucin is considered to be the protection of intestinal epithelial surface, whereas the regulatory mechanism of MUC2 mucin production in immune response is not completely understood. We have studied the effects of cytokines, IL-4, IL-13 and TNF-alpha, on the regulation of MUC2 mRNA in the human colonic cancer cell lines, LS174T and HT29. The quantitative reverse transcription-polymerase chain reaction showed that single addition of IL-4, IL-13 and TNF-alpha to cell culture induced about two-fold increase of MUC2 mRNA level in LS174T cells. Interleukin-4 and IL-13 activated phosphorylation of mitogen-activated protein kinase in LS174T cells. A specific inhibitor of mitogen-activated protein kinase pathway, U0126, totally inhibited the increase of MUC2 mRNA by IL-4 or IL-13 in those cells. Therefore, mitogen-activated protein activation of kinase is required for the increase of MUC2 mRNA by IL-4 or IL-13 in LS174T cells. In contrast to LS174T cells, only TNF-alpha increased MUC2 mRNA through a mitogen-activated protein kinase pathway in HT29 cells that express low levels of MUC2 mRNA. These findings sustain a novel phenomenon that MUC2 mRNA expression is differently controlled by IL-4, IL-13, or TNF-alpha in LS174T and HT29 cells, whereas the mitogen-activated protein kinase pathway plays a role in the MUC2 mRNA expression induced by those cytokines in both cell lines.

Background: Knowledge regarding the morphology of the glenoid rim is important when patients with recurrent anterior glenohumeral instability are assessed. Ordinary imaging techniques are not always sensitive enough to demonstrate the morphology of the glenoid rim accurately. We developed a method of three-dimensionally reconstructed computed tomography with elimination of the humeral head to evaluate glenoid morphology. The purpose of the present study was to quantify glenoid osseous defects and to define their characteristics in patients with recurrent anterior instability., Methods: The morphology of the glenoid rim in 100 consecutive shoulders with recurrent unilateral anterior glenohumeral instability was evaluated on three-dimensionally reconstructed computed tomography images with the humeral head eliminated. The configuration of the glenoid rim was evaluated on both en face and oblique views. Concurrently, we also investigated seventy-five normal glenoids, including both glenoids in ten normal volunteers. Shoulders without an osseous fragment at the anteroinferior portion of the glenoid were compared with the contralateral shoulder in the same patient to determine if the glenoid morphology was normal. In shoulders with an osseous fragment, the fragment was evaluated quantitatively and its size was classified as large (>20% of the glenoid fossa), medium (5% to 20%), or small (<5%). Finally, all 100 shoulders were evaluated arthroscopically to confirm the presence of the lesion at the glenoid rim that had been identified with three-dimensionally reconstructed computed tomography., Results: Investigation of the normal glenoids revealed no side-to-side differences. Investigation of the affected glenoids revealed an abnormal configuration in ninety shoulders. Fifty glenoids had an osseous fragment. One fragment was large (26.9% of the glenoid fossa), twenty-seven fragments were medium (10.6% of the glenoid fossa, on the average), and twenty-two were small (2.9% of the glenoid fossa, on the average). In the forty shoulders without an osseous fragment, the anteroinferior portion of the glenoid appeared straight on the en face view and it appeared obtuse or slightly rounded, compared with the normally sharp contour of the normal glenoid rim, on the oblique view, suggesting erosion or a mild compression fracture at this site. Arthroscopic investigation revealed a Bankart lesion in ninety-seven of the 100 shoulders and an osseous fragment in forty-five of the fifty shoulders in which an osseous Bankart lesion had been identified with the three-dimensionally reconstructed computed tomography. In the shoulders with distinctly abnormal morphology on three-dimensionally reconstructed computed tomography, the arthroscopic appearance of the anteroinferior portion of the glenoid rim was compatible with the appearance demonstrated by the three-dimensionally reconstructed computed tomography., Conclusions: We introduced a method to evaluate the morphology of the glenoid rim and to quantify the osseous defect in a simple and practical manner with three-dimensionally reconstructed computed tomography with elimination of the humeral head. Fifty percent of the shoulders with recurrent anterior glenohumeral instability had an osseous Bankart lesion; 40% did not have an osseous fragment but demonstrated loss of the normal circular configuration on the en face view and an obtuse contour on the oblique view, suggesting erosion or compression of the glenoid rim.

Study Design: Prospective descriptive biomechanical analysis of shoulder motion in golf., Objective: To characterize normal shoulder motion during the driving swing in male recreational golfers of various age groups., Background: Shoulder trauma accounts for approximately 12% of all golf-related injuries. To design sport-specific rehabilitation programs for the injured golfer and exercise programs for the healthy golfer, clinicians and teachers need quantitative information describing range of motion requirements about the shoulder for the amateur player. METHOD AND MEASURE: Sixty-five male golfers were divided into 3 age groups: college, middle, and senior. A high-speed, 6-camera motion analysis system recorded 3-dimensional bilateral shoulder motion (vertical elevation, horizontal adduction, external rotation, and shoulder tum) for 3 swings of the driver. Group means for ranges and functional end points of motion were compared using a single-factor 1-way ANOVA (alpha = 0.05)., Results: All maximum values of shoulder motion were lower in the senior group than in the other 2 groups. At peak backswing, senior golfers exhibited 38 degrees less right-side shoulder external rotation than college golfers. However, from address, seniors horizontally abduct the right arm 18 degrees more than college golfers. In the older golfers, total range of motion was reduced for both shoulders in the vertical plane and for the left shoulder in the horizontal plane., Conclusions: This study describes shoulder motion for asymptomatic golfers of various age groups. These data may serve as a baseline reference for assessing disease- or injury-related changes in the golf swing and for designing sport-specific exercise and rehabilitation programs.

A human case of creeping eruption due to Gnathostoma nipponicum was confirmed indirectly by finding infective advanced third-stage larvae in leftover largemouth bass meat. This is the first report indicating that the largemouth bass (Micropterus salmoides) serves as a source of G. nipponicum infection in humans.

To determine the possible role of eosinophils in Angiostrongylus costaricensis infection, both interleukin-5 (IL-5) transgenic (Tg) and non-transgenic (non-Tg) C3H/HeN mice were infected with A. costaricensis third-stage larvae. IL-5 Tg mice demonstrated greater resistance than non-Tg mice to A. costaricensis, as shown by lower adult worm recovery, smaller adults, fewer eggs in the intestinal wall and fewer larvae passed in the feces. Both mice showed similar antigen-specific IgA and IgGI antibody responses, although IgA was more prominent than IgG1. Egg deposition and inflammatory responses in the intestinal walls were milder in IL-5 Tg mice than in non-Tg mice. The eggs with developed larvae, deposited in the intestinal walls of IL-5 Tg mice, were surrounded by numerous degranulating eosinophils and sometimes with Splendore-Hoeppli deposits. The data suggest that eosinophils are involved in the resistance of the mouse during primary infection with A. costaricensis.

A sensitive method for measuring the serum level of protein-induced by vitamin K absence or antagonist II (PIVKA-II) has become so widely available that it is now used for the clinical diagnosis of small hepatocellular carcinoma (HCC). It is known that serum PIVKA-II can be a prognostic indicator for HCC, but there have been no detailed investigations concerning the tissue expression of PIVKA-II. The present study assessed the relationship between serum or tissue PIVKA-II and the biological malignant potential of HCC. The subjects were 25 patients with histologically confirmed HCC, that were solitary and 3 cm or less in diameter. Tissue PIVKA-II was detected by immunostaining using MU-3 as the primary antibody. The biological malignant potential of the tumors was evaluated on the basis of the Ki-67 labeling index of HCC cells and the tumor arterial vascularity assesed by angiography and CO(2) enhanced ultrasonography. The recurrence-free period after treatment was also evaluated. Among the 25 patients, eight were positive for tissue PIVKA-II. Serum PIVKA-II levels were significantly higher in the tissue PIVKA-II-positive patients compared with the negative patients, but serum and tissue PIVKA-II expressions were not consistently parallel. Tumor cell proliferation was closely correlated with the tissue PIVKA-II expression, while the recurrence-free period was correlated with the serum PIVKA-II level. Tumor arterial vascularity showed a strong correlation with the expression of both serum and tissue PIVKA-II. In conclusion, serum and tissue PIVKA-II expression reflect the biological malignant potential of HCC and thus may be useful indicators for the prognosis of small HCC.

Angiostrongylus costaricensis is a nematode parasitic of rodents. Man may become infected by ingestion of the third stage larvae produced within the intermediate hosts, usually slugs from the family Veronicellidae. An epidemiological study carried out in a locality in southern Brazil (western Santa Catarina State) where these slugs are a crop pest and an important vector for A. costaricensis has documented for the first time the natural infection of Deroceras laeve with metastrongylid larvae. This small limacid slug is frequently found amid the folds of vegetable leaves and may be inadvertently ingested. Therefore D. laeve may have an important role in transmission of A. costaricensis to man.

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO(4)) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15-36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15-34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x(L) mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.

Infection with the helminth parasite Schistosoma mansoni is associated with a pathogenic granulomatous response to parasite eggs. Multiple cell types constitute the granuloma with eosinophils achieving numerical dominance. We hypothesize that eosinophil dominance is achieved by selective apoptosis in lymphocytes. We report here that lymphocytes from both the spleens and granulomas of S. mansoni-infected mice undergo apoptosis. We also show that granuloma lymphocytes are more susceptible to Fas-FasL-mediated apoptosis than spleen lymphocytes and this apoptosis may be related to antigen concentration. Conversely, eosinophils from the granuloma and spleens of S. mansoni-infected mice are resistant to apoptosis in vivo and are protected in vitro from Fas-FasL-mediated apoptosis by the absence of FasL expression in the presence of Fas expression. Finally, the apoptotic regulatory molecules Bcl-2, Bcl-xL, and Bax, do not appear to play a significant role in the regulation of eosinophil apoptosis in the schistosome granuloma.

In the present study, we performed an immunohistochemical examination of the expression of transforming growth factor-beta (TGF-beta), TGF-beta type II receptor (TGF-beta RII) and a trefoil peptide, pS2, in the several types of gastric cancer. The TGF-beta:TGF-beta RII ratios of IIc-like type and Ménétrier type closely resembled that of linitis type. pS2 was intensely expressed in cytoplasm of the superficial and foveolar epithelium, as well as in scirrhous type gastric cancers which retained the gastric or intestinal phenotype. However, pS2 expression was significantly (p < 0.05) reduced in linitis type gastric cancers (1 of 6; 17%) when compared with other scirrhous types of gastric cancers (13 of 22; 59%). A decrease in the TGF-beta:TGF-beta RII ratios in linitis type, IIc-like type and Ménétrier type gastric cancers may be associated with the fibrosis seen in these types of cancer. Furthermore, reduction of pS2 expression in linitis type gastric cancer may represent a dedifferentiation of the cancer from gastric or intestinal phenotype. Judging by the expression patterns of TGF-beta, TGF-beta RII and pS2, it is possible that IIc-like type and Ménétrier type gastric cancers are precursor lesions of linitis type gastric cancer., (Copyright 2002 S. Karger AG, Basel)

Five hundred fifty patients hospitalized to our hospital during 1990 to 1999 were studied. Subjects consisted of 413 males and 102 females and mean age was 62.1 years. Association with HBV infection, HCV infection and both infection was 11.1%, 78.4% and 2.5% respectively. According to the criteria based in Liver Cancer Study of Japan, 5 year survival rate in clinical stage I, II, III was 42.3%, 38.8% and 17.5%. Tumor morphology was nodular type in 78.9%, massive type in 9.1% and diffuse type in 10.5% of cases. Portal tumor thrombus and distal metastasis were observed in 19.9% and 6.3% of all cases. The 1-, 3- and 5-year survival rate in patients received any therapy was 80.8%, 44.6% and 28.7%, respectively. Analysis by the proportional hazard model showed that HBV infection, advanced clinical stage, multiple tumors, a tumor diameter in excess of 5 cm and AFP positivity were shown as significant factors on poor prognosis.

C.B-17-SCID/SCID (SCID) mice infected with Angiostrongylus cantonensis yielded a high percentage of worm recovery and did not show any body weight loss until day 24 postinfection. Unexpectedly, C.B-17-+/+(+/+) mice also produced a similar worm burden containing well-developed worms. This is probably attributable to the observation that +/+ mice failed to induce eosinophilia in cerebrospinal fluid (CSF) despite their production of antigen-specific IgA and IgGI; +/+ mice have defective bone-marrow eosinopoiesis, which in turn results in reduced blood and CSF eosinophilia. Interleukin 5 (IL-5) production in +/+ mice is similar to that in BALB/c and C57BL/6 mice. However, bone-marrow eosinopoiesis in response to IL-5 is markedly suppressed in +/+ mice. This is probably associated with impaired expression of common beta-chain mRNA in bone-marrow cells of +/+ mice, which leads to the failure of bone-marrow eosinopoiesis. Hence, +/+ mice may serve as a useful model for the elucidation of eosinophil production in the mouse and for determination of the relationship between parasite infection and the eosinophil.

Aims/background: Although serum PIVKA-II has been widely used as a tumor marker for hepatocellular carcinoma (HCC), little information is available concerning tissue PIVKA-II, and detection of tissue PIVKA-II in paraffin-embedded tissue specimens of HCC is also considered difficult., Methods: Paraffin-embedded HCC tissues obtained at autopsy from 22 patients were subjected to immunohistochemical staining using the antibody MU-3 (Eisai Co., Ltd.) after microwave antigen retrieval., Results: PIVKA-II was mainly detected in the cytoplasm of HCC cells. The intensity of tissue PIVKA-II staining was not correlated with serum PIVKA-II levels or with the histotogical differentiation of HCC. However PIVKA-II staining tended to be more intense in tissue from patients with portal tumor thrombus, distant metastases, or a longer duration of HCC., Conclusion: This method of immunohistochemical staining is easy and simple to use and may be helpful for detecting tissue PIVKA-II in paraffin-embedded HCC specimens.

To elucidate a role of TTV infection in patients with acute liver injury, TTV-DNA in the sera from 97 patients with acute liver injury of various etiology were determined according to Okamoto's method. Out of 77 patients with acute liver injury of determined etiology, 31 patients(40.3%) showed TTV-DNA positive, and out of 15 patients with acute liver injury of undetermined etiology, 8 patients(53.3%) showed TTV-DNA positive. These results suggested no evident role of TTV in patients with acute liver injury was shown. Further study including genotype and quantitative determination of TTV-DNA and antibody assay is needed.

Background: Polaprezinc has been shown to exert an anti-oxidant property in a tube experiment, protect gastric mucosa from experimental ulcerations in vivo, and accelerate the healing of gastric ulcer in humans., Aim: To examine a possible protective effect of polaprezinc on oxidant-mediated injury in primary monolayer cultures of rat gastric fundic mucosa., Methods: Cytotoxicity was quantified by measuring 51Cr release. Whether or not polaprezinc exerts an antioxidant property was investigated by determining the effect of this agent on hydrogen peroxide (H2O2)-induced injury. The effects of polaprezinc on superoxide (O2-. ) generation as well as on ethanol (EtOH)-induced injury were also examined. Generation of O2-. was assessed by the reduction in cytochrome c., Results: H2O2 caused a time- and dose-dependent increase in 51Cr release. The dose-response curve of 51Cr release by H2O2 shifted to the right in the presence of polaprezinc. Polaprezinc, at submillimolar concentrations, prevented H2O2-induced 51Cr release. EtOH also caused a dose-dependent increase in 51Cr release, which was prevented by the addition of polaprezinc. The incubation of cells with EtOH caused an increase in cytochrome c reduction, as the concentrations of EtOH increased. Polaprezinc inhibited EtOH-induced cytochrome c reduction. Protection by polaprezinc was microscopically associated with the prevention of monolayer disruption., Conclusions: Polaprezinc is antioxidative and directly protects gastric mucosal cells from noxious agents through its antioxidant properties in vitro. This finding may provide the theoretical basis for the usage of an antiulcer drug with antioxidant properties for the treatment of gastric inflammation, such as that induced by ethanol.

Eosinophils are a numerically dominant cell population within the schistosome granuloma. These granuloma eosinophils can produce a variety of cytokines, including IL-2, IL-4, IL-5, and IFN-gamma. Therefore, eosinophils may play a key role in the determination of the unique cytokine microenvironment within the granuloma milieu. These studies investigated the potential role of eosinophils in the regulation of granuloma immunopathology. We have characterized spleen- and granuloma-derived eosinophils based on cellular activation and cytokine production during the development of murine schistosomiasis. Based on the criteria of hypodensity and CD69 expression, granuloma eosinophils were highly activated and very homogeneous at 7 and 11 wk postinfection. Splenic eosinophils were also activated at 7 wk postinfection, but were much more heterogeneous than their granuloma counterparts. By 11 wk postinfection, few hypodense splenic eosinophils were observed. Eosinophils represented the majority of cytokine-producing cells in the granuloma and were a dominant source of IL-4. Eosinophils also produced IL-2, IL-5, and IFN-gamma, using the criteria of mRNA in situ hybridization and intracellular cytokine staining by FACS. Granuloma eosinophil activation and cytokine production were greatest at the time of maximum granuloma formation, i.e., 10-12 wk after initial cercarial exposure. Therefore, locally activated eosinophils, not Th2 lymphocytes, produce the majority of Th2 cytokines in the granuloma milieu and may be important determinators of immunopathology in schistosomiasis.

Granuloma formation and its regulation are dependent on lymphocytes. Therefore, we compared the characteristics of lymphocytes derived from the spleens and granulomas of Schistosoma mansoni-infected mice during the course of their disease. We examined lymphocyte cell cycle kinetics, migration, expression of activation Ags (CD69 and IL-2R), cytokine production (IL-2, IL-4, IFN-gamma), and apoptosis. Lymphocytes in the G2/M phase of the cell cycle and high levels of lymphocyte intracellular IL-2 were found in the spleen but not in the granuloma. Cell trafficking experiments showed Ag-specific recruitment of schistosomal egg Ag (SEA)-reactive lymphoblasts into granulomas in vivo, as well as recruitment to, residence within, and egress from granulomas in vitro. Granuloma-derived lymphocytes were more highly activated than splenic lymphocytes based on higher levels of CD69 and IL-2R expression. While the granuloma microenvironment was rich in Th2 cytokines, during peak granuloma formation, the lymphocytes per se from the spleen and granuloma did not exhibit a dominant Th1 or Th2 cytokine profile, producing low but similar levels of IL-4 and IFN-gamma. The discrepancy between high IL-2R expression and low levels of IL-2 protein production by granuloma lymphocytes was associated with increased apoptosis in the granuloma compared with the spleen. These findings support the hypothesis that granulomas may play a role in the regulation of systemic pathology in schistosomiasis by adversely affecting the survival of SEA-reactive, immunopathogenic T lymphocytes.

A 63-year-old woman who had received hemodialysis therapy since she fell acute on chronic renal failure 4 years ago presented with multiple joint pain. Nephrocalcinosis was not detected by abdominal X-ray when hemodialysis therapy was initiated. Laboratory testing showed azotemia, anemia, hypoproteinemia and mild liver dysfunction but no liver cirrhosis. Biopsied bone tissue demonstrated numerous calcium oxalate crystal depositions. Laparoscopy revealed black liver in macroscopic view. Histological studies showed numerous lipofuscin-like dark brown granules were deposited in hepatocytes. The activity of alanine : glyoxylate aminotransferase (AGT) was less than 0.1 U/g in biopsied patient's liver tissue. Generally, clinical symptoms demonstrated by Japanese primary hyperoxaluria type I (PH-I) patients are milder than those of European patients. Some PH-I patients may successfully avoid urinary tract calcification unless they fall into oliguria by some other causes. The lipofuscin granules are most likely the source of the dark color. Massive deposition of the lipofuscin granules indicated that the duration of the liver metabolic abnormality had lasted for long time. Thus, black liver may be related to a mild form of PH-I.

T cell populations and IL-3 mRNA expression were analysed in mesenteric lymph node cells and intestinal intraepithelial lymphocytes (IEL) in Strongyloides ratti-infected mice. On days 7 and 12 post-infection, 2.6 times as many mesenteric lymph node cells were present in S. ratti-infected mice compared with uninfected mice. Although the percentages of CD3+, CD4+ and CD8+ cells decreased during infection, the absolute numbers of these cell types increased on day 7 due to an overall increase in the mesenteric lymph node cell number. The CD4/CD8 ratio in IEL was increased on day 5, whereas no significant change in the CD4/CD8 ratio was observed in the mesenteric lymph node cells. Expression of IL-3 mRNA, which is an important cytokine for the induction of murine mucosal mastocytosis and S. ratti-expulsion, was examined in mesenteric lymph nodes and IEL of uninfected and infected mice. IL-3 mRNA was detected in mesenteric lymph nodes of S. ratti-infected mice but not detected in the lymph nodes of uninfected mice. IL-3 mRNA was detected in IEL from both infected and uninfected mice with an 20-fold increase in expression in IEL of infected mice. Overall, IL-3 mRNA levels were higher in IEL than in mesenteric lymph nodes following S. ratti-infection. Expression of IL-4, IL-10, stem cell factor (SCF or c-kit ligand) and IFN-gamma mRNA was also examined in these two tissues. IL-10 mRNA was not detected in any tissue examined and IFN-gamma mRNA levels were unaltered as a result of an S. ratti-infection. Elevated expression of mRNA for SCF (5-fold) and IL-4 (20-fold) was observed in the mesenteric lymph nodes of infected mice. In contrast, SCF mRNA levels were similar in IEL of uninfected and infected animals and only a modest increase in IL-4 mRNA was observed in IEL of infected mice.

Strain-dependent differences in host morbidity and mortality due to Angiostrongylus cantonensis infection have been established between C57BL/6 and BALB/c mice; C57BL/6 mice show rapid worm killing with low morbidity, whereas BALB/c mice indicate slow worm killing with high morbidity and mortality. To determine the possible roles of CD4+ and CD8+ T-cells in host morbidity and innate resistance to A. cantonensis infection we treated C57BL/6 and BALB/c mice with anti-CD4 or anti-CD8 monoclonal antibody and examined the changes in host morbidity and worm-killing activity. Our study indicates that anti-CD4 antibody treatment interferes with worm killing and improves the morbidity of A. cantonensis-infected BALB/c mice, whereas anti-CD8 antibody treatment fails to improve the morbidity. Tumor necrosis factor-alpha (TNF-alpha, or cachectin) production in infected mice was not correlated with host morbidity. Anti-IL-5 monoclonal antibody treatment also failed to affect the morbidity of infected BALB/c mice, although their worm-killing activity was restrained as shown in anti-CD4-treated mice. These findings clearly indicate that the morbidity of infected BALB/c mice is regulated by some unknown CD4+ T-cell-dependent mechanism but not by an IL-5-, eosinophil-, or TNF-alpha-dependent mechanism.

For determination of the kinetics of cytokine production and its possible role in host resistance to Angiostrongylus cantonensis in the mouse, Th1 [interleukin 2 (IL-2) and interferon gamma (IFN-gamma] and Th2 (IL-5 and IL-4) cytokine production in the cerebrospinal fluid (CSF), sera, and culture supernatants of spleen cells (SC) or cervical lymph-node cells (CLNC) of infected BALB/c and C57BL/6 mice was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA). IL-5 and IL-4 were detected in CSF of both strains, with a peak response occurring at around days 12-15 and 20 postinfection (p.i.), respectively. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay also revealed prominent IL-5 and IL-4 mRNA expression in T-cells but not in eosinophils in CSF. SC and CLNC stimulated with A. cantonensis young adult-worm antigen released IL-5 in vitro at and after day 20 p.i. Contrarily, IFN-gamma production in CSF and SC or CLNC culture supernatants was almost negligible before day 30 p.i. IL-5, IL-4, and IL-2 production in culture supernatants was rather prominent in resistant C57BL/6 mice as opposed to susceptible BALB/c mice as assessed by the magnitude of increase over preinfection levels. Antigen-specific IgG1 (but not IgG2a) responses were more prominent in C57BL/6 mice than in BALB/c mice. These data suggest that systemic and local Th2 cytokine responses, especially those involving IL-5, are predominant in A. cantonensis-infected mice and that IL-5 is an important cytokine underlying the innate resistance of the mouse against A. cantonensis.

We infected interleukin-5 (IL-5)-transgenic (IL-5-Tg) and IL-5 receptor alpha knockout (IL-5R alpha -/-) mice with Angiostrongylus cantonensis to determine the possible roles of IL-5 and eosinophils in A. cantonensis infection in mice. IL-5-Tg mice demonstrated significantly higher eosinophilia in bone marrow, blood and cerebrospinal fluid (CSF), lower intracranial worm recovery and smaller female worms than naive C3H/HeN mice. Both IL-5-Tg and C3H/HeN mice evoked antigen-specific serum and CSF IgA antibody responses as early as days 5 and 7 postinfection, respectively. Prominent eosinophil infiltration was noted around intracranial worms in the subarachnoid spaces of the mouse brains; eosinophils adhering to the worm surface were degranulated. In contrast, IL-5R alpha -/- mice yielded a higher worm recovery than wild-type or heterozygous mice at day 20 postinfection and failed to provoke CSF eosinophilia. These findings indicate that A. cantonensis infection in the mouse causes IL-5 production and subsequent CSF eosinophilia, the latter probably being involved in the killing of intracranial worms.

Resistance to Angiostrongylus cantonensis is contingent upon the generation of an eosinophilic response in the CSF of infected hosts. We have studied the parameters required for the generation of this CSF eosinophilia in normally permissive rats. We initially induced a marked peripheral eosinophilia in rats by infection with either Mesocestoides corti or Angiostrongylus cantonensis or the surgical transfer of A. cantonensis young adult worms (YA) into their pulmonary arteries. Next, we injected various antigens into the ventricles of these rats. A. cantonensis-preinfected rats demonstrated significant CSF eosinophilia following injection of A. cantonensis egg antigen, 1st-stage larval (L1) antigen, or M. corti antigen, but not following YA antigen inoculation. A. cantonensis egg and M. corti antigens were potent chemoattractants for eosinophils in an in vitro chemotaxis assay. These data indicate that peripheral eosinophilia, meningeal stimulation by A. cantonensis infection and the presence of potent chemoattractants, e.g., egg and L1 antigens are prerequisites for CSF eosinophil accumulation in permissive rat hosts.

The kinetics of eosinophil growth and/or survival stimulating factor (Eo-stimulating factor) production in Angiostrongylus cantonensis-infected rats was assessed by in vitro marrow cultures. When lymphocytes, obtained from cervical, mediastinal or mesenteric lymph nodes of infected WKAH rats, were cultured with A. cantonensis antigens, Eo-stimulating factor activity was detected in the conditioned media obtained only from mediastinal lymph node cells at and after 35 days p.i. Eo-stimulating factor activity in the conditioned media was not detected before or at 20 days p.i. The kinetics of the factor was, therefore, almost identical to those of eosinophilia in bone marrow and peripheral blood. This Eo-stimulating factor activity was inhibited (maximum inhibition = 73%) by anti-mouse IL-5 monoclonal antibody in a dose-dependent fashion, indicating that the factor would be, at least in part, identical to IL-5. Similar Eo-stimulating factors were also detected in the conditioned media which were obtained by stimulating spleen cells from Wistar rats at 35-45 days p.i. with Con A. These data suggest that lymphocytes in the mediastinal lymph nodes, local lymph nodes near the adult worm habitat, could play an essential role in induction of eosinophilia in A. cantonensis-infected rats.

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

We studied the effect of percutaneous ethanol injection therapy (PEI) on glucose tolerance in liver cirrhosis patients with hepatocellular carcinoma. All of 10 patients underwent PEI and aspiration biopsy of the tumor on separate day. Two-time oral glucose tolerance tests (OGTT), before and after PEI, were performed in all patients. There were no significant changes in blood glucose and insulin chronologically measured on aspiration biopsy and PEI. To detect changes in glucose tolerance, we compared the results of OGTT before PEI with those of OGTT after PEI. On the basis of results of OGTT before PEI, patients were classified to impaired glucose tolerance group (4) and diabetes mellitus group (6). Blood glucose at 180 minutes on OGTT after PEI showed significantly higher value than that of OGTT before PEI, but insulin response was not suppressed. From these experiments we speculate that exaggerated insulin resistance due to injected ethanol may be one of the factors influencing glucose tolerance after PEI.