Proteomics analysis of site- and stage-specific protein expression after peripheral nerve injury.

BACKGROUND: The peripheral nervous system has greater regenerative potential than the CNS. This fact suggests the existence of molecules that act as key factors in nerve regeneration during molecular changes in the peripheral nervous system. METHODS: The right sciatic nerve of female Sprague-Dawley rats was exposed and transected at the mid-thigh level. Animals were sacrificed at 5, 10 or 35 days after nerve transection. Proximal and distal nerve segments (1-cm in length) were dissected. We then sought to observe overall molecular changes after peripheral nerve injury using a proteomic approach. For an overview of the identified proteins, each protein was classified according to its biological and molecular functions. We identified a number of proteins showing site- and stage-specific patterns of expression. RESULTS: Both proximal and distal molecular changes at 5, 10 and 35 days after nerve transection were investigated, and a total of 2353 proteins were identified. Among the various expression patterns observed, aFGF and GAP-43 were found to increase in the proximal stump at 10 days after transection, and PN-1, RPL9 and prosaposin increased in the distal stump at 5 days after transection. Among these proteins, aFGF, GAP-43, PN-1 and prosaposin were found to be associated with nerve regeneration. CONCLUSION: We demonstrated that aFGF, GAP-43, PN-1 and prosaposin expression increased at specific stages and in specific sites, such as the proximal or distal stump, after nerve transection by comprehensive measurement using proteomics analysis. We believe that these specific expression patterns might play important roles during regeneration after nerve injury.

Evaluation of the Japanese Metabolic Syndrome Risk Score (JAMRISC): a newly developed questionnaire used as a screening tool for diagnosing metabolic syndrome and insulin resistance in Japan.

OBJECTIVES: To prevent the onset of lifestyle-related diseases associated with metabolic syndrome (MetS) in Japan, research into the development of a useful screening method is strongly desired. We developed a new screening questionnaire (JAMRISC) utilizing a logistic regression model and evaluated its ability to predict the development of MetS, type 2 diabetes and other lifestyle-related diseases in Japanese populace. METHODS: JAMRISC questionnaire was sent to 1,850 individuals in Rumoi, a small city in Hokkaido. We received a total of 1,054 valid responses. To maximize the target individuals accurately diagnosed with MetS, logistic regression analysis was used to generate a unique metabolic syndrome score calculation formula as taking into consideration the clinical relevance of each question item as individual coefficients. RESULTS: The results of our comparative research utilizing both JAMRISC and Finnish Diabetes Risk Score (FINDRISC) questionnaires revealed the usefulness of JAMRISC for its ability to detect risks for MetS, pre-MetS, diabetes, and pre-diabetes. Study of disease risk detection via JAMRISC questionnaire targeting the 4283 residents of Rumoi indicated a high detection rate for pre-MetS (98.8 %), MetS (94.2 %), pre-diabetes (85.1 %) and type 2 diabetes (94.9 %). In addition, JAMRISC was useful not only as a MetS risk score test, but also as a screening tool for diagnosing insulin resistance. CONCLUSIONS: JAMRISC questionnaire is a useful instrument for the detection of early risk of not only MetS and type 2 diabetes but also insulin resistance.

Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma.

Allergic rhinitis (AR), the most common allergic disorder of the airway, is often accompanied by bronchial asthma. However, little is known about the mechanism by which AR advances to AR comorbid with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results showed polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, the %Breg cells in total B cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Moreover, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that relative decrease in Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.

Deficiency of NOX1/nicotinamide adenine dinucleotide phosphate, reduced form oxidase leads to pulmonary vascular remodeling.

OBJECTIVE: Involvement of reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology. APPROACH AND RESULTS: Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene. CONCLUSIONS: These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.

Significant increase of plasma tetranectin in ovx mice as defined by proteomics analysis.

BACKGROUND: Proteomics is recognized as a useful tool in the dynamic screening of plasma protein expression. This study aimed to identify increased expressions of novel plasma proteins in ovariectomized mice (ovx) using selective reaction monitoring (SRM) validation in combination with electrospray ionized-quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) screening. MATERIALS AND METHODS: Twenty-week-old female C57BL/6 mice were ovariectomized or subjected to surgical exposure of the ovaries alone (sham). Blood plasma protein at 4 weeks after these operations was pooled for the ovx and sham group each and separated on SDS-PAGE, and then digested by peptides, which were first differentially displayed by ESI-Q-TOF-MS analysis. Mass spectra of peptides upregulated more than twofold in ovx compared to sham mice were selected for protein identification by ESI-Q-TOF-MS. The selected peptides were further validated in independent samples by SRM using electrospray ionized-triple quadrupole-linear ion trap mass spectrometry (ESI-QqLIT-MS). Optimum transitions for SRM were manually chosen for their high specificity in identifying peptides derived from the candidate proteins. RESULTS: Differential analysis of peptides revealed 1,108 upregulated peptides in ovx compared with sham control mice. Among the upregulated peptides, 231 nonredundant proteins were identified. Validation analysis for the potential use of these proteins as markers of bone turnover was performed using ESI-QqLIT-MS. The four proteins from the plasma samples, namely mannose-binding lectin-C, major urinary protein 2, type I collagen alpha 2 chain, and tetranectin, were evaluated in a blinded manner. A statistically significant elevation of all four proteins in the plasma of ovx mice was confirmed by SRM. Of the four upregulated plasma proteins, tetranectin increased by almost 50 times in the ovx mice compared with the sham mice. CONCLUSIONS: On the basis of proteomics analysis, this study demonstrated that four plasma proteins were significantly elevated in the ovx mice; of these, tetranectin was markedly upregulated by almost 50 times compared with the sham mice.

Innervation pattern at the undersurface of the extensor carpi radialis brevis tendon in recalcitrant tennis elbow.

PURPOSE: We investigated the general, sensory, and sympathetic innervation patterns at the undersurface of the extensor carpi radialis brevis (ECRB) origin in patients with recalcitrant tennis elbow. METHODS: Eight elbows in eight consecutive patients (6 females and 2 males) with tennis elbow who underwent arthroscopic surgery were included in this study. The mean age was 45 years (38-66 years), and the mean duration of symptoms before surgery was 23 months (13-52 months). Operative treatment consisted of an arthroscopic inspection and debridement of the ECRB origin. Control tissues were obtained from biopsy of the ECRB capsule in two patients with osteochondritis dissecance of the capitellum who underwent arthroscopic resection of loose bodies. The tissue specimens were investigated immunohistochemically with antibodies delineating general (PGP9.5), sensory (SP/CGRP), and sympathetic (NPY) nerve patterns. RESULTS: In the non-tendinosis control tissue, SP/CGRP and NPY immunoreactions were heterogeneously distributed in association with blood vessels. Pathologic evaluation of the biopsy tissue showed atypical fibrous granulation containing numerous vessels and nerve structures in all eight patients. Marked reactions to PGP 9.5 took the form of nerve fibers associated with arteries and arterioles in the atypical granulation. Most of the perivascular innervation was found to express NPY. The immunoreactions for SP and CGRP were invariably weak. CONCLUSION: Increased perivascular sympathetic innervation accompanied with loss of sensory innervation at the undersurface of the ECRB tendon may play a role in chronic pain generation in recalcitrant tennis elbow. LEVEL OF EVIDENCE: Diagnostic, Level IV.

CCL8 deficiency in the host abrogates early mortality of acute graft-versus-host disease in mice with dysregulated IL-6 expression.

Although allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for diverse malignant and nonmalignant diseases, acute graft-versus-host disease (aGVHD) is strongly linked to mortality caused by HSCT. We previously reported that CC chemokine ligand 8 (CCL8) is closely correlated to aGVHD mortality in both humans and mice. To study the role of CCL8 in aGVHD, CCL8 knockout (CCL8-/-) mice were transplanted with fully allogeneic marrow grafts. These mice exhibited a significant reduction in mortality (90.0% vs. 23.4% survival for CCL8-/- vs. wild-type recipients at day 28, p < 0.0001). As a result, apparent prolonged median survival from 9 days in wild-type mice to 45 days in CCL8-/- mice was observed. Acute GVHD pathology and liver dysfunction in CCL8-/- mice were significantly attenuated compared with those in wild-type mice. In association with the reduced mortality, a surge of plasma interleukin (IL)-6 was observed in CCL8-/- recipients with allogeneic marrow, which was significantly increased compared with wild-type mice that received allografts. Donor T-cell expansion and plasma levels of interferon-γ and TNF-α during aGVHD were similar in both types of mice. Collectively, these findings indicate that CCL8 plays a major role in aGVHD pathogenesis with possible involvement of an IL-6 signaling cascade.

Activation of glial cells in the spinal cord of a model of lumbar radiculopathy.

PURPOSE: Glial cells in the spinal cord of a lumbar radiculopathy model were investigated using immunohistochemical methods. Neuropathic pain is a consequence of neural plasticity. In models of neuropathic pain models, roles for glial cells in the development of pain behaviors have been reported. Accumulating evidence suggests that activation of p38 mitogen-activated protein kinase (p38) in glial cells contributes to the pathogenesis of neuropathic pain. We examined whether activation of glial cells is involved in the development of neuropathic pain-like behavior observed in a model of lumbar radicular pain that we developed. However, the pathogenesis of lumbar radiculopathy and in particular the effect of spinal glial activation on pain transmission in the dorsal horn of the spinal cord are still not fully known. METHODS: The left L5 spinal root of Sprague-Dawley rats was ligated proximal to the DRG to produce models of lumbar radiculopathy. Protein levels of phosphorylated-p38 (p-p38) in the spinal cord were quantified by Western blot analysis. Double-immunofluorescense studies of p-p38 and specific markers of glia and neurons were performed to determine when and which types of cells were activated in the spinal cord. RESULTS: We observed p38 activation in hyperactive microglia in the dorsal horn ipsilateral to surgery at 1 and 7 days after root constriction, but not in astrocytes or neurons. CONCLUSIONS: Constriction of the lumbar root activated microglia in the spinal cord at 1 and 7 days after surgery, and then returned to normal state at 28 days after surgery, while pain behavior continued. These findings suggest that development of lumbar radicular pain may be initiated by activation of microglia.

Investigation of annexin A5 as a biomarker for Alzheimer's disease using neuronal cell culture and mouse model.

Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Abeta). Abeta, a proteolytic product of amyloid precursor proteins (APP), has a toxic effect on neuronal cells, which involves perturbation of their Ca(2+) homeostasis. This effect implies that changes of protein expression in neuronal cells with calcium stress should provide a molecular marker for this disease. In the present study, we used the supernatant from a neuronal cell culture after incubation with or without Abeta and isolated a Ca(2+)-dependent acidic phospholipid binding fraction to perform a proteomic study. Several unique proteins were identified after incubation with Abeta. We focused on annexin A5, among these proteins, because it binds both Ca(2+) and lipids likely to be involved in calcium homeostasis. Tg2576 transgenic mice (AD model) overexpressing mutant human APP showed a significant increase of annexin A5 in the brain cortex but not in other organs, including liver, kidney, lung, and intestine. In human plasma samples, the level of annexin A5 was significantly increased in a proportion of AD patients compared with a control group (P < 0.0001 in the logistic regression analysis). From the receiver operating characteristic (ROC) curve with plasma annexin A5 concentrations, the mean area under the curve (AUC 0.898) suggests that annexin A5 is a favorable marker for AD.

Proteomics analysis in 28 patients with systemic IgG4-related plasmacytic syndrome.

It is considered that autoimmune pancreatitis (AIP), Mikulicz's disease (MD) and IgG4-related tubulointerstitial nephritis (TIN) comprise systemic IgG4-related plasmacytic syndrome (SIPS), of which the origin remains unknown. We analyzed these patients with focus on serological aspects to invest whether there are autoantigens in SIPS. We evaluated 28 patients with SIPS who presented at Sapporo Medical University Hospital and the collaborated institutions. They were mainly middle-aged (eight male), and consisted of 26 patients with MD and two patients with AIP. The three among 26 patients diagnosed with MD were complicated to AIP, and another three patients had IgG4-related TIN. As a control, healthy volunteers and the patients with Sjögren's syndrome were examined. At first, we measured the levels of serum complements and circulating immune complexes in these patients. Next, immune complexes were collected from the serum of patients and healthy controls by immunoprecipitation. They were divided into immunoglobulin and the antigens by glycine-HCl solution. The divided samples including the antigens were analyzed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Nine patients had hypocomplementemia and 15 had elevated levels of circulating immune complexes in the group of SIPS. In the groups of healthy volunteers and SS, all showed that the levels of serum complements and circulating immune complexes were normal. SELDI-TOF-MS detected a 13.1-kDa protein from all samples of SIPS, and not in normal control and SS. It is possible that the 13.1-kDa protein is one of the autoantigens of SIPS.

Anti-tumor effect of orally administered spinach glycolipid fraction on implanted cancer cells, colon-26, in mice.

We succeeded in purifying a major glycolipid fraction from a green vegetable, spinach. This fraction consists mainly of three glycolipids: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG). In a previous study, we found that the glycolipid fraction inhibited DNA polymerase activity, cancer cell growth and tumor growth with subcutaneous injection. We aimed to clarify oral administration of the glycolipid fraction, suppressing colon adenocarcinoma (colon-26) tumor growth in mice. A tumor graft study showed that oral administration of 20 mg/kg glycolipid fraction for 2 weeks induced a 56.1% decrease in the solid tumor volume (P < 0.05) without any side-effects, such as loss of body weight or major organ failure, in mice. The glycolipid fraction induced the suppression of colon-26 tumor growth with inhibition of angiogenesis and the expression of cell proliferation marker proteins such as Ki-67, proliferating cell nuclear antigen (PCNA), and Cyclin E in the tumor tissue. These results suggest that the orally administered glycolipid fraction from spinach could suppress colon tumor growth in mice by inhibiting the activities of neovascularization and cancer cellular proliferation in tumor tissue.

Effect of collagen oligopeptide injection on rabbit tenositis.

Effects of the collagen oligopeptide (COP) were examined by its repeat injection into the inflammatory rabbit Achilles tendon (Tendo calcaneus communis), in which tenositis was induced by injection of bacterial collagenase. COP was evaluated 5 times over a period of 3 weeks to 1 month after injection of collagenase. At 1 month after treatment, the therapeutic effect of COP was evaluated by examining the structure of collagen fibrils, amount and components of glycosaminoglycans (GAGs) and matrix metalloproteinases (MMPs), and compared with the saline injection, control, and normal groups. The Achilles tendon of rabbit in the control group (no COP injection) and saline injection group showed a notable increase in the number of fine collagen fibrils, a change in GAG composition and increase in the amount of pro-MMP-2, indicating the weakening of the tendon. In contrast, the size distribution of collagen fibrils, GAG composition and the amount of pro-MMP-2 was similar to those in the normal group. These results suggest that COP injection promotes healing processes of the tendon injury.

Mikulicz's disease and systemic IgG4-related plasmacytic syndrome (SIPS).

Mikulicz's disease represents persistent enlargement of the lacrimal and salivary glands, and autoimmune pancreatitis is shown with diffuse pancreatic swelling. Both diseases are characterized with elevated IgG4 concentrations in the serum and prominent infiltration by plasmacytes expressing IgG4 in the glands. Clinical analyses were performed in 40 patients with systemic IgG4-related plasmacytic syndrome (SIPS) who consulted the doctors in Sapporo Medical University Hospital. Our patients were mainly middle-aged or elderly females. The average age was 58.9 years. The diagnosis was following ; 33 cases with Mikulicz's disease, 3 cases with Küttner's tumor, and 4 cases with IgG4-related dacryoadenitis. Slight dysfunction of lacrimal and salivary gland was observed in about 60% of them. Antinuclear antibodies were detected in only 15% of the cases with SIPS. Almost all, except one case, did not have anti-SS-A or anti-SS-B antibodies. Interestingly, hypocomplementemia was revealed in 30% of them. The complications of SIPS include autoimmune pancreatitis, tubulointerstitial nephritis, retroperitoneal fibrosis, prostatitis, and so on. SIPS is mainly treated by the administration of steroids. We started to prescribe much quantity of prednisolone to the patients with organ failure. The recurrence was admitted in the 3 patients for the followed 16 years. We present here the problems and prospects in SIPS.

Effects of dietary supplementation with milk fat globule membrane on the physical performance of community-dwelling Japanese adults: a randomised, double-blind, placebo-controlled trial.

We conducted a randomised, double-blind, placebo-controlled trial to elucidate the effects of dietary milk fat globule membrane (MFGM) on the physical performance of community-dwelling Japanese adults. For this 24-week study, 115 middle-aged subjects (range 50-70 years old) were invited, of whom 113 (seventy-two women, forty-one men) completed the trial. Participants were then divided into either the placebo control or MFGM group. Measurements of physical performance (without undertaking any mandatory exercise) examining muscle strength, agility and balance were tested every 6 weeks until 24 weeks. Analyses were performed using the intention-to-treat method for all participants. Although the effects of MFGM on muscle strength and agility were not significant, we noted that the parameter for balance (such as the ability to stand on one leg with eyes closed for longer durations) increased in the MFGM group (mean 10·1 (95 % CI 8·25, 12·4) s) compared with the placebo (mean 7·53 (95 % CI 6·11, 9·30) s) (P = 0·046). Similarly, application of the mixed-effect model for repeated measures under unstructured covariance also revealed that the effect of MFGM was significant when compared with the placebo (10·2 (95 % CI 8·33, 12·4) v. 7·61 (95 % CI 6·17, 9·30) s) (P = 0·045). In conclusion, we demonstrated that MFGM had an effect on the physical performance of community-dwelling Japanese adults despite mandatory exercise. However, studies using larger cohorts of individuals from different demographic backgrounds are required to further elucidate the mechanisms underlying these effects and to extend the application of MFGM.

[Plasma analysis of rheumatoid arthritis by SELDI].

To identify protein biomarkers linking to disease activity and treatment responses of patients with rheumatoid arthritis (RA), proteomic study using mass spectrometric analysis of plasma proteins was performed. Proteomic profiling technologies can simultaneously resolve and analyze multiple proteins in plasma. Evaluation of multiple proteins of the plasma will be essential to discover protein biomarkers. In this study, we used protein chip surface enhanced laser desorption/ionization time of flight mass spectrometry approach (SELDI-TOF MS). Through differential profiling of plasma proteins, we selected two prospective candidate biomarkers. One mass spectrometric peak distinguished patients with RA from healthy controls was transthyretin (TTR) and the other distinguished inactive patients with RA from patients with active RA was Serum Amyloid A (SAA). This study demonstrates that proteomic profiling using mass spectrometry of plasma greatly facilitates global discovery and verify clinically relevant sets of disease biomarker directly links to disease activity and treatment responses.

Reduction of HbA1c levels by fucoxanthin-enriched akamoku oil possibly involves the thrifty allele of uncoupling protein 1 (UCP1): a randomised controlled trial in normal-weight and obese Japanese adults.

Lifestyle-related problems are becoming a major health threat in East Asian countries. Therefore, finding an efficacious nutraceutical for this population is important. One candidate is fucoxanthin (Fx), a carotenoid abundantly found in edible brown seaweed that has been associated with a number of valuable health-promoting benefits. Unfortunately, clinical studies of Fx are limited. In the present study, we aimed to evaluate the effects of Fx on obesity-related parameters in Japanese subjects harbouring an SNP associated with lifestyle-related problems. In all, sixty normal-weight and obese Japanese adults with BMI over 22 kg/m2 were single-blinded and randomly assigned to three Fx-dose cohorts and administered Fx-enriched akamoku oil containing Fx at 0, 1 or 2 mg/d for 8 weeks (n 20 per group). Parameters relating to obesity and serum Fx metabolites were measured before and after intervention, but no significant differences were observed between and within the groups. Despite no changes in visceral fat areas and resting energy expenditures after intervention, we observed a significant decline in HbA1c levels in the 2 mg/d Fx group compared with that in the 0 mg/d group (P < 0·05), which was correlated with an increase in serum fucoxanthinol (Fx metabolite) levels. In addition, HbA1c levels declined more significantly in subjects with G/G alleles of the uncoupling protein 1 (UCP1) gene than in those with the A/A and A/G alleles (P < 0·05). We conclude that although Fx supplementation does not affect visceral fat areas, it may reduce HbA1c levels in those harbouring the thrifty allele of UCP1-3826A/G.

HLA-A24 ligandome analysis of colon and lung cancer cells identifies a novel cancer-testis antigen and a neoantigen that elicits specific and strong CTL responses.

This study focused on HLA-A24 and comprehensively analyzed the ligandome of colon and lung cancer cells without the use of MHC-binding in silico prediction algorithms. Affinity purification using the antibody specific to HLA-A24 followed by LC-MS/MS sequencing was used to detect peptides, which harbored the known characteristics of HLA-A24 peptides in terms of length and anchor motifs. Ligandome analysis demonstrated the natural presentation of two different types of novel tumor-associated antigens. The ligandome contained a peptide derived from SUV39H2, a gene found to be expressed in a variety of cancers but not in normal tissues (except for the testis). The SUV39H2 peptide is immunogenic and elicits cytotoxic CD8+ T-cell (CTL) responses against cancer cells and is thus a novel cancer-testis antigen. Moreover, we found that microsatellite instability (MSI)-colon cancer cells displayed a neoepitope with an amino-acid substitution, while microsatellite stable (MSS)-colon and lung cancer cells displayed its counterpart peptide without the substitution. Structure modeling of peptide-HLA-A24 complexes predicted that the mutated residue at P8 was accessible to T-cell receptors. The neoepitope readily elicited CTL responses, which discriminated it from its wild-type counterpart, and the CTLs exhibited considerably high cytotoxicity against MSS-colon cancer cells carrying the responsible gene mutation. The specific and strong CTL lysis observed in this study fosters our understanding of immune surveillance against neoantigens.

Occludin expression decreases with the progression of human endometrial carcinoma.

The tight junctions of the glandular epithelium are crucial for the maintenance of cell polarity, separating the plasma membrane into apical and basolateral domains. Thus abnormalities of the tight junctions may result in the structural disturbances of glandular epithelial neoplasia. In this study we introduced an anti-occludin monoclonal antibody for semiquantitative assay of the occludin expression in tissue sections of human normal and neoplastic endometrial epithelia using the Adobe Photoshop and NIH Image programs. Normal endometrial glands and samples of endometrial hyperplasia and endometrioid carcinoma grade 1 fully expressed occludin at the apical cell border. In endometrioid carcinomas grades 2 and 3, however, occludin disappeared in solid areas of the carcinomatous tissues. Occludin was also found at the apical borders of the cancer cells that formed glandular structures. Occludin expression decreased progressively in parallel with the increase in carcinoma grade, and the decreased occludin expression correlated with myometrial invasion and lymph node metastasis. These results suggest that the loss of tight junctions has a close relationship with structural atypia in the progression of human endometrial carcinomas and their malignant potential.

Abdominal small round cell tumor with osteoid and EWS/FLI1.

In this report, we described a case of multiple intraperitoneal tumors. Histologically, the tumors were composed of small round cells with malignant phenotype, necrotic areas, and islands of osteoid matrix in the stroma. In immunohistochemical and molecular analyses, the tumors expressed CD99 and EWS-Fli1 fusion gene. Production of osteoid by small round tumor cells was consistent with the histologic criteria of small-cell osteosarcoma, whereas expression of EWS-Fli1 was a characteristic genetic feature of Ewing's sarcoma family of tumor. Such tumors have been limited to a case in which histologically proven small-cell osteosarcoma of the scapula showed a chromosomal translocation, t(11;22)(q24;q12).