Incorporation, intracellular trafficking and processing of extracellular heparanase by mast cells: Involvement of syndecan-4-dependent pathway.

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.

Decreased 16:0/20:4-phosphatidylinositol level in the post-mortem prefrontal cortex of elderly patients with schizophrenia.

The etiology of schizophrenia includes phospholipid abnormalities. Phospholipids are bioactive substances essential for brain function. To analyze differences in the quantity and types of phospholipids present in the brain tissue of patients with schizophrenia, we performed a global analysis of phospholipids in multiple brain samples using liquid chromatography electrospray ionization mass/mass spectrometry (LC-ESI/MS/MS) and imaging mass spectrometry (IMS). We found significantly decreased 16:0/20:4-phosphatidylinositol (PI) levels in the prefrontal cortex (PFC) in the brains from patients with schizophrenia in the LC-ESI/MS/MS, and that the 16:0/20:4-PI in grey matter was most prominently diminished according to the IMS experiments. Previous reports investigating PI pathology of schizophrenia did not identify differences in the sn-1 and sn-2 fatty acyl chains. This study is the first to clear the fatty acid composition of PI in brains from patients with schizophrenia. Alteration in the characteristic fatty acid composition of PI may also affect neuronal function, and could play a role in the etiology of schizophrenia. Although further studies are necessary to understand the role of reduced 16:0/20:4-PI levels within the prefrontal cortex in the etiology of schizophrenia, our results provide insight into the development of a novel therapy for the clinical treatment of schizophrenia.

Accumulation of arachidonic acid-containing phosphatidylinositol at the outer edge of colorectal cancer.

Accumulating evidence indicates that cancer cells show specific alterations in phospholipid metabolism that contribute to tumour progression in several types of cancer, including colorectal cancer. Questions still remain as to what lipids characterize the outer edge of cancer tissues and whether those cancer outer edge-specific lipid compositions emerge autonomously in cancer cells. Cancer tissue-originated spheroids (CTOSs) that are composed of pure primary cancer cells have been developed. In this study, we aimed to seek out the cancer cell-autonomous acquisition of cancer outer edge-characterizing lipids in colorectal cancer by analysing phospholipids in CTOSs derived from colorectal cancer patients with matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS). A signal at m/z 885.5 in negative ion mode was detected specifically at the surface regions. The signal was identified as an arachidonic acid (AA)-containing phosphatidylinositol (PI), PI(18:0/20:4), by tandem mass spectrometry analysis. Quantitative analysis revealed that the amount of PI(18:0/20:4) in the surface region of CTOSs was two-fold higher than that in the medial region. Finally, PI(18:0/20:4) was enriched at the cancer cells/stromal interface in colorectal cancer patients. These data imply a possible importance of AA-containing PI for colorectal cancer progression, and suggest cells expressing AA-containing PI as potential targets for anti-cancer therapy.

Heparanase augments inflammatory chemokine production from colorectal carcinoma cell lines.

To explore possible roles of heparanase in cancer-host crosstalk, we examined whether heparanase influences expression of inflammatory chemokines in colorectal cancer cells. Murine colorectal carcinoma cells incubated with heparanase upregulated MCP-1, KC, and RANTES genes and released MCP-1 and KC proteins. Heparanase-dependent production of IL-8 was detected in two human colorectal carcinoma cell lines. Addition of a heparanase inhibitor Heparastatin (SF4) did not influence MCP-1 production, while both latent and mature forms of heparanase augmented MCP-1 release, suggesting that heparanase catalytic activity was dispensable for MCP-1 production. In contrast, addition of heparin to the medium suppressed MCP-1 release in a dose-dependent manner. Similarly, targeted suppression of Ext1 by RNAi significantly suppressed cell surface expression of heparan sulfate and MCP-1 production in colon 26 cells. Taken together, it is concluded that colon 26 cells transduce the heparanase-mediated signal through heparan sulfate binding. We propose a novel function for heparanase independent of its endoglycosidase activity, namely as a stimulant for chemokine production.

E-cadherin-negative acinar cell carcinoma of the pancreas: report of a case showing a solid pseudopapillary growth pattern.

E-cadherin expression patterns in acinar cell carcinomas (ACCs) of the pancreas have not been well documented. Herein, we present a hitherto undescribed case of E-cadherin-negative ACC with a solid pseudopapillary growth pattern in a 65-year-old man. We used an antibody against the extracellular domain of E-cadherin. As a further unusual status in ACC, faint ß-catenin expression was observed in the cytoplasm of carcinoma cells. Morphological distinction from a solid pseudopapillary neoplasm (SPN) of the pancreas might be problematic in such a case, because of their similarities concerned with the growth pattern and E-cadherin negativity. Without nuclear accumulation of ß-catenin, a diagnosis of SPN was almost excluded. Immunoreactivity for trypsin and BCL10 made an accurate diagnosis of ACC to this case. The tumor recurred 10 months post-surgery as rapidly enlarging masses in the liver, presumably indicating the aggressiveness of the E-cadherin-negative phenotype among ACCs.

Cystic apocrine ductal carcinoma in situ with increased EGFR expression, trisomy 7, and associated focal invasion.

A 71-year-old woman was admitted to our hospital with a lump in her right breast. Mammography revealed an internal high-density mass in the lower right breast, which was larger than it was 2 years ago. Considering the findings from ultrasonography, computed tomography, and cytology, an intracystic carcinoma could not be ruled out. The patient underwent excisional biopsy, which revealed an apocrine ductal carcinoma in situ (ADCIS) with focal invasive apocrine carcinoma (IAC). The diagnosis was based on morphology and immunohistochemistry (IHC), which was negative for the estrogen and progesterone receptors, and positive for the androgen receptor. Expression of the human epidermal growth factor receptor 2 and the epidermal growth factor receptor (EGFR) received an immunohistochemical score of 2+. Trisomy of chromosome 7, including multiple CEP 7 and EGFR signals, was observed in ADCIS by fluorescence in situ hybridization (FISH). IAC exhibited similar results for IHC and FISH. This is the first reported case showing trisomy 7 resulting in EGFR copy number gain and increased EGFR expression in ADCIS.

Pleomorphic leiomyosarcoma with a dedifferentiation-like appearance in the kidney: case report and literature review.

Although primary leiomyosarcoma of the kidney is extremely rare, it is the most common sarcoma of the kidney. Leiomyosarcoma with a large pleomorphic component is designated as pleomorphic leiomyosarcoma. The pleomorphic component is usually similar to undifferentiated high-grade pleomorphic sarcoma, although it variably expresses smooth muscle markers on immunohistochemistry. In the few reported cases of pleomorphic leiomyosarcoma of the kidney, cases with the pleomorphic component showing distinct nodularity similar to dedifferentiated leiomyosarcoma have not been described, to the best of our knowledge. Herein, we present a case of a 49-year-old woman with pleomorphic leiomyosarcoma in the kidney showing distinct nodularity of smooth muscle marker-expressing pleomorphic cells within a background of classic leiomyosarcoma. Along with the classification as a pleomorphic leiomyosarcoma, suggesting aggressive clinical behavior, the renal origin itself might also be a predictor of poor prognosis, as shown in a previous study. This case also involved concomitant distant metastases, already present during the initial detection of the renal tumor.

A case of Miller Fisher syndrome during preoperative chemotherapy for breast cancer.

A 53-year-old woman with breast cancer received FEC treatment (5FU: 500 mg/m(2), epirubicin: 100 mg/m(2), and cyclophosphamide: 500 mg/m(2)) every 3 weeks as preoperative chemotherapy. Fifteen days after her third cycle of FEC, she developed a cold. Diplopia occurred 4 days after developing the cold, and progressive paresthesia of the hands and weakness of the limbs occurred. She had ophthalmoplegia, ataxia, and are flexia and was diagnosed with Miller Fisher Syndrome (MFS). The cause of MFS during chemotherapy is believed to be caused by an immunological response to infection, or drug neurotoxicity. In our case, since the patient underwent an antecedent upper respiratory infection in the period of myelosuppression, her MFS was probably induced by the immunoreaction associated with this infection. Our patient underwent intravenous immunoglobulin therapy. After initiation of the treatment, her neurological symptoms improved, then, she received a fourth cycle of FEC and her remaining neurological symptoms did not worsen. Thus, we report a rare case of MFS developed in immunosuppression by chemotherapy and remind physicians of the alarming triad of MFS symptoms.

Direct profiling of the phospholipid composition of adult Caenorhabditis elegans using whole-body imaging mass spectrometry.

A protocol for the direct analysis of the phospholipid composition in the whole body of adult soil nematode, Caenorhabditis elegans (C. elegans), was developed, which combined freeze-cracking of the exoskeletal cuticle and matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Biomolecules in the m/z range from 700 to 900 were more effectively detected in the freeze-cracked than from simple frozen adult nematode bodies. Different distribution of biomolecules was observed in a nematode body when the matrix was applied with a sublimation deposition method. The whole-body IMS technique was applied on genetically deficient mutant C. elegans to combine whole-body lipidomics and genetics, by comparing the fatty acid compositions, especially of the phosphatidylcholine (PC) species, between the wild-type and fat-1 mutants, which lack the gene encoding an n-3 fatty acid desaturase. A significant reduction of PC(20:5/20:5) and PC(20:4/20:5) and a marked increase of PC(20:4/20:4), PC(20:3/20:4), and PC(20:3/20:3) were detected in the fat-1 mutants in positive ion mode. In addition, phospholipid compositions other than PCs were analyzed in negative ion mode. A loss of a possible phosphatidylinositol (PI) with 18:0/20:5 and a compensative accumulation of putative PI(18:0/20:4) were detected in the fat-1 mutants. In conclusion, the whole-body MALDI-IMS technique is useful for the profiling of multiple biomolecules in C. elegans in both intra- and inter-individual levels.

TSLP Directly Interacts with Skin-Homing Th2 Cells Highly Expressing its Receptor to Enhance IL-4 Production in Atopic Dermatitis.

Thymic stromal lymphopoietin (TSLP) is overtly expressed on skin lesions of atopic dermatitis (AD), and the initiative role of TSLP-activated DCs in AD has gained much attention in the past few years, while its actions on other immune cells such as T cells have been given less notice. We aimed to clarify whether TSLP receptor (TSLPR) is expressed on certain populations of T cells and whether TSLP possesses the capability to directly interact with T cells from AD patients. Peripheral lymphocytes from 51 AD patients are analyzed by flow cytometry, and ex vivo experiments using peripheral blood and lesional skin-derived T cells were conducted. TSLPR expression was defined to CD4+ T cells, and CD4+CCR4+CXCR3-CCR7-CCR10+CLA+ T cells in AD patients exhibited enhanced TSLPR expression. The frequency of TSLPR+CD4+ T cells correlated with disease activity. CD4+ T cells from AD patients directly interacted with TSLP to produce a higher amount of IL-4 than those from normal subjects, and this action was attenuated with anti-TSLPR antibody. The importance of IL-4 in the induction of TSLPR expression was found in AD T cells. Our findings indicate that T cells from AD patients possess strong potential to directly interact with TSLP to promote Th2 response.

Decreased level of phosphatidylcholine (16:0/20:4) in multiple myeloma cells compared to plasma cells: a single-cell MALDI-IMS approach.

Lipid metabolic changes under diseased conditions, particularly in solid tumors, are attracting increased attention. However, in non-solid tumors, including most hematopoietic tumors, lipid analyses are scarce. Multiple myeloma (MM) is a plasma cell disorder arising from bone marrow, and the lipid status of MM cells has not been reported yet. In this study, we analyzed flow cytometry-sorted single MM cells and normal plasma cells (NPCs) using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), a two-dimensional label-free mass spectrometry technique for biomolecular analysis, to obtain specific lipid information. We isolated 1.31-5.77% of MM cells and 0.03-0.24% of NPCs using fluorescence-activated cell sorting (FACS). Analysis of purified cells using MALDI-IMS at the single-cell level revealed that the peak intensity and ion signals of phosphatidylcholine [PC (16:0/20:4) + H](+) at m/z 782.5 were significantly decreased in MM cells compared to NPCs. By examining particular cell populations rather than cell mixtures, our method can become a suitable tool for the analysis of rare cell populations at the single-cell level and advance the understanding of MM progression.

Massive hemoperitoneum following spontaneous rupture of an arterial aneurysm overlying a uterine myoma.

Intraperitoneal hemorrhage caused by a uterine myoma is rare. A 54-year-old woman was admitted to the emergency room; on admission, she was in cardiopulmonary arrest with pulseless electrical activity. Transabdominal ultrasonography revealed hyperechoic fluid filled almost the entire abdominal cavity. On contrast-enhanced computed tomography, extravasation of contrast material was observed inside the fluid, although the bleeding site was not identifiable. An emergency operation was performed to stabilize the patient. There was pulsating bleeding from a subserosal myoma on the posterior wall of the uterus; the myoma measured approximately 6 cm in maximum diameter. After resection of the myoma, the bleeding stopped. Pathological assessment of the resected specimen revealed a ruptured arterial aneurysm, approximately 8 mm in diameter, situated on the surface of a leiomyoma without degeneration. Spontaneous rupture of a vein or an artery overlying a myoma has been documented in the English literature, though it is extremely rare. Rupture of a vein is a more frequent occurrence than the rupture of an artery. This is the first reported case documenting a ruptured arterial aneurysm overlying a myoma.

Palmitic acid, verified by lipid profiling using secondary ion mass spectrometry, demonstrates anti-multiple myeloma activity.

Recent studies indicate that lipid metabolic changes affect the survival of multiple myeloma (MM) cells. Time-of-flight secondary ion mass spectrometry (TOF-SIMS), an imaging mass spectrometry technique, is used to visualize the subcellular distribution of biomolecules including lipids. We therefore applied this method to human clinical specimens to analyze the membrane fatty acid composition and determine candidate molecules for MM therapies. We isolated MM cells and normal plasma cells (PCs) from bone marrow aspirates of MM patients and healthy volunteers, respectively, and these separated cells were analyzed by TOF-SIMS. Multiple ions including fatty acids were detected and their ion counts were estimated. In MM cells, the mean intensity of palmitic acid was significantly lower than the mean intensity in PCs. In a cell death assay, palmitic acid reduced U266 cell viability dose-dependently at doses between 50 and 1000 µM. The percentage of apoptotic cells increased from 24h after palmitic acid administration. In contrast, palmitic acid had no effect on the viability of normal peripheral blood mononuclear cells (PBMCs). The results of this study indicated that palmitic acid is a potential candidate for novel therapeutic agents that specifically attack MM cells.

Cystic partially differentiated nephroblastoma in an adult: a case imitating the process of normal nephrogenesis along with corresponding WT1 expression.

Cystic partially differentiated nephroblastoma (CPDN) is extremely rare in adults. Only 2 cases have been documented in the English literature. Herein, we present a third case of CPDN with unique morphological and immunohistochemical features. A 45-year-old man had a multicystic right renal mass, with a maximum diameter of 3 cm on magnetic resonance imaging. Being unable to rule out malignancy, partial nephrectomy was performed. The surgically resected specimen contained a multicystic mass, 3 × 3 × 2.5 cm in size, without an expansile solid nodule. Histopathological examination revealed nephroblastomatous elements without identifiable blastema; transition from cap-mesenchyme-like cells to an immature glomerulus was observed and maturing tubules and a glomerulus were present. Despite the lack of a blastema, the diagnosis of CPDN was the most appropriate. Immunohistochemical WT1 expression imitated the pattern of ongoing normal nephrogenesis. Therefore, we believe that the blastema disappeared because of maturation.

Xanthogranulomatous gastritis associated with actinomycosis: report of a case presenting as a large submucosal mass.

Xanthogranulomatous gastritis (XGG) is a rarely encountered condition, and its causative mechanism is still unclear. Given that some types of xanthogranulomatous inflammation (XGI) are associated with pathogens, infection should be considered as a possible cause of XGG. Herein, we report a case of an 86-year-old woman presenting with a large, bleeding lesion resembling a submucosal tumor. Distal gastrectomy was performed, and the surgically resected specimen revealed a mass measuring 6 × 4.5 × 3 cm and appearing yellowish on the cut surface. Histopathological examination revealed a few Actinomyces "sulfur granules" and cellular composition characteristic of XGI, supporting a diagnosis of XGG associated with actinomycosis. Gastric actinomycosis is a rare condition and has not previously been reported in association with XGG, although rare cases of XGI associated with actinomycosis have been documented in other organs.

Acquired cystic disease-associated renal cell carcinoma with a focal sarcomatoid component: Report of a case showing more pronounced polysomy of chromosomes 3 and 16 in the sarcomatoid component.

Acquired cystic disease (ACD)-associated renal cell carcinoma (RCC) has recently been established. Herein we report the sixth case of ACD-associated RCC with a sarcomatoid change. The patient was a 77-year-old man who regularly underwent hemodialysis for 14 years due to chronic renal failure resulting from IgA nephropathy. On computed tomography, a large right RCC was observed with contrast enhancement in the arterial phase. A nodular protrusion into the perirenal fat was detected. Right nephrectomy was performed under laparoscopy. Surgically resected specimens revealed a tan-to-yellow tumor (95 × 75 × 55 mm) with a whitish nodule (20 × 15 × 15 mm) invading into the perirenal fat. Histopathologically, the large carcinoma component of the tumor displayed a cribriform or microcystic growth pattern with deposition of oxalate crystals. The whitish nodule corresponded to the sarcomatoid component, and the spindled and pleomorphic tumor cells showed diffuse positivity of p53 on immunohistochemistry. Fluorescence in situ hybridization revealed trisomy of chromosomes 3 and 16 in the carcinoma component, as was expected from the literature. In addition, increased polysomy of these chromosomes was also observed in the sarcomatoid component. This finding may be related to the development of the sarcomatoid component along with the TP53 mutation.

Nanoparticle-assisted laser desorption/ionization for metabolite imaging.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has enabled the spatial analysis of various molecules, including peptides, nucleic acids, lipids, and drug molecules. To expand the capabilities of MALDI-IMS, we have established an imaging technique using metal nanoparticles (NPs) to visualize metabolites, termed nanoparticle-assisted laser desorption/ionization imaging mass spectrometry (nano-PALDI-IMS). By utilizing Ag-, Fe-, Au-, and TiO2-derived NPs, we have succeeded in visualizing various metabolites, including fatty acid and glycosphingolipids, with higher sensitivity and spatial resolution than conventional techniques. Herein, we describe the practical experimental procedures and methods associated with nano-PALDI-IMS for the visualization of these molecules.

Granulocyte colony-stimulating factor-producing undifferentiated carcinoma of the colon mimicking a pulmonary giant cell carcinoma: a case showing overexpression of CD44 along with highly proliferating nestin-positive tumor vessels.

Granulocyte colony-stimulating factor (G-CSF)-producing tumors are known for their aggressive behavior. Only four cases of G-CSF-producing colorectal carcinoma have been previously reported. Herein, we present a case of an undifferentiated carcinoma of the descending colon showing G-CSF production and giant cell carcinoma morphology in a 93-year-old woman. A tumor with a diameter of 80 mm was identified in the descending colon via computed tomography. Descending colectomy was performed involving the abdominal wall where tumor invasion was observed. The white blood cell count, which was elevated before resection, decreased to normal levels after intervention. However, local recurrence at the resected site was detected 39 days after surgery. Upon recurrence, increased white blood cell counts and serum G-CSF were seen. The patient died because of respiratory failure 98 days after colectomy. By using immunohistochemistry, G-CSF expression was detected in tumor cells in the resected specimen, along with overexpression of CD44 and highly proliferating nestin-positive tumor vessels. The poor clinical outcome of this patient is consistent with previous reports that the expression of these three molecules predict poor prognosis. While G-CSF can be a therapeutic target considering its auto/paracrine function to induce tumor growth via the G-CSF receptor, CD44 and nestin may also be possible candidate therapeutic targets. Further studies are required to assess the efficacy of treatments targeting these three molecules.

Single-cell time-of-flight secondary ion mass spectrometry reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells.

Lipids comprise the primary component of cell membranes. Imaging mass spectrometry is increasingly being used to visualize membranous lipids in clinical specimens, and it has revealed that abnormal lipid metabolism is related to the development of diseases. To characterize cell populations which are rare and sparsely localized in tissues, we conducted time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of individual cells sorted by fluorescence activated cell sorting (FACS) and applied the method to analyze breast cancer stem cells (CSCs). TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CD45(-)/CD44(+)/CD24(-) CSCs, and these ions are suspected to have originated from membranous phospholipids as they were uniformly detected from the locus where the cells attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] normalized by phosphoric acid signals were decreased significantly in CSCs as compared to that of CD45(-)/CD44(-)/CD24(+) non-stem cancer cells (NSCCs). This finding was supported by liquid chromatography coupled electrospray ionization-tandem mass spectrometry analysis, which revealed phosphatidylcholine (PC)(16:0/16:1) to be less abundant and PC(16:0/16:0) to be more abundant in CSCs as compared to NSCCs. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified by the expression of a complex combination of cell-surface markers. The lipid compositions of CSCs originating from the heterogeneous cellular populations of clinical specimens were successfully characterized by this method.