Differential diagnosis of benign or malignant intraductal papillary mucinous neoplasm of the pancreas by multidetector row helical computed tomography: evaluation of predictive factors by logistic regression analysis

OBJECTIVE: The purpose of this study is to evaluate predictive factors for discriminating benign from malignant intraductal mucin-producing neoplasm (IPMN) of the pancreas on multidetector row computed tomography (MDCT). MATERIALS AND METHODS: Fifty-three patients with IPMN underwent MDCT, and the imaging and pathological findings were evaluated. In patients with branch duct-type tumors, sex and age of the patient, location, shape, size and multiplicity of the cystic lesion, presence of mural nodule, and maximum diameter of main pancreatic duct (MPD) dilatation were evaluated by logistic regression analysis. RESULTS: Tumors were classified as main duct-type (n = 7) and branch duct-type (n = 46). Among main duct-type tumors, all 7 lesions were diagnosed as malignant. Among 46 lesions of branch-type IPMN, 8 lesions were malignant, and 38 lesions were benign. On adjusted logistic regression analysis, combination factor of main duct dilatation and mural nodule or large cystic size had statistical significance for the risk of malignancy in branch duct-type IPMN. CONCLUSIONS: Main duct-type IPMN is highly suggestive for malignancy. Combination factors of main ductal dilation and mural nodule, and main ductal dilation, and large cystic tumor size are thought to be predictive factors for malignant branch-type IPMN.     

Diverse mutations in the ftsI gene in ampicillin-resistant Haemophilus influenzae isolates from pediatric patients with acute otitis media

To clarify molecular changes in β-lactamase-nonproducing, ampicillin-resistant (BLNAR) Haemophilus influenzae, which is increasing in pediatric patients with acute otitis media (AOM) in Japan, we identified amino acid (aa) substitutions in penicillin-binding protein 3 for the BLNAR strains. Of 191 H. influenzae strains isolated from middle ear fluid of pediatric AOM patients between October 2005 and March 2008, BLNAR strains determined by PCR accounted for 49.2%. Of the BLNAR strains, 91.5% possessed 4 aa substitutions: Met377Ile, Ser385Thr, Leu389Phe, and either Asn526Lys or Arg517His. Additionally, the emergence of BLNAR strains possessing a new aa substitution of Val329Ala in the conserved aa motif of Ser327-Thr-Val-Lys, or Val511Ala adjacent to the conserved aa motif of Lys512-Thr-Gly, was noted. Transformation of the ftsI gene into the Rd reference strain (ATCC 51907) demonstrated that these two aa substitutions reduced susceptibility to amoxicillin more than to cephalosporins. Pulsed-field gel electrophoretic profiles of BLNAR strains were highly diverse. These results suggested that inadequate antibiotic use may increase BLNAR strains by selecting mutations in the ftsI gene and that such use may have favored the new aa substitutions.     

Erythema multiforme after radiotherapy with aromatase inhibitor administration in breast-conservation treatment for breast cancer

Generalized eruptions associated with radiotherapy such as erythema multiforme (EM), Steven-Johnson syndrome and toxic epidermal necrolysis are uncommon reactions. A few cases of generalized eruptions during and after radiotherapy have been reported with the use of anticonvulsants and anticancer drugs. However, no reports have described mucocutaneous reactions associated with radiotherapy and concurrent use of anastrozole, an aromatase inhibitor. This report describes EM occurring after radiotherapy performed during breast-conserving treatment for breast cancer in a patient who was taking oral anastrozole.     

IgG/IgA pemphigus with dyskeratotic acantholysis and intraepidermal neutrophilic microabscesses

Herpetiform vesicles and erythema with erosion developed on the trunk of a 48-year-old Japanese man. Acantholysis was observed in the spinous layer of the lesional epidermis and direct immunofluorescence revealed cell surface deposition of immunoglobulin IgG and C3. Indirect immunofluorescence could not detect circulating anti-cell surface antibodies. Immunoblot analysis detected neither anti-desmoglein (Dsg)1 antibody nor anti-Dsg3 antibody. Enzyme-linked immunosorbent assay could detect IgG and IgA anti-Dsg1 antibodies and IgA anti-Dsg3 antibody, in addition to a gray zone titer of IgG anti-Dsg3 antibody. Intraepidermal neutrophilic infiltration with neutrophilic microabscesses and intense dyskeratotic cells were histopathologically characteristic in this case. Skin lesions improved within 1 month and remission has continued for 9 years under oral administration of dapsone.     

The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor–dependent metabolism in myeloid cells

Extracellular adenosine triphosphate (ATP) released by mucosal immune cells and by microbiota in the intestinal lumen elicits diverse immune responses that mediate the intestinal homeostasis via P2 purinergic receptors, while overactivation of ATP signaling leads to mucosal immune system disruption, which leads to pathogenesis of intestinal inflammation. In the small intestine, hydrolysis of luminal ATP by ectonucleoside triphosphate diphosphohydrolase (E-NTPD)7 in epithelial cells is essential for control of the number of T helper 17 (Th17) cells. However, the molecular mechanism by which microbiota-derived ATP in the colon is regulated remains poorly understood. Here, we show that E-NTPD8 is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8^−/− mice develop more severe dextran sodium sulfate–induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes by injecting with anti–Gr-1 antibody or the introduction of P2rx4 deficiency into hematopoietic cells. An increased level of luminal ATP in the colon of Entpd8^−/− mice promotes glycolysis in neutrophils through P2x4 receptor–dependent Ca²⁺ influx, which is linked to prolonged survival and elevated reactive oxygen species production in these cells. Thus, E-NTPD8 limits intestinal inflammation by controlling metabolic alteration toward glycolysis via the P2X4 receptor in myeloid cells.

The intestinal microbiota contribute to reinforcing epithelial integrity and shaping the immune system via metabolically derived signaling molecules (1–3). However, alterations in microbial metabolites and their translocation following intestinal dysbiosis are implicated in the pathogenesis of chronic disorders, such as inflammatory bowel diseases (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC) (4, 5). Extracellular adenosine triphosphate (ATP) is released by microbes and immune cells in the intestine (6–8) and drives immune responses through the P2X_1-7 and P2Y_{1, 2, 11} receptors (9). To avoid inappropriate immune reactions in the intestine, luminal ATP is strictly controlled by epithelial ATP-hydrolyzing ectoenzymes, such as ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPs) and ectonucleoside triphosphate diphosphohydrolases (E-NTPDases). E-NPP3 on the epithelial cells depresses the apoptosis of plasmacytoid dendritic cells (DCs) in the small intestine and Peyer’s patches (10). In addition, E-NTPD7 in small-intestinal epithelial cells hydrolyzes luminal ATP, thus inhibiting excessive T helper 17 (Th17) responses (11). However, how the concentration of luminal ATP produced by commensal bacteria is regulated in the large intestine remains undetermined.Although intestinal phagocytes such as monocytes, macrophages (Mϕ), DCs, and neutrophils have some protective effects, these cells can also function in pathological conditions (12–14). In patients with IBD, inflamed sites of the intestinal mucosa have more inflammatory DCs and Mϕ, many of which are dysfunctional (15). In addition, an enhanced neutrophil accumulation in the intestinal mucosa of UC patients correlates with disease severity (16–18). Accordingly, experimental murine colitis can be abrogated by inhibiting the recruitment of monocytes and neutrophils to the intestinal lamina propria by using anti-CCR2 (19) or -Gr-1 (20–22) antibody or by the targeted deletion of CCR2 (23) or β7-integrin (24). Thus, the number of intestinal phagocytes and their physiological functions must be tightly tuned to prevent the intestinal inflammation. However, the mechanisms by which the activity of monocytes and neutrophils that have infiltrated into the intestinal mucosa are regulated remain poorly understood.Different immune cell populations have distinct nutrient utilizations and cellular metabolisms (25), which are involved in their differentiation, proliferation, functions, longevity, and epigenetic modification (25–27). Microbial components and metabolites, such as lipopolysaccharide (28) and short chain fatty acids (29), can switch to glycolysis in monocytes and T cells, respectively. The small molecule dimethyl fumarate (DMF), which suppresses glycolysis in lymphocytes and myeloid cells (30), abrogates chemically induced colitis in mice (31, 32), which indicates that inadequate glycolysis activation is involved in the pathogenesis of intestinal inflammation. However, the mechanism underlying the adaptation of intestinal myeloid cells to environmental factors that fuel glycolysis is unclear.In this study, we investigated the immunomodulatory function of ATP-hydrolyzing ectoenzyme E-NTPD8 in maintenance of the gut homeostasis. Mice with Entpd8 deficiency had an increased concentration of luminal ATP in their colons, which led to the prolonged survival of neutrophils owing to the facilitation of glycolysis by the P2X4 receptor (P2X4R), thereby exacerbating dextran sodium sulfate (DSS)-induced colitis. Therefore, the clearance of extracellular ATP by E-NTPD8 is essential for the prevention of innate intestinal pathology by inhibiting a metabolic alteration toward glycolysis in myeloid cells.     

Shear stress activates mitochondrial oxidative phosphorylation by reducing plasma membrane cholesterol in vascular endothelial cells

Vascular endothelial cells (ECs) sense and respond to hemodynamic shear stress, which is critical for circulatory homeostasis and the pathophysiology of vascular diseases. The mechanisms of shear stress mechanotransduction, however, remain elusive. We previously demonstrated a direct role of mitochondria in the purinergic signaling of shear stress: shear stress increases mitochondrial adenosine triphosphate (ATP) production, triggering ATP release and Ca²⁺ signaling via EC purinoceptors. Here, we showed that shear stress rapidly decreases cholesterol in the plasma membrane, thereby activating mitochondrial ATP production. Imaging using domain 4 mutant-derived cholesterol biosensors showed that the application of shear stress to cultured ECs markedly decreased cholesterol levels in both the outer and inner plasma membrane bilayers. Flow cytometry showed that the cholesterol levels in the outer bilayer decreased rapidly after the onset of shear stress, reached a minimum (around 60% of the control level) at 10 min, and plateaued thereafter. After the shear stress ceased, the decreased cholesterol levels returned to those seen in the control. A biochemical analysis showed that shear stress caused both the efflux and the internalization of plasma membrane cholesterol. ATP biosensor imaging demonstrated that shear stress significantly increased mitochondrial ATP production. Similarly, the treatment of cells with methyl-β-cyclodextrin (MβCD), a membrane cholesterol-depleting agent, increased mitochondrial ATP production. The addition of cholesterol to cells inhibited the increasing effects of both shear stress and MβCD on mitochondrial ATP production in a dose-dependent manner. These findings indicate that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs.

Vascular endothelial cells (ECs) recognize shear stress, a biomechanical force generated by flowing blood, and transduce it into intracellular biochemical signals, thereby causing responses such as changes in cell morphology, function, and gene expression (1). These EC responses play crucial roles in maintaining the homeostasis of the circulatory system, and their impairments cause various vascular diseases such as hypertension, aneurysm, and atherosclerosis (2–4). To date, numerous studies have elucidated the mechanism of EC mechanotransduction and have revealed a unique feature: shear stress activates multiple signal transduction pathways through a variety of membrane molecules, including ion channels, receptors, and adhesion proteins, almost simultaneously (5). Recently, it has become apparent that the plasma membrane itself plays an important role in EC mechanotransduction. EC plasma membranes rapidly respond to shear stress by altering their physical properties, such as fluidity, viscosity, and lipid order (6, 7). We previously demonstrated that shear stress decreases the lipid order of not only EC plasma membranes but also, artificial lipid bilayer membranes, thereby causing a transition from the liquid-ordered state to the liquid-disordered state, along with an increase in membrane fluidity (8). These changes in membrane physical properties were linked to downstream signaling pathways, such as the activation of mitogen-activated protein kinase (9) and the phosphorylation of vascular endothelial growth factor receptors (VEGFRs) (10). This mechanism, in which changes in membrane physical properties occur initially and are then followed by the activation of membrane molecules, may characterize the EC mechanotransduction described above (11).Ca²⁺ signaling is known to play a critical role in EC mechanotransduction (12). ECs rapidly release intrinsic adenosine triphosphate (ATP) in response to shear stress (13, 14), and this ATP activates purinoceptors, such as ligand-gated channel P2X and G protein-coupled P2Y receptors, located in the plasma membranes; these purinoceptors are responsible for extracellular Ca²⁺ influx and Ca²⁺ release from the endoplasmic reticulum (15–17). The increase in cytoplasmic Ca²⁺ activates a variety of EC functions. In P2X4-knockout mice, ECs exhibited neither Ca²⁺ signaling nor the production of a potent vasodilator, nitric oxide, in response to shear stress, thereby impairing the blood flow-dependent vasodilator response and vascular remodeling and resulting in hypertension (4). Our recent study revealed that mitochondria play an important role in shear stress-induced ATP release and Ca²⁺ signaling in ECs (18). Upon shear stress stimulation, the ECs rapidly augmented their mitochondrial ATP production, triggering ATP release and subsequent Ca²⁺ signaling. However, the means by which shear stress acting on the plasma membrane affects mitochondrial oxidative phosphorylation in ECs remains unknown.Cholesterol plays a dominant role in determining the mechanical properties of plasma membranes by affecting the membrane lipid order, fluidity, bending modulus, thickness, stiffness, and bilayer pressure profile; in this manner, cholesterol modulates the conformation and function of membrane proteins (19). Plasma membrane cholesterol is involved in not only the control of various cell functions, such as signaling, adhesion, motility, and remodeling of the cytoskeleton (20), but also EC responses to biomechanical forces (10, 21–23). For instance, the depletion of EC plasma membrane cholesterol inhibits the shear stress-induced activation of extracellular signal-regulated kinase (22), whereas the addition of cholesterol to EC plasma membranes suppresses the shear stress-induced phosphorylation of VEGFRs (10). However, the roles of plasma membrane cholesterol in EC mechanotransduction have not been fully elucidated, partly because of a lack of information concerning how plasma membrane cholesterol behaves in ECs under shear stress.In the last decade, domain 4 (D4) of perfringolysin O (PFO), a cholesterol-binding toxin, has been widely used as a cholesterol-specific molecular sensor for measuring and imaging cholesterol in cellular membranes (24). When D4 and its mutants labeled with fluorophores are added to the extracellular medium, cholesterol in the outer leaflet of the plasma membrane can be visualized in living cells; when these sensors are introduced into the cells by gene transfer or microinjection, cholesterol in the inner leaflets of the membrane can be visualized. A recent study showed that these D4-based cholesterol sensors did not lyse the cells or cross the plasma membrane, allowing trans-bilayer asymmetries in plasma membrane cholesterol to be identified in various mammalian cells (25).In the present study, we applied controlled levels of shear stress to cultured human pulmonary aortic endothelial cells (HPAECs) and examined changes in the amounts and distribution of plasma membrane cholesterol using fluorescence imaging and flow cytometry with D4 mutant-based cholesterol biosensors. We also used biochemical measurements to analyze changes in the amounts of cholesterol in whole cells and isolated plasma membranes. Furthermore, to determine whether changes in plasma membrane cholesterol affect mitochondrial oxidative phosphorylation, we examined changes in mitochondrial ATP concentrations using real-time imaging with a fluorescence resonance energy transfer (FRET)-based ATP biosensor (26).     

Age-related changes in intracellular cytokine profiles and Th2 dominance in allergic children

The unbalanced T helper response has been pointed out in allergic diseases. Especially in childhood, it is important to consider the development of acquired immunity. We investigated the relationship between age and Th1, Th2, Tc1 or Tc2 cells. In addition, Th1, Th2, Tc1 or Tc2 cells in allergic diseases were compared with control subjects. Thirty-four healthy controls (0-40 years old), 200 samples of cord blood, nine patients with atopic dermatitis (AD) (1-3 years old) and five patients with bronchial asthma (BA) (2-6 years old) were studied. Surface staining with CD4, CD8 and intracellular staining with anti-interferon-gamma (IFN-gamma) and anti-interleukin (IL)-4 were carried out, and analyzed by using flow cytometry. In the healthy controls, the percentages of Th1, Tc1 or Th2 showed positive correlation with age. The absolute numbers of Th1 or Tc1 also correlated with age. Cord blood with a family history of allergic disease showed no significant difference compared to that without a family history. The percentage of Th2 in AD and BA patients was significantly higher than in the age-matched healthy controls. The increase in Th1, Th2 and Tc1 with age might reflect on the development of acquired immunity. Age matching is important when evaluating the cytokine profiles of T cells. In allergic diseases, although cord blood showed a Th1-dominant pattern, it changed to Th2 dominance in childhood, and this may reflect on some genetic background.     

Establishment of human oral-cancer cell lines (KOSC-2 and -3) carrying p53 and c-myc abnormalities by geneticin treatment

Two cultured cell lines derived from human squamous-cell carcinomas were established through xenografted tumors in nude mice by ?Geneticin”? treatment, which allows to eliminate contaminated mouse fibroblasts and obtain enriched tumor cells at the early stage of cultivation. Line KOSC-2 and KOSC-3 were each derived from a squamous-cell carcinoma of the oral floor and of the lower gingiva, respectively. Both lines grew in a cobblestone pattern, demonstrating their epithelial heritage. Ciemsa-banding patterns by chromosome analysis confirmed that both lines are of human origin. Molecular analysis of cancer-related genes, including the Ha-ras, c-myc and p53 genes, was performed. KOSC-3 cells showed co-over-expression of p53 and c-myc mRNA, in addition to p53 point mutation at codon 248 with transition from CGG to TGG. However, loss of heterozygosfty (LOH) on chromosome 17 was detected in both lines by Southern blotting. These cell lines provide a model for elucidating the mechanism involving p53 inactivation and c-myc-gene over-expression.