Gastritis cystica profunda is associated with aberrant p53 and Epstein–Barr virus in gastric cancer: A clinicopathological,immunohistochemical and in situ hybridization study

Gastritis cystica profunda (GCP) is a lesion characterized by cystic gastric glands within the submucosa. Some studies have reported that GCP is a precancerous lesion. Here, we investigated the association between GCP and gastric cancer. Gastric cancer specimens were taken from 1432 patients undergoing surgery or endoscopic submucosal resection and were classified as GCP or non-GCP. The clinicopathological features, immunohistochemistry and in situ hybridization expression of p53, Ki-67, KCNE2, Epstein–Barr virus (EBV) and programmed death ligand 1 (PD-L1) were compared between the two groups, as well as between GCPs and normal pyloric glands. One hundred and eighty patients (12.6%) had GCPs. In the GCP group, no cancerous lesions were found within the GCPs, but 13% were linked to GCPs and 60.2% were located above or near GCPs. Aberrant p53 expression, EBV-positive cancer cells and PD-L1 scores were significantly higher in the GCP group. The p53 score and Ki-67 labelling index were significantly higher and the KCNE2 score was significantly lower in GCPs than in pyloric glands. Although we suggest GCP is paracancerous, GCP has high proliferation activity and gastric cancer with GCP is associated with aberrant p53 and EBV. GCP is associated with aberrant p53 expression and EBV.     

Additive effect of low-frequency ultrasound and endothelial monocyte-activating polypeptide II on blood–tumor barrier in rats with brain glioma

Brain glioma is a malignant tumor which needs surgery followed by chemotherapy. Low-frequency ultrasound (LFU) and Optison could open blood–tumor barrier (BTB) selectively and noninvasively and thus increase the permeability of BTB. Endothelial monocyte-activating polypeptide II (EMAP-II) induces cytoskeletal remodeling in endothelial cells. In this study, we asked whether LFU, Optison, and/or EMAP-II used in combination have additive effects on increasing the permeability of BTB by tight junction (TJ)-associated protein-dependent manner and thus help understand the possible mechanisms for TJ-based drug delivery to the central nervous system through BTB. Evans Blue assay was used to measure the permeability of BTB in rat model of C6 glioma. The mRNA and protein levels of TJ-associated proteins, claudin-5, occludin, and ZO-1, were determined. Results showed that Evans blue content significantly increased and the mRNA and protein levels of claudin-5, occludin, and ZO-1 significantly reduced after the treatment in groups treated with EMAP-II and LFU combined with or without Optison (LFU + EMAP-II and LFU + Optison + EMAP-II groups) and in the group treated with LFU and Optison (LFU + Optison group). In conclusion, LFU and EMAP-II used in combination have additive effects on increasing the permeability of BTB and remodeling of TJ-associated proteins.     

Transfection of VEGF(165) genes into endothelial progenitor cells and in vivo imaging using quantum dots in an ischemia hind limb model

Endothelial progenitor cells (EPCs) were transfected with fluorescently labeled quantum dot nanoparticles (QD NPs) with or without VEGF(165) plasmid DNA (pDNA) to probe the EPCs after in?vivo transplantation and to test whether they presented as differentiated endothelial cells (ECs). Bare QD NPs and QD NPs coated with PEI or PEI?+?VEGF(165) genes were characterized by dynamic light scattering, scanning electron microscopy, and atomic force microscopy. Transfection of EPCs with VEGF(165) led to the expression of specific genes and proteins for mature ECs. A hind limb ischemia model was generated in nude mice, and VEGF(165) gene-transfected EPCs were transplanted intramuscularly into the ischemic limbs. At 28 days after transplantation, the VEGF(165) gene-transfected EPCs significantly increased the number of differentiated ECs compared with the injection of medium or bare EPCs without VEGF(165) genes. Laser Doppler imaging revealed that blood perfusion levels were increased significantly by VEGF(165) gene-transfected EPCs compared to EPCs without VEGF(165). Moreover, the transplantation of VEGF(165) gene-transfected EPCs increased the specific gene and protein expression levels of mature EC markers and angiogenic factors in the animal model.     

Correlation of emmprin expression in vascular endothelial cells with blood–brain-barrier function: a study using magnetic resonance imaging enhanced by Gd-DTPA and immunohistochemistry in brain tumors

In a previous study, we demonstrated that the expression levels in tumor cells of emmprin (CD147) correlated with the grade of astrocytic tumors. Also, we found that emmprin was expressed in vascular endothelial cells of the non-neoplastic brain and hypothesized that emmprin expression could be associated with normal blood-brain-barrier (BBB) function of vascular endothelial cells. In this study, this possibility was examined in non-neoplastic brain, glioma and metastatic carcinoma tissues by comparing emmprin immunohistochemistry with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) enhancement of magnetic resonance imaging (MRI), which is a clinical indicator of the BBB function. This study included 10 cases of non-neoplastic brain tissues, 7 of metastatic carcinoma, 7 of diffuse astrocytoma, 4 of anaplastic astrocytoma and 13 of glioblastoma multiforme. In all the cases, MRI with administration of Gd-DTPA was performed. The lesions were resected using the microdissection method with the help of ultrasonography and a neuronavigator. The tissues from Gd-DTPA-enhanced or non-enhanced areas were processed into frozen sections and subjected to immunohistochemistry with anti-emmprin antibody. The expression of emmprin in brain vascular endothelial cells inversely correlated with Gd-DTPA-enhancement of MRI: emmprin was positive in tissues not enhanced by Gd-DTPA and was negative in DTPA-enhanced tissues. Since BBB function presumably remains unimpaired in regions in which MR images are not Gd-DTPA-enhanced, emmprin expression appears to be associated with unimpaired BBB function. This is the first report to demonstrate a possible correlation between emmprin expression and BBB function in humans.     

Molecular and clinical findings and their correlations in Silver-Russell syndrome: implications for a positive role of IGF2 in growth determination and differential imprinting regulation of the IGF2–H19 domain in bodies and placentas

Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features and is frequently caused by hypomethylation (epimutation) of the H19-DMR. Although molecular and clinical studies have extensively been performed for SRS patients themselves, such studies have not been carried out for placentas. We identified 20 epimutation-positive and 40 epimutation-negative Japanese SRS patients and obtained placental weight data from 12 epimutation-positive and ten epimutation-negative patients and paraffin-embedded placental tissues for molecular and histological examinations from three epimutation-positive and two epimutation-negative patients. Methylation patterns were comparable between leukocytes and placentas in both epimutation-positive and epimutation-negative patients. Epimutations resulted in virtually no IGF2 expression and biallelic slight H19 expression in the leukocytes and obviously reduced IGF2 expression of paternal origin and nearly normal H19 expression of maternal origin in the placentas. Epimutation-positive patients had characteristic body phenotype and small placentas with hypoplastic chorionic villi, and epimutation-negative patients had somewhat small placentas with hypoplastic chorionic villi or massive infarction. Furthermore, significant correlations were identified between the H19-DMR methylation index and the body and placental sizes and between the placental weight and the body size in the epimutation-positive patients, whereas such correlations were not detected for the head circumference. These results suggest (1) characteristic phenotype and reduced IGF2 expression in the epimutation-positive placentas; (2) similarities and differences in the epigenetic control of the IGF2–H19 domain between leukocytes and placentas; (3) a positive role of the IGF2 expression level, as reflected by the methylation index, in the determination of body and placental growth in epimutation-positive patients, except for the brain where IGF2 is expressed biallelically; (4) involvement of placental dysfunction in prenatal growth failure; and (5) relevance of both (epi)genetic factor(s) and environmental factor(s) to SRS in epimutation-negative patients. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin–Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells

Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.     

Systems biology of host–mycobiota interactions: Dissecting Dectin-1 and Dectin-2 signalling in immune cells with DC-ATLAS

Modelling the networks sustaining the fruitful coexistence between fungi and their mammalian hosts is becoming increasingly important to control emerging fungal pathogens.     

The characteristics and transfection efficiency of cationic poly (ester–co–urethane) – short chain PEI conjugates self-assembled with DNA

To improve the transfection efficiency of polycations with DNA, we synthesized poly(ester–co–urethane)(PEU-g-PEI800) with short chain PEI800 in the side chain, and poly(ester–co–urethane)(PEU) without short chain PEI800. Both PEU-g-PEI800 and PEU, readily self-assembled with plasmid DNA (pCMV-βgal) in a HEPES buffer, were characterized by dynamic light scattering and zeta-potential. The results reveal that PEU-g-PEI800 and PEU were able to self-assemble particles with DNA and yield nano-sized complexes (<200 nm) with positive charge at N/P ratios of 20/1 and 120/1, respectively. The degradation studies indicate that the half-life of PEU-g-PEI800 and PEU in the HEPES buffer were 14 and 35 h at pH 7.4, respectively. Titration studies were performed to determine the buffering capacities of the polymers. The COS-7 cell viabilities in the presence of PEU-g-PEI800/DNA, PEU/DNA, and PEI25k/DNA were studied. In addition, The PEU-g-PEI800/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier PEI25k. The results indicate that PEU-g-PEI800 is an attractive cationic poly (ester–co–urethane) for gene delivery and an interesting candidate for further study.     

A critical review of vascular endothelial growth factor (VEGF) analysis in peripheral blood: is the current literature meaningful?

Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor with a key role in many physiological and pathological processes. Investigation into the implications of circulating levels of this cytokine is progressing at an exponential rate. However, there are important inconsistencies between reports ranging from method of sample collection, processing, software manipulation and data interpretation and controversy as to whether plasma, serum or whole blood will provide the best prognostic information. Different techniques of centrifugation and temperature on sample handling and the impact of in vitro collection of blood on subsequent VEGF results have not been fully appreciated. We provide a critical review of the literature, report the results of our further investigations, suggest a uniform protocol for handling blood samples and highlight previously unsuspected problems in data interpretation. This revised version was published online in July 2006 with corrections to the Cover Date.     

The stimulation of CD8+ T cells by dendritic cells pulsed with polyketal microparticles containing ion-paired protein antigen and poly(inosinic acid)–poly(cytidylic acid)

New adjuvants and delivery strategies are needed to optimize the ability of protein-based vaccines to elicit CD8⁺ T cell responses. We have developed a model vaccine formulation containing ovalbumin (OVA) and the double-stranded RNA analog poly(inosinic acid)–poly(cytidylic acid) (poly(I:C)), a TLR3 agonist. OVA and poly(I:C) were each ion-paired to cetyltrimethylammonium bromide (CTAB) to produce hydrophobic complexes, which were co-encapsulated in pH-sensitive polyketal (PK3) microparticles (1–3 μm) using a single emulsion method. Loading levels ranged from 13.6 to 18.8 μg/mg OVA and 4.8 to 10.3 μg/mg poly(I:C). Murine splenic dendritic cells (DCs) pulsed with PK3-OVA–poly(I:C) microparticles, at antigen doses of 0.01 and 0.1 μg/mL, induced a higher percentage of IFNγ-producing CD8⁺ T cells than DCs treated with PK3-OVA particles or soluble OVA/poly(I:C). A higher antigen dose (1 μg/mL) was less effective, which can be attributed to CTAB toxicity. At the lowest antigen dose (0.01 μg/mL), PK3-OVA–poly(I:C) microparticles also enhanced TNF-α and IL-2 production in CD8⁺ T cells. These data demonstrate the potential of polyketal microparticles in formulating effective CD8⁺ T cell-inducing vaccines comprising protein antigens and dsRNA adjuvants.     

Error-free replicative bypass of (6–4) photoproducts by DNA polymerase ζ in mouse and human cells

The ultraviolet (UV)-induced (6–4) pyrimidine–pyrimidone photoproduct [(6–4) PP] confers a large structural distortion in DNA. Here we examine in human cells the roles of translesion synthesis (TLS) DNA polymerases (Pols) in promoting replication through a (6–4) TT photoproduct carried on a duplex plasmid where bidirectional replication initiates from an origin of replication. We show that TLS contributes to a large fraction of lesion bypass and that it is mostly error-free. We find that, whereas Pol η and Pol ι provide alternate pathways for mutagenic TLS, surprisingly, Pol ζ functions independently of these Pols and in a predominantly error-free manner. We verify and extend these observations in mouse cells and conclude that, in human cells, TLS during replication can be markedly error-free even opposite a highly distorting DNA lesion.     

Cytocomposition of the vitellarium in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae): another caryophyllidean species with lamellar bodies and lipids

The vitellarium of the invasive caryophyllidean tapeworm Khawia sinensis Hsü, 1935 from carp Cyprinus carpio L. was examined by means of transmission electron microscopy and cytochemical staining for glycogen with periodic acid–thiosemicarbazide–silver proteinate (PA-TSC-SP). A vitellarium consists of numerous follicles of irregular size that are interconnected by a net of vitelline ducts. Vitelline follicles are composed of vitelline cells at various stages of development that are interconnected by interstitial tissue. Vitelline follicles are surrounded by a cytoplasmic sheath associated with an intercellular matrix. Extensive development of the granular endoplasmic reticulum and Golgi complexes are both involved in the production of shell globules/shell globule clusters and characterise cytodifferentiation of vitellocytes. Nuclear and nucleolar transformation lead to the formation and storage of intranuclear glycogen, a feature specific for the Caryophyllidea. Newly observed within the mature vitellocytes of Khawia sp. is the presence of lamellar bodies and a few lipid droplets. These cytoplasmic inclusions first occur in the mature cells within the follicles and persist in the vitelline cells within vitelloducts and intrauterine eggs. Two types of lamellar bodies are detected: regular lamellar-structured body and irregular lamellar-structured body. None of the lamellar bodies are membrane bound. Results of the present study indicate that the formation of lamellar bodies may be closely related to the endoplasmic reticulum or shell globule clusters. Some of the shell globule clusters are transformed into lamellar body clusters. Ultrastructural features of vitellocytes in K. sinensis are compared with those of other monopleuroid, polypleuroid, and strobilated cestodes.     

Activation of epithelial and myoepithelial cells in the salivary glands of patients with Sjögren's syndrome: high expression of intercellular adhesion molecule-1 (ICAM.1) in biopsy specimens and cultured cells

ICAM.1 (CD54) is a surface protein expressed on epithelial and other nonhematopoietic cells upon activation and is known to play an important role in the stimulation of T cells by the provision of cellular adhesion and costimulatory support. Sjogren's syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. To address the contribution of ICAM.1 in the pathogenesis of SS, the expression of this protein was studied by immunohistochemistry and flow cytometry in minor salivary gland (SG) biopsies as well as in cultured SG epithelial cell (SGEC) lines obtained from 18 SS patients and 16 controls. In biopsies from SS patients (but not controls), strong ICAM.1 was expressed by infiltrating mononuclear cells (52%) and by a significant proportion of periacinar myoepithelial cells (18%). In addition, a patchy pattern of moderate ICAM.1 expression was detected in 31% of ductal epithelia of SS patients. These ICAM.1-expressing epithelial and myoepithelial cells were observed throughout glandular tissues and were not confined in areas proximal to lymphoid infiltrates. In support to an intrinsic activation profile of SGEC in SS, long-term cultured non-neoplastic SGEC lines derived from SS patients displayed significantly upregulated spontaneous expression of ICAM.1, compared to controls (P < 0.05). The high expression of ICAM.1 protein by the salivary epithelium of SS patients is likely suggestive of its important role in the pathogenesis of the disorder. Further, our results support a model of intrinsic activation of salivary epithelial and myoepithelial cells in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients.     

Differential modulating effect of natural killer (NK) T cells on interferon-γ production and cytotoxic function of NK cells and its relationship with NK subsets in Chlamydia muridarum infection

Natural killer T (NKT) cells are a newly identified T-cell population with potential immunomodulatory functions. Several studies have shown modulating effects of NKT cells activated by α-galactosylceramide, a model antigen, on NK cell function. We here report a differential modulating effect of NKT cells on the interferon-γ (IFN-γ) production and cytolytic function of NK cells in a chlamydial infection model, using NKT-cell-deficient mice and antibody blocking (anti-CD1d monoclonal antibody) approaches. Our results showed that both NKT and NK cells became activated and produced IFN-γ following Chlamydia muridarum infection in vitro and in vivo. The NK cells in NKT-cell-deficient mice and CD1d-blocked mice showed decreased CD69 expression, cellular expansion and IFN-γ production but surprisingly showed increased cytolytic activity (degranulation) of immature and more mature NK cell subsets, suggesting an inhibitory role of NKT cells on NK cell killing activity. The results suggest that NKT cells preferentially promote IFN-γ production but are inhibitory for the cytotoxic function of NK cells in this infection model. Furthermore, the differential modulating effect of NKT cells on the IFN-γ production and cytotoxicity of NK cells was observed in immature and mature NK cell subsets, although it was more dramatic in the relatively mature CD11b(high) CD27(high) NK cell subset. This finding demonstrates the complexity of innate cell interactions in infection and the possible differential impact of NKT cells on the variable functional aspects of other cell(s) even in one infection setting.     

Vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1ɑ) in lacrimal gland Adenoid cystic carcinoma: Correlation with clinical outcome

PurposeVEGF and HIF-1α are important regulators of angiogenesis, overexpressed in various tumors. Lacrimal gland Adenoid cystic carcinoma (ACC) is a malignant tumor whose angiogenic properties remain unexplored. This study was designed to evaluate the expression of HIF-1α and VEGF in lacrimal gland ACC.MethodsVEGF and HIF-1α immunoexpression was undertaken in 30 lacrimal gland ACC cases. mRNA expression of VEGF and HIF-1α was analysed in 17 cases by quantitative real time PCR. The results obtained were correlated with clinicopathological features and survival of the patients to determine the prognostic significance.ResultsImmunoexpression of HIF-1α and VEGF was seen in 36.6% and 46.6% ACC cases. HIF-1α expression showed significant association with advanced T-stage (P = 0.001) and VEGF with intracranial extension (P = 0.014) and solid histological pattern (P = 0.045). HIF-1α mRNA expression was seen in 29.4% cases and showed significant association with perineural invasion (P = 0.027). Recurrence occurred in 60%, distant metastasis in 20% and death in 20% cases. Survival analysis revealed that patients with HIF-1α, VEGF immunoexpression, solid histological pattern, perineural invasion, bone erosion, intracranial extension, metastasis, advanced T-stage, and exenteration had poor survival. On multivariate analysis VEGF immunoexpression (hazard ratio, 16.785; 95% confidence interval, 1.872–150.495; P = 0.012) was the most significant poor prognostic factor.ConclusionsThis study demonstrates that VEGF is a potential predictor for poor clinical outcome in lacrimal gland Adenoid cystic carcinoma.     

Lymphocyte recognition of human parvovirus B19 non-structural (NS1) protein: associations with occurrence of acute and chronic arthropathy?

Immune recognition of recombinant parvovirus B19 non-structural (rNS1) protein was studied by immunoblot and lymphoproliferative assays in blood from the following B19 seropositive groups: B19 infected (n = 14), B19 exposed but non-infected (n = 16), other illness with rash (n = 3), chronic arthropathy of unknown aetiology (n = 4) and healthy controls (n = 7). Sera from 11 B19 seronegative subjects were also studied. Sera collected at initial diagnosis or at the time of accidental B19 exposure in pregnancy were tested for NS1 antibody and evidence of B19 DNA by nested PCR. Follow-up specimens were obtained 3-12 months later for serological, PCR and proliferation studies. B19 DNA was detected sporadically in early specimens and in one follow-up specimen from a subject who developed chronic arthropathy after B19 infection. There was no correlation with development of arthropathy. NS1-specific IgG was detected in early sera from B19-infected and exposed subjects but to a lesser degree in follow-up specimens, and in only one healthy control serum. No correlation with the presence of NS1-specific antibodies was found with development of acute or chronic arthropathy. Although lymphocyte proliferation in response to stimulation with rNS1 in vitro occurred at a higher frequency in patients who developed acute and chronic joint manifestations after B19 infection, suggesting an association with this outcome, NS1-reactive lymphocytes were also found in three B19 seronegative patients, two of whom had recently been exposed to B19 but had no illness. Hence, immune recognition of NS1 may be more indicative of recent infection with, or exposure to, parvovirus B19 than associated with development of arthropathy as previously reported.