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Accelerated fetal myocardial growth with altered cardiac function is a well-documented complication of human diabetic pregnancy, but its pathophysiology is still largely unknown. Our aim was to explore the mechanisms of fetal cardiac remodeling and cardiovascular hemodynamics in a rat model of maternal pregestational streptozotocin-induced hyperglycemia. The hyperglycemic group comprised 107 fetuses (10 dams) and the control group 219 fetuses (20 dams). Fetal cardiac function was assessed serially by Doppler ultrasonography. Fetal cardiac to thoracic area ratio, newborn heart weight, myocardial cell proliferative and apoptotic activities, and cardiac gene expression patterns were determined. Maternal hyperglycemia was associated with increased cardiac size, proliferative, apoptotic and mitotic activities, upregulation of genes encoding A- and B-type natriuretic peptides, myosin heavy chain types 2 and 3, uncoupling proteins 2 and 3, and the angiogenetic tumor necrosis factor receptor superfamily member 12A. The genes encoding Kv channel-interacting protein 2, a regulator of electrical cardiac phenotype, and the insulin-regulated glucose transporter 4 were downregulated. The heart rate was lower in fetuses of hyperglycemic dams. At 13-14 gestational days, 98% of fetuses of hyperglycemic dams had holosystolic atrioventricular valve regurgitation and decreased outflow mean velocity, indicating diminished cardiac output. Maternal hyperglycemia may lead to accelerated fetal myocardial growth by cardiomyocyte hyperplasia. In fetuses of hyperglycemic dams, expression of key genes that control and regulate cardiomyocyte electrophysiological properties, contractility, and metabolism are altered and may lead to major functional and clinical implications on the fetal heart.

Objectives: This study investigated the effects of age, duration of a high-fat diet, and type 2 diabetes on atherosclerotic plaque development and uptake of (18)F-fluorodeoxyglucose ((18)F-FDG) in 2 mouse models., Background: The animal's age and start time and duration of a high-fat diet have effects on plaque composition in atherosclerotic mice., Methods: The aortas of atherosclerotic low-density lipoprotein receptor deficient mice expressing only apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) and atherosclerotic and diabetic mice overexpressing insulin-like growth factor II (IGF-II/LDLR(-/-)ApoB(100/100)) were investigated at 4, 6, and 12 months of age and older after varying durations of high-fat diet. C57BL/6N mice on normal chow served as controls. Plaque size (intima-to-media ratio), macrophage density (Mac-3 staining), and plaque uptake of (18)F-FDG were studied by means of in vivo positron emission tomography/computed tomography by ex vivo autoradiography and by histological and immunohistochemical methods., Results: From the ages of 4 to 6 months and 12 months and older, the plaque size increased and the macrophage density decreased. Compared with the controls, the in vivo imaging showed increased aortic (18)F-FDG uptake at 4 and 6 months, but not at 12 months and older. Autoradiography showed focal (18)F-FDG uptake in plaques at all time points (average plaque-to-normal vessel wall ratio: 2.4 ± 0.4, p < 0.001) with the highest uptake in plaques with high macrophage density. There were no differences in the plaque size, macrophage density, or uptake of (18)F-FDG between LDLR(-/-)ApoB(100/100) and IGF-II/LDLR(-/-)ApoB(100/100) mice at any time point., Conclusions: The 6-month-old LDLR(-/-)ApoB(100/100) and IGF-II/LDLR(-/-)ApoB(100/100) mice demonstrated highly inflamed, large, and extensive atherosclerotic plaques after 4 months of a high-fat diet, presenting a suitable model for studying the imaging of atherosclerotic plaque inflammation with (18)F-FDG. The presence of type 2 diabetes did not confound evaluation of plaque inflammation with (18)F-FDG., (Copyright © 2011 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Atherosclerosis is a chronic inflammatory disease of artery wall characterized by infiltration of monocytes into subendothelial space and their differentiation into macrophages. Since rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophage content may provide a useful tool for the evaluation of plaque vulnerability. The purpose of this study was to explore the uptake of 68gallium (68Ga) in atherosclerotic plaques in mice., Methods: Uptake of ionic 68Ga was investigated in atherosclerotic LDLR-/-ApoB100/100 and C57BL/6N control mice at 3 h after injection. The ex vivo biodistribution of the 68Ga was assessed and autoradiography of aortic cryosections was defined. In vivo imaging of 68Ga was performed using a small animal positron emission tomography PET/CT scanner., Results: Our results revealed that the uptake of 68Ga-radioactivity was higher in atherosclerotic plaques than in healthy vessel wall (ratio 1.8 ± 0.2, p = 0.0002) and adventitia (ratio 1.3 ± 0.2, p = 0.0011). The autoradiography signal co-localized with macrophages prominently as demonstrated by Mac-3 staining. In both mice strains, the highest level of radioactivity was found in the blood., Conclusions: We observed a moderate but significantly elevated 68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages. While the uptake of 68Ga was promising in this animal model, the slow blood clearance may limit the usability of 68Ga as a PET tracer for clinical imaging of atherosclerotic plaques.

Objective: Atherosclerotic plaques with large lipid cores and inflammation contain regions of hypoxia. We examined the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide ([18F]EF5), a specific marker of hypoxia labeled for positron emission tomography, in mouse atherosclerotic plaques., Methods and Results: Atherosclerotic mice of 2 different genetic backgrounds (low-density lipoprotein receptor-/- apolipoprotein B100/100 and insulin-like growth factor II/low-density lipoprotein receptor-/- apolipoprotein B100/100) were first fed a Western diet to induce development of plaques with variable phenotypes and then injected with [18F]EF5. C57BL/6N mice served as controls. Aortas were dissected for biodistribution studies, autoradiography, histology, and immunohistochemistry. Uptake of [18F]EF5 was significantly higher in the aortas of mice with large atherosclerotic plaques than in the C57BL/6N controls. Furthermore, autoradiography demonstrated, on average, 2.0-fold higher [18F]EF5 uptake in atherosclerotic plaques than in the adjacent normal vessel wall. Hypoxia in plaques was verified by using an EF5 adduct-specific antibody and pimonidazole. The blood clearance of [18F]EF5 was slow, with blood radioactivity remaining relatively high up to 180 minutes after injection., Conclusions: Large atherosclerotic plaques in mice contained hypoxic areas and showed uptake of [18F]EF5. Despite its slow blood clearance, the high uptake of [18F]EF5 in plaques suggested that plaque hypoxia is a potential target for identifying high-risk plaques noninvasively.

Unlabelled: The purpose of this study was to explore the feasibility of (11)C-choline in the assessment of the degree of inflammation in atherosclerotic plaques., Methods: Uptake of (11)C-choline was studied ex vivo in tissue samples and aortic sections excised from 6 atherosclerotic mice deficient for both low-density lipoprotein receptor and apolipoprotein B48 (LDLR(-/-)ApoB(100/100)) and 5 control mice. The autoradiographs were compared with the immunohistology of the arterial sites., Results: The uptake of (11)C-choline (percentage of the injected activity per gram of tissue) in the atherosclerotic aortas of the LDLR(-/-)ApoB(100/100) mice was significantly higher (1.9-fold, P = 0.0016) than that in the aortas of the control mice. The autoradiography analysis showed significantly higher uptake of (11)C-choline in the plaques than in healthy vessel wall (mean ratio, 2.3 +/- 0.6; P = 0.014), prominently in inflamed plaques, compared with noninflamed plaque areas., Conclusion: We observed a high (11)C-choline uptake in the aortic plaques of atherosclerotic mice. Our data suggest that macrophages may be responsible for the uptake of (11)C-choline in the plaques.

Among mammalian secreted phospholipases A2 (sPLA(2)s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA(2) [mouse (m)GX] is one of the most highly expressed PLA(2) in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA(2)s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA(2) inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA(2)alpha and M-type sPLA(2) receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA(2) mitogenic effects. Together, our results indicate that group X sPLA(2) may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression.

Group IIA Phospholipase A(2) (PLA2-IIA), a key enzyme in arachidonic acid and eicosanoid metabolism, participates in a variety of inflammatory processes but possibly also plays a role in tumor progression in vivo. Our aim was to determine the mRNA and protein expression of PLA2-IIA during prostate cancer progression in localized and metastatic prostate tumors. We evaluated the prognostic significance of PLA2-IIA expression in biochemical recurrence, clinical recurrence and disease-specific survival after surgical treatment. The expression of PLA2-IIA was examined by immunohistochemistry and chromogenic in situ hybridization in tissue microarrays of radical prostatectomy specimens and advanced/metastatic carcinomas. The expression data were analyzed in conjunction with clinical follow-up information and clinicopathological variables. The mRNA and protein expression of PLA2-IIA was significantly increased in Gleason pattern grade 2-4 carcinomas compared with benign prostate (p-values 0.042-0.001). In metastases, the expression was significantly lower than in local cancers (p=0.001). The PLA2-IIA expression correlated positively with Ki-67 and alpha-methylacyl CoA racemase (AMACR) expression. The prognostic evaluation revealed decreased PLA2-IIA protein expression among patients who had died of prostate cancer. In conclusion, PLA2-IIA expression is increased in carcinoma when compared with benign prostate. However, metastatic carcinoma showed decreased expression of PLA2-IIA when compared with primary carcinomas. PLA2-IIA may serve as a marker for highly proliferating, possibly poorly differentiated prostate carcinomas. The protein expression of PLA2-IIA may be diminished in patients who consequently die of prostate cancer.

Purpose: The ligand [(11)C]PK11195 binds with high affinity and selectivity to peripheral benzodiazepine receptor, expressed in high amounts in macrophages. In humans, [(11)C]PK11195 has been used successfully for the in vivo imaging of inflammatory processes of brain tissue. The purpose of this study was to explore the feasibility of [(11)C]PK11195 in imaging inflammation in the atherosclerotic plaques., Methods: The presence of PK11195 binding sites in the atherosclerotic plaques was verified by examining the in vitro binding of [(3)H]PK11195 onto mouse aortic sections. Uptake of intravenously administered [(11)C]PK11195 was studied ex vivo in excised tissue samples and aortic sections of a LDLR/ApoB48 atherosclerotic mice. Accumulation of the tracer was compared between the atherosclerotic plaques and non-atherosclerotic arterial sites by autoradiography and histological analyses., Results: The [(3)H]PK11195 was found to bind to both the atherosclerotic plaques and the healthy wall. The autoradiography analysis revealed that the uptake of [(11)C]PK11195 to inflamed regions in plaques was more prominent (p = 0.011) than to non-inflamed plaque regions, but overall it was not higher than the uptake to the healthy vessel wall. Also, the accumulation of (11)C radioactivity into the aorta of the atherosclerotic mice was not increased compared to the healthy control mice., Conclusions: Our results indicate that the uptake of [(11)C]PK11195 is higher in inflamed atherosclerotic plaques containing a large number of inflammatory cells than in the non-inflamed plaques. However, the tracer uptake to other structures of the artery wall was also prominent and may limit the use of [(11)C]PK11195 in clinical imaging of atherosclerotic plaques.

Strong expression of many matrix metalloproteinases (MMPs) has been related to poor survival of colorectal cancer (CRC) patients. The expression of tissue inhibitors of metalloproteinases (TIMPs) has been associated with both a beneficial and a poor outcome and there is thus a need to further clarify the significance of MMPs and TIMPs in CRC. The prognostic significance of 4 MMPs and TIMPs in CRC was evaluated. Formalin-fixed, paraffin-embedded tissue arrayed samples of 351 patients with primary colon or rectal cancer of Dukes' stages A-D were selected for immunohistochemical staining of MMP-1, -2, -7 and -13, and TIMP-1, -2, -3 and -4. High expression of MMP-2 in the malignant epithelium as well as in the surrounding stroma was associated with reduced survival of colon cancer patients. Strong epithelial and stromal cytoplasmic staining of TIMP-3 was associated with a longer survival in rectal cancer patients, and here the interobserver variation for evaluating the degree of staining was lower than for epithelial staining. Strong stromal cytoplasmic staining of TIMP-4 predicted longer survival of rectal cancer patients. Multivariate analysis showed that stromal cytoplasmic TIMP-3 staining was the only marker of independent prognostic value. MMP-2 might be a useful prognostic marker in colon cancer, and TIMP-3 and TIMP-4 in rectal cancer, but the findings associated with stromal staining should be interpreted with some caution. Different biologic behavior or different genetic development may explain the differences between colon and rectal cancers regarding the expression of MMP-2, TIMP-3 and TIMP-4.

Purpose: [(18)F]FDG has been used as an inflammation marker and shown to accumulate in inflammatory atherosclerotic plaques. The aim of this study was to investigate the uptake and location of [(18)F]FDG in atherosclerotic plaque compartments., Methods: The biodistribution of intravenously administered [(18)F]FDG was analysed in atherosclerotic LDLR/ApoB48 mice (n=11) and control mice (n=9). Digital autoradiography was used to detect the ex vivo distribution in frozen aortic sections. In vitro binding of [(18)F]FDG in human atherosclerotic arteries was also examined., Results: The uptake of [(18)F]FDG was significantly higher in the aorta of atherosclerotic mice as compared with the control mice. Autoradiography of excised arteries showed higher [(18)F]FDG uptake in the plaques than in the healthy vessel wall (mean ratio +/-SD 2.7+/-1.1). The uptake of [(18)F]FDG in the necrotic, calcified sites of the advanced atherosclerotic lesions was 6.2+/-3.2 times higher than that in the healthy vessel wall. The in vitro studies of human arterial sections showed marked binding of [(18)F]FDG to the calcifications but not to other structures of the artery wall., Conclusion: In agreement with previous studies, we observed [(18)F]FDG uptake in atherosclerotic plaques. However, prominent non-specific binding to calcified structures was found. This finding warrants further studies to clarify the significance of this non-specific binding in human plaques in vivo.

Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.

Group IIA phospholipase A2 (PLA2-IIA) is an enzyme which has important roles in inflammation and infection. Recently, a novel human secretory PLA2 called group XIIA PLA2 (PLA2-XIIA) has been identified. Both PLA2-IIA and PLA2-XIIA are bactericidal against Gram-positive bacteria like many other secretory PLA2s. However, PLA2-XIIA is the only known PLA2 displaying significant bactericidal activity against the Gram-negative bacterium Escherichia coli. We examined the antibacterial properties of recombinant human PLA2-IIA and PLA2-XIIA against Helicobacter pylori, a Gram-negative bacterium, in vitro. PLA2-IIA was not bactericidal against H. pylori, whereas PLA2-XIIA effectively killed H. pylori at a concentration of 50 microg/ml but was not bactericidal at concentrations of 0.5 microg/ml and 5 microg/ml.

The ErbB1 and ErbB2 receptors are oncogenes with therapeutic significance in human cancer, whereas the transforming potential of the related ErbB4 receptor has remained controversial. Here, we have addressed whether four alternatively spliced ErbB4 isoforms differ in regulating cellular responses relevant for tumor growth. We show that the two tumor necrosis factor-alpha converting enzyme (TACE)-cleavable ErbB4 isoforms (the juxtamembrane [JM]-a isoforms) were overexpressed in a subset of primary human breast cancers together with TACE. The overexpression of the JM-a cytoplasmic (CYT)-2 ErbB4 isoform promoted ErbB4 phosphorylation, survival of interleukin-3-dependent cells, and proliferation of breast cancer cells even in the absence of ligand stimulation, whereas activation of the other three ErbB4 isoforms required ligand stimulation. Ligand-independent cellular responses to ErbB4 JM-a CYT-2 overexpression were regulated by both tyrosine kinase activity and a two-step proteolytic generation of an intracellular receptor fragment involving first a TACE-like proteinase, followed by gamma-secretase activity. These data suggest a novel transforming mechanism for the ErbB4 receptor in human breast cancer that is 1) specific for a single receptor isoform and 2) depends on proteinase cleavage and kinase activity but not ligand activation of the receptor.

Purpose: Clinicopathologic staging is even today the best prognostic factor in both colon and rectal cancers. There is still considerable variation in survival within the stages. To find other prognostic indicators we investigated six biologic markers associated with apoptosis and cell proliferation., Methods: Formalin-fixed, paraffin-embedded tissue samples of 363 patients with primary colon or rectal cancer of Dukes Stages A to D were chosen for immunohistochemical staining of five tumor markers: bcl-2, p53, Ki-67, cyclin D1, and carcinoembryonic antigen. Also, the number of apoptotic cells was studied by the terminal deoxynucleotidyl transferase-mediated D: -UTP nick end labeling method in 347 cases. The study was done on specially prepared tissue arrays., Results: In rectal cancer, patients with a Ki-67 labeling index of 5 percent or higher had a better prognosis than those with a lower index. Also, positive cytoplasmic p53 expression predicted a favorable outcome in rectal cancer. In colon cancer, positive nuclear staining of cyclin D1 reflected better survival. Weak and moderate staining of carcinoembryonic antigen correlated with better prognosis than strong staining, but negative staining predicted poor outcome. High apoptotic index of 100 or higher correlated with poor prognosis in colon cancer. However, in rectal cancer, the trend was the opposite. Bcl-2 staining tended to be more intense in samples of patients living 5 years or longer compared with those with worse prognosis., Conclusions: Colon cancer and rectal cancer seem to have different biologic behavior, at least with respect to apoptosis, cytoplasmic p53 expression, and perhaps Ki-67 and carcinoembryonic antigen. Further studies are needed to clarify the significance of these factors.

The complement system is regarded as an important component of the innate defence system against invading bacteria. However, synergistic actions between the complement and the other components of innate immunity are incompletely known. Human group IIA phospholipase A(2) (hGIIA PLA(2)) is an effective antibacterial enzyme in serum of patients with severe bacterial infections. Our aim was to investigate the significance of complement and hGIIA PLA(2) in acute phase serum. Serum samples were collected from patients with acute bacterial infections and from healthy control subjects. We prepared hGIIA PLA(2)-depleted serum by immunoadsorption and inhibited the activity of complement by a specific inhibitor, compstatin. The bactericidal effects of treated and untreated serum were compared by incubating Staphylococcus aureus and Listeria monocytogenes in the presence of serum. Acute phase serum effectively killed S. aureus and L. monocytogenes, and depletion of hGIIA PLA(2) significantly reduced the antibacterial effect. Complement had a weak bactericidal effect against L. monocytogenes. We conclude that hGIIA PLA(2) is the major antibacterial factor in human acute phase serum against the gram-positive bacteria S. aureus and L. monocytogenes, exceeding complement in efficiency.

Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A(2) (PLA(2)), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein. Antibodies were raised in rabbits against recombinant human PLA(2) proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 microg/L for the TR-FIA for GIB PLA(2), 1 microg/L (GIIA), 35 microg/L (GIID), 3 microg/L (GIIE), 4 microg/L (GIIF), 14 microg/L (GIII), 11 microg/L (GV), 2 microg/L (GX), 92 microg/L (GXIIA) and 242 microg/L (GXIIB). All secreted PLA(2)s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA(2) in serum (median 19.0 microg/L, range 13.1-33.7 microg/L, n = 4) as compared to the healthy blood donors (median 1.8 microg/L, range 0.8-3.4 microg/L, n = 28, P < 0.0001). The concentration of GIIA PLA(2) in the sera of septic patients (median 315.7 microg/L, range 15.9-979.6 microg/L, n = 34) was highly elevated as compared to that of the blood donors (median 1.8 microg/L, range 0.8-5.8 microg/L, n = 28, P < 0.0001). Our current results confirmed elevated concentrations of GIB and GIIA PLA(2) in the sera of patients suffering from acute pancreatitis or septic infections, respectively, as compared to healthy subjects. However, in the same serum samples, the concentrations of the other secreted PLA(2)s, viz. GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were below the respective analytical sensitivities of the TR-FIAs. It is concluded that generalized bacterial infections do not lead to elevated serum levels of GIIE, GIIF, GIII, GV and GX PLA(2)s above the detection limits of the current TR-FIAs.

Background: Both various virulence factors of Helicobacter pylori and host factors influence the clinical outcome of H. pylori infection. In animal experiments with Helicobacter felis, large variations in the severity of disease have been observed between different mouse strains infected with a single isolate of H. felis. C57BL/6 J mouse strain that lacks the expression of group IIA phospholipase A2 has been shown to develop more severe gastric inflammation than other mouse strains. Thus, group IIA phospholipase A2 has been suggested to play a role in regulating inflammation in gastric mucosa. The aim of this study was to examine the possible role of group IIA phospholipase A2 in experimental Helicobacter infection., Materials and Methods: Transgenic mice expressing human group IIA phospholipase A2 and their group IIA phospholipase A2 deficient nontransgenic C57BL/6 J littermates were infected with H. felis. The mice were killed 3, 8, and 19 weeks after inoculation of bacteria to determine the histopathological changes in gastric mucosa., Results: The infected mice developed chronic inflammation in gastric mucosa. We found no differences in the colonization of bacteria between transgenic and nontransgenic mice. At 3 and 8 weeks, no difference was found in the severity of inflammation between the two groups. Nineteen weeks after the administration of bacteria the inflammation was more marked in nontransgenic than transgenic mice. Group IIA phospholipase A2 was expressed by in situ hybridization in the neck cells of the glandular stomach in transgenic mice., Conclusions: The results of the present study suggest that the endogenous expression of group IIA phospholipase A2 diminishes chronic inflammation in gastric mucosa in experimental H. felis infection in mice.

Recruitment of vascular smooth muscle cells (SMC) by endothelial cells (EC) is essential for angiogenesis. Endothelial-derived heparin binding EGF-like growth factor (HB-EGF) was shown to mediate this process by signaling via ErbB1 and ErbB2 receptors in SMCs. 1) Analysis of ErbB-ligands demonstrated that primary ECs expressed only HB-EGF and neuregulin-1. 2) Primary SMCs expressed ErbB1 and ErbB2, but not ErbB3 or ErbB4. 3) Consistent with their known receptor specificities, recombinant HB-EGF, but not neuregulin-1, stimulated tyrosine phosphorylation of ErbB1 and ErbB2 and migration in SMCs. 4) Neutralization of HB-EGF or inhibition of ErbB1 or ErbB2 blocked 70-90% of the potential of ECs to stimulate SMC migration. Moreover, 5) angiopoietin-1, an EC effector with a role in recruitment of SMC-like cells to vascular structures in vivo, enhanced EC-stimulated SMC migration by a mechanism involving up-regulation of endothelial HB-EGF. Finally, 6) immunohistochemical analysis of developing human tissues demonstrated that HB-EGF was expressed in vivo in ECs associated with SMCs or pericytes but not in ECs of the hyaloid vessels not associated with SMCs. These results suggest an important role for HB-EGF and ErbB receptors in the recruitment of SMCs by ECs and elaborate on the mechanism by which angiopoietins exert their vascular effects.

Introduction: There is circumstantial and contradictory evidence of the role of group IIA phospholipase A2 (PLA2) in acute pancreatitis., Aim: To examine the severity of acute experimental pancreatitis in transgenic mice expressing human PLA2 compared with mice not expressing PLA2., Methods: The study involved 12 young female CB57/bl mice not expressing group IIA PLA2 (wild-type mice) and 12 transgenic female CB57/bl mice expressing human group IIA PLA2 (transgenic mice). A choline-deficient, 0.5% ethionine-supplemented diet induced acute pancreatitis for 72 hours after 12 hours of fasting. Mice were killed 4 and 10 days after induction of acute pancreatitis. Pancreas, lung, kidney, and liver were examined histologically, and apoptosis in pancreas and liver was evaluated by DNA nick-end labeling (TUNEL)., Results: On day 4, there were no significant differences in pancreatic apoptosis or total pancreatitis score. Liver damage was similar in both groups. On day 10, pancreatic damage was less but apoptosis more severe than on day 4, and neither hepatic damage nor apoptosis was seen. All mice expressing human group IIA PLA2 but none of the mice not expressing human group IIA PLA2 had marked pancreatic fibrosis. No significant pulmonary or renal damage was found at any time., Conclusion: Pancreatitis in mice expressing human group IIA PLA2 is not more severe than in normal mice. Expression of group IIA PLA2 per se is not a major determinant of severity in experimental acute pancreatitis.

Group IIA phospholipase A2 (PLA2-IIA) is a newly recognized antibacterial acute phase protein. The concentration of PLA2-IIA increases up to 500-fold in the blood plasma of patients with severe acute diseases, compared with healthy persons. Despite numerous studies, the exact roles of this enzyme in human diseases are unknown. This study investigated the antibacterial properties of PLA2-IIA in human acute phase serum. PLA2-IIA in serum samples of patients with bacterial infections was capable of killing 90% of Staphylococcus aureus and 99% of Listeria monocytogenes in vitro after incubation for 2 h. At concentrations found in normal human serum, PLA2-IIA killed 90% of L. monocytogenes but did not kill S. aureus or Escherichia coli. The bactericidal effects of acute phase and normal human serum were abolished after depletion of PLA2-IIA by immunoadsorption.

Cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis, is expressed in constitutive (COX-1) and inducible (COX-2) isoforms. The COX-2 has been proposed to be involved in development of autoimmune type 1 diabetes (T1D). We examined COX-2 expression in the gut-associated lymphoid tissue (GALT), and found COX-2 was strongly expressed in goblet cells of non-obese diabetic (NOD) mice at the apical villi at the age of 2.5 weeks, clearly before the onset of insulitis, while the expression in the control BALB/c mice was weak or absent at all ages (P < 0.001). Lipopolysaccharide (LPS) given intraperitoneally slightly increased COX-2 expression in the goblet cells and epithelium of both NOD and BALB/c mice. High-resolution confocal microscopy showed that the surroundings of the goblet cells contained no COX-2, implying that the enzyme is synthesized by the goblet cells. The COX-2 is secreted from goblet cells into the intestinal lumen along with mucins. The COX-2 concentration in the goblet cell of BALB/c and especially of NOD mice was markedly higher than that in the intraepithelial lymphocytes or lamina propria macrophages. High mucin COX-2 from goblet cells may increase luminal prostaglandin synthesis, alter epithelial permeability, modulate intestinal immune responses and modify functional properties of the lymphocytes in the GALT, which all may be important for the initiation of the autoimmune phenomenon in the NOD mice.

Group IIA secreted phospholipase A(2) (sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of recombinant murine and human groups I, II, V, X, and XII sPLA2s on Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. The rank order potency among human sPLA2s against Gram-positive bacteria is group IIA > X > V > XII > IIE > IB, IIF (for murine sPLA2s: IIA > IID > V > IIE > IIC, X > IB, IIF), and only human group XII displays detectable bactericidal activity against the Gram-negative bacterium E. coli. These studies show that highly basic sPLA2s display potent bactericidal activity with the exception of the ability of the acidic human group X sPLA2 to kill Gram-positive bacteria. By studying the Bacillus subtilis and S. aureus bactericidal potencies of a large panel of human group IIA mutants in which basic residues were mutated to acidic residues, it was found that: 1) the overall positive charge of the sPLA2 is the dominant factor in dictating bactericidal potency; 2) basic residues on the putative membrane binding surface of the sPLA2 are modestly more important for bactericidal activity than are other basic residues; 3) relative bactericidal potency tracks well with the ability of these mutants to degrade phospholipids in the bacterial membrane; and 4) exposure of the bacterial membrane of Gram-positive bacteria by disruption of the cell wall dramatically reduces the negative effect of charge reversal mutagenesis on bactericidal potency.

Group V phospholipase A(2) (PLA(2)) is a recently characterized 14-kDa secretory PLA(2) of mammalian heart and macrophage-derived cells. Group IIA PLA(2), which is structurally close to group V PLA(2), has been shown to kill Gram-positive bacteria in vitro and to prevent symptoms of Gram-positive infection in vivo. We studied the antibacterial properties of fully active recombinant rat group IIA and V PLA(2)s. Both group IIA and V PLA(2)s were highly bactericidal against Gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Only high concentrations of group IIA PLA(2) showed some bactericidal effect against the Gram-negative bacterium Escherichia coli. Our results confirm that group IIA PLA(2) is a potent antibacterial enzyme against Gram-positive bacteria. Moreover, we show here that group V PLA(2) is a novel antibacterial mammalian protein, but is less potent than group IIA PLA(2). Both enzymes may be considered as future therapeutic agents against bacterial infections.

Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.

Group IIA phospholipase A2 (PLA2) is a newly recognized acute phase protein with marked antibacterial properties. We have shown previously that transgenic C57BL/6 J mice expressing human group IIA PLA2 (PLA2+ mice) are more resistant to bacterial infections than nontransgenic C57BL/6 J mice that, among mice, are unusual in that they lack the mouse analogue of group IIA PLA2 (PLA2- mice). To elucidate the possible mechanisms involved in the host response of these mice in bacterial infection, peripheral inflammatory cell responses of PLA2+ and PLA2- mice were studied after i.p. administration of Escherichia coli, E. coli lipopolysaccharide or Staphylococcus aureus. Uninfected PLA2+ mice had higher numbers of lymphocytes and polymorphonuclear neutrophil leukocytes (PMNs) in their blood than PLA2- mice. In PLA2+ mice, the number of PMNs increased in peripheral blood in parallel with the concentration of group IIA PLA2 after the administration of bacteria, whereas these responses were not seen in PLA2- mice. High concentrations of group IIA PLA2 in PLA2+ mice may increase the synthesis of bioactive molecules, such as prostaglandins, which in turn may mobilize PMNs into circulation. Our results support the hypothesis that group IIA PLA2 is an important inflammatory mediator in bacterial infections.

Objective: To find out if two immunomodulatory drugs used in organ transplantation (FK506 (tacrolimus) and OKT3 (Orthoclone) would reduce early inflammatory complications in experimental acute pancreatitis., Design: Laboratory study., Setting: University hospital, Germany., Animals: 36 Balb/c mice., Interventions: Pancreatitis induced by 7 intraperitoneal injections of cerulein 50 microg/kg at hourly intervals followed by FK506 0.32 mg/kg, OKT3 0.6 mg/kg, or 0.9% sodium chloride (controls) (n = 12 in each group). 12 hours after induction of pancreatitis the animals were killed., Main Outcome Measures: Serum amylase activity and interleukin-6 (IL-6) concentrations; histological damage to pancreas and lungs, apoptotic cells in pancreas; and myeloperoxidase activity in lungs., Results: No animal died during the experiment. At 12h serum amylase activity and IL-6 concentrations were increased in all 3 groups, but highest in the OKT3 group. The pancreatic histological score, apoptosis, and inflammatory infiltration were lower in the two experimental groups than controls, but the degree of vacuolisation of acinar cells was similar. Packed cell volume was higher in the control than the experimental groups, and pulmonary damage and myeloperoxidase activity were less in the experimental groups than the controls., Conclusion: Single therapeutic doses of FK506 and OKT3 reduced the early severity of pancreatitis, pulmonary damage, and haemoconcentration in mice. Single doses of FK506 or OKT3 may therefore be effective in preventing the early complications of pancreatitis.

We present a modification of mRNA differential display in which increased throughput results from the use of an automated fluorescent sequencer. The sequence analysis is performed directly on purified fragments without further cloning. The amplified fragments carry a T7 RNA polymerase promoter sequence tag for in vitro transcription of riboprobes for nonradioactive in situ hybridization. We compared changes in gene expression in the liver and colon of group II phospholipase A2 transgenic and group II phospholipase A2 deficient mice during the course of experimental Escherichia coli infection. Fluorescent mRNA differential display comprising a 7 x 24 set of primers was used to study a total of 31,257 amplified cDNA fragments. Sequence analysis of the displayed fragments associated with infection identified classical acute-phase proteins in the liver and host defense proteins in the colon. The displayed mRNAs associated to transgenicity were the transgene itself, i.e., human group II phospholipase A2, and glutathione-S-transferase in the liver. In the colon, the displayed mRNAs associated with transgenicity were the pancreatitis-associated protein and mucin. The results show that fluorescent mRNA differential display is a reliable method to identify differences in the expression of the genes of acute-phase proteins.

Group II phospholipase A2 (PLA2) is a newly recognized antibacterial acute-phase protein. Recently we observed that transgenic mice expressing group II PLA2 (PLA2(+) mice) were able to resist experimental Staphylococcus aureus infection by killing the bacteria, as indicated by improved survival and by the small numbers of live bacteria in their tissues (V. J. O. Laine, D. S. Grass, and T. J. Nevalainen, J. Immunol. 162:7402-7408, 1999). To establish the role of group II PLA2 in Escherichia coli infection, the host responses of PLA2(+) mice and their PLA2-deficient C57BL/6J littermates (PLA2(-) mice) were studied after intraperitoneal administration of E. coli. The levels of group II PLA2 in sera of PLA2(+) mice increased after the administration of E. coli, and the concentration of group II PLA2 correlated significantly with the catalytic activity of PLA2 in serum. PLA2(+) mice showed lower rates of mortality and less bacterial growth in peritoneal lavage fluid, blood, and spleen and liver tissues than PLA2(-) mice. Unlike the observations with staphylococcal infection, serum and peritoneal lavage fluid did not inhibit the growth of E. coli in vitro. The results indicate that expression of the group II PLA2 transgene improves the host defense of mice against E. coli infection.

To examine the role of lymphocyte activation in the development of local and systemic complications in acute pancreatitis, we compared disease severity of choline-deficient, 0.5% ethionine supplemented (CDE) diet-induced acute pancreatitis in T- and B-cell deficient SCID mice and immunocompetent C.B-17 mice. Twenty-five female SCID and 17 female C.B-17 mice were fasted for 24 h and fed a CDE diet for 72 h. Twenty SCID and 12 C.B-17 mice were bled and their organs removed for histologic evaluation. Five control animals of both kinds were fed a regular diet for 6 days. Lung, kidney, and pancreas were examined microscopically, and pancreatic damage scored. Apoptosis was detected by DNA nick-end labeling and confirmed by DNA laddering. Trypsinogen-activation peptide was measured by enzyme-linked immunosorbent assay (ELISA), and the catalytic activity of PLA2 was determined by a radiometric assay. Four-day mortality was 10% in SCID and 33% in C.B-17 mice, and 10-day mortality was 0 in SCID and 60% in C.B-17 mice. SCID mice had mild pulmonary damage, whereas pulmonary injury was severe in C.B-17 mice. Pancreatic damage was severe in both groups. Even though in situ staining of apoptotic cells was found in all pancreatitis animals, apoptosis was confirmed by DNA laddering only in C.B-17 mice. In SCID mice, apoptotic cell staining positively correlated with necrosis (r = 0.91; p < 0.001). Plasma TAP and PLA2 catalytic activity did not differ significantly between the groups. In conclusion, the absence of T and B lymphocytes prevents severe pulmonary injury resulting from acute pancreatitis but does not influence pancreatic or renal damage. Our results suggest that systemic lymphocyte activation does not affect the initiating events that trigger pancreatic injury but modulates the systemic response, in particular, pulmonary injury caused by acute pancreatitis.

Group II phospholipase A2 (PLA2) is an enzyme that has marked antibacterial properties in vitro. To define the role of group II PLA2 in the defense against Staphylococcus aureus, we studied host responses in transgenic mice expressing human group II PLA2 and group II PLA2-deficient C57BL/6J mice in experimental S. aureus infection. After the administration of S. aureus, the transgenic mice showed increased expression of group II PLA2 mRNA in the liver and increased concentration of group II PLA2 in serum, whereas the PLA2-deficient mice completely lacked the PLA2 response. Expression of human group II PLA2 resulted in reduced mortality and improved the resistance of the mice by killing the bacteria as indicated by low numbers of live bacteria in their tissues. Human group II PLA2 was responsible for the bactericidal activity of transgenic mouse serum. These results suggest a possible role for group II PLA2 in the innate immunity against S. aureus infection.

Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohistochemistry, human group II PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group II PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group II PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2.

Background: Bacterial lipopolysaccharide (LPS) has been proposed to participate in the pathogenesis of pancreatic inflammatory disease., Aims: This study investigated the role of endotoxaemia in the pathogenesis of pancreatic acinar cell injury., Methods: Sixty eight male Spraque-Dawley rats were used in the study. Escherichia coli LPS (5 mg/kg) was injected into the peritoneal cavity of the rats. The concentration of pancreatic phospholipase A2 (PLA2) in plasma was measured and pancreatic tissue examined by histology, in situ detection of free DNA 3'-ends, and electrophoretic DNA analysis., Results: The concentration of pancreatic PLA2 increased in plasma and the catalytic activity of PLA2 increased in pancreatic tissue after an LPS injection. Apoptosis in pancreatic acinar cells and fragmentation of DNA typical of apoptosis in pancreatic tissue was seen 24 hours after an LPS injection. Pancreatic acinar atrophy was seen 72 hours after the LPS injection., Conclusions: These data show that LPS causes release of pancreatic PLA2 into blood plasma, activation of PLA2 in pancreatic tissue, and apoptosis of acinar cells.

Phospholipase A2 values increase in serum in various inflammatory states, infections, and postoperatively in surgical patients. Several organs, including the liver and spleen have been suggested as sources of circulating phospholipase A2. The purpose of the present work was to examine the possible role of the spleen as a source of elevated serum concentrations of phospholipase A2 after surgery. Pre- and postoperative serum samples of patients undergoing splenectomy were studied for group I phospholipase A2, group II phospholipase A2, and C-reactive protein mass concentrations and catalytic activity concentration of phospholipase A2. The catalytic activity concentration of phospholipase A2 and the mass concentrations of group II phospholipase A2 and C-reactive protein increased postoperatively (8.08 +/- 1.40 U/l vs. 3.96 +/- 0.89 U/l (mean +/- SEM) for phospholipase A2 catalytic concentration (p < 0.03), and 154.8 +/- 32.1 micrograms/l vs. 47.5 +/- 14.7 micrograms/l (mean +/- SEM) for group II phospholipase A2 mass concentration (p < 0.02, n = 7). The mass concentration of group I phospholipase A2 remained unchanged. The catalytic concentration of phospholipase A2 correlated well with the mass concentration of group II phospholipase A2 (p < 0.001, r = 0.846, n = 43). The concentration of C-reactive protein correlated well with the mass concentration of group II phospholipase A2 (p < 0.001, r = 0.566, n = 43) in serum. The results indicate that group II phospholipase A2 is released into the circulation after splenectomy, and the spleen seems not to be the source of circulating group II phospholipase A2.

The effects of chronic alcohol intake and carbachol stimulation on pancreatic muscarinic receptor binding and insulin concentrations were studied in the rat pancreas. There was a strong correlation between the number of muscarinic receptors and the concentration of insulin in the pancreas. The concentration of insulin decreased in the pancreas after long-term ethanol exposure and increased after carbachol stimulation. These results indicate that the secretion of insulin is mediated via the muscarinic receptor pathway, and that the changes in the number of muscarinic receptors may have a role in insulin deficiency after long-term alcohol consumption.

Immunohistochemical characteristics of a mucinous islet-cell carcinoma of the pancreas are described. The tumour presented with jaundice in a 59-year-old male. It consisted of polygonal atypical cells forming a reticular pattern, and invaded the common bile duct. In DNA flow cytometry, the tumour cells showed a clear-cut aneuploid peak. Intercellular mucin was abundant. A panel of antisera and monoclonal markers was applied in the immunohistochemical analysis. In addition to general epithelial and endocrine markers, the tumour cells showed a focal positive immunoreaction with anti-glucagon, anti-insulin, anti-vasoactive intestinal polypeptide, anti-pancreatic secretory trypsin inhibitor and anti-phospholipase A2 antigen. At the ultrastructural level, mucous and neuroendocrine granules were demonstrated in the same tumour cells.