DIFFERENTIAL EXPRESSION OF GLUTARYL-CoA DEHYDROGENASE IN ADULT RAT CNS, PERIPHERAL TISSUES AND DURING EMBRYONIC DEVELOPMENT

Glutaryl-CoA dehydrogenase (GCDH, EC 1.3.99.7) deficiency, known as glutaric acidemia type I, is one of the more common organic acidurias. To investigate the role of this pathway in different organs we studied the tissue-specific expression pattern of rat Gcdh. The open reading frame cDNA of the rat Gcdh gene was cloned from rat brain mRNA by RT-PCR, allowing the synthesis of digoxigenin-labeled in situ hybridization (ISH) riboprobes. Gcdh mRNA expression was analyzed by ISH on cryosections of adult rat brain, kidney, liver, spleen and heart muscle, as well as on E15 and E18 rat embryos. Gcdh was found expressed in the whole rat brain, almost exclusively in neurons. Gcdh was absent from astrocytes but expressed in rare oligodendrocytes. Strong Gcdh expression was found in liver and spleen, where expression appears predominant to lymphatic nodules. In kidney, the highest Gcdh expression is found in the juxtamedullar cortex (but not in glomerula), and at lower levels in medulla. Heart muscle was negative. During embryonic development, Gcdh was found well expressed in liver, intestinal mucosa and skin, as well as at lower levels in CNS. Further studies are ongoing to provide evidence on the presence of the entire pathway in CNS in order to understand the mechanisms leading to neurotoxicity in glutaric aciduria. The high expression of Gcdh in kidney may explain why certain patients with residual enzyme activity are low excretors at the urine metabolite level.

Semi-supervised segmentation of ultrasound images based on patch representation and continuous min cut.

Ultrasound segmentation is a challenging problem due to the inherent speckle and some artifacts like shadows, attenuation and signal dropout. Existing methods need to include strong priors like shape priors or analytical intensity models to succeed in the segmentation. However, such priors tend to limit these methods to a specific target or imaging settings, and they are not always applicable to pathological cases. This work introduces a semi-supervised segmentation framework for ultrasound imaging that alleviates the limitation of fully automatic segmentation, that is, it is applicable to any kind of target and imaging settings. Our methodology uses a graph of image patches to represent the ultrasound image and user-assisted initialization with labels, which acts as soft priors. The segmentation problem is formulated as a continuous minimum cut problem and solved with an efficient optimization algorithm. We validate our segmentation framework on clinical ultrasound imaging (prostate, fetus, and tumors of the liver and eye). We obtain high similarity agreement with the ground truth provided by medical expert delineations in all applications (94% DICE values in average) and the proposed algorithm performs favorably with the literature.

VEGF is required for growth and survival in neonatal mice.

We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development.

Fixed rings and strictures in Eosinophilic Esophagitis develop due to continuing inflammation over time

Background and Aims: Two distinct e ndoscopic phenotypes of E osinophilic Esophagitis (EoE) h ave been identified: t he inflammatory (IP) a nd the stenosing (SP) p henotype. I t is not known whether these EoE-associated phenotypes are reflective of different phases during disease course. We aimed to assess the phenotype a t initial EoE p resentation and d iagnosis and to evaluate if SP increases over time. Methods: R etrospective a nalysis of t he Swiss EoE Database (SEED) extended b y a review of p atients charts, endoscopy and pathology records. Results: F orty-four E oE p atients were a nalyzed (33 males, mean age at index visit 41 ± 14 years, all Caucasians). Median follow-up t ime was 3.1 years (IQR 1-4, r ange 1 -18 years). Median diagnostic delay w as 5 y ears (IQR 2-16, range 0-34 years). A t first diagnosis, 3 2% ( 14/44) o f EoE patients h ad already presented w ith a stenosis. T he mean d iameter o f the stenoses w as 1 0 ± 2 mm, and the mean length was 2 .8 ± 2 .9 cm. Peak e osinophil count d id n ot c hange over t ime (48 ± 39 eos/HPF at index visit vs. 59 ± 41 eos/HPF at end of follow-up, n=44). The risk of the presence of a stenosis at index visit was 0% f or a d isease duration of 0 -4 y ears, 37% f or a d isease duration between 5-10 years and 67% f or a d isease duration >10 years (p = 0.0035, trend test). Conclusions: T he frequency of e sophageal stenoses i s proportional to the disease duration, whereas the inflammatory activity does n ot s ignificantly c hange over t ime. O ur f indings underscore the necessity to reduce diagnostic delay in EoE and to control the underlying inflammatory processes to prevent esophageal remodeling.

Continuous monitoring of liver oxygenation with near infrared spectroscopy during naso-gastric tube feeding in neonates.

Near infrared spectroscopy (NIRS) is a non-invasive method of estimating the haemoglobin concentration changes in certain tissues. It is frequently used to monitor oxygenation of the brain in neonates. At present it is not clear whether near infrared spectroscopy of other organs (e.g. the liver as a corresponding site in the splanchnic region, which reacts very sensitively to haemodynamic instability) provides reliable values on their tissue oxygenation. The aim of the study was to test near infrared spectroscopy by measuring known physiologic changes in tissue oxygenation of the liver in newborn infants during and after feeding via a naso-gastric tube. The test-retest variability of such measurements was also determined. On 28 occasions in 25 infants we measured the tissue oxygenation index (TOI) of the liver and the brain continuously before, during and 30 minutes after feeding via a gastric tube. Simultaneously we measured arterial oxygen saturation (SaO2), heart rate (HR) and mean arterial blood pressure (MAP). In 10 other newborn infants we performed a test-retest analysis of the liver tissue oxygenation index to estimate the variability in repeated intra-individual measurements. The tissue oxygenation index of the liver increased significantly from 56.7 +/- 7.5% before to 60.3 +/- 5.6% after feeding (p < 0.005), and remained unchanged for the next 30 minutes. The tissue oxygenation index of the brain (62.1 +/- 9.7%), SaO2 (94.4 +/- 7.1%), heart rate (145 +/- 17.3 min-1) and mean arterial blood pressure (52.8 +/- 10.2 mm Hg) did not change significantly. The test-retest variability for intra-individual measurements was 2.7 +/- 2.1%. After bolus feeding the tissue oxygenation index of the liver increased as expected. This indicates that near infrared spectroscopy is suitable for monitoring changes in tissue oxygenation of the liver in newborn infants.

Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice.

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Establishment of HEV RNA reverse transcriptase PCR assays for the diagnosis of acute and chronic hepatitis E

Background and aim: H epatitis E v irus (HEV) infection has emerged as a c ause o f travel-related a nd autochthonous a cute hepatitis as well as chronic hepatitis in immunosuppressed patients. While t ravel-related cases a re c aused primarily b y infections w ith HEV of g enotype 1 ( HEV-1), autochthonous c ases a nd chronic cases a re d ue t o genotype 3 (HEV-3), which is s hared between humans and diverse animal species. The aim of this study was to establish HEV RNA detection assays f or q uantitative v iral load testing and genotyping. Methods: V iral RNA was p urified from plasma or s erum a nd converted to cDNA prior to (1) multiplex real-time PCR for HEV RNA quantification and (2) multiplex PCR coupled to DNA sequencing for HEV genotype determination. Real-time PCR was d esigned to match a ll known HEV genotypes available i n Genbank while PCR was designed using conserved primers flanking a variable region of the HEV RNA. Results: In a validation panel, the newly developed assays allowed for the reliable detection and genotyping of HEV-1 or HEV-3. Cases of t ravel-related and a utochthonous a cute h epatitis E a s well a s chronic hepatitis E i n immunosuppressed patients have b een identified using t hese a ssays a nd will be p resented in detail. Anti- HEV antibodies were n egative i n three well-characterized patients with chronic hepatitis E after organ transplantation. Conclusions: We developed and validated a quantitative HEV RNA detection assay that c an now be o ffered on a r outine basis (www.chuv.ch/imul/imu-collaborations-viral_hepatitis). Genotyping can also be offered on selected cases. HEV RNA detection is key in diagnosing chronic hepatitis E i n immunosuppressed patients with unexplained transaminase elevations, as serology can be negative in these patients.

Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.

Expression of two important glucose transporter proteins, GLUT 2 (which is the typical glucose transporter in hepatocytes of adult liver) and the erythroid/brain type glucose transporter GLUT 1 (representing the typical glucose transporter in fetal liver parenchyma), was studied immunocytochemically during hepatocarcinogenesis in rats at different time points between 7 and 65 wk after cessation of 7-wk administration of 12 mg/kg of body weight of N-nitrosomorpholine p.o. (stop model). Foci of altered hepatocytes excessively storing glycogen (GSF) and mixed cell foci (MCF) composed of both glycogenotic and glycogen-poor cells were present at all time points studied. Seven wk after withdrawal of the carcinogen, GSF were the predominant type of focus of altered hepatocytes. Morphometrical evaluation of the focal lesions revealed that the number and volume fraction of GSF increased steadily until Wk 65. MCF were rare at 7 wk, increased slightly in number and size until Wk 37, but showed a pronounced elevation in their number and volume fraction from Wk 37 to Wk 65. In both GSF and MCF, GLUT 2 was generally decreased or partially absent at all time points. Consequently, foci of decreased GLUT 2 expression showed a steady increase in number and volume fraction from Wk 7 to Wk 65. GLUT 1 was lacking in GSF but occurred in some MCF from Wk 50 onward. The liver type glucose transporter GLUT 2 was decreased in all adenomas and hepatocellular carcinomas (HCC). In three of seven adenomas and 10 of 12 carcinomas, expression of GLUT 1 was increased compared with normal liver parenchyma. In two cases of adenoid HCC, cells of ductular formations coexpressed GLUT 2 and GLUT 1. In contrast, normal bile ducts, bile duct proliferations, and cystic cholangiomas expressed only GLUT 1. Seven of 12 HCC contained many microvessels intensely stained for GLUT 1, a phenomenon never observed in normal liver. Whenever adenoid tumor formations occurred, GLUT 1-positive microvessels were located in the immediate vicinity of these formations. Only in one HCC were such microvessels found in the absence of adenoid formations. Our studies indicate that a reduction of GLUT 2 expression occurs already in early preneoplastic hepatic foci and is maintained throughout hepatocarcinogenesis, including benign and malignant neoplasms. Reexpression of GLUT 1, however, appears in a few MCF and in the majority of adenomas and carcinomas.

Proline- and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPAR alpha) activity.

In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or activity oscillate during the day. The clock-controlled proline- and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.

Cloning and expression of rat pancreatic beta-cell malonyl-CoA decarboxylase.

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

Comparison of copper-67- and iodine-125-labeled anti-CEA monoclonal antibody biodistribution in patients with colorectal tumors.

Copper-67 has comparable beta-particle emissions to that of 131I, but it displays more favorable gamma emission characteristics for application in radioimmunotherapy (RIT). This study investigates the potential of 67Cu-labeled monoclonal antibody (MAb) 35 for RIT of colorectal carcinoma. METHODS: Biokinetics of simultaneously injected 67Cu- and 125I-labeled MAb35 were studied in six patients scheduled for surgery of primary colorectal cancer. RESULTS: Whole-body clearance (T 1/2) of 67Cu, estimated from sequential anterior and posterior whole-body scans and corrected for decay of 67Cu, was 41 hr. Serum clearance of 67Cu was faster (27.41 hr) than that of 125I (38.33 hr). Mean tumor uptake of the 67Cu-labeled compound (0.0133% ID/g) exceeded that of 125I (0.0095% ID/g), and tumor-to-blood ratios were higher for 67Cu than for 125I, with averages of 6.07 and 2.41, respectively. The average 67Cu/125I ratio was 1.9 for tumor uptake, 0.7 for blood and 2.6 for tumor-to-blood ratios. Nonspecific liver uptake of 67Cu as calculated from whole-body scans was high in four patients, up to 25% of residual whole-body activity at 48 hr, but did not increase with time. We also observed some nonspecific bowel activity, as well as moderate to high uptake in benign polyps. CONCLUSION: Copper-67-labeled MAb35 is more favorable than its radioiodine-labeled counterpart for RIT of colorectal carcinoma due to higher tumor-to-blood ratios, but the problem of nonspecific liver and bowel uptake must first be overcome. The absolute accumulation of activity in tumor remains low, however, so the probability of cure with this compound alone is questionable. The use of 67Cu as one component of a multimodality adjuvant treatment seems to remain the most appropriate application for RIT.

Prevalence and pathology of the nematode Heterakis gallinarum, the trematode Paratanaisia bragai, and the protozoan Histomonas meleagridis in the turkey, Meleagris gallopavo

The prevalence of infection and associated pathology induced by two helminth and one protozoan species infecting Brazilian turkeys are reported. The intestinal nematode Heterakis gallinarum appeared with a prevalence of 70% in the infected birds, without gross lesions when not associated to the protozoan Histomonas meleagridis. Histological findings in the ceca were represented by the presence of H. gallinarum worms, intense chronic diffuse inflammatory processes with mononuclear and polymorphonuclear (heterophils) leucocyte infiltrations. The prevalence of the protozoan H. meleagridis associated to H. gallinarum was of 2.5% and microscopic examination revealed a severe inflammatory process in the liver and cecum with the presence of small clear areas with round eosinophilic parasites. Gross lesions were absent in turkeys infected with the renal digenetic trematode Paratanaisia bragai; the parasite was prevalent in 20% of the cases and cross-sections of the kidneys showed a remarkable distension of the collecting ducts with several worms in the lumen. The walls of the ducts presented a discrete heterophilic infiltrate among mononuclear cells.

Effects of dietary protein type on oxidized cholesterol-induced alteration in age-related modulation of lipid metabolism and indices of immune function in rats.

Exogenous oxidized cholesterol disturbs both lipid metabolism and immune functions. Therefore, it may perturb these modulations with ageing. Effects of the dietary protein type on oxidized cholesterol-induced modulations of age-related changes in lipid metabolism and immune function was examined using differently aged (4 weeks versus 8 months) male Sprague-Dawley rats when casein, soybean protein or milk whey protein isolate (WPI) was the dietary protein source, respectively. The rats were given one of the three proteins in diet containing 0.2% oxidized cholesterols mixture. Soybean protein, as compared with the other two proteins, significantly lowered both the serum thiobarbituric acid reactive substances value and cholesterol, whereas it elevated the ratio of high density lipoprotein-cholesterol/cholesterol in young rats, but not in adult. Moreover, soybean protein, but not casein and WPI, suppressed the elevation of Delta6 desaturation indices of phospholipids in both liver and spleen, particularly in young. On the other hand, WPI, compared to the other two proteins, inhibited the leukotriene B4 production of spleen, irrespective of age. Soybean protein reduced the ratio of CD4(+)/CD8(+) T-cells in splenic lymphocytes. Therefore, the levels of immunoglobulin (Ig)A, IgE and IgG in serum were lowered in rats given soybean protein in both age groups except for IgA in adult, although these observations were not shown in rats given other proteins. Thus, various perturbations of lipid metabolism and immune function caused by oxidized cholesterol were modified depending on the type of dietary protein. The moderation by soybean protein on the change of lipid metabolism seems to be susceptible in young rats whose homeostatic ability is immature. These observations may be exerted through both the promotion of oxidized cholesterol excretion to feces and the change of hormonal release, while WPI may suppress the disturbance of immune function by oxidized cholesterol in both ages. This alleviation may be associated with a large amount of lactoglobulin in WPI. These results thus showed a possibility that oxidized cholesterol-induced perturbations of age-related changes of lipid metabolism and immune function can be moderated by both the selection and combination of dietary protein.