Current Nuclear-medicine News Results

SPECT/CT hybrid imaging; with which CT?

Contrast Media Mol Imaging. 2010 Jul; 5(4): 208-12
Bach-Gansmo T, Schwarzlmüller T, Jøraholmen V, Salbu J, Biermann M, Naum A, Kleven-Madsen N

AIM: The aim of this study is to show the practical use of, and to discuss the rationale for, high-end computed tomography (CT) integrated with intrinsic low-resolution single-photon emission tomography (SPECT). MATERIALS AND METHODS: All examinations performed on three new SPECT/CT systems with diagnostic CT capabilities were recorded retrospectively. The use of CT was classified as low-dose, using the CT with restraint as to the tube current and radiation dose, or diagnostic, with an optimum use of the CT, using CT protocols as used in ordinary radiological practice. The number of low-dose CT was compared with the number of diagnostic CT examinations. The report is based on 436 patient examinations from three hospitals in Norway with recently installed SPECT/CT systems, the time of use varying from 6 months to 2 years. The examinations performed were myocardial perfusion (45%), various tumors (thyroid, parathyroid, neuroendocrine 37%), malignant skeletal disease (12%), brain perfusion (4%), sentinel nodes in breast cancer (1%) and gastrointestinal bleeding (1%). RESULTS: Of the 436 patients, 431 had a low-dose CT for attenuation correction, anatomic localisation and, also for diagnosis, whereas five patients had a diagnostic CT. In these series, as was found in recent literature, the diagnostic potential of the CT was seldom used to its capacity and always in predetermined diagnostic situations. CONCLUSION: There is a low degree of utilization of the diagnostic capabilities of the CT in the SPECT/CT context, for a number of reasons. This raises questions about the cost-benefit of investing in high-end CT for SPECT/CT applications. Copyright (c) 2010 John Wiley & Sons, Ltd.

Synthesis and Evaluation of (99m)Tc-Labelled Monoclonal Antibody 1D09C3 for Molecular Imaging of Major Histocompatibility Complex Class II Protein Expression.

Mol Imaging Biol. 2010 Sep 2;
Malviya G, de Vries EF, Dierckx RA, Signore A

PURPOSE: It is known that major histocompatibility complex class II protein HLA-DR is highly expressed in B-cell lymphomas and in a variety of autoimmune and inflammatory diseases. Therefore, a radiolabelled fully humanized IgG4 monoclonal antibody (mAb) can provide useful prognostic and diagnostic information. Aims of the present study were to radiolabel an anti-HLA-DR mAb with technetium-99m and to evaluate its binding specificity, tissue distribution and targeting potential. PROCEDURES: For labelling, we compared a direct method, after 2-mercaptoethanol (2-ME) reduction of disulphide bonds, with a two-step labelling method, using a heterobifunctional succinimidyl-6-hydrazinonicotinate hydrochloride chelator. Several in vitro quality controls and in vivo experiments in mice were performed. RESULTS: We obtained highest labelling efficiency (LE, >98%) and specific activity (SA; 5,550 MBq/mg) via the direct method. In vitro quality control showed good stability, structural integrity and retention of the binding properties of the labelled mAb. The biodistribution in mice showed high and persistent uptake in spleen and suggests kidney and liver-mediated clearance pathways. In tumour targeting experiments, we observed high uptake in HLA-DR-positive xenografts compared to controls. In vivo binding was proportional to the number of injected cells. In the in vivo blocking assay, uptake of radiolabelled mAb was significantly decreased in mice pre-injected with 100-fold molar excess of unlabelled mAb. CONCLUSION: We efficiently labelled a humanized anti-HLA-DR mAb with (99m)Tc using a direct labelling method. Radiolabelled mAb binds to human HLA-DR antigens and therefore warrants further evaluation as a prognostic and diagnostic tool for patients with lymphoma or autoimmune diseases.

Detection of RTK Pathway Activation in Drosophila Using Anti-dpERK Immunofluorescence Staining.

Methods Mol Biol. 2010; 661: 401-8
Helman A, Paroush Z

In Drosophila, like in other metazoans, receptor tyrosine kinase (RTK) signaling pathways control diverse cellular processes such as migration, growth, fate determination, and differentiation (Shilo, Development 132:4017-4027, 2005). Activation of RTKs by their extracellular ligands triggers a signal transduction cascade, mediated by the Ras/Raf/MEK cassette, which ultimately leads to dual phosphorylation and activation of the mitogen-activated protein kinase/extracellularly regulated kinase (MAPK/Erk). Once active, MAPK/Erk phosphorylates its cytoplasmic and nuclear substrates, consequently modulating (i.e., stimulating or inhibiting) their biological function (Murphy and Blenis, Trends in Biochemical Sciences 31:268-275, 2006). The currently available antibody specific for the doubly phosphorylated form of MAPK/Erk (dpERK) (Yung et al., FEBS Letters 408:292-296, 1997) provides a valuable readout for RTK signaling: it enables the spatiotemporal detection of RTK pathway activity in the developing organism, in situ (Gabay et al., Development 124:3535-3541, 1997; Gabay et al., Science 277:1103-1106, 1997). Here, we present a detailed protocol for anti-dpERK immunofluorescent staining that can be applied to the analysis of MAPK/Erk signaling in Drosophila embryogenesis.

Protective effect of the dopamine D3 receptor agonist (7-OH-PIPAT) against apoptosis in malignant peripheral nerve sheath tumor (MPNST) cells.

Int J Oncol. 2010 Oct; 37(4): 927-34
Castorina A, Giunta S, D'Agata V

Emerging evidence indicates that the dopamine D3 receptor (D3R) mediates protective roles both in neuronal and non-neuronal cell lines. In a previous study we proposed that neurofibromin, a large tumor suppressor protein encoded by the neurofibromatosis type 1 gene (NF1), may increase susceptibility to apoptosis after serum deprivation in malignant peripheral nerve sheath tumor (MPNST) cells, thus acting as a proapoptotic gene. In addition, it has been observed that D3Rs are functionally correlated to neurofibromin. In this study, we examined whether 7-OH-PIPAT, a potent dopamine D3R agonist, exerts an antiapoptotic role under the same culture conditions and then correlated this effect to changes in NF1 expression. Results showed that serum deprivation caused a significant reduction of cell viability (MTT assay) both after 24 and 48 h (p<0.001). Treatment with increasing concentrations of 7-OH-PIPAT (10(-9)-10(-5) M) induced a progressive increase in cell viability both after 24 and 48 h as compared to vehicle-treated cells, with significant changes at the highest concentrations tested (10(-6) and 10(-5) M). Consistently, at the latter two concentrations, a significant reduction in oligonucleosomes formation was observed, thus suggesting an antiapoptotic role of 7-OH-PIPAT. These results were confirmed by Hoechst 33254 nuclear staining. To investigate whether these effects were correlated to changes in NF1 transcript and protein expression, quantitative real-time PCR, Western blot and immunofluorescence analyses were performed. Results demonstrated that the upregulation of NF1 transcripts and protein levels induced by serum withdrawal were remarkably attenuated by 10(-6) and 10(-5) M agonist treatment within 24 h (p<0.01 and p<0.001, respectively), whereas similar effects were observed already at a lower concentration (10(-7) M) after 48 h treatment (p<0.001). In conclusion, these results suggest that D3R might mediate the protective response to serum deprivation in MPNST cells through the inhibition of NF1 gene expression, further underlying a subtle role of these receptors in MPNST development.

RhoA protein is generally distributed in the nuclei of cancer cells.

Oncol Rep. 2010 Oct; 24(4): 1005-9
Li Y, Chen Y, Tao Y, Xu J, Chen M

The aim of this study was to elucidate the localization of RhoA in human cancer tissues and cancer cell lines. Immunohistochemistry and immunofluorescence microscopy were used to determine the localization of RhoA in cancer tissues and cell lines, respectively. Western blotting was used to determine the quantity of RhoA in cell nucleus, cytosol and membrane. Immunofluorescence microscopy revealed that in all cell lines examined in this experiment (including SGC-7901, Hela, A549 and SW480), RhoA was localized not only on the membrane, in the cytosol, but also in the nucleus. Within the nucleus its precise localization was in the nucleolus. In the transformed gastric epithelial cell line GES, RhoA was also found to be localized on the membrane, in the cytosol and the nucleus, but the nuclear location was less than that of cancer cell lines. Immunohistochemistry microscopy revealed that in cancer tissues the nuclear RhoA was obviously more than that of para-cancer tissue; in the cancer tissues undergoing inflammation and necrosis, nuclear RhoA was even higher. RhoA is generally distributed in the nucleus of cells and the distribution increases when the cells undergo tumorigenesis, suggesting a new role for RhoA.

Transcriptional Regulation of Human and Rat Hepatic Lipid Metabolism by the Grapefruit Flavonoid Naringenin: Role of PPARalpha, PPARgamma and LXRalpha.

PLoS One. 2010; 5(8):
Goldwasser J, Cohen PY, Yang E, Balaguer P, Yarmush ML, Nahmias Y

Disruption of lipid and carbohydrate homeostasis is an important factor in the development of prevalent metabolic diseases such as diabetes, obesity, and atherosclerosis. Therefore, small molecules that could reduce insulin dependence and regulate dyslipidemia could have a dramatic effect on public health. The grapefruit flavonoid naringenin has been shown to normalize lipids in diabetes and hypercholesterolemia, as well as inhibit the production of HCV. Here, we demonstrate that naringenin regulates the activity of nuclear receptors PPARalpha, PPARgamma, and LXRalpha. We show it activates the ligand-binding domain of both PPARalpha and PPARgamma, while inhibiting LXRalpha in GAL4-fusion reporters. Using TR-FRET, we show that naringenin is a partial agonist of LXRalpha, inhibiting its association with Trap220 co-activator in the presence of TO901317. In addition, naringenin induces the expression of PPARalpha co-activator, PGC1alpha. The flavonoid activates PPAR response element (PPRE) while suppressing LXRalpha response element (LXRE) in human hepatocytes, translating into the induction of PPAR-regulated fatty acid oxidation genes such as CYP4A11, ACOX, UCP1 and ApoAI, and inhibition of LXRalpha-regulated lipogenesis genes, such as FAS, ABCA1, ABCG1, and HMGR. This effect results in the induction of a fasted-like state in primary rat hepatocytes in which fatty acid oxidation increases, while cholesterol and bile acid production decreases. Our findings explain the myriad effects of naringenin and support its continued clinical development. Of note, this is the first description of a non-toxic, naturally occurring LXRalpha inhibitor.

Bilateral Renal Agenesis/Hypoplasia/Dysplasia (BRAHD): Postmortem Analysis of 45 Cases with Breakpoint Mapping of Two De Novo Translocations.

PLoS One. 2010; 5(8):
Harewood L, Liu M, Keeling J, Howatson A, Whiteford M, Branney P, Evans M, Fantes J, Fitzpatrick DR

BACKGROUND: Bilateral renal agenesis/hypoplasia/dysplasia (BRAHD) is a relatively common, lethal malformation in humans. Established clinical risk factors include maternal insulin dependent diabetes mellitus and male sex of the fetus. In the majority of cases, no specific etiology can be established, although teratogenic, syndromal and single gene causes can be assigned to some cases. METHODOLOGY/PRINCIPAL FINDINGS: 45 unrelated fetuses, stillbirths or infants with lethal BRAHD were ascertained through a single regional paediatric pathology service (maleratiofemale 34ratio11 or 3.1ratio1). The previously reported phenotypic overlaps with VACTERL, caudal dysgenesis, hemifacial microsomia and Müllerian defects were confirmed. A new finding is that 16/45 (35.6%; mratiof 13ratio3 or 4.3ratio1) BRAHD cases had one or more extrarenal malformations indicative of a disoder of laterality determination including; incomplete lobulation of right lung (seven cases), malrotation of the gut (seven cases) and persistence of the left superior vena cava (five cases). One such case with multiple laterality defects and sirelomelia was found to have a de novo apparently balanced reciprocal translocation 46,XY,t(2;6)(p22.3;q12). Translocation breakpoint mapping was performed by interphase fluorescent in-situ hybridization (FISH) using nuclei extracted from archival tissue sections in both this case and an isolated bilateral renal agenesis case associated with a de novo 46,XY,t(1;2)(q41;p25.3). Both t(2;6) breakpoints mapped to gene-free regions with no strong evidence of cis-regulatory potential. Ten genes localized within 500 kb of the t(1;2) breakpoints. Wholemount in-situ expression analyses of the mouse orthologs of these genes in embryonic mouse kidneys showed strong expression of Esrrg, encoding a nuclear steroid hormone receptor. Immunohistochemical analysis showed that Esrrg was restricted to proximal ductal tissue within the embryonic kidney. CONCLUSIONS/SIGNIFICANCE: The previously unreported association of BRAHD with laterality defects suggests that renal agenesis may share a common etiology with heterotaxy in some cases. Translocation breakpoint mapping identified ESRRG as a plausible candidate gene for BRAHD.

Nanotargeted radionuclides for cancer nuclear imaging and internal radiotherapy.

J Biomed Biotechnol. 2010; 2010:
Ting G, Chang CH, Wang HE, Lee TW

Current progress in nanomedicine has exploited the possibility of designing tumor-targeted nanocarriers being able to deliver radionuclide payloads in a site or molecular selective manner to improve the efficacy and safety of cancer imaging and therapy. Radionuclides of auger electron-, alpha-, beta-, and gamma-radiation emitters have been surface-bioconjugated or after-loaded in nanoparticles to improve the efficacy and reduce the toxicity of cancer imaging and therapy in preclinical and clinical studies. This article provides a brief overview of current status of applications, advantages, problems, up-to-date research and development, and future prospects of nanotargeted radionuclides in cancer nuclear imaging and radiotherapy. Passive and active nanotargeting delivery of radionuclides with illustrating examples for tumor imaging and therapy are reviewed and summarized. Research on combing different modes of selective delivery of radionuclides through nanocarriers targeted delivery for tumor imaging and therapy offers the new possibility of large increases in cancer diagnostic efficacy and therapeutic index. However, further efforts and challenges in preclinical and clinical efficacy and toxicity studies are required to translate those advanced technologies to the clinical applications for cancer patients.

Differential modulation of valence and arousal in high- alexithymic and low-alexithymic individuals.

Neuroreport. 2010 Aug 31;
Heinzel A, Schäfer R, Müller HW, Schieffer A, Ingenhag A, Northoff G, Franz M, Hautzel H

High-alexithymic individuals are characterized by an impaired ability to identify and communicate emotions whereas low-alexithymic individuals have a wide-ranging ability to deal with emotions. This study examined the hypothesis that valence and arousal modifications of emotional stimuli differentially modulate cortical regions in high-alexithymic and low-alexithymic individuals. To this end, 28 high-alexithymic and 25 low-alexithymic individuals were investigated with event-related fMRI using visual emotional stimuli. We found differential neural activations in the dorsal anterior cingulate, the insula and the amygdala. We suggest that these differences may account for the impaired ability of high-alexithymic individuals to appropriately handle emotional stimuli.

General nuclear medicine.

J Nucl Med. 2010 Sep; 51(9): 25N-30N
Ziessman HA

Hepatitis C virus infection impairs IRF-7 translocation and interferon-{alpha} synthesis in immortalized human hepatocytes.

J Virol. 2010 Sep 1;
Raychoudhuri A, Shrivastava S, Steele R, Dash S, Kanda T, Ray R, Ray RB

Hepatitis C virus (HCV) establishes chronic infection in a significant number of infected humans, although the mechanisms for chronicity remain largely unknown. We have previously shown that HCV infection in immortalized human hepatocytes (IHH) induces interferon (IFN)-beta expression (Kanda et al., 2007). However, the regulation of downstream signaling pathway for IFN-alpha production by HCV is not clearly understood. In this study, the regulation of IFN signaling pathway following HCV genotype 1a (clone H77) or genotype 2a (clone JFH1) infection of IHH was examined. HCV infection upregulated expression of total STAT1, but failed to induce phosphorylation and efficient nuclear translocation. Subsequent study revealed that HCV infection induces ISRE activation as evident from upregulation of OAS1. However, nuclear translocation of IRF-7 was impaired following HCV infection. In HCV infected IHH, IFN-alpha expression was initially increased (up to 24 h) and decreased at later time points, and IFI27 was not induced. Interestingly, HCV infection blocked IRF-7 nuclear translocation upon poly (I-C) or IFN-alpha treatment of IHH. Together, our data suggest that HCV infection enhances STAT1 expression, but impairs nuclear translocation of IRF-7 and its downstream molecules. These impairments in the IFN-alpha signaling pathway may, in part, be responsible for establishment of chronic HCV infection.

Epstein-Barr virus SM protein utilizes cellular splicing factor SRp20 to mediate alternative splicing.

J Virol. 2010 Sep 1;
Verma D, Bais S, Gaillard M, Swaminathan S

Epstein Barr virus (EBV) SM protein is an essential nuclear protein produced during the lytic cycle of EBV replication. SM is an RNA binding protein with multiple mechanisms of action. SM enhances expression of EBV genes by stabilizing mRNA and facilitating nuclear export. SM also influences splicing of both EBV and cellular pre-mRNAs. SM modulates splice site selection of the host cell STAT1 pre-mRNA, directing utilization of a novel 5' splice site that is used only in the presence of SM. SM activates splicing in the manner of SR proteins but does not contain canonical RS domains typical of cellular splicing factors. Affinity purification and mass spectrometry of SM complexes from SM-transfected cells led to the identification of the cellular SR splicing factor SRp20 as an SM-interacting protein. The regions of SM and SRp20 required for interaction were mapped by in vitro and in vivo assays. The SRp20 interaction was shown to be important for the effects of SM on alternative splicing by the use of STAT1 splicing assays. Overexpression of SRp20 enhanced SM-mediated alternative splicing and knockdown of SRp20 inhibited the SM effect on splicing. These data suggest a model whereby SM, a viral protein, recruits and co-opts the function of cellular SRp20 in alternative splicing.

Estrogen Receptor-{beta} Prevents Cardiac Fibrosis.

Mol Endocrinol. 2010 Sep 1;
Pedram A, Razandi M, O'Mahony F, Lubahn D, Levin ER

Development of cardiac fibrosis portends the transition and deterioration from hypertrophy to dilation and heart failure. Here we examined how estrogen blocks this important development. Angiotensin II (AngII) and endothelin-1 induce cardiac hypertrophy and fibrosis in humans. and we find that these agents directly stimulate the transition of the cardiac fibroblast to a myofibroblast. AngII and endothelin-1 stimulated TGFbeta1 synthesis in the fibroblast, an inducer of fibrosis that signaled via c-jun kinase to Sma- and Mad-related protein 3 phosphorylation and nuclear translocation in myofibroblasts. As a result, mesenchymal proteins fibronectin and vimentin were produced, as were collagens I and III, the major forms found in fibrotic hearts. 17beta-Estradiol (E2) or dipropylnitrile, an estrogen receptor (ER)beta agonist, comparably blocked all these events, reversed by estrogen receptor (ER)beta small interfering RNA. E2 and dipropylnitrile signaling through cAMP and protein kinase A prevented myofibroblast formation and blocked activation of c-jun kinase and important events of fibrosis. In the hearts of ovariectomized female mice, cardiac hypertrophy and fibrosis were induced by AngII infusion and prevented by E2 administration to wild type but not ERbeta knockout rodents. Our results establish the cardiac fibroblast as an important target for hypertrophic/fibrosis-inducing peptides the actions of which were mitigated by E2/ERbeta acting in these stromal cells.

Metformin Suppresses Colorectal Aberrant Crypt Foci in a Short-term Clinical Trial.

Cancer Prev Res (Phila Pa). 2010 Sep 1;
Hosono K, Endo H, Takahashi H, Sugiyama M, Sakai E, Uchiyama T, Suzuki K, Iida H, Sakamoto Y, Yoneda K, Koide T, Tokoro C, Abe Y, Inamori M, Nakagama H, Nakajima A

The biguanide metformin is widely used for treating diabetes mellitus. We previously showed the chemopreventive effect of metformin in two rodent models of colorectal carcinogenesis. However, besides epidemiologic studies, little is known about the effects of metformin on human colorectal carcinogenesis. The objective of this pilot study was to evaluate the chemopreventive effect of metformin on rectal aberrant crypt foci (ACF), which are an endoscopic surrogate marker of colorectal cancer. We prospectively randomized 26 nondiabetic patients with ACF to treatment with metformin (250 mg/d, n = 12) or no treatment (control, n = 14); 23 patients were evaluable for end point analyses (9 metformin and 14 control); the two groups were similar in ACF number and other baseline clinical characteristics. Magnifying colonoscopy determined the number of rectal ACF in each patient at baseline and after 1 month in a blinded fashion (as were all laboratory end point analyses). We also examined proliferative activity in colonic epithelium (via proliferating cell nuclear antigen labeling index) and apoptotic activity (via terminal deoxynucleotidyl transferase dUTP nick-end labeling). At 1 month, the metformin group had a significant decrease in the mean number of ACF per patient (8.78 +/- 6.45 before treatment versus 5.11 +/- 4.99 at 1 month, P = 0.007), whereas the mean ACF number did not change significantly in the control group (7.23 +/- 6.65 versus 7.56 +/- 6.75, P = 0.609). The proliferating cell nuclear antigen index was significantly decreased and the apoptotic cell index remained unaltered in normal rectal epithelium in metformin patients. This first reported trial of metformin for inhibiting colorectal carcinogenesis in humans provides preliminary evidence that metformin suppresses colonic epithelial proliferation and rectal ACF formation in humans, suggesting its promise for the chemoprevention of colorectal cancer. Cancer Prev Res; 3(9); 1077-83. (c)2010 AACR.

A plasma membrane wound proteome: reversible externalization of intracellular proteins following reparable mechanical damage.

J Biol Chem. 2010 Sep 1;
Mellgren RL

Cells in mechanically active tissues undergo constant plasma membrane damage that must be repaired to allow survival. To identify wound-associated proteins, a cell impermeant, thiol-reactive biotinylation reagent was used to label and subsequently isolate intracellular proteins that become exposed on the surface of cultured cells after plasma membrane damage induced by scraping from substratum, or crushing with glass beads. Scrape-damaged cells survived injury and were capable of forming viable colonies. Proteins that were exposed to the cell surface were degraded or internalized a few seconds to several minutes after damage, except for vimentin, which was detectable on the cell surface for at least an hour after injury. Seven major biotinylated protein bands were identified on SDS-PAGE gels. Mass spectrometric studies identified cytoskeletal proteins (caldesmon-1, vimentin), endoplasmic reticulum proteins (ERp57, ERp5, HSP47), and nuclear proteins (lamin C, hnRNPF, nucleophosmin-1) as major proteins exposed after injury. While caldesmon was a major wound-associated protein in calpain small subunit knockout fibroblasts, it was rapidly degraded in wild-type cells, probably by calpains. Lamin C exposure after wounding was most likely the consequence of nuclear envelope damage. These studies document major intracellular proteins associated with the cell surface of reversibly damaged somatic cells. The studies also show that externalization of some proteins reported to have physiologic or pathologic roles on the cell surface can occur in cells undergoing plasma membrane damage and subsequent repair.

The Nuclear Angiotensin-(1-7) Receptor is Functionally Coupled to the Formation of Nitric Oxide.

Am J Physiol Renal Physiol. 2010 Sep 1;
Gwathmey TM, Westwood BM, Pirro NT, Tang L, Rose JC, Diz DI, Chappell MC

The kidney is an important target for the actions of the renin-angiotensin system (RAS) and this tissue contains a complete local RAS that expresses the bioactive peptides angiotensin II (Ang II) and Ang-(1-7). We find both angiotensin type 1 (AT(1)R) and type 2 (AT(2)R) receptors expressed on renal nuclei that stimulate reactive oxygen species (ROS) and nitric oxide (NO), respectively. Since Ang-(1-7) also exhibits actions within the kidney and the Ang-(1-7)/Mas receptor protein contains a nuclear localization sequence, we determined the expression of Ang-(1-7) receptors (AT7R) in nuclei isolated from the kidneys of young adult sheep. Binding studies with (125)I-[Sar(1)Thr(8)]-Ang II revealed sites sensitive to the AT7R antagonist [D-Ala(7)]-Ang-(1-7) (DALA, A779), as well as to AT(2) and AT(1) antagonists. Incubation of Ang-(1-7) [10(-15) to 10(-9) M] with isolated cortical nuclei elicited a dose-dependent increase in the fluorescence of the nitric oxide (NO) indicator [4-amino-5-methylamino-2',7']-difluorofluorescein diacetate (DAF). The NO response to Ang-(1-7) was abolished by the NO inhibitor N-nitro-L-arginine methyl ester and DALA, but not the AT(1) antagonist losartan or the AT(2) blocker PD123319. Immunofluorescent studies utilizing the AT7/Mas receptor antibody revealed immunolabeling of the proximal tubules but not staining within the glomerulus in cortical sections of the sheep kidney. In the nuclear fraction of isolated proximal tubules, immunoblots revealed the precursor angiotensinogen and renin, as well as functional activity for ACE, ACE2 and neprilysin. We conclude that renal nuclei express AT7/Mas receptors that are functionally linked to NO formation. The marked sensitivity of the intracellular NO response to Ang-(1-7) implicates a functional role of the Ang-(1-7)-ACE2-AT7R axis within the nucleus. Moreover, evidence for the precursor and enzymatic components of the RAS within the nuclear compartment of the proximal tubules provides a potential pathway for the intracellular generation of Ang-(1-7).

The Transcription Factor HNF1{alpha} Regulates the Expression of the Chloride-Proton Exchanger ClC-5 in the Renal Proximal Tubule.

Am J Physiol Renal Physiol. 2010 Sep 1;
Tanaka K, Terryn S, Geffers L, Garbay S, Pontoglio M, Devuyst O

The Cl(-)/H(+) exchanger ClC-5 is essential for the endocytic activity of the proximal tubule cells and the tubular clearance of proteins filtered in the glomeruli. The mechanisms that regulate the expression of ClC-5 in general and its specific expression in the proximal tubule are unknown. In this study, we investigated the hypothesis that the hepatocyte nuclear transcription factor HNF1alpha, which is predominantly expressed in proximal tubule segments, may directly regulate the expression of ClC-5. In situ hybridisation demonstrated that the expression of Clcn5 strikingly overlaps with that of Hnf1alpha in the developing kidney as well as in absorptive epithelia including the digestive tract and yolk sac. Multiple binding sites for HNF1 were mapped in the 5'-regulatory sequences of the mouse and human Clcn5/CLCN5 genes. The transactivation of the Clcn5/CLCN5 promoter by HNF1alpha was verified in vitro, and the binding of HNF1alpha to the Clcn5 promoter in vivo was confirmed by chromatin immunoprecipitation in mouse kidney. The expression of Clcn5 was reduced in the proximal tubule segments of HNF1alpha-null kidneys and it was rescued upon transfection of HNF1alpha-null cells with wild-type but not with mutant HNF1alpha. These data demonstrate that HNF1alpha directly regulates the expression of ClC-5 in the renal proximal tubule and yield insights into the mechanisms governing epithelial differentiation and specialized transport activities in the kidney.

Suppression of NF-{kappa}B Increases Bone Formation and Ameliorates Osteopenia in Ovariectomized Mice.

Endocrinology. 2010 Sep 1;
Alles N, Soysa NS, Hayashi J, Khan M, Shimoda A, Shimokawa H, Ritzeler O, Akiyoshi K, Aoki K, Ohya K

Bone degenerative diseases, including osteoporosis, impair the fine balance between osteoclast bone resorption and osteoblast bone formation. Single-agent therapy for anabolic and anticatabolic effects is attractive as a drug target to ameliorate such conditions. Inhibition of nuclear factor (NF)-kappaB reduces the osteoclast bone resorption. The role of NF-kappaB inhibitors on osteoblasts and bone formation, however, is minimal and not well investigated. Using an established NF-kappaB inhibitor named S1627, we demonstrated that inhibition of NF-kappaB increases osteoblast differentiation and bone formation in vitro by up-regulating the mRNAs of osteoblast-specific genes like type I collagen, alkaline phosphatase, and osteopontin. In addition, S1627 was able to increase bone formation and repair bone defect in a murine calvarial defect model. To determine the effect of NF-kappaB on a model of osteoporosis, we injected two doses of inhibitor (25 and 50 mg/kg . d) twice a day in sham-operated or ovariectomized 12-wk-old mice and killed them after 4 wk. The anabolic effect of S1627 on trabecular bone was determined by micro focal computed tomography and histomorphometry. Bone mineral density of inhibitor-treated ovariectomized animals was significantly increased compared with sham-operated mice. Osteoblast-related indices like osteoblast surface, mineral apposition rate, and bone formation rate were increased in S1627-treated animals in a dose-dependent manner. NF-kappaB inhibition by S1627 increased the trabecular bone volume in ovariectomized mice. Furthermore, S1627 could inhibit the osteoclast number, and osteoclast surface to bone surface. In vitro osteoclastogenesis and bone resorbing activity were dose-dependently reduced by NF-kappaB inhibitor S1627. Taken collectively, our results suggest that NF-kappaB inhibitors are effective in treating bone-related diseases due to their dual anabolic and antiresorptive activities.

Clinical activity of bortezomib in relapsed/refractory MALT lymphomas: results of a phase II study of the International Extranodal Lymphoma Study Group (IELSG).

Ann Oncol. 2010 Sep 1;
Conconi A, Martinelli G, Lopez-Guillermo A, Zinzani PL, Ferreri AJ, Rigacci L, Devizzi L, Vitolo U, Luminari S, Cavalli F, Zucca E

BACKGROUND: The nuclear factor-kappa B activation in mucosa-associated lymphoid tissue (MALT) lymphoma pathogenesis provided the rationale for the evaluation of bortezomib in this malignancy. PATIENTS AND METHODS: Thirty-two patients with relapsed/refractory MALT lymphoma were enrolled. Thirty-one patients received bortezomib 1.3 mg/m(2) i.v., on days 1, 4, 8, and 11, for up to six 21-day cycles. RESULTS: Median age was 63 years (range, 37-82 years). Median number of prior therapies was 2 (range, 1-4). Nine patients had Ann Arbor stage I, 7 patients had stage II, and 16 patients had stage IV. Primary lymphoma localization was the stomach in 14 patients; multiple extranodal sites were present in 10 patients. Among the 29 patients assessable for response, the overall response rate was 48% [95% confidence interval (CI) 29% to 67%], with 9 complete and 5 partial responses. Nine patients experienced stable disease and six had disease progression during therapy. The most relevant adverse events were fatigue, thrombocytopenia, neutropenia, and peripheral neuropathy. After a median follow-up of 24 months, the median duration of response was not reached yet. Five deaths were reported, in two patients due to disease progression. CONCLUSION: Bortezomib is active in relapsed MALT lymphomas. Further investigations to identify optimal bortezomib dose, schedule, and combination regimens are needed since the frequent detection of dose-limiting peripheral neuropathy.

Attenuation-based characterization of coronary atherosclerotic plaque: Comparison of dual source and dual energy CT with single-source CT and histopathology.

Eur J Radiol. 2010 Aug 30;
Henzler T, Porubsky S, Kayed H, Harder N, Krissak UR, Meyer M, Sueselbeck T, Marx A, Michaely H, Schoepf UJ, Schoenberg SO, Fink C

OBJECTIVE: To compare different CT acquisition techniques regarding for attenuation-based characterization of coronary atherosclerotic plaques using histopathology as the standard of reference. MATERIALS AND METHODS: In a post mortem study 17 human hearts were studied with dual-source CT (DSCT) and dual energy CT (DECT) mode on a DSCT as well as with 16-slice single-source CT (SSCT). At autopsy, atherosclerotic lesions were cut at 5mum sections. Histopathologic classification of the plaques according to the American Heart Association (AHA) criteria was performed by two pathologists. Attenuation values of all plaques were measured in DSCT, DECT and SSCT studies, respectively and classified based on attenuation according to modified AHA criteria. RESULTS: 58 coronary plaques were identified at autopsy. Regardless of the CT technique only 52/58 plaques were found at CT (sensitivity=89.6%). There was no significant difference between the mean attenuation values of different plaque types between DSCT, DECT, and SSCT: type IV: 11HU/8HU/19HU; type Va: 44HU/45HU/52HU; type Vb: 1088HU/966HU/1079HU). The sensitivity for correct classification varied depending on the plaque type (type II=0%, type III=0%, type IV=43%, type Va=58%, Vb=97%). CONCLUSION: Independent of the used acquisition technique, SSCT, DSCT and DECT show similar results for attenuation-based characterization of atherosclerotic coronary plaques.