Vessel adherent growth represents a major challenge in the surgical resection of neuroblastoma and Is associated with adverse outcome.

PURPOSE: Neuroblastoma (NB) is the most common extracranial, solid tumor in childhood, with a peak incidence in children under 6 years of age. Due to its variable course of disease, which ranges from spontaneous regression to metastatic spread, NB still represents a significant therapeutic challenge. Strikingly, a certain number of NBs intraoperatively show vessel adhesion and/or infiltrative growth, which is often not visible in pre-operative imaging. We proposed the term unexpected vessel infiltration of NB (UVIN) to denote this phenomenon. UVIN represents a major surgical challenge. METHODS: In this study, we determined frequency and clinical relevance of UVIN in a cohort of 100 NB-patients with subsequent correlation to several unfavorable characteristics of disease. RNA expression levels of MYCN and its co-regulated antisense transcript MYCNOS to identify markers was measured by PCR. RESULTS: We found UVIN to be present in 34% of cases and significantly correlated with incomplete resection, MYCN amplification, complications, neoadjuvant therapy, tumor grade and MYCNOS expression levels. MYCN expression levels showed no significant results with UVIN. CONCLUSION: Collectively, our data show that UVIN represents a frequent surgical problem associated with a poor outcome in NB patients. MYCN and MYCNOS seem to be no appropriate markers for UVIN. TYPE OF STUDY: Prognosis study. LEVEL OF EVIDENCE: Level III.

Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.

In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

Low expression of N-myc downstream-regulated gene 2 (NDRG2) correlates with poor prognosis in hepatoblastoma.

BACKGROUND/PURPOSE OF THE STUDY: Despite tremendous progress in therapy, about 30% of patients with hepatoblastoma still succumb to the disease. Thus, the development of improved therapies as well as the identification of prognostic factors are urgently needed. METHODS: In the present study, expression and promoter methylation of the N-myc downstream-regulated gene (NDRG2), a tumor suppressor gene contributing to the regulation of the Wnt signalling pathway, was analysed in 38 hepatoblastoma samples by real-time reverse transcription-PCR and pyrosequencing, respectively. RESULTS: The NDRG2 gene was highly expressed in normal pediatric liver tissue, but was significantly downregulated in heptoblastoma primary tumors. Detailed methylation analysis of CpG sites in the NDRG2 promoter region revealed a general high degree of DNA methylation in hepatoblastoma, which correlated with the suppression of NDRG2. By analyzing clinicopathological features we could demonstrate a strong association between low NDRG2 expression and tumor metastasis. Importantly, the overall survival analysis by Kaplan-Meier revealed that high NDRG2 expression was correlated with a higher survival rate in hepatoblastoma patients. CONCLUSION: Our data show that downregulation of NDRG2 may play an important role in advanced hepatoblastomas.

Oxyphil cell metaplasia in the parathyroids is characterized by somatic mitochondrial DNA mutations in NADH dehydrogenase genes and cytochrome c oxidase activity-impairing genes.

Oxyphil cell transformation of epithelial cells due to the accumulation of mitochondria occurs often during cellular aging. To understand the pathogenic mechanisms, we studied mitochondrial DNA (mtDNA) alterations in the three cell types of the parathyroids using multiplex real-time PCR and next-generation sequencing. mtDNA was analyzed from cytochrome c oxidase (COX)-positive and COX-negative areas of 19 parathyroids. Mitochondria-rich pre-oxyphil/oxyphil cells were more prone to develop COX defects than the mitochondria-poor clear chief cells (P < 0.001). mtDNA increased approximately 2.5-fold from clear chief to oxyphil cells. In COX deficiency, the increase was even more pronounced, and COX-negative oxyphil cells had approximately two times more mtDNA than COX-positive oxyphil cells (P < 0.001), illustrating the influence of COX deficiency on mtDNA biosynthesis, probably as a consequence of insufficient ATP synthesis. Next-generation sequencing revealed a broad spectrum of putative pathogenic mtDNA point mutations affecting NADH dehydrogenase and COX genes as well as regulatory elements of mtDNA. NADH dehydrogenase gene mutations preferentially accumulated in COX-positive pre-oxyphil/oxyphil cells and, therefore, could be essential for inducing oxyphil cell transformation by increasing mtDNA/mitochondrial biogenesis. In contrast, COX-negative cells predominantly harbored mutations in the MT-CO1 and MT-CO3 genes and in regulatory mtDNA elements, but only rarely NADH dehydrogenase mutations. Thus, multiple hits in NADH dehydrogenase and COX activity-impairing genes represent the molecular basis of oxyphil cell transformation in the parathyroids.

The cytokine midkine supports neutrophil trafficking during acute inflammation by promoting adhesion via ß2 integrins (CD11/CD18).

Emerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK(-/-) mice compared with MK(+/+) animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK(-/-) mice. In a hind limb ischemia model, leukocyte accumulation in MK(-/-) mice was significantly diminished compared with MK(+/+) animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of ß2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of ß2 integrins, thereby facilitating PMN trafficking during acute inflammation.

Epigallocatechin-3-gallate inhibits hepatoblastoma growth by reactivating the Wnt inhibitor SFRP1.

Activation of Wnt signaling plays a central role in the formation of hepatoblastoma (HB), the most common pediatric liver cancer. Blocking this pathway with specific inhibitors is currently the target of various research endeavours. This study provides evidence that the naturally occurring flavonoid epigallocatechin-3-gallate (EGCG) is highly effective against HB growth through inhibition of Wnt signaling. We demonstrate that EGCG has a strong cytotoxic effect on HB cells in a time- and dose-dependent manner by impinging on cell viability, while leaving normal fibroblasts unaffected. Apoptotic features, including morphological changes, caspase 3 activity, and proteolytic cleavage of poly(ADP-ribose) polymerase, were frequently found in EGCG-treated HB cells, thereby suggesting involvement of the mitochondrial intrinsic apoptotic pathway. We furthermore show that EGCG effectively inhibits Wnt signaling, as evidenced by down-regulation of Wnt-responsive reporter gene activity and expression of the Wnt target genes MYC and CCND1. Interestingly, EGCG induced reexpression of the tumor suppressor gene SFRP1, which is transcriptionally silenced in HB cells and known to down-regulate Wnt signaling. Considering the lack of toxic effects on normal cells, EGCG should be preclinically validated as an adjuvant therapy in vivo with the ultimate goal of determining its efficacy in human trials.

Selective methylation of CpGs at regulatory binding sites controls NNAT expression in Wilms tumors.

Aberrant expression of imprinted genes, such as those coding for the insulin-like growth factor 2 (IGF2) and neuronatin (NNAT), is a characteristic of a variety of embryonic neoplasms, including Wilms tumor (WT). In case of IGF2, it is generally accepted that loss of imprinting in a differentially methylated region of the IGF2/H19 locus results in biallelic expression and, thus, upregulation of the gene. In this study we examined methylation pattern at potential regulatory elements of the paternally expressed NNAT gene in a cohort of WT patients in order to further characterize the molecular mechanism causing overexpression of this regulatory gene. We demonstrate that transcriptional upregulation of NNAT in WT is grossly independent of the bladder cancer-associated protein (BLCAP) gene, an imprinted gene within the imprinted domain of the NNAT locus. However, expression of the BLCAP transcript isoform v2a formerly known to be selectively expressed from the paternal allele in brain was associated with high expression of NNAT. This contrasts the situation we found at the IGF2/H19 locus, which shows high overexpression of IGF2 and inversely correlated expression of the H19 gene in WT. An analysis of DNA methylation in two potential regulatory regions of the NNAT locus by pyrosequencing revealed significant hypomethylation of the tumors compared to normal kidney tissue. Interestingly, the difference in DNA methylation was highest at CpGs that were observed within three putative binding sites of the CCCTC-binding factor CTCF. Most importantly, hypomethylation of both NNAT regulatory regions is significantly associated with the upregulation of NNAT expression and the BLCAP_v2a transcript. Our data indicate that the methylation status of a not-yet-described regulatory element within the NNAT locus that contains four potential CTCF binding sites determines the expression level of NNAT and the nearby located BLCAP_v2a transcript, thereby suggesting a functional role in the aberrant upregulation of NNAT in WT.

Novel mutation in Hermansky-Pudlak syndrome type 2 with mild immunological phenotype.

Patients with Hermansky-Pudlak syndrome type 2 (HPS2) present with oculocutaneous albinism, nystagmus, prolonged bleeding time, and increased susceptibility to infections. Twelve HPS2 patients with mutations in the ß3A-subunit of the cytosolic adaptor-related protein complex 3 (AP3B1, also called HPS2) have been described so far. Here, we report on a patient with oculocutaneous albinism who developed a life-threatening bleeding after tonsillectomy. She presented with moderate neutropenia and reduced granulopoiesis. Analyzing patient's impaired platelet function using electron microscopy and flow cytometry led to the diagnosis of HPS2. Flow cytometric analysis of the patient's platelets showed already elevated CD63 expression on resting platelets with no further increase after thrombin stimulation. Natural killer (NK) cell degranulation was partially impaired but target cell lysis of NK cells and cytotoxic T-lymphocytes (CTLs) were normal and the patient did not develop signs of hemophagocytic syndrome. Molecular genetic analyses revealed a novel 2 bp-deletion (c.3222_3223delTG) in the last exon of AP3B1 causing a frameshift and a prolonged altered protein. The location of the deletion at the very C-terminal end may prevent a complete loss of the HPS2 protein leading to a less pronounced severity of immunodeficiency than in other HPS2 patients.

ß-Catenin mutations in 2 nested stromal epithelial tumors of the liver--a neoplasia with defective mesenchymal-epithelial transition.

Nested stromal epithelial tumor of the liver is a rare neoplasm of early childhood and adolescence with a characteristic nested morphology of spindle and epithelioid cells. Histogenesis and pathogenesis of this neoplasm are, however, still unclear. Because the characteristic nested morphology with spindle mesenchymal and epithelioid cells is suggestive of altered mesenchymal-epithelial transition and ß-catenin mutations are rather common in other liver tumors such as hepatoblastomas, we investigated the ß-catenin gene in 2 nested stromal epithelial tumors of the liver and analyzed additional factors involved in mesenchymal-epithelial transition, such as E-cadherin, vimentin, c-Met, TWIST, SNAIL, and SLUG by molecular genetic and immunohistochemical methods. Mutation analysis of both cases revealed large deletions in exon 3 of the ß-catenin gene (155 and 228 base pairs), resulting in an accumulation of ß-catenin in the cytoplasm and nuclei of tumor cells, as evidenced by immunohistochemistry. The expression of the mesenchymal-epithelial transition factors SNAIL, SLUG, TWIST, c-Met, vimentin, and ß-catenin was generally increased, whereas E-cadherin was decreased. Morphological and immunohistochemical analysis, however, showed a variable expression pattern of various epithelial and mesenchymal markers both in the spindle and epithelioid cell compartments of the tumors, thus illustrating the transitional status of the tumor cells. In conclusion, our data clearly identify protein stabilizing mutations of the ß-catenin gene as a common feature of nested stromal epithelial tumors of the liver, similarly as in hepatoblastomas. Therefore, nested stromal epithelial tumors of the liver may be regarded as a variant of hepatoblastoma, despite differing from it in clinical and morphological aspects. The characteristic epithelioid-spindle morphology along with the incomplete epithelial differentiation proposes impaired mesenchymal-epithelial transition as a possible pathogenetic mechanism of this rare tumor. However, because only 2 cases were studied, this hypothesis awaits further validation.

Midkine acts as proangiogenic cytokine in hypoxia-induced angiogenesis.

The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identified the source of MK in the vascular system under hypoxic conditions and demonstrated the relevance of MK during ischemia of normal tissue. Hypoxia increased MK protein expression in human polymorphonuclear neutrophils (PMN), monocytes, and human umbilical vein endothelial cells (HUVEC) compared with normoxia. Immunoelectron microscopy showed elevated cell surface expression of MK in PMN and monocytes during hypoxia. However, only HUVEC released significant amounts of soluble MK during hypoxia compared with normoxia (301 ± 81 pg/ml vs. 158 ± 45 pg/ml; P < 0.05). Exogenous MK induced neovascularization in a chorioallantoic membrane (CAM) assay compared with negative control as measured by counting the number of branching points per visual field (1,074 ± 54 vs. 211 ± 70; P < 0.05). In a hind limb ischemia model, the angiogenic response was almost completely absent in MK-deficient mice, whereas control animals showed a profound angiogenic response measured as proliferating endothelial cells per visual field (45 ± 30 vs. 169 ± 34; P < 0.01). These unanticipated results identified endothelial cells as the source of soluble MK in the vascular system during hypoxia and defined MK as a pivotal player of angiogenesis during ischemia in nonmalignant tissue.

IGFBP3 impedes aggressive growth of pediatric liver cancer and is epigenetically silenced in vascular invasive and metastatic tumors.

BACKGROUND: Hepatoblastoma (HB) is an embryonal liver neoplasm of early childhood with a poor prognosis for patients with distant metastases and vascular invasion. We and others have previously shown that the overexpression of insulin-like growth factor 2 (IGF2), loss of imprinting at the IGF2/H19 locus, and amplification of pleomorphic adenoma gene 1 (PLAG1) are common features in HB, suggesting a critical role of the IGF axis in hepatoblastomagenesis. In this study, we investigated the role of the insulin-like growth factor binding protein 3 (IGFBP3), a known competitor of the IGF axis, in pediatric liver cancers. RESULTS: The IGFBP3 gene was highly expressed in normal pediatric livers but was heavily downregulated in four HB cell lines and the majority of HB primary tumors (26/36). Detailed methylation analysis of CpG sites in the IGFBP3 promoter region by bisulfite sequencing revealed a high degree of DNA methylation, which is causatively associated with the suppression of IGFBP3 in HB cell lines. Consequently, the treatment of HB cell lines with 5-aza-2'-deoxycytidine resulted in DNA demethylation and reactivation of the epigenetically silenced IGFBP3 expression. Interestingly, IGFBP3 promoter methylation predominantly occurred in metastatic HB with vascular invasion. Restoring IGFBP3 expression in HB cells resulted in reduced colony formation, migration, and invasion. CONCLUSION: This study provides the first direct evidence that the reactivation of IGFBP3 decreases aggressive properties of pediatric liver cancer cells and that IGFBP3 promoter methylation might be used as an indicator for vessel-invasive tumor growth in HB patients.

Rapamycin blocks hepatoblastoma growth in vitro and in vivo implicating new treatment options in high-risk patients.

Activation of the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway plays a central role in the formation of hepatoblastoma (HB), the most common liver cancer in childhood. Blocking this pathway with specific mTOR inhibitors such as the immunosuppressant rapamycin is being currently tested for a variety of cancers. Here, we report that rapamycin treatment induced a significant dose-dependent inhibition of cell viability and promoted apoptosis in HB cells in vitro. Moreover, rapamycin inhibited AKT/mTOR signalling by dephosphorylation of the downstream target p70S6 kinase (p70S6K). Most importantly, treating subcutaneous HUH6 xenograft tumour bearing mice orally with 5mg/kg/day rapamycin for three weeks resulted in a striking reduction of tumour growth, as evidenced by reduced volume and weight, and moderately lowered tumour-specific alpha-fetoprotein (AFP) serum levels. The anti-tumourigenic effect was primarily ascribed to a significantly reduced proliferation rate upon p70S6K dephosphorylation, as microvascular density of rapamycin-treated compared to vehicle-treated tumours stayed grossly unchanged. Of uttermost clinical importance, we found no evidence for a feedback-loop activation of AKT in vivo. In conclusion, we demonstrate that rapamycin effectively inhibits HB growth both in vitro and in vivo by blocking AKT/mTOR signalling at the level of p70S6K and that rapamycin should be considered to treat HB patients especially those to be indicated for liver transplantation to benefit from its anti-tumourigenic and immunosuppressive properties.

Novel desmoplakin mutation: juvenile biventricular cardiomyopathy with left ventricular non-compaction and acantholytic palmoplantar keratoderma.

Two sons of a consanguineous marriage developed biventricular cardiomyopathy. One boy died of severe heart failure at the age of 6 years, the other was transplanted because of severe heart failure at the age of 10 years. In addition, focal palmoplantar keratoderma and woolly hair were apparent in both boys. As similar phenotypes have been described in Naxos disease and Carvajal syndrome, respectively, the genes for plakoglobin (JUP) and desmoplakin (DSP) were screened for mutations using direct genomic sequencing. A novel homozygous 2 bp deletion was identified in an alternatively spliced region of DSP. The deletion 5208_5209delAG led to a frameshift downstream of amino acid 1,736 with a premature truncation of the predominant cardiac isoform DSP-1. This novel homozygous truncating mutation in the isoform-1 specific region of the DSP C-terminus caused Carvajal syndrome comprising severe early-onset heart failure with features of non-compaction cardiomyopathy, woolly hair and an acantholytic form of palmoplantar keratoderma in our patient. Congenital hair abnormality and manifestation of the cutaneous phenotype in toddler age can help to identify children at risk for cardiac death.

Dysregulation of the cell cycle and chromosomal imbalances in juxtaglomerular cell tumors - a comparative study with endocrine tumors of the pancreas.

Two juxtaglomerular cell tumors (JGCTs) were investigated in comparison with 14 endocrine tumors of the pancreas (ETPs), focusing on the cell cycle, apoptosis, and cytogenetic changes. JGCTs revealed nuclear accumulation of Cyclin D(1), together with the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1). In contrast, no accumulation of Cyclin D(3), p53, p16(INK4a), or Mdm-2 was seen. Bcl-2 protein was intensively, but Rb only moderately, expressed. This immunoreactive profile was not found in the ETPs, which were negative for Bcl-2, p27(Kip1), p21(Cip1/Waf1), and - with one exception - for Cyclin D(1) (1/14) but expressed Cyclin D(3) in 7/14 cases. JGCTs displayed characteristic genetic alterations with combined losses of chromosomes 9, 11, 15, and 21 and gains of chromosome 18. In contrast, no characteristic pattern of genetic alterations was found in ETPs. In both, the amount of chromosomal aberrations correlated with tumor size. In small ETPs and JGCTs, genetic losses dominated over gains of chromosomes, whereas in large/malignant ETPs, gains and losses were equally affected. Thus, JGCTs represent a special type of renal endocrine neoplasm characterized by deregulation of cell cycle components and a typical profile of chromosomal aberrations. Since only two JCTs were investigated, further studies for validation of these results are, however, necessary.

Altered expression of imprinted genes in Wilms tumors.

Overexpression of insulin-like growth factor 2 (IGF2), an imprinted gene located on chromosome 11p15, has been reported as a characteristic feature in various embryonal tumors, including Wilms tumor (WT). Recent studies specified loss of imprinting (LOI) in a differential methylated region (DMR) of the IGF2/H19 cluster or loss of heterozygosity (LOH), respectively, uniparental disomy (UPD) being responsible for this overexpression. However, the role of other imprinted genes in the genesis of WT is still unknown. In the current study, we analyzed transcriptional activity of the imprinted genes IGF2, H19, NNAT, DLK1, RTL1, MEG3, and MEST as well as the methylation status of the DMR of the IGF2/H19 cluster in a panel of 32 WTs. Except for H19, we detected massive overexpression of all genes in the majority of WTs compared to normal renal tissue, which was most prominent for the paternally expressed genes IGF2, NNAT, and MEST. Alterations of the H19DMR were found in two-thirds of the WTs. Moreover, we have seen a strong correlation between the transcriptional activity of IGF2, NNAT and MEST and LOI/LOH of H19DMR, which was inverse for H19. Expression of DLK1, RTL1 and MEG3 does not correlate with LOI/LOH of H19DMR. Altogether, our findings suggest that over-expression of imprinted genes is common in WTs and correlates at least for some imprinted genes with LOI of H19DMR. Thus, it may be speculated that alterations of the DNA modification machinery drive erroneous setting of methylation marks in imprinting regions throughout the genome, which leads to the concomitant activation of imprinted genes in blastomagenesis.

A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation.

BACKGROUND: E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease. METHODS AND FINDINGS: To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen. CONCLUSION: These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.

Facioscapulohumeral muscular dystrophy presenting with unusual phenotypes and atypical morphological features of vacuolar myopathy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy and usually follows an autosomal dominant trait. Clinically, FSHD affects facial muscles and proximal upper limb and girdle muscles, but may present with variable clinical phenotypes even within the same family. Most genetically confirmed FSHD patients exhibit unspecific morphological signs of a degenerative myopathy. We report on five unrelated patients who carried the pathogenic FSHD mutation on chromosome 4q35. Muscle biopsies revealed numerous rimmed vacuoles and filamentous cytoplasmic inclusions in all cases. Clinically, the patients suffered from weakness and atrophy predominantly of the lower limb muscles. In conclusion, we suggest considering FSHD in the differential diagnosis of adult-onset distal myopathies with rimmed vacuoles.

The p.G154S mutation of the alpha-B crystallin gene (CRYAB) causes late-onset distal myopathy.

Mutations in alpha-B crystallin gene (CRYAB) have been described to cause congenital cataracts, dilated cardiomyopathy and myofibrillar myopathy. For skeletal myopathy, only three different mutations have been reported within the last decade. Here we describe for the first time the missense mutation p.Gly154Ser to be associated with a late-onset distal vacuolar myopathy with protein aggregates without respiratory or cardiac dysfunction, and without significant cataracts. The mutation affects a residue in a highly preserved domain of alpha-B crystallin and has been identified earlier in patients with isolated cardiomyopathy.

Myocardial perfusion imaging is feasible for infarct size quantification in mice using a clinical single-photon emission computed tomography system equipped with pinhole collimators.

INTRODUCTION: The aim of this study is to evaluate a non-invasive method for measuring myocardial perfusion defect size in mice using a clinical single-photon emission computed tomography system equipped with pinhole collimators (pinhole SPECT). MATERIALS AND METHODS: Thirty days after ligation of the left anterior descending coronary artery, 13 mice (C57BL/6J) were imaged following intravenous injection of 370 MBq [99mTc]sestamibi. Eight control mice without myocardial infarction were likewise investigated. Image quality optimization had been achieved by repeated scanning of a multiple point phantom, with varying zoom factors, number of projection angles, and pinhole diameter. Volumetric sampling was used to generate polar maps, in which intensity was normalized to that of a standard septal region of interest (ROI), which was set at 100%. Receiver operating characteristic analyses were performed to define an optimal threshold as compared to histologically measured defect sizes, which were considered as gold standard. RESULTS: A spatial resolution of 1.9 mm was achieved using a pinhole diameter of 0.5 mm, a zoom factor of 2, and 6 degrees projection angles. Histological results were best reproduced by a 60% threshold relative to the septal reference ROI. By applying this threshold, SPECT perfusion defect sizes revealed very high correlation to the histological results (R(2) = 0.867) with excellent intra- and interobserver reproducibility (intraclass correlation coefficients of 0.84 and 0.82). CONCLUSIONS: We achieved a spatial resolution of 1.9 mm in myocardial perfusion imaging in mice using a clinical SPECT system mounted with pinhole collimators. Compared to a histological gold standard, the infarct sizes were accurately estimated, indicating that this method shows promise to monitor experimental cardiac interventions in mice.

Blocking the hedgehog pathway inhibits hepatoblastoma growth.

UNLABELLED: Recent evidence has indicated that Hedgehog (Hh) signaling significantly contributes to liver development and regeneration and that activation of the pathway may contribute to growth of hepatocellular carcinoma (HCC) in adults. However, the role of Hh signaling in pediatric liver tumors remains to be elucidated. In this study, we show that Hh signaling is activated in hepatoblastoma (HB), the most common liver tumor in childhood, with most occurrences before the age of 3 years. The Hh target genes glioma-associated oncogene homolog 1 (GLI1) and Patched (PTCH1) showed increased transcript levels in 65% and 30% of HB samples, respectively, compared with normal liver tissues. Most interestingly, the gene encoding the hedgehog interacting protein (HHIP) is transcriptionally silenced by cytosine-phospho-guanosine (CpG) island promoter hypermethylation in 26% of HB cases and treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine partially restored HHIP expression. Blocking Hh signaling with the antagonist cyclopamine had a strong inhibitory effect on cell proliferation of HB cell lines with an activated pathway. We further demonstrate that this decrease in cell viability is caused by a massive induction of apoptosis, as shown by morphological changes and phosphatidylserine membrane asymmetry. In cyclopamine-exposed HB cells, caspase 3 and poly(adenosine diphosphate-ribose) polymerase proteins were specifically activated by their proteolytic cleavage. CONCLUSION: This study demonstrates, for the first time, the frequent occurrence of GLI1 and PTCH1 overexpression and HHIP promoter methylation in early childhood HB, thus indicating a key role for Hh signaling activation in the malignant transformation of embryonal liver cells.