Glucagon-like peptide-1 receptor imaging for the localisation of insulinomas: a prospective multicentre imaging study

BACKGROUND Small benign insulinomas are hard to localise, leading to difficulties in planning of surgical interventions. We aimed to prospectively assess the insulinoma detection rate of single-photon emission CT in combination with CT (SPECT/CT) with a glucagon-like peptide-1 receptor avid radiotracer, and compare detection rates with conventional CT/MRI techniques. METHODS In our prospective imaging study, we enrolled adults aged 25-81 years at centres in Germany, Switzerland, and the UK. Eligible patients had proven clinical and biochemical endogenous hyperinsulinaemic hypoglycaemia and no evidence for metastatic disease on conventional imaging. CT/MRI imaging was done at referring centres according to standard protocols. At three tertiary nuclear medicine centres, we used whole body planar images and SPECT/CT of the abdomen up to 168 h after injection of (111)In-[Lys40(Ahx-DTPA-(111)In)NH2]-exendin-4 ((111)In-DTPA-exendin-4) to identify insulinomas. Consenting patients underwent surgery and imaging findings were confirmed histologically. FINDINGS Between Oct 1, 2008, and Dec 31, 2011, we recruited 30 patients. All patients underwent (111)In-DTPA-exendin-4 imaging, 25 patients underwent surgery (with histological analysis), and 27 patients were assessed with CT/MRI. (111)In-DTPA-exendin-4 SPECT/CT correctly detected 19 insulinomas and four additional positive lesions (two islet-cell hyperplasia and two uncharacterised lesions) resulting in a positive predictive value of 83% (95% CI 62-94). One true negative (islet-cell hyperplasia) and one false negative (malignant insulinoma) result was identified in separate patients by (111)In-DTPA-exendin-4 SPECT/CT. Seven patients (23%) were referred to surgery on the basis of (111)In-DTPA-exendin-4 imaging alone. For 23 assessable patients, (111)In-DTPA-exendin-4 SPECT/CT had a higher sensitivity (95% [95% CI 74-100]) than did CT/MRI (47% [27-68]; p=0·011). INTERPRETATION (111)In-DTPA-exendin-4 SPECT/CT could provide a good second-line imaging strategy for patients with negative results on initial imaging with CT/MRI. FUNDING Oncosuisse, the Swiss National Science Foundation, and UK Department of Health.

CD8+ T cells and NK cells from blister fluid of an EEM/SJS overlap highly express Fas ligand and Granulysin

INTRODUCTION Erythema exsudativum multiforme majus (EEMM) and Stevens-Johnson Syndrome (SJS) are severe cutaneous reaction patterns caused by infections or drug hypersensitivity. The mechanism by which widespread keratinocyte death is mediated by the immune system in EEMM/SJS are still to be elucidated. Here, we characterized the blister cells isolated from a patient with EEMM/SJS overlap and investigated its cause. METHODS Clinical classification of the cutaneous eruption was done according to the consensus definition of severe blistering skin reactions and histological analysis. Common infectious causes of EEMM were investigated using standard clinical techniques. T cell reactivity for potentially causative drugs was assessed by lymphocyte transformation tests (LTT). Lymphocytes isolated from blister fluid were analyzed for their expression of activation markers and cytotoxic molecules using flow cytometry. RESULTS The healthy 58 year-old woman suffered from mild respiratory tract infection and therefore started treatment with the secretolytic drug Ambroxol. One week later, she presented with large palmar and plantar blisters, painful mucosal erosions, and flat atypical target lesions and maculae on the trunc, thus showing the clinical picture of an EEMM/SJS overlap (Fig. 1). This diagnosis was supported by histology, where also eosinophils were found to infiltrate the upper dermis, thus pointing towards a cutaneous adverse drug reaction (cADR). Analysis of blister cells showed that they mainly consisted of CD8+ and CD4+ T cells and a smaller population of NK cells. Both the CD8+ T cells and the NK cells were highly activated and expressed Fas ligand and the cytotoxic molecule granulysin (Fig. 2). In addition, in comparison to NK cells from PBMC, NK cells in blister fluids strongly upregulated the expression of the skin-homing chemokine receptor CCR4 (Fig 4). Surprisingly, the LTT performed on PBMCs in the acute phase was positive for Ambroxol (SI=2.9) whereas a LTT from a healthy but exposed individual did not show unspecific proliferation. Laboratory tests for common infectious causes of EEMM were negative (HSV-1/-2, M. pneumoniae, Parvovirus B19). However, 6 weeks later, specific proliferation to Ambroxol could no longer be observed in the LTT (Fig 4.).

CA125/MUC16 is dispensable for mouse development and reproduction.

Cancer antigen 125 (CA125) is a blood biomarker that is routinely used to monitor the progression of human epithelial ovarian cancer (EOC) and is encoded by MUC16, a member of the mucin gene family. The biological function of CA125/MUC16 and its potential role in EOC are poorly understood. Here we report the targeted disruption of the of the Muc16 gene in the mouse. To generate Muc16 knockout mice, 6.0 kb was deleted that included the majority of exon 3 and a portion of intron 3 and replaced with a lacZ reporter cassette. Loss of Muc16 protein expression suggests that Muc16 homozygous mutant mice are null mutants. Muc16 homozygous mutant mice are viable, fertile, and develop normally. Histological analysis shows that Muc16 homozygous mutant tissues are normal. By the age of 1 year, Muc16 homozygous mutant mice appear normal. Downregulation of transcripts from another mucin gene (Muc1) was detected in the Muc16 homozygous mutant uterus. Lack of any prominent abnormal phenotype in these Muc16 knockout mice suggests that CA125/MUC16 is not required for normal development or reproduction. These knockout mice provide a unique platform for future studies to identify the role of CA125/MUC16 in organ homeostasis and ovarian cancer.

Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

CD8+ Pagetoid Reticulosis Presenting as a Solitary Foot Plaque in a Young Woman.

Background: Pagetoid reticulosis is a rare variant of mycosis fungoides. This rare condition typically presents as a solitary plaque located on the extremities with an indolent clinical course (Woringer-Kolopp disease) or as a more generalized presentation with diffuse cutaneous involvement and a more aggressive clinical course (Ketron-Goodman disease). Purpose: To review the cutaneous manifestations, pathology, and treatment of localized pagetoid reticulosis. Methods: The authors describe a 24-year-old woman with a slowly enlarging, localized plaque of seven months duration, representing the localized form of pagetoid reticulosis with CD8+ immunophenotype. Results: The histological, immunohistochemical, and clinical features of the patient's skin lesion were characteristic for a diagnosis of Woringer-Kolopp disease. Systemic work-up for lymphoma was negative. Conclusion: Woringer-Kolopp disease is most commonly seen in middle-aged men as a solitary lesion of the extremities, and it should always be considered in the differential diagnosis when a patient presents with such a lesion. A histological analysis demonstrated atypical lymphocytes preferentially localized to the epidermis with a CD4+, CD8+, or CD4-/CD8- phenotype. The treatment of choice for a solitary lesion may be localized radiation therapy, but newer therapies, such as bexarotene, may warrant further investigation.

Annexin A5-Conjugated Polymeric Micelles For Dual SPECT and Optical Detection of Apoptosis

Apoptosis, a form of programmed cell death, is critical to homoeostasis, normal development, and physiology. Dysregulation of apoptosis can lead to the accumulation of unwanted cells, such as occurs in cancer, and the removal of needed cells or disorders of normal tissues, such as heart, neurodegenerative, and autoimmune diseases. Noninvasive detection of apoptosis may play an important role in the evaluation of disease states and response to therapeutic intervention for a variety of diseases. It is desirable to have an imaging method to accurately detect and monitor this process in patients. In this study, we developed annexin A5-conjugated polymeric micellar nanoparticles dual-labeled with a near-infrared fluorescence fluorophores (Cy7) and a radioisotope (111In), named as 111In-labeled annexin A5-CCPM. In vitro studies demonstrated that annexin A5-CCPM could strongly and specifically bind to apoptotic cells. In vivo studies showed that apoptotic tissues could be clearly visualized by both single photon emission computed tomography (SPECT) and fluorescence molecular tomography (FMT) after intravenous injection of 111In-labeled Annexin A5-CCPM in 6 different apoptosis models. In contrast, there was little signal in respective healthy tissues. All the biodistribution data confirmed imaging results. Moreover, histological analysis revealed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals co-localized with the region of apoptosis. In sum, 111In-labeled annexin A5-CCPM allowed visualization of apoptosis by both nuclear and optical imaging techniques. The complementary information acquired with multiple imaging techniques should be advantageous in improving diagnostics and management of patients.

Cardiovascular magnetization transfer ratio imaging compared with histology: A postmortem study

Cardiovascular magnetization transfer ratio (MTR) imaging by steady state free precession is a promising imaging method to assess microstructural changes within the myocardium. Hence, MTR imaging was correlated to histological analysis. Three postmortem cases were selected based on a suspicion of myocardial infarction. MTR and T2 -weighted (T2w ) imaging was performed, followed by autopsy and histological analysis. All tissue abnormalities, identified by autopsy or histology, were retrospectively selected on visually matched MTR and T2w images, and corresponding MTR values compared with normal appearing tissue. Regions of elevated MTR (up to approximately 20%, as compared to normal tissue), appearing hypo-intense in T2w -images, revealed the presence of fibrous tissue in microscopic histological analysis. Macroscopic observation (autopsy) described scar tissue only in one case. Regions of reduced MTR (up to approximately 20%) corresponded either to (i) the presence of edema, appearing hyperintense in T2w -images and confirmed by autopsy, or to (ii) inflammatory granulocyte infiltration at a microscopic level, appearing as hypo-intense T2w -signal, but not observed by autopsy. Findings from cardiovascular MTR imaging corresponded to histology results. In contrast to T2w -imaging, MTR imaging discriminated between normal myocardium, scar tissue and regions of acute myocardial infarction in all three cases. J. Magn. Reson. Imaging 2013. © 2013 Wiley Periodicals, Inc.

Effects of enamel matrix proteins in combination with a bovine-derived natural bone mineral for the repair of bone defects.

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

Oil-soluble vitamins: illegal use for lip augmentation.

Fillers for lip augmentation have become more and more popular in recent years and seem to be indispensable in the cosmetic market nowadays. A series of six young females is presented who developed massive swellings and pain after vitamins A and/or E lip augmentation. The vitamins were extracted from gelatinous capsules (Gericaps [Adipharm EAD, Sofia, Bulgaria], Geritamins [Actavis EAD, Balkanpharma-Dubnitsa AD, Bulgaria], or vitamin E yellow gel capsules) and injected by unprofessional physicians and beauticians in different cosmetic centers. Physical examination revealed firm indurations of the lips and perioral skin, tenderness, erythema, and hard dermal nodules. Histological analysis revealed numerous round-to-ovoid cavities of varying sizes, resulting in a Swiss cheese-like appearance, consistent with lipogranulomas. The patients were treated with systemic and intralesional triamcinolone injections and broad-spectrum antibiotics with good clinical response. In conclusion, these cases demonstrate the danger of the use of unregistered products as fillers injected by unprofessional physicians and beauticians.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand on NK Cells Protects From Hepatic Ischemia-Reperfusion Injury

BACKGROUND: Ischemia-reperfusion injury (IRI) significantly contributes to graft dysfunction after liver transplantation. Natural killer (NK) cells are crucial innate effector cells in the liver and express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent inducer of hepatocyte cell death. Here, we investigated if TRAIL expression on NK cells contributes to hepatic IRI. METHODS: The outcome after partial hepatic IRI was assessed in TRAIL-null mice and contrasted to C57BL/6J wild-type mice and after NK cell adoptive transfer in RAG2/common gamma-null mice that lack T, B, and NK cells. Liver IRI was assessed by histological analysis, alanine aminotransferase, hepatic neutrophil activation by myeloperoxidase activity, and cytokine secretion at specific time points. NK cell cytotoxicity and differentiation were assessed in vivo and in vitro. RESULTS: Twenty-four hours after reperfusion, TRAIL-null mice exhibited significantly higher serum transaminases, histological signs of necrosis, neutrophil infiltration, and serum levels of interleukin-6 compared to wild-type animals. Adoptive transfer of TRAIL-null NK cells into immunodeficient RAG2/common gamma-null mice was associated with significantly elevated liver damage compared to transfer of wild-type NK cells. In TRAIL-null mice, NK cells exhibit higher cytotoxicity and decreased differentiation compared to wild-type mice. In vitro, cytotoxicity against YAC-1 and secretion of interferon gamma by TRAIL-null NK cells were significantly increased compared to wild-type controls. CONCLUSIONS: These experiments reveal that expression of TRAIL on NK cells is protective in a murine model of hepatic IRI through modulation of NK cell cytotoxicity and NK cell differentiation.

Healing of localized gingival recessions treated with coronally advanced flap alone or combined with either a resorbable collagen matrix or subepithelial connective tissue graft: A preclinical study.

OBJECTIVES To histologically evaluate the effectiveness of a porcine derived collagen matrix (CM) and a subepithelial connective tissue graft (CTG) for coverage of localized gingival recessions. MATERIALS AND METHODS Chronic single Miller Class I-like recessions were created at the buccal at the canines and at the third and fourth premolars in the upper and lower jaws of six beagle dogs. The defects were randomly treated with (1) coronally advanced flap surgery (CAF) + CM, (2) CAF + CTG, or (3) CAF alone. At 12 weeks, histometric measurements were made, e.g., between a reference point (N) - and the gingival margin (GM) - and the outer contour of the adjacent soft tissue (gingival thickness [GT]). RESULTS The postoperative healing was uneventful in all animals. No complications such as allergic reactions, abscesses or infections were noted throughout the entire study period. All three treatments resulted in coverage of localized gingival recessions. The histological analysis failed to identify any residues of CM or CTG. The histometric measurements revealed comparable outcomes for N-GM and GT values for all three groups (CAF + CM: 1.04 ± 0.69 mm/0.68 ± 0.33 mm; CAF + CTG: 1.15 ± 1.12 mm/0.76 ± 0.37 mm; CAF: 1.43 ± 0.45 mm/0.79 ± 0.24 mm). CONCLUSIONS In the used defect model, the application of CTG or CM in conjunction with CAF did not have an advantage over the use of CAF alone. CLINICAL RELEVANCE The use of CAF alone is a valuable option for the treatment localized Miller Class I recessions.

Conditional expression of Sry, and Wnt4 by gene-targeting: Studies in sexual and skeletal development

Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) locus by gene targeting in embryonic stem (ES) cells to create a conditional gene expression system in mice. In the targeted alleles, expression of these cDNAs should be blocked by a neomycin resistance selection cassette that is flanked by loxP sites. Transgene expression should be activated after the blocking cassette is deleted by Cre recombinase. ^ To test this conditional expression system, I have bred R26-stop- Sry and R26-stop-Wnt4 heterozygotes with a MisRII-Cre mouse line that expresses Cre in the gonads of both sexes. Analysis of these two types of bigenic heterozygotes indicated that their gonads developed normally like those of wild types. However, one XX R26-Sry/R26-Sry; MisR2-Cre/+ showed epididymis-like structures resembling those of males. In contrast, only normal phenotypes were observed in XY R26-Wnt4/R26-Wnt4; MisR2-Cre /+ mice. To interpret these results, I have tested for Cre recombinase activity by Southern blot and transcription of the Sry and Wnt4 transgenes by RT-PCR. Results showed that bigenic mutants had insufficient activation of the transgenes in their gonads at E12.5 and E13.5. Therefore, the failure to observe mutant phenotypes may have resulted from low activity of MisR2-Cre recombination at the appropriate time. ^ Col2a1-Cre transgenic mice express Cre in differentiating chondrocytes. R26-Wnt4; Col2a1-Cre bigenic heterozygous mice were found to exhibit a dramatic alteration in growth presumably caused by Wnt4 overexpression during chondrogenesis. R26-Wnt4; Col2a1-Cre mice exhibited dwarfism beginning approximately 10 days after birth. In addition, they also had craniofacial abnormalities, and had delayed ossification of the lumbar vertebrate and pelvic bones. Histological analysis of the growth plates of R26-Wnt4; Col2a1-Cre mice revealed less structural organization and a delay in onset of the primary and secondary ossification centers. Molecular studies confirmed that overexpression of Wnt4 causes decreased proliferation and early maturation of chondrocytes. In addition, R26-Wnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF), suggesting that defects in vascularization may contribute to the dwarf phenotype. Finally, 9-month-old R26-Wnt4; Col2a1-Cre mice had significantly more fat cells in the marrow cavities of their metaphysis long bones, implying that long-term overexpression of Wnt4may cause bone marrow pathologies. In conclusion, Wnt4 was activated by Col2a1-Cre recombinase and was overexpressed in the growth plate, resulting in aberrant proliferation and differentiation of chondrocytes, and ultimately leads to dwarfism in mice. ^

Behavioral Implications of Knockout for the Dyslexia-Risk Gene Dcdc2 in Mice

Several genetic linkage and epidemiological studies have provided strong evidence that DCDC2 is a candidate gene for developmental dyslexia, a disorder that impairs a person’s reading ability despite adequate intelligence, education, and socio-economic status. Studies investigating embryonic intra-ventricular RNA interference (RNAi) of Dcdc2, a rat homolog of the DCDC2 gene in humans, indicate disruptions in neuronal migration in the rat cortex during development. Interestingly, these anatomical anomalies are consistent with post mortem histological analysis of human dyslexic patients. Other rodent models of cortical developmental disruption have shown impairment in rapid auditory processing and learning maze tasks in affected subjects. The current study investigates the rapid auditory processing abilities of mice heterozygous for Dcdc2 (one functioning Dcdc2 allele) and mice with a homozygous knockout of Dcdc2 (no functioning Dcdc2 allele). It is important to note that this genetic model for behavioral assessment is still in the pilot stage. However, preliminary results suggest that mice with a genetic mutation of Dcdc2 have impaired rapid auditory processing, as well as non-spatial maze learning and memory ability, as compared to wildtypes. By genetically knocking out Dcdc2 in mice, behavioral features associated with Dcdc2 can be characterized, along with other neurological abnormalities that may arise due to the loss of the functioning gene.

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

CARD9 functions as a key regulator in monocyte homeostasis and pathogen clearance

Mononuclear phagocytes are designed to neutralize systemic bacterial and fungal infections. However, the exact regulation of these functions are largely unknown. CARD9 was first identified as an immune-specific adaptor protein of unclear function. Here, we have found that Card9 is specifically expressed in monocyte-origin cell populations. To better understand the biological function of Card9, we have generated Card9-deficient (Card9-/-) mice. Hematologic profiling and histological analysis of Card9-/- mice revealed a decreased leukocyte/myeloid cell count, delayed monocyte maturation in bone marrow as well as monocyte counts in the peripheral blood. Upon M-CSF stimulation, Card9-/- macrophages further exhibit a partial loss in IKK phosphorylation. As a consequence, in vivo challenge with Listeria monocytogenes in Card9-/- mice results in a higher susceptibility to infection-associated inflammation and fatality. Collectively, these data suggest that CARD9 is required for monocyte development and function. ^ At the cellular level, Card9-/- macrophages are defective in killing Listeria and the production of pro-inflammatory cytokines. Molecular characterizations have further demonstrated that CARD9 inducibly interacts with NOD2, controls p38 MAPK activation, and regulates ROS production during Listeria infections. Cytotrap screening showed that CARD9 could physically associate with various g&barbelow;uanine e&barbelow;xchange f&barbelow;actor (GEF) proteins that are essential for regulating ROS production. In summary, we have first identified and provided genetic evidence that CARD9 functions as a novel regulator during monocyte development and serves as an essential protein adaptor for p38 MAPK activation during bacterial clearance processes in macrophages. ^