Deregulated ephrin-B2 expression in the mammary gland interferes with the development of both the glandular epithelium and vasculature and promotes metastasis formation

Eph receptor tyrosine kinases and their membrane-bound ephrin ligands play key roles during morphogenesis and adult tissue homeostasis. Receptor-ligand interactions result in forward and reverse signalling from the receptor and ligand respectively. To delineate the role(s) of forward and reverse signalling in mammary gland biology we have established transgenic mice exhibiting mammary epithelial-specific overexpression of either the native ephrin-B2 or a dominant negative ephrin-B2 mutant incapable of reverse signalling. During pregnancy and lactation overexpression of the native ephrin-B2 resulted in precocious differentiation, whereas overexpression of mutated ephrin-B2 caused delayed epithelial differentiation and in disturbed tissue architecture. Both transgenes affected also mammary vascularisation. Whereas ephrin-B2 induced superfluous but organised capillaries, mutant ephrin-B2 overexpression resulted in an irregular vasculature with blind-ending capillaries. Mammary tumours were not observed in either transgenic line, however, the crossing with NeuT transgenic animals revealed that mutated ephrin-B2 expression significantly accelerated tumour growth and imposed a metastatic phenotype.

A novel succinate dehydrogenase subunit B gene mutation, H132P, causes familial malignant sympathetic extraadrenal paragangliomas

We report a family with malignant sympathetic paragangliomas (PGL) exhibiting a new type of germline mutation in the succinate dehydrogenase subunit B (SDHB) gene. Two affected brothers, presenting with symptoms at the ages of 25 and 52 yr, suffered from malignant abdominal extraadrenal sympathetic PGL. They died of their disease at ages 43 and 61 yr. Their mother had the same history of signs and symptoms, suggesting a catecholamine-producing tumor at the age of 55 yr. Analysis of the germline DNA from these three patients revealed a novel mutation in exon 4 (H132P) of the SDHB gene. This mutation was absent in 160 control chromosomes. Loss of heterozygosity analysis of the tumors showed a loss of one SDHB allele, and RT-PCR-based expression analysis confirmed the exclusive expression of the mutated allele in both tumors. A review of the published PGL families revealed malignant tumors in seven of 12 well-documented families with SDHB mutation-associated extraadrenal PGL. These findings, as well as findings of the family reported here, suggest a strong causal relationship of SDHB germline mutations with malignant extraadrenal abdominal PGL and imply the necessity of a close follow-up of affected individuals and family members.

Conserved leucine residue in the head region of morbillivirus fusion protein regulates the large conformational change during fusion activity

Paramyxovirus cell entry is controlled by the concerted action of two viral envelope glycoproteins, the fusion (F) and the receptor-binding (H) proteins, which together with a cell surface receptor mediate plasma membrane fusion activity. The paramyxovirus F protein belongs to class I viral fusion proteins which typically contain two heptad repeat regions (HR). Particular to paramyxovirus F proteins is a long intervening sequence (IS) located between both HR domains. To investigate the role of the IS domain in regulating fusogenicity, we mutated in the canine distemper virus (CDV) F protein IS domain a highly conserved leucine residue (L372) previously reported to cause a hyperfusogenic phenotype. Beside one F mutant, which elicited significant defects in processing, transport competence, and fusogenicity, all remaining mutants were characterized by enhanced fusion activity despite normal or slightly impaired processing and cell surface targeting. Using anti-CDV-F monoclonal antibodies, modified conformational F states were detected in F mutants compared to the parental protein. Despite these structural differences, coimmunoprecipitation assays did not reveal any drastic modulation in F/H avidity of interaction. However, we found that F mutants had significantly enhanced fusogenicity at low temperature only, suggesting that they folded into conformations requiring less energy to activate fusion. Together, these data provide strong biochemical and functional evidence that the conserved leucine 372 at the base of the HRA coiled-coil of F(wt) controls the stabilization of the prefusogenic state, restraining the conformational switch and thereby preventing extensive cell-cell fusion activity.

Polyganglioradiculoneuritis in a young cat: clinical and histopathological findings

An 18-month-old European shorthair cat was presented with a two week history of progressive decrease in consciousness, ambulatory tetraparesis, moderate ataxia and generalised decreased-to-absent postural reactions. Bilateral facial and nasal hypalgesia, absent menace response and anisocoria were found, and segmental spinal reflexes were normal. Neurological signs progressed to nonambulatory tetraparesis, tremor and spinal hyperalgesia. Histopathological examination revealed a mild-to-moderate lymphoplasmacytic and histiocytic infiltration, predominantly in the dorsal spinal roots, cranial nerves and ganglia in association with marked demyelination and proliferation of Schwann cells. Neurons and axons were preserved. Lesions were multi-focal and varied in severity. A predominantly sensory polyganglioradiculoneuritis was diagnosed. This lesion has not been reported previously in cats. Rabies, herpesviruses, feline infectious peritonitis, feline immunodeficiency virus, Toxoplasma gondii and feline leukaemia virus were excluded as possible aetiologies. Infections by other viruses or an autoimmune disease are discussed.

Foxo-mediated Bim transcription is dispensable for the apoptosis of hematopoietic cells that is mediated by this BH3-only protein

The BH3-only protein Bim is a critical initiator of apoptosis in hematopoietic cells. Bim is upregulated in response to growth factor withdrawal and in vitro studies have implicated the transcription factor Foxo3a as a critical inducer. To test the importance of this regulation in vivo, we generated mice with mutated Foxo-binding sites within the Bim promoters (Bim(ΔFoxo/ΔFoxo)). Contrary to Bim-deficient mice, Bim(ΔFoxo/ΔFoxo) mice had a normal hematopoietic system. Moreover, cytokine-dependent haematopoietic cells from Bim(ΔFoxo/ΔFoxo) and wt mice died at similar rates. These results indicate that regulation of Bim by Foxo transcription factors is not critical for the killing of hematopoietic cells.

Loss-of-function mutation of the SCN3B-encoded sodium channel {beta}3 subunit associated with a case of idiopathic ventricular fibrillation.

AIMS Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. METHODS AND RESULTS Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. CONCLUSION This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.

A novel C-terminal truncation SCN5A mutation from a patient with sick sinus syndrome, conduction disorder and ventricular tachycardia.

OBJECTIVES Individual mutations in the SCN5A-encoding cardiac sodium channel alpha-subunit cause single cardiac arrhythmia disorders, but a few cause multiple distinct disorders. Here we report a family harboring an SCN5A mutation (L1821fs/10) causing a truncation of the C-terminus with a marked and complex biophysical phenotype and a corresponding variable and complex clinical phenotype with variable penetrance. METHODS AND RESULTS A 12-year-old male with congenital sick sinus syndrome (SSS), cardiac conduction disorder (CCD), and recurrent monomorphic ventricular tachycardia (VT) had mutational analysis that identified a 4 base pair deletion (TCTG) at position 5464-5467 in exon 28 of SCN5A. The mutation was also present in six asymptomatic family members only two of which showed mild ECG phenotypes. The deletion caused a frame-shift mutation (L1821fs/10) with truncation of the C-terminus after 10 missense amino acid substitutions. When expressed in HEK-293 cells for patch-clamp study, the current density of L1821fs/10 was reduced by 90% compared with WT. In addition, gating kinetic analysis showed a 5-mV positive shift in activation, a 12-mV negative shift of inactivation and enhanced intermediate inactivation, all of which would tend to reduce peak and early sodium current. Late sodium current, however, was increased in the mutated channels. CONCLUSIONS The L1821fs/10 mutation causes the most severe disruption of SCN5A structure for a naturally occurring mutation that still produces current. It has a marked loss-of-function and unique phenotype of SSS, CCD and VT with incomplete penetrance.

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

Magnetic resonance imaging and genetic investigation of a case of Rottweiler leukoencephalomyelopathy.

BACKGROUND Leukoencephalomyelopathy is an inherited neurodegenerative disorder that affects the white matter of the spinal cord and brain and is known to occur in the Rottweiler breed. Due to the lack of a genetic test for this disorder, post mortem neuropathological examinations are required to confirm the diagnosis. Leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate levels is a rare, autosomal recessive disorder in humans that was recently described to have clinical features and magnetic resonance imaging (MRI) findings that are similar to the histopathologic lesions that define leukoencephalomyelopathy in Rottweilers. Leukoencephalopathy with brain stem and spinal cord involvement is caused by mutations in the DARS2 gene, which encodes a mitochondrial aspartyl-tRNA synthetase. The objective of this case report is to present the results of MRI and candidate gene analysis of a case of Rottweiler leukoencephalomyelopathy to investigate the hypothesis that leukoencephalomyelopathy in Rottweilers could serve as an animal model of human leukoencephalopathy with brain stem and spinal cord involvement. CASE PRESENTATION A two-and-a-half-year-old male purebred Rottweiler was evaluated for generalised progressive ataxia with hypermetria that was most evident in the thoracic limbs. MRI (T2-weighted) demonstrated well-circumscribed hyperintense signals within both lateral funiculi that extended from the level of the first to the sixth cervical vertebral body. A neurodegenerative disorder was suspected based on the progressive clinical course and MRI findings, and Rottweiler leukoencephalomyelopathy was subsequently confirmed via histopathology. The DARS2 gene was investigated as a causative candidate, but a sequence analysis failed to identify any disease-associated variants in the DNA sequence. CONCLUSION It was concluded that MRI may aid in the pre-mortem diagnosis of suspected cases of leukoencephalomyelopathy. Genes other than DARS2 may be involved in Rottweiler leukoencephalomyelopathy and may also be relevant in human leukoencephalopathy with brain stem and spinal cord involvement.

Intraductal papillary neoplasms of the bile duct: stepwise progression to carcinoma involves common molecular pathways

Intraductal papillary neoplasms of the bile duct are still poorly characterized regarding (1) their molecular alterations during the development to invasive carcinomas, (2) their subtype stratification and (3) their biological behavior. We performed a multicenter study that analyzed these issues in a large European cohort. Intraductal papillary neoplasms of the bile duct from 45 patients were graded and subtyped using mucin markers and CDX2. In addition, tumors were analyzed for common oncogenic pathways, and the findings were correlated with subtype and grade. Data were compared with those from 22 extra- and intrahepatic cholangiocarcinomas. Intraductal papillary neoplasms showed a development from preinvasive low- to high-grade intraepithelial neoplasia to invasive carcinoma. Molecular and immunohistochemical analysis revealed mutated KRAS, overexpression of TP53 and loss of p16 in low-grade intraepithelial neoplasia, whereas loss of SMAD4 was found in late phases of tumor development. Alterations of HER2, EGFR, β-catenin and GNAS were rare events. Among the subtypes, pancreato-biliary (36%) and intestinal (29%) were the most common, followed by gastric (18%) and oncocytic (13%) subtypes. Patients with intraductal papillary neoplasm of the bile duct showed a slightly better overall survival than patients with cholangiocarcinoma (hazard ratio (cholangiocarcinoma versus intraductal papillary neoplasm of the bile duct): 1.40; 95% confidence interval: 0.46-4.30; P=0.552). The development of biliary intraductal papillary neoplasms of the bile duct follows an adenoma-carcinoma sequence that correlates with the stepwise activation of common oncogenic pathways. Further large trials are needed to investigate and verify the finding of a better prognosis of intraductal papillary neoplasms compared with conventional cholangiocarcinoma.

Myoepithelioma of the cerebellopontine angle: a previously not documented benign salivary gland-type neoplasm within the cranium

Myoepithelioma is a dimorphic neoplasm with contractile-epithelial phenotype, originally interpreted as deriving from, but not actually restricted to the salivary glands. As a novel addition to the list of exquisitely rare intracranial salivary gland-type tumors and tumor-like lesions, we report on an example of myoepithelioma encountered in the left cerebellopontine angle of a 32-year-old male. Clinically presenting with ataxia and dizziness, this extraaxial mass of 4 × 3.5 × 3 cm was surgically resected, and the patient is alive 6 years postoperatively. Histologically, the tumor exhibited a continuum ranging from compact fascicles of spindle cells to epithelial nests and trabeculae partitioned by hyalinized septa, while lacking tubular differentiation. Regardless of architectural variations, there was robust immunoexpression of S100 protein, smooth muscle actin, GFAP, cytokeratin, and vimentin. Cytologic atypia tended to be modest throughout, and the MIB1 labeling index averaged less than 1%. Fluorescent in situ hybridization indicated no rearrangement of the EWSR1 locus. We interpret these results to suggest that myoepithelioma of the posterior fossa - along with related salivary epithelial tumors in this ostensibly incongruous locale - may possibly represent analogous neoplasms to their orthotopic counterparts, ones arising within aberrant salivary anlagen. The presence of the latter lends itself to being mechanistically accounted for by either postulating placodal remnants in the wake of branchial arch development, or linking them to exocrine glandular nests within endodermal cysts. Alternatively, myoepithelioma at this site could be regarded as a non tissue-specific lesion similar to its relatives ubiquitously occurring in the soft parts.

The Coin Rotation Task: A Valid Test for Manual Dexterity in Multiple Sclerosis

BACKGROUND: Impaired manual dexterity is frequent and disabling in patients with multiple sclerosis (MS). Therefore, convenient, quick and validated tests for manual dexterity in MS patients are needed. OBJECTIVE: The aim of this study was to validate the Coin Rotation task (CRT) to examine manual dexterity in patients with MS. DESIGN: Cross-sectional study. METHODS: 101 outpatients with MS were assessed with the CRT, the Expanded Disability Status Scale (EDSS), the Scale for the assessment and rating of ataxia (SARA), the Modified Ashworth Scale (MAS), and their muscle strength and sensory deficits of the hands were noted. Concurrent validity and diagnostic accuracy of the CRT were determined by comparison with the Nine Hole Peg Test (9HPT). Construct validity was determined by comparison with a valid dexterity questionnaire. Multiple regression analysis was done to explore correlations of the CRT with the EDSS, SARA, MAS, muscle strength and sensory deficits. RESULTS: The CRT correlated significantly with the 9HPT (r=.73, p<.0001) indicating good concurrent validity. The cut-off values for the CRT relative to the 9HPT were 18.75 seconds for the dominant (sensitivity: 81.5%; specificity 80.0%) and 19.25 seconds for the non-dominant hand (sensitivity: 90.3%; specificity: 81.8%) demonstrating good diagnostic accuracy. Furthermore, the CRT correlated significantly with the dexterity questionnaire (r=-.49, p<.0001) indicating moderate construct validity. Multiple regression analyses revealed that the EDSS was the strongest predictor for impaired dexterity. LIMITATIONS: Mostly relapsing-remitting MS patients with an EDSS up to 7 were examined. CONCLUSIONS: This study validates the CRT as a test that can be used easily and quickly to evaluate manual dexterity in patients with MS.

Intracranial meningiomas associated with cervical syringohydromyelia in a cat

Eine dreizehnjährige, weibliche, nicht kastrierte Hauskatze wurde zur chirurgischen Therapie eines multiplen Meningeoms in der Kleintierklinik des Tierspital Bern vorgestellt. Eine leichtgradige generalisierte Ataxie wurde beobachtet, die Propriozeption war generalisiert herabgesetzt und der Drohreflex war beidseitig reduziert. Es wurden eine prä- und eine postoperative Magnetrezonanztomographie durchgeführt. Drei supratentoriale extra-axiale Raumforderungen wurden diagnostiziert. Eine vierte Masse wurde infratentorial extra-axial über der linken zerebellären Hemisphere lokalisiert. Eine Herniation des kaudoventralen Kleinhirn (Vermis) von ungefähr einem Zentimeter Länge durch das Foramen magnum wurde beobachtet. Eine zervikale Syringohydromyelie wurde als Zufallsbefund diagnostiziert. Die Meningeome wurden durch 3 Kraniotomiestellen entfernt. Zwei Jahre nach der Operation ist die Katze normal. Anhand der vorhandenen Literatur wird die tumor-assoziierte Syringohydromyelie besprochen. Die Therapie der Syringohydromyelie sollte gegen den kausalen pathologischen Prozess (z.B. intrakranieller Tumor) für die Liquorzirkulationsstörung gerichtet sein.

Viral escape in the CNS with multidrug-resistant HIV-1.

Introduction: HIV-1 viral escape in the cerebrospinal fluid (CSF) despite viral suppression in plasma is rare [1,2]. We describe the case of a 50-year-old HIV-1 infected patient who was diagnosed with HIV-1 in 1995. Antiretroviral therapy (ART) was started in 1998 with a CD4 T cell count of 71 cells/ìL and HIV-viremia of 46,000 copies/mL. ART with zidovudine (AZT), lamivudine (3TC) and efavirenz achieved full viral suppression. After the patient had interrupted ART for two years, treatment was re-introduced with tenofovir (TDF), emtricitabin (FTC) and ritonavir boosted atazanavir (ATVr). This regimen suppressed HIV-1 in plasma for nine years and CD4 cells stabilized around 600 cells/ìL. Since July 2013, the patient complained about severe gait ataxia and decreased concentration. Materials and Methods: Additionally to a neurological examination, two lumbar punctures, a cerebral MRI and a neuropsycological test were performed. HIV-1 viral load in plasma and in CSF was quantified using Cobas TaqMan HIV-1 version 2.0 (Cobas Ampliprep, Roche diagnostic, Basel, Switzerland) with a detection limit of 20 copies/mL. Drug resistance mutations in HIV-1 reverse transcriptase and protease were evaluated using bulk sequencing. Results: The CSF in January 2014 showed a pleocytosis with 75 cells/ìL (100% mononuclear) and 1,184 HIV-1 RNA copies/mL, while HIV-1 in plasma was below 20 copies/mL. The resistance testing of the CSF-HIV-1 RNA showed two NRTI resistance-associated mutations (M184V and K65R) and one NNRTI resistance-associated mutation (K103N). The cerebral MRI showed increased signal on T2-weighted images in the subcortical and periventricular white matter, in the basal ganglia and thalamus. Four months after ART intensification with AZT, 3TC, boosted darunavir and raltegravir, the pleocytosis in CSF cell count normalized to 1 cell/ìL and HIV viral load was suppressed. The neurological symptoms improved; however, equilibrium disturbances and impaired memory persisted. The neuro-psychological evaluation confirmed neurocognitive impairments in executive functions, attention, working and nonverbal memory, speed of information processing, visuospatial abilities and motor skills. Conclusions: HIV-1 infected patients with neurological complaints prompt further investigations of the CSF including measurement of HIV viral load and genotypic resistance testing since isolated replication of HIV with drug resistant variants can rarely occur despite viral suppression in plasma. Optimizing ART by using drugs with improved CNS penetration may achieve viral suppression in CSF with improvement of neurological symptoms.

A pilot study for targeted surveillance of bovine spongiform encephalopathy in Nigeria.

Bovine spongiform encephalopathy (BSE), popularly known as 'mad cow disease', led to an epidemic in Europe that peaked in the mid-1990s. Its impact on developing countries, such as Nigeria, has not been fully established as information on livestock and surveillance has eluded those in charge of this task. The BSE risk to Nigeria's cattle population currently remains undetermined, which has resulted in international trade restrictions on commodities from the cattle population. This is mainly because of a lack of updated BSE risk assessments and disease surveillance data. To evaluate the feasibility of BSE surveillance in Nigeria, we carried out a pilot study targeting cattle that were presented for emergency or casualty slaughter. In total, 1551 cattle of local breeds, aged 24 months and above were clinically examined. Ataxia, recumbency and other neurological signs were topmost on our list of criteria. A total of 96 cattle, which correspond to 6.2%, presented clinical signs that supported a suspect of BSE. The caudal brainstem tissues of these animals were collected post-mortem and analysed for the disease-specific form of the prion protein using a rapid test approved by the International Animal Health Organization (OIE). None of the samples were positive for BSE. Although our findings do not exclude the presence of BSE in Nigeria, they do demonstrate that targeted sampling of clinically suspected cases of BSE is feasible in developing countries. In addition, these findings point to the possibility of implementing clinical monitoring schemes for BSE and potentially other diseases with grave economic and public health consequences.