Magnetic resonance imaging features of canine gliomatosis cerebri.

A retrospective, case series study was undertaken to identify magnetic resonance imaging (MRI) characteristics of gliomatosis cerebri in dogs. Fourteen dogs were included by review of histopathological records and contemporaneous MRI. On MRI, all lesions presented as ill-defined, intraaxial lesions within the left and right forebrain hemispheres with involvement of white and gray matter. Lesions presented as hyperintense areas on T2-weighted and FLAIR sequences and as hypointense or isointense areas on T1-weighted images, with mild parenchymal contrast enhancement in three dogs. Signal changes were noted in three to 10 cerebral lobes. Other most commonly affected structures were the thalamus (13), caudate nucleus (13), interthalamic adhesion (11), hypothalamus (11), callosal commissure (10), hippocampus (9), and quadrigeminal plate (8). Abnormalities within the caudal fossa were noted in 10 dogs. Solid tumor portions were identified in five dogs. The histopathological examination demonstrated in all dogs a widespread diffuse infiltration with neoplastic glial cells in white and gray matter with meningeal infiltration. Comparison between MRI and histopathology showed that all areas with signal changes on MRI corresponded to diffuse and dense infiltration with neoplastic cells. The signal intensity on T2-weighted and FLAIR images reflected the density of neoplastic cells. In all dogs, MRI underestimated lesion extent and meningeal infiltration. Involvement of the caudal fossa was not seen on MRI in three dogs. Despite this, MRI allowed identification of lesions extending into at least three cerebral lobes and therefore satisfying the criteria used for diagnosis of diffuse glioma with gliomatosis cerebri growth pattern in humans.

Longitudinal extension of myelomalacia by intramedullary and subdural hemorrhage in a canine model of spinal cord injury.

BACKGROUND CONTEXT: In canine intervertebral disc (IVD) extrusion, a spontaneous animal model of spinal cord injury, hemorrhage is a consistent finding. In rodent models, hemorrhage might be involved in secondary tissue destruction by biochemical mechanisms. PURPOSE: This study aimed to investigate a causal association between the extents of intramedullary, subdural and epidural hemorrhage and the severity of spinal cord damage following IVD extrusion in dogs. STUDY DESIGN/SETTING: A retrospective study using histologic spinal cord sections from 83 dogs euthanized following IVD extrusion was carried out. METHODS: The degree of hemorrhage (intramedullary, subdural, epidural), the degree of spinal cord damage in the epicenter (white and gray matter), and the longitudinal extent of myelomalacia were graded. Associations between the extent of hemorrhage and the degree of spinal cord damage were evaluated statistically. RESULTS: Intramedullary and subdural hemorrhages were significantly associated with the degree of white (p<.001/ p=.004) and gray (both p<.001) matter damage, and with the longitudinal extension of myelomalacia (p<.001/p=.005). Intriguingly, accumulation of hemorrhagic cord debris inside or dorsal to a distended and ruptured central canal in segments distant to the epicenter of the lesion was observed exhibiting a wave-like pattern on longitudinal assessment. The occurrence of this debris accumulation was associated with high degrees of tissue destruction (all p<.001). CONCLUSIONS: Tissue liquefaction and increased intramedullary pressure associated with hemorrhage are involved in the progression of spinal cord destruction in a canine model of spinal cord injury and ascending or descending myelomalacia. Functional and dynamic studies are needed to investigate this concept further.

Dominance of chemokine ligand 2 and matrix metalloproteinase-2 and -9 and suppression of pro-inflammatory cytokines in the epidural compartment after intervertebral disc extrusion in a canine model.

BACKGROUND CONTEXT: In canine intervertebral disc (IVD) disease, a useful animal model, only little is known about the inflammatory response in the epidural space. PURPOSE: To determine messenger RNA (mRNA) expressions of selected cytokines, chemokines, and matrix metalloproteinases (MMPs) qualitatively and semiquantitatively over the course of the disease and to correlate results to neurologic status and outcome. STUDY DESIGN/SETTING: Prospective study using extruded IVD material of dogs with thoracolumbar IVD extrusion. PATIENT SAMPLE: Seventy affected and 13 control (24 samples) dogs. OUTCOME MEASURES: Duration of neurologic signs, pretreatment, neurologic grade, severity of pain, and outcome were recorded. After diagnostic imaging, decompressive surgery was performed. METHODS: Messenger RNA expressions of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF), interferon (IFN)γ, MMP-2, MMP-9, chemokine ligand (CCL)2, CCL3, and three housekeeping genes was determined in the collected epidural material by Panomics 2.0 QuantiGene Plex technology. Relative mRNA expression and fold changes were calculated. Relative mRNA expression was correlated statistically to clinical parameters. RESULTS: Fold changes of TNF, IL-1ß, IL-2, IL-4, IL-6, IL-10, IFNγ, and CCL3 were clearly downregulated in all stages of the disease. MMP-9 was downregulated in the acute stage and upregulated in the subacute and chronic phase. Interleukin-8 was upregulated in acute cases. MMP-2 showed mild and CCL2 strong upregulation over the whole course of the disease. In dogs with severe pain, CCL3 and IFNγ were significantly higher compared with dogs without pain (p=.017/.020). Dogs pretreated with nonsteroidal anti-inflammatory drugs revealed significantly lower mRNA expression of IL-8 (p=.017). CONCLUSIONS: The high CCL2 levels and upregulated MMPs combined with downregulated T-cell cytokines and suppressed pro-inflammatory genes in extruded canine disc material indicate that the epidural reaction is dominated by infiltrating monocytes differentiating into macrophages with tissue remodeling functions. These results will help to understand the pathogenic processes representing the basis for novel therapeutic approaches. The canine IVD disease model will be rewarding in this process.

Mechanism for active membrane fusion triggering by morbillivirus attachment protein.

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

Structural rearrangements of the central region of the morbillivirus attachment protein stalk domain trigger F protein refolding for membrane fusion.

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

Investigation of a unique short open reading frame within the 3' untranslated region of the canine distemper virus matrix messenger RNA.

Increasing evidence suggest that the long "untranslated" region (UTR) between the matrix (M) and the fusion (F) proteins of morbilliviruses has a functional role. In canine distemper virus (CDV), the F 5' UTR was recently shown to code for a long F signal peptide (Fsp). Subsequently, it was reported that the M/F UTRs combined with the long Fsp were synergistically regulating the F mRNA and protein expression, thereby modulating virulence. Unique to CDV, a short putative open reading frame (ORF) has been identified within the wild-type CDV-M 3' UTR (termed M2). Here, we investigated whether M2 was expressed from the genome of the virulent and demyelinating A75/17-CDV strain. An expression plasmid encoding the M2 ORF tagged both at its N-terminal (HA) and C-terminal domains (RFP), was first constructed. Then, a recombinant virus with its putative M2 ORF replaced by HA-M2-RFP was successfully recovered from cDNA (termed recA75/17(green)-HA-M2-RFP). M2 expression in cells transfected or infected with these mutants was studied by immunoprecipitation, immunofluorescence, immunoblot and flow cytometry analyses. Although fluorescence was readily detected in HA-M2-RFP-transfected cells, absence of red fluorescence emission in several recA75/17(green)-HA-M2-RFP-infected cell types suggested lack of M2 biosynthesis, which was confirmed by the other techniques. Consistent with these data, no functional role of the short polypeptide was revealed by infecting various cell types with HA-M2-RFP over-expressing or M2-knockout recombinant viruses. Thus, in sharp contrast to the CDV-F 5' UTR reported to translate a long Fsp, our data provided evidence that the CDV-M 3' UTR does not express any polypeptides.

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.

Immunophenotypical characterization of monocytes in canine distemper virus infection.

Canine distemper virus (CDV) infection induces multifocal demyelination in the central nervous system (CNS). It is thought that the resident macrophages of the CNS, the microglia, as well as invading monocytes associated with the inflammatory reaction may play a central role in the demyelinating process. To evaluate changes in peripheral monocytes in CDV infection their immunophenotype was characterized by flow cytometry during the course of an experimental CDV infection in dogs. The highest number of CDV-infected monocytes was found in dogs developing demyelinating lesions. In CD18, CD45, CD44, and CD14 neither up- nor down-regulation was observed. Marked up-regulation occurred in a number of surface molecules including CD1c, B7-1 and B7-2, MHC I, and CD11b. Peak expression was found at 4-5 weeks post-infection (PI), regardless of clinical outcome. All these molecules play an important role in the host's immune response, notably antigen presentation and cell adhesion. These results demonstrate that CDV infection in vivo may enhance several macrophage functions. This could lead to more effective clearance of the virus but may also increase demyelination through a bystander effect in animals that accumulated significant amounts of CDV in the CNS.

Retrospective analysis of seizures associated with feline infectious peritonitis in cats.

Seizures have been reported frequently in feline infectious peritonitis (FIP) but have not been studied in detail in association with this disease. The purpose of this study was to perform a retrospective analysis of neurological signs in a population of 55 cats with a histopathologically confirmed neurological form of FIP. Seizure patterns were determined and it was attempted to relate occurrence of seizures with age, breed, sex and neuropathological features. Fourteen cats had seizure(s), while 41 cats had no history of seizure(s). Generalised tonic-clonic seizures were seen in nine cats; and complex focal seizures were observed in four patients. The exact type of seizure could not be determined in one cat. Status epilepticus was observed in one patient but seizure clusters were not encountered. Occurrence of seizures was not related to age, sex, breed or intensity of the inflammation in the central nervous system. However, seizures were significantly more frequent in animals with marked extension of the inflammatory lesions to the forebrain (P=0.038). Thus, the occurrence of seizures in FIP indicates extensive brain damage and can, therefore, be considered to be an unfavourable prognostic sign.

Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus.

Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity.

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Multiple meningiomas: clinical, radiological, surgical, and pathological findings with outcome in four cats.

The present report describes the clinical signs, magnetic resonance imaging (MRI) findings, surgical procedure, pathological findings and follow-up in four cats with multiple meningiomas; three castrated male and one spayed female domestic shorthair indoor cats, ranging in age from 11 to 14 years. In three of four cats, clinical signs at presentation were suggestive of a focal lesion. Three cats had two meningiomas and one had four meningiomas. Most of the tumours were supratentorial, one arose from the tentorium and one was infratentorial. The duration of presenting signs before surgery ranged from 10 days to 11 months. Postoperative MRI revealed complete gross tumour removal in three cases. In one cat with two cranial fossa meningiomas, subtotal excision with a small basal remnant (2 x 2 mm) of the ventral part of one meningioma lying on the floor of the skull, was observed. Based on histopathological architecture, six tumours revealed features of a transitional subtype meningioma, and four of a meningotheliomatous meningioma. In each cat, the multiple meningiomas were all assigned to the same histopathological group. The preoperative presenting signs had resolved by the follow-up examinations 4 weeks after surgery in two cats. Long-term follow-up evaluation revealed that surgically-induced or exacerbated neurological deficits in two cats had completely or almost completely resolved within 8 weeks of surgery. All patients are still alive 12 to 21 months after surgery and no clinical signs of recurrence could be detected at that time.

Prenatal intervention for isolated congenital diaphragmatic hernia.

PURPOSE OF REVIEW: We aim to review the recent literature regarding early prenatal prediction of outcome in babies diagnosed with isolated congenital diaphragmatic hernia, as well as results of fetal therapy for this condition. RECENT FINDINGS: Current survival rates in population-based studies are around 55-70%. Highly specialized centers report 80% and more, but discount the hidden mortality, mainly in the antenatal period. Fetuses presenting with liver herniation and a lung-to-head ratio of less than 1.0 measured in midgestation have a poor prognosis. Other volumetric techniques are being evaluated for use in midtrimester. Recently, a randomized trial failed to show benefit from prenatal therapy, but lacked power to document the potential advantage of prenatal therapy in severe cases. We proposed percutaneous fetal endoluminal tracheal occlusion with a balloon at 26-28 weeks through a 3.3 mm incision. In severe cases, fetal endoluminal tracheal occlusion increased lung size as well as survival, with an early (7 day) survival, late neonatal (28 day) survival and survival at discharge of 75, 58 and 50%, respectively, comparing favorably with 9% in contemporary controls. Airways can be restored prior to birth improving neonatal survival (83.3% compared with 33.3%). The procedure carries a risk for preterm prelabour rupture of the fetal membranes, although that may decrease with experience. SUMMARY: Fetuses with severe congenital diaphragmatic hernia can be identified in the second trimester. Fetal endoluminal tracheal occlusion can be considered as a minimally invasive fetal therapy, improving outcome in such highly selected cases.

Prenatal intervention for isolated congenital diaphragmatic hernia.

PURPOSE OF REVIEW: We aim to review the recent literature regarding early prenatal prediction of outcome in babies diagnosed with isolated congenital diaphragmatic hernia, as well as results of fetal therapy for this condition. RECENT FINDINGS: Current survival rates in population-based studies are around 55-70%. Highly specialized centers report 80% and more, but discount the hidden mortality, mainly in the antenatal period. Fetuses presenting with liver herniation and a lung-to-head ratio of less than 1.0 measured in midgestation have a poor prognosis. Other volumetric techniques are being evaluated for use in midtrimester. Recently, a randomized trial failed to show benefit from prenatal therapy, but lacked power to document the potential advantage of prenatal therapy in severe cases. We proposed percutaneous fetal endoluminal tracheal occlusion with a balloon at 26-28 weeks through a 3.3 mm incision. In severe cases, fetal endoluminal tracheal occlusion increased lung size as well as survival, with an early (7 day) survival, late neonatal (28 day) survival and survival at discharge of 75, 58 and 50%, respectively, comparing favorably with 9% in contemporary controls. Airways can be restored prior to birth improving neonatal survival (83.3% compared with 33.3%). The procedure carries a risk for preterm prelabour rupture of the fetal membranes, although that may decrease with experience. SUMMARY: Fetuses with severe congenital diaphragmatic hernia can be identified in the second trimester. Fetal endoluminal tracheal occlusion can be considered as a minimally invasive fetal therapy, improving outcome in such highly selected cases.

Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion protein.