Neuronal ceroid lipofuscinosis in Salukis is caused by a single base pair insertion in CLN8.

Neuronal ceroid lipofuscinoses (NCLs) are heterogenic inherited lysosomal storage diseases that have been described in a number of species including humans, sheep, cattle, cats and a number of different dog breeds, including Salukis. Here we present a novel genetic variant associated with the disease in this particular breed of dog. In a clinical case, a Saluki developed progressive neurological signs, including disorientation, anxiety, difficulties in eating, seizures and loss of vision, and for welfare reasons, was euthanized at 22 months of age. Microscopy showed aggregation of autofluorescent storage material in the neurons of several brain regions and also in the retina. The aggregates showed positive staining with Sudan black B and periodic acid Schiff, all features consistent with NCL. Whole genome sequencing of the case and both its parents, followed by variant calling in candidate genes, identified a new variant in the CLN8 gene: a single bp insertion (c.349dupT) in exon 2, introducing an immediate stop codon (p.Glu117*). The case was homozygous for the insertion, and both parents were heterozygous. A retrospective study of a Saluki from Australia diagnosed with NCL identified this case as being homozygous for the same mutation. This is the fourth variant identified in CLN8 that causes NCL in dogs.

LPS-induced systemic inflammation reveals an immunomodulatory role for the prion protein at the blood-brain interface.

BACKGROUND: The cellular prion protein (PrPC) is an evolutionary conserved protein abundantly expressed not only in the central nervous system but also peripherally including the immune system. A line of Norwegian dairy goats naturally devoid of PrPC (PRNP Ter/Ter) provides a novel model for studying PrPC physiology. METHODS: In order to explore putative roles for PrPC in acute inflammatory responses, we performed a lipopolysaccharide (LPS, Escherichia coli O26:B6) challenge of 16 goats (8 PRNP +/+ and 8 PRNP Ter/Ter) and included 10 saline-treated controls (5 of each PRNP genotype). Clinical examinations were performed continuously, and blood samples were collected throughout the trial. Genome-wide transcription profiles of the choroid plexus, which is at the blood-brain interface, and the hippocampus were analyzed by RNA sequencing, and the same tissues were histologically evaluated. RESULTS: All LPS-treated goats displayed clinical signs of sickness behavior, which were of significantly (p < 0.01) longer duration in animals without PrPC. In the choroid plexus, a substantial alteration of the transcriptome and activation of Iba1-positive cells were observed. This response included genotype-dependent differential expression of several genes associated with the immune response, such as ISG15, CXCL12, CXCL14, and acute phase proteins, among others. Activation of cytokine-responsive genes was skewed towards a more profound type I interferon response, and a less obvious type II response, in PrPC-deficient goats. The magnitude of gene expression in response to LPS was smaller in the hippocampus than in the choroid plexus. Resting state expression profiles revealed a few differences between the PRNP genotypes. CONCLUSIONS: Our data suggest that PrPC acts as a modulator of certain pathways of innate immunity signaling, particularly downstream of interferons, and probably contributes to protection of vulnerable tissues against inflammatory damage.

Glomerular Collagen V Codeposition and Hepatic Perisinusoidal Collagen III Accumulation in Canine Collagen Type III Glomerulopathy.

Collagen type III glomerulopathy, also known as collagenofibrotic glomerulopathy, is a rare renal disease of unknown pathogenesis. The disease occurs in humans and animals and is characterized by massive glomerular accumulations of collagen type III. In the present study, we describe a Drever dog litter affected by an early onset variant of this glomerular disease, where 4 of 9 puppies developed renal failure within 50 days of age. Necropsy specimens of kidney from the 4 affected cases were studied by light microscopy, electron microscopy, and immunohistochemistry, and characteristic lesions compatible with a diagnosis of collagen type III glomerulopathy were found. In addition, 2 cases showed atypical epithelium in the collecting ducts of the medulla, so-called adenomatoid change. Immunohistochemistry of renal specimens from collagen type III glomerulopathy-affected dogs (n = 10) originating from two different dog strains, the Drever dogs and a mixed-breed strain, demonstrated that the deposited glomerular collagen is composed of a mixture of collagen III and collagen V. The distribution of the collagen V corresponded to the localization of collagen III; however, differences in staining intensity showed that collagen type III is the dominating component. Immunohistochemistry for collagen III (n = 9) and a transmission electron microscopic study (n = 1) showed hepatic perisinusoidal collagen type III deposition in affected cases from both dog strains. This is the first report documenting glomerular accumulations of collagen type V and perisinusoidal liver collagen III deposition in canine collagen type III glomerulopathy.

Pancreatitis associated with hyperlipoproteinaemia type I in mink (Mustela vison): earliest detectable changes occur in mitochondria of exocrine cells.

Pancreatic tissue from young mink homozygous for a mutation in the lipoprotein lipase gene was studied by light and electron microscopy, with the aim of describing the earliest detectable changes in a process which rapidly progresses into overt pancreatitis. The mutation leads to hyperlipoproteinaemia, corresponding to hyperlipoproteinaemia type I in man. Assessment of relevant hepatic and pancreatic enzymes were included in the investigation. The earliest detectable changes consisted of widespread swelling and vacuolation of exocrine cells, arising mainly from swollen mitochondria. To a lesser extent, vesiculation of endoplasmic reticulum occurred. Mitochondria exhibited various changes, including cavitation and dilution of the matrix, with shortened and disorganized cristae displaced towards the periphery. Lamellar figures that developed within mitochondria were numerous. Acinar lumina were somewhat dilated, while plasma membranes were relatively well preserved and secretory granules seemed unchanged. Exfoliative processes progressively occurred, resulting in total necrosis of groups of parenchymal cells, while intercalated ducts were spared. The necrosis was rapidly followed by inflammatory reactions. The activity of the mitochondrial enzyme carnitine O-palmitoyltransferase, essential for the transport of fatty acids into the mitochondria, was lower in the pancreas than in the liver. The activity of the peroxisomal fatty acid beta-oxidation was high in the liver and low in the pancreas of both lipoprotein lipase-deficient and control mink. It is concluded that pancreatic lesions associated with hyperlipoproteinaemia start in exocrine cells, and are most probably the result of a metabolic disturbance, possibly a toxic effect of an excess of free fatty acids.

Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep.

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.

Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie.

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.

Filter function and immune complex trapping in splenic ellipsoids.

The role of splenic ellipsoids in the trapping of particulate material and immune complexes was investigated in mink (Mustela vison). The ellipsoids were prominent, with typical features such as a permeable endothelium and a discontinuous basement membrane surrounded by a sheath of macrophages and reticular cells. Ellipsoidal trapping of circulating particles was demonstrated 10 min after intracardiac injection of colloidal carbon and fluorescent microspheres. Preformed peroxidase-antiperoxidase immune complexes were detected in ellipsoids 10 min and also 1 h after intracardiac injection. Erythrocytes were frequently observed in the ellipsoidal sheath, and many phagocytized fragments of erythrocytes were found in the ellipsoidal macrophages. It was concluded that mink ellipsoids are effective blood filters with a role in retention of circulating particulate material, and that mammalian splenic ellipsoids also have the ability to trap immune complexes.

Mapping PrPSc propagation in experimental and natural scrapie in sheep with different PrP genotypes.

Twenty-one orally inoculated and seven naturally infected sheep with scrapie were examined for PrP(Sc) in peripheral tissues and in the central nervous system (CNS), using immunohistochemistry. In the inoculated group, VRQ (valine at codon 136, arginine at codon 154 and glutamine at codon 171)/VRQ sheep generally had a greater accumulation of the pathologic form of prion protein (PrP(Sc)) in peripheral tissues, as compared with VRQ/ARQ (alanine at codon 136, arginine at codon 154, and glutamine at codon 171) animals at corresponding time points after inoculation. PrP(Sc) was not detected in the ileal Peyer's patch, the spleen, the superficial cervical lymph node, and peripheral nervous tissues of several inoculated VRQ/ARQ animals. All inoculated VRQ/VRQ sheep, but only one of eight inoculated VRQ/ARQ animals, were PrP(Sc)-positive in the CNS. Thus, the propagation of PrP(Sc) seemed slower and more limited in VRQ/ARQ animals. Tissue and cellular localization of PrP(Sc) suggested that PrP(Sc) was disseminated through three different routes. PrP(Sc)-positive cells in lymph node sinuses and in lymphatics indicated spreading by lymph. The sequential appearance of PrP(Sc) in the peripheral nervous system and the CNS, with satellite cells as early targets, suggested the periaxonal transportation of PrP(Sc) through supportive cells. Focal areas of vascular amyloid-like PrP(Sc) in the brain of five sheep, suggested the hematogenous dissemination of PrP(Sc). There was a poor correlation between the amount of PrP(Sc) in the CNS and clinical signs. One subclinically affected sheep showed widespread PrP(Sc) accumulation in the CNS, whereas three sheep had early clinical signs without detectable PrP(Sc) in the CNS. A VV(136) (homozygous for valine at codon 136) sheep inoculated with ARQ/ARR (alanine at codon 136, arginine at codon 154, and arginine at codon 171) tissue succumbed to disease, demonstrating successful heterologous transmission. Less susceptible sheep receiving VRQ/VRQ or ARQ/ARR material were PrP(Sc)-negative by immunohistochemistry, enzyme-linked immunosorbent assay, and western blot.

The PrP-like protein Doppel gene in sheep and cattle: cDNA sequence and expression.

cDNAs encoding the ovine and bovine prion protein-like protein Doppel (Dpl) have been cloned. Sequencing revealed cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular tissue, in accordance with observations of single transcripts of 3.2 and 3.6 kb on Northern blots. Sequence alignments showed a very high degree of identity between the sheep and cattle Dpl cDNAs, except for a 0.4-kb stretch in the bovine 3' untranslated region and the terminal 3' end of the sequences. The expression pattern of the Dpl gene (Prnd) in adult tissues from both species was compared by Northern blot and RT-PCR analyses. The Prnd gene was expressed strongly in testicular tissue, while low levels of expression were seen in other tissues. The open reading frame of the ovine and bovine sequences encodes a 178-amino acid protein with 95% sequence identity between the two species. Predicted structural features are in close agreement with previous reports for mouse, human, and rat Dpl.

Apoptosis in phagocytotic cells of lymphoid tissues in rainbow trout (Oncorhynchus mykiss) following administration of clodronate liposomes.

Macrophage function has been studied in vivo by using liposomes that contain dichloromethylene-bisphosphonate (clodronate liposomes) to deplete macrophage subpopulations. In the present study, the effects of intravenously injected clodronate liposomes on the head kidney and spleen of the rainbow trout (Oncorhynchus mykiss) were studied from 1 h to 7 days after injection. The rapid trapping of liposomes in the splenic ellipsoids was followed by depletion of ellipsoidal sheath macrophages and accumulation of particulate material and IgM in the ellipsoidal wall, findings illustrating the importance of ellipsoidal macrophages in the clearance of filtered substances trapped in the reticular matrix of the ellipsoidal wall. A reduced reactivity for acid phosphatase in the spleen and ultrastructural evidence of cell death in phagocytotic cells of the head kidney and spleen supported the selective effect of clodronate liposomes on macrophages in rainbow trout. Apoptotic bodies were prominent ultrastructural features in tissues collected from clodronate-liposome-treated rainbow trout. The increased presence of apoptotic cells in clodronate-liposome-treated trout compared with trout given liposomes containing phosphate-buffered saline was confirmed by using terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate nick-end-labelling of cells with extensive DNA fragmentation. The characterization of liposome-mediated macrophage depletion in fish provides a useful model for further investigation of piscine macrophage function.

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalization of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In non-immunized fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and non-immunized trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.