Recently, novel inborn errors of metabolism were identified because of mutations in V-ATPase assembly factors TMEM199 and CCDC115. Patients are characterized by generalized protein glycosylation ...defects, hypercholesterolemia, and fatty liver disease. Here, we set out to characterize the lipid and fatty liver phenotype in human plasma, cell models, and a mouse model.
Patients with TMEM199 and CCDC115 mutations displayed hyperlipidemia, characterized by increased levels of lipoproteins in the very low density lipoprotein range. HepG2 hepatoma cells, in which the expression of TMEM199 and CCDC115 was silenced, and induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells from patients with TMEM199 mutations showed markedly increased secretion of apolipoprotein B (apoB) compared with controls. A mouse model for TMEM199 deficiency with a CRISPR/Cas9-mediated knock-in of the human A7E mutation had marked hepatic steatosis on chow diet. Plasma N-glycans were hypogalactosylated, consistent with the patient phenotype, but no clear plasma lipid abnormalities were observed in the mouse model. In the siTMEM199 and siCCDC115 HepG2 hepatocyte models, increased numbers and size of lipid droplets were observed, including abnormally large lipid droplets, which colocalized with lysosomes. Excessive de novo lipogenesis, failing oxidative capacity, and elevated lipid uptake were not observed. Further investigation of lysosomal function revealed impaired acidification combined with impaired autophagic capacity.
Our data suggest that the hypercholesterolemia in TMEM199 and CCDC115 deficiency is due to increased secretion of apoB-containing particles. This may in turn be secondary to the hepatic steatosis observed in these patients as well as in the mouse model. Mechanistically, we observed impaired lysosomal function characterized by reduced acidification, autophagy, and increased lysosomal lipid accumulation. These findings could explain the hepatic steatosis seen in patients and highlight the importance of lipophagy in fatty liver disease. Because this pathway remains understudied and its regulation is largely untargeted, further exploration of this pathway may offer novel strategies for therapeutic interventions to reduce lipotoxicity in fatty liver disease.
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Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was first detected in Europe in 1996 co-incident with the introduction of a live attenuated vaccine. Since then, only limited ORF5 ...and ORF7 sequences of Type 2 PRRS viruses have been reported throughout Europe. In the present study, the genetic and antigenic diversity of 11 complete genomes and 49 ORF5 and 55 ORF7 nucleotide sequences obtained from 57 viruses in Denmark from 2003 to 2012 were examined. The genetic identity of the 11 complete genomes to the vaccine strain (Ingelvac PRRS MLV) ranged between 93.6 and 99.6% while the 49 ORF5 sequences examined were 94.0–99.8% identical to the vaccine strain. Among the Danish sequences, the pairwise nucleotide identity was 90.9–100% and 93.0–100.0% for ORF5 and ORF7, respectively. Analysis of the genetic region encoding NSP2 revealed high diversity among the Danish viruses with an 86.6–98.9% range in similarity. Furthermore, several of the sequenced viruses harbored deletions in the NSP2 coding region. Phylogenetic analysis in a global Type 2 PRRSV framework classified all Danish isolates to a single cluster (sub-lineage 5.1) which comprised strains closely-related to the Type 2 prototype isolate VR2332.
The reverse zoonotic events that introduced the 2009 pandemic influenza virus into pigs have drastically increased the diversity of swine influenza viruses in Europe. The pandemic potential of these ...novel reassortments is still unclear, necessitating enhanced surveillance of European pigs with additional focus on risk assessment of these new viruses. In this study, four European swine influenza viruses were assessed for their zoonotic potential. Two of the four viruses were enzootic viruses of subtype H1N2 (with avian-like H1) and H3N2, and two were new reassortants, one with avian-like H1 and human-like N2 and one with 2009 pandemic H1 and swine-like N2. All viruses replicated to high titres in nasal wash and nasal turbinate samples from inoculated ferrets and transmitted efficiently by direct contact. Only the H3N2 virus transmitted to naïve ferrets via the airborne route. Growth kinetics using a differentiated human bronchial epithelial cell line showed that all four viruses were able to replicate to high titres. Further, the viruses revealed preferential binding to the 2,6-α-silalylated glycans and investigation of the antiviral susceptibility of the viruses revealed that all were sensitive to neuraminidase inhibitors. These findings suggested that these viruses have the potential to infect humans and further underline the need for continued surveillance as well as biological characterization of new influenza A viruses.
BACKGROUND: Neonatal diarrhoea is a frequent clinical condition in commercial swine herds, previously regarded to be uncomplicated to treat. However, since 2008 it seems that a new neonatal ...diarrhoeic syndrome unresponsive to antibiotics and common management practices has emerged. Routine laboratory examinations have not detected any pathogen related to this syndrome. The primary purpose of this study was to evaluate if well-known enteric pathogens could be associated with outbreaks of neonatal diarrhoea, thus question the hypotheses of a new syndrome. Furthermore, we wanted to evaluate macroscopic and microscopic findings associated with these outbreaks and if possible propose a preliminary piglet-level case-definition on syndrome New Neonatal Porcine Diarrhoea syndrome (NNPDS). RESULTS: Four well-managed herds experiencing neonatal diarrhoea with no previously established laboratory conclusion and suspected to suffer from New Neonatal Porcine Diarrhoea Syndrome, were selected. Within these herds, 51 diarrhoeic and 50 non-diarrhoeic piglets at the age of three to seven days were necropsied and subjected to histological and microbiological examination. Faeces were non-haemorrhagic. Neither enterotoxigenic E. coli, Clostridium perfringens type A or C, Clostridium difficile, rotavirus, coronavirus, Cryptosporidium spp, Giardia spp, Cystoisospora suis nor Strongyloides ransomi were associated with diarrhoea in the investigated outbreaks. Macroscopically, the diarrhoeic piglets were characterized by filled stomachs and flaccid intestines without mucosal changes. The predominant histological lesions were villous atrophy in jejunum and ileum. Epithelial lesions in colon were seen in one third of the case piglets. CONCLUSIONS: The results of the study supported the hypothesis that a new neonatal porcine diarrhoea was present in the investigated herds, since no known pathogen(s) or management factors could explain the diarrhoeal outbreaks. Based on the findings in the four herds the following case-definition of NNPDS was suggested: Non-haemorrhagic diarrhoea during the first week of life, without detection of known infectious pathogens, characterized by milk-filled stomachs and flaccid intestines at necropsy.
Porcine circovirus type 2 (PCV2) diagnostics in live pigs often involves pooled serum and/or oral fluid samples for group-level determination of viral load by quantitative real-time polymerase chain ...reaction (qPCR). The purpose of the study was to compare the PCV2 viral load determined by qPCR of paired samples at the pen level of pools of sera (SP) from 4 to 5 pigs and the collective oral fluid (OF) from around 30 pigs corresponding to one rope put in the same pen. Pigs in pens of 2 finishing herds were sampled by cross-sectional (Herd 1) and cross-sectional with follow-up (Herd 2) study designs. In Herd 1, 50 sample pairs consisting of SP from 4 to 5 pigs and OF from around 23 pigs were collected. In Herd 2, 65 sample pairs consisting of 4 (SP) and around 30 (OF) pigs were collected 4 times at 3-week intervals.
A higher proportion of PCV2-positive pens (86% vs. 80% and 100% vs. 91%) and higher viral loads (mean difference: 2.10 and 1.83 log(10) PCV2 copies per ml) were found in OF versus SP in both herds. The OF cut-off value corresponding to a positive SP (>3 log(10) PCV2 copies per ml) was estimated to 6.5 and 7.36 log(10) PCV2 copies per ml for Herds 1 and 2, respectively. Significant correlations between SP and OF results were found in Herd 1 (rho = 0.69) and the first sampling in Herd 2 (rho = 0.39), but not for the subsequent consecutive 3 samplings in Herd 2.
The proportion and viral loads of PCV2 positive pens were higher in collective OF (including up to 30 pigs) compared to SP (including 4-5 pigs) of the same pens. Also, OF seemed to detect the PCV2 infection earlier with OF values just below 6.5 (Herd 1) and 7.36 (Herd 2) log(10) being associated with a negative SP for the same pen. Nevertheless, a statistically significant correlation between SP and OF could not be found for all sampling time points, probably due to a high within-pen variation in individual pig viral load becoming very evident in SP of only four or five pigs. Consequently, the results imply that OF is well suited for detecting presence of PCV2 but less so for determining the specific viral load of pigs in a pen.
An ankle-foot orthosis is suggested to actively assist dorsiflexion of the foot by induction of a positive proprioceptive impact to ankle dorsiflexor muscles. However, an objective proof is missing.
...To assess the proprioceptive impact of an ankle-foot orthosis application by Hoffmann reflex recordings of the soleus muscle under static conditions. It was hypothesized that the use of an ankle-foot orthosis facilitated dorsiflexor motor function and thus a decreased the soleus Hoffmann reflex.
Experimental study in healthy volunteers, pre-post test design.
In all, 20 healthy volunteers were enrolled in order to assess the proprioceptive impact of orthosis application. The Hoffmann reflex was recorded before, during, and after orthosis application.
Under orthosis application, the Hoffmann reflex significantly decreased as compared to before (p < 0.05) and after application (p < 0.05).
Findings indicate an inhibition of plantarflexors probably induced by facilitation of ankle dorsiflexors under static conditions. At first glance, it seems that foot orthoses primarily have a stabilizing effect on ankle joints in terms of simple mechanical bandages. However, the present results suggest an additional active impact on proprioceptive control.
The putative neuromodulatory effect on motor control may support the application of such ankle-foot orthoses in, for example, drop foot. Furthermore, the objective assessment of a neurophysiological mode of action of orthoses by Hoffmann reflex recordings might be an appropriate primary outcome parameter in clinical trials.
As a part of a prospective cohort study in four herds, a nested case control study was carried out. Five slow growing pigs (cases) and five fast growing pigs (controls) out of 60 pigs were selected ...for euthanasia and laboratory examination at the end of the study in each herd. A total of 238 pigs, all approximately 12 weeks old, were included in the study during the first week in the grower–finisher barn. In each herd, approximately 60 pigs from four pens were individually ear tagged. The pigs were weighed at the beginning of the study and at the end of the 6–8 weeks observation period. Clinical data, blood and faecal samples were serially collected from the 60 selected piglets every second week in the observation period. In the killed pigs serum was examined for antibodies against Lawsonia intracellularis (LI) and procine circovirus type 2 (PCV2) and in addition PCV2 viral DNA content was quantified. In faeces the quantity of LI cells/g faeces and number of PCV2 copies/g faeces was measured by qPCR. The objective of the study was to examine if growth rate in grower-finishing pig is associated with the detection of LI and PCV2 infection or clinical data. This study has shown that diarrhoea is a significant risk factor for low growth rate and that one log10 unit increase in LI load increases the odds ratio for a pig to have a low growth rate by 2.0 times. Gross lesions in the small intestine and LI load>log106/g were significant risk factors for low growth. No association between PCV2 virus and low growth was found.
Psoriasis is a chronic inflammatory skin disease arising from poorly defined pathological cross-talk between keratinocytes and the immune system. BCL10 (B cell lymphoma/leukemia 10) and MALT1 ...(mucosa-associated lymphoid tissue lymphoma translocation protein 1) are ubiquitously expressed inflammatory signaling proteins that can interact with the psoriasis susceptibility factor CARD14, but their functions in psoriasis are insufficiently understood. We report that although keratinocyte-intrinsic BCL10/MALT1 deletions completely rescue inflammatory skin pathology triggered by germline
gain-of-function mutation in mice, the BCL10/MALT1 signalosome is unexpectedly not involved in the CARD14-dependent interleukin-17 receptor (IL-17R) proximal pathway. Instead, it plays a more pleiotropic role by amplifying keratinocyte responses to a series of inflammatory cytokines, including IL-17A, IL-1β, and TNF. Moreover, selective keratinocyte-intrinsic activation of BCL10/MALT1 signaling with an artificial engager molecule is sufficient to initiate lymphocyte-mediated psoriasiform skin inflammation, and aberrant BCL10/MALT1 activity is frequently detected in the skin of human sporadic psoriasis. Together, these results establish that BCL10/MALT1 signalosomes can act as initiators and crucial amplifiers of psoriatic skin inflammation and indicate a critical function for this complex in sporadic psoriasis.
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