Expression of CD25 in Individuals With Obesity With or Without Insulin Resistance After Following a North American Diet

Objectives Obesity and insulin resistance (IR) are associated with systemic inflammation, lower immune function, and a higher risk of infection. We previously reported that individuals with obesity and type 2 diabetes have an impaired T cell response (i.e., lower IL-2 production, a marker of proliferation) upon T cell stimulation despite having more activated T cells compared to normoglycemic (NG) individuals with obesity. It remains unclear if the immune dysfunction is caused by adiposity, hyperglycemia and/or dietary patterns. The aim of this study was to investigate the effect of consuming an isocaloric North American-type diet on the IL-2 receptor (CD25) expression and cardiometabolic risk factors in lean, obese-NG, and obese-IR individuals. Methods This is a three parallel-arm trial in controlled feeding conditions being conducted at the Human Nutrition Research Unit, at the University of Alberta. Three groups of adults: Lean-NG (n = 7), Obese-NG (n = 8), and Obese-IR (n = 9) consumed an isocaloric standardized diet containing 35% fat, 48% carbohydrate, and 17% protein for 4 weeks. All meals were provided to participants. Blood samples were collected in the fasting state before and after the intervention and cardiometabolic risk factors were measured. Peripheral blood mononuclear cells (PBMCs) were isolated and the proportion of total immune cells expressing CD25 was determined by flow cytometry. Results At baseline and post-intervention, Obese-IR had higher levels of glucose, insulin, and HOMA-IR and lower levels of HDL-C compared to both Lean-NG and Obese-NG groups (P < 0.05). At baseline, the proportion of PBMCs expressing CD25 tended to be lower in the Lean-NG (17.6 ± 2.7) compared to both Obese-NG (23.3 ± 4.8) and Obese-IR (23.2 ± 4.8) (P = 0.08). Post-intervention, the expression of CD25 was reduced in Lean-NG and Obese-IR groups (P < 0.01) but similar trends were still observed among all groups (P = 0.07). Conclusions Our preliminary data suggest that obesity, independent of IR, is associated with greater activation of immune cells and consuming a North American-type diet lowers the expression of the IL-2 receptor in individuals with and without obesity. Therefore, both excess adiposity and dietary pattern appear to modulate the function of immune cells in obesity. Funding Sources Canadian Institutes of Health Research.

Pyruvate Carboxylase Deficiency Type C: A Rare Cause of Acute Transient Flaccid Paralysis with Ketoacidosis

Pyruvate carboxylase (PC) is a biotin-containing enzyme that is responsible for the adenosine triphosphate-dependent carboxylation of pyruvate to oxaloacetate, a key intermediate in the tricarboxylic acid cycle. PC deficiency (OMIM 266150) is a rare autosomal recessive metabolic disease, causing elevation of pyruvate, lactate, and alanine. Three types of PC deficiency have been described in the literature; A, B, and C. Type A PC deficiency, also called infantile or North American type, is characterized by infantile onset acidosis, failure to thrive, and developmental delay. The second subtype or type B, the neonatal or French form, presents usually in the neonatal period, mostly in the first 72 hours of life with severe lactic acidosis, truncal hypotonia, and seizures. The third type is called type C, is extremely rare with few cases published in the literature. In this case report, we present an 11-month-old girl who presented with acute flaccid paralysis, lethargy, and constipation with elevated ketones and lactate. She was confirmed genetically and biochemically to have PC deficiency type C. The patient's unusual presentation expands the clinical phenotype of this extremely rare disease.

Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid bySpeI andXhoI sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesizedin vitroand amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.

Simultaneous Detection and Differentiation of Highly Virulent and Classical Chinese-Type Isolation of PRRSV by Real-Time RT-PCR

Porcine reproductive and respiratory syndrome (PRRS) is a leading disease in pig industry worldwide and can result in serious economic losses each year. The PRRS epidemic situation in China has been very complicated since the unprecedented large-scale highly pathogenic PRRS (HP-PRRS) outbreaks in 2006. And now the HP-PRRS virus (HP-PRRSV) and classical North American type PRRSV strains have coexisted in China. Rapid differential detection of the two strains of PRRSV is very important for effective PRRS control. The real-time RT-PCR for simultaneous detection and differentiation of HP-PRRSV and PRRSV by using both SYBR Green and TaqMan probes was developed and validated. Both assays can be used for rapid detection and strain-specific identification of HP-PRRSV and PRRSV. However, the TaqMan probe method had the highest detection rate whereas the conventional RT-PCR was the lowest. The real-time RT-PCR developed based on SYBR Green and TaqMan probe could be used for simultaneous detection and differentiation of HP-PRRSV and PRRSV in China, which will benefit much the PRRS control and research.

Phylogeny-Based Evolutionary, Demographical, and Geographical Dissection of North American Type 2 Porcine Reproductive and Respiratory Syndrome Viruses

ABSTRACT Type 2 (or North American-like) porcine reproductive and respiratory syndrome virus (PRRSV) was first recorded in 1987 in the United States and now occurs in most commercial swine industries throughout the world. In this study, we investigated the epidemiological and evolutionary behaviors of type 2 PRRSV. Based on phylogenetic analyses of 8,624 ORF5 sequences, we described a comprehensive picture of the diversity of type 2 PRRSVs and systematically classified all available sequences into lineages and sublineages, including a number of previously undescribed lineages. With the rapid growth of sequence deposition into the databases, it would be technically difficult for veterinary researchers to genotype their sequences by reanalyzing all sequences in the databases. To this end, a set of reference sequences was established based on our classification system, which represents the principal diversity of all available sequences and can readily be used for further genotyping studies. In addition, we further investigated the demographic histories of these lineages and sublineages by using Bayesian coalescence analyses, providing evolutionary insights into several important epidemiological events of type 2 PRRSV. Moreover, by using a phylogeographic approach, we were able to estimate the transmission frequencies between the pig-producing states in the United States and identified several states as the major sources of viral spread, i.e., “transmission centers.” In summary, this study represents the most extensive phylogenetic analyses of type 2 PRRSV to date, providing a basis for future genotyping studies and dissecting the epidemiology of type 2 PRRSV from phylogenetic perspectives.