Haptoglobin baseline value in jennies and the effect of ovariectomy on its serum concentration

The study was conducted to determine the baseline concentration of serum haptoglobin (Hp) in jennies during the breeding and nonbreeding season and to evaluate the effects of ovariectomy on serum Hp concentrations in jennies. Eighteen adult jennies were divided in three groups: nine jennies (OVA) were ovariectomized using laparoscopic surgery, six jennies (LAP) were exploratory examined by laparoscopic surgery, and three jennies were used as a control group. Blood samples were collected from the animals at Day -6, -2, -1, 0, 1, 2, 5, 8, 15, 22, 29 and 36 of surgery. Serum samples were analyzed by an ELISA specifically developed for determining equine Hp. The mean weekly Hp concentration ranged between 149.76 ± 7.55 and 178.94 ± 6.67 mg/L. The Hp concentrations of clinically healthy jennies revealed no significant variation among time, and there was no effect of reproductive season on Hp concentrations in jennies. Serum Hp concentration was elevated at the first day after operations in the OVA and LAP group. Five days after the operation, the Hp concentration reached the maximum in the LAP and OVA group (278.84 ± 34.22 and 359.88 ± 35.45 mg/L, respectively) and decreased at Day 8 after the operations. On Day 22, 29 and 32 after the operations, the concentration of Hp in LAP and OVA animals was close to its concentration in the control group. In conclusion, Hp is not related to reproductive status of jennies and it can be used as an indicator for cell and tissue damage after surgical operations.     

Role of tartrate-resistant acid phosphatase (TRAP) in long bone development

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.     

Dietary cystine level affects metabolic rate and glycaemic control in adult mice

Plasma total cysteine (tCys) is strongly and independently associated with obesity in large human cohorts, but whether the association is causal is unknown. Dietary cyst(e)ine increases weight gain in some rodent models. We investigated the body composition, metabolic rate and metabolic phenotype of mature C3H/HeH mice assigned to low-cystine (LC) or high-cystine (HC) diets for 12 weeks.Compared to LC mice, HC mice gained more weight (P=.004 for 12-week weight gain %), with increased fat mass and lean mass, and lowered O₂ consumption and CO₂ production by calorimetry. The HC mice had 30% increase in intestinal fat/body weight % (P=.003) and ～twofold elevated hepatic triglycerides (P=.046), with increased expression of hepatic lipogenic factors, peroxisome proliferator-activated receptor-γ and sterol regulatory element binding protein-1. Gene expression of both basal and catecholamine-stimulated lipolytic enzymes, adipose triglyceride lipase and hormone-sensitive lipase was inhibited in HC mice adipose tissue. The HC mice also had elevated fasting glucose (7.0 vs. 4.5 mmol/L, P<.001) and a greater area under the curve (P<.001) in intraperitoneal glucose tolerance tests, with enhanced expression of the negative regulator of insulin signaling, protein tyrosine phosphatase-1B, in liver and adipose tissue.Overall, high cystine intake promotes adiposity and an adverse metabolic phenotype in mice, indicating that the positive association of plasma tCys with obesity in humans may be causal.     

Importance of human leukocyte antigen (HLA) class I and II alleles on the risk of multiple sclerosis

Multiple sclerosis (MS) is a complex disease of the central nervous system of unknown etiology. The human leukocyte antigen (HLA) locus on chromosome 6 confers a considerable part of the susceptibility to MS, and the most important factor is the class II allele HLA-DRB1*15:01. In addition, we and others have previously established a protective effect of HLA-A*02. Here, we genotyped 1,784 patients and 1,660 healthy controls from Scandinavia for the HLA-A, HLA-B, HLA-C and HLA-DRB1 genes and investigated their effects on MS risk by logistic regression. Several allele groups were found to exert effects independently of DRB1*15 and A*02, in particular DRB1*01 (OR = 0.82, p = 0.034) and B*12 (including B*44/45, OR = 0.76, p = 0.0028), confirming previous reports. Furthermore, we observed interaction between allele groups: DRB1*15 and DRB1*01 (multiplicative: OR = 0.54, p = 0.0041; additive: AP = 0.47, p = 4 × 10(-06)), DRB1*15 and C*12 (multiplicative: OR = 0.37, p = 0.00035; additive: AP = 0.58, p = 2.6 × 10(-05)), indicating that the effect size of these allele groups varies when taking DRB1*15 into account. Analysis of inferred haplotypes showed that almost all DRB1*15 bearing haplotypes were risk haplotypes, and that all A*02 bearing haplotypes were protective as long as they did not carry DRB1*15. In contrast, we found one class I haplotype, carrying A*02-C*05-B*12, which abolished the risk of DRB1*15. In conclusion, these results confirms a complex role of HLA class I and II genes that goes beyond DRB1*15 and A*02, in particular by including all three classical HLA class I genes as well as functional interactions between DRB1*15 and several alleles of DRB1 and class I genes.     

Plasmodium berghei Δp52&p36 Parasites Develop Independent of a Parasitophorous Vacuole Membrane in Huh-7 Liver Cells

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.     

The Association of Cysteine with Obesity, Inflammatory Cytokines and Insulin Resistance in Hispanic Children and Adolescents

Context

Plasma total cysteine (tCys) independently relates to fat mass in adults. Dietary cyst(e)ine promotes adiposity and decreases glucose tolerance in some rodent models, but alleviates insulin resistance in others.

Objective

To investigate whether the association of tCys with body fat extends to children at particular risk of obesity, and whether tCys is associated with insulin resistance and obesity-associated inflammation.

Methods

We explored the cross-sectional relations of fasting plasma tCys and related metabolites with body composition measured by dual-energy X-ray absorptiometry in 984 Hispanic children and adolescents aged 4–19 years from the Viva La Familia Study. Linear and logistic regression and dose-response curves were used to evaluate relations of tCys with obesity, insulin resistance and inflammatory markers including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein (CRP).

Results

tCys, methionine and total homocysteine (tHcy) increased with age. Upper tCys quartile was independently associated with a 5-fold increased risk of obesity (95% CI 3.5–8.0, P<0.001), and 2-fold risk of insulin resistance (95% CI: 1.6-5.0, P<0.001; adjusted for body fat%). Within the overweight/obese subgroup, but not in normal-weight children, tCys accounted for 9% of the variability in body fat% (partial r = 0.30, P<0.001; adjusted for age and gender). tCys correlated positively with serum non-esterified fatty acids and leptin, partly independent of body fat, but was not associated with serum IL-6, TNF-α or MCP-1. A positive correlation with CRP disappeared after adjustment for BMI.

Conclusion

tCys is independently associated with obesity and insulin resistance in Hispanic children and adolescents, highlighting a previously underappreciated link between the sulfur amino acid metabolic pathway and obesity and cardiometabolic risk.     

Humoral immune responses to a single allele PfAMA1 vaccine in healthy malaria-naïve adults

Plasmodium falciparum: apical membrane antigen 1 (AMA1) is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. The polymorphic nature of AMA1 may compromise vaccine induced protection. The humoral response induced by two dosages (10 and 50 μg) of a single allele AMA1 antigen (FVO) formulated with Alhydrogel, Montanide ISA 720 or AS02 was investigated in 47 malaria-na?ve adult volunteers. Volunteers were vaccinated 3 times at 4 weekly intervals and serum samples obtained four weeks after the third immunization were analysed for (i) Antibody responses to various allelic variants, (ii) Domain specificity, (iii) Avidity, (iv) IgG subclass levels, by ELISA and (v) functionality of antibody responses by Growth Inhibition Assay (GIA). About half of the antibodies induced by vaccination cross reacted with heterologous AMA1 alleles. The choice of adjuvant determined the magnitude of the antibody response, but had only a marginal influence on specificity, avidity, domain recognition or subclass responses. The highest antibody responses were observed for AMA1 formulated with AS02. The Growth Inhibition Assay activity of the antibodies was proportional to the amount of antigen specific IgG and the functional capacity of the antibodies was similar for heterologous AMA1-expressing laboratory strains. Trial registration: ClinicalTrials.gov NCT00730782.     

Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Plant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY‐2 cells expressing the human antibody M12 by selecting the co‐expressed fluorescent marker protein DsRed located on the same T‐DNA. Separation yielded ～35% wells containing single protoplasts and ～15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90–96% in the six monoclonal lines resulted in an up to 13‐fold increase in M12 production that remained stable for 10–12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.     

The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women's cancer

Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.     

The interplay between toxin-releasing β-glucosidase and plant iridoid glycosides impairs larval development in a generalist caterpillar, Grammia incorrupta (Arctiidae)

Herbivores with polyphagous feeding habits must cope with a diet that varies in quality. One of the most important sources of this variation in host plant suitability is plant secondary chemistry. We examined how feeding on plants containing one such group of compounds, the iridoid glycosides, might affect the growth and enzymatic activity in a polyphagous caterpillar that feeds on over 80 plant species in 50 different families. Larvae of the polyphagous arctiid, Grammia incorrupta, were reared exclusively on one of two plant species, one of which contains iridoid glycosides (Plantago lanceolata, Plantaginaceae) while the other does not (Taraxacum officinale, Asteraceae). Larval weight was measured on the two host plants, and midgut homogenates of last instar larvae were then assayed for activity and kinetic properties of β-glucosidases, using both a standard substrate, 4-nitrophenyl-β-D-glucose (NPβGlc), and the iridoid glycoside aucubin, one of the two main iridoid glycosides in P. lanceolata. Larvae feeding on P. lanceolata weighed significantly less and developed more slowly compared to larvae on T. officinale. While the larval midgut β-glucosidase activity determined with NPβGlc was significantly decreased when fed on P. lanceolata, aucubin was substantially hydrolyzed and the larval β-glucosidase activity towards both substrates correlated negatively with larval weight. Our results demonstrate that host plants containing high concentrations of iridoid glycosides have a negative impact on larval development of this generalist insect herbivore. This is most likely due to the hydrolysis of plant glycosides in the larval midgut which results in the release of toxic aglycones. Linking the reduced larval weight to the toxin-releasing action of an iridoid glycoside cleaving β-glucosidase, our results thus support the detoxification limitation hypothesis, suggesting fitness costs for the larvae feeding solely on P. lanceolata. Thus, in addition to the adaptive regulation of midgut β-glucosidase activity, host plant switching as a behavioral adaptation might be a prerequisite for generalist herbivores that allows them to circumvent the negative effects of plant secondary compounds.