IL-17 and IL-22 elicited by a DNA vaccine encoding ROP13 associated with protection against Toxoplasma gondii in BALB/c mice

Toxoplasma gondii, an intracellular parasitic protozoan, is capable of infecting man and all warm-blooded animals. Cell-mediated immunity is vital in mounting protective responses against T. gondii infection. Recent studies have shown that T-helper (Th) 17 responses may play a key role in parasite control. In this current study, we constructed a DNA vaccine encoding T. gondii ROP13 in a pcDNA vector. Groups of BALB/c mice were immunized intramuscularly with pcROP13 or controls and challenged with the RH strain of T. gondii. The results showed that immunization with pcROP13 could elicit an antibody response against T. gondii. The expression of the canonical Th17 cytokines, interleukin (IL)-17 and IL-22, were significantly increased after immunization with pcROP13 compared with control groups ( p < 0.05). Furthermore, vaccination resulted in a significant decrease in parasite load ( p < 0.05). The induction of Th17 related cytokines, using a ROP13 DNA vaccine, against T. gondii should be considered as a potential vaccine approach for the control of toxoplasmosis.     

Possible involvement of miRNAs in tropism of Parvovirus B19

Human Parvovirus B19 (PVB19) is one of the most important pathogens that targets erythroid lineage. Many factors were mentioned for restriction to erythroid progenitor cells (EPCs). Previous studies showed that in non-permissive cells VP1 and VP2 (structural proteins) mRNAs were detected but could not translate to proteins. A bioinformatics study showed that this inhibition might be due to specific microRNAs (miRNAs) present in non-permissive cells but not in permissive EPCs. To confirm the hypothesis, we evaluated the effect of miRNAs on VP expression. CD34⁺ HSCs were separated from cord blood. Then, CD34⁺ cells were treated with differentiation medium to obtain CD36⁺ EPCs. To evaluate the effect of miRNAs on VP expression in MCF7 and HEK-293 cell lines (non-permissive cells) and CD36⁺ EPCs, dual luciferase assay was performed in presence of shRNAs against Dicer and Drosha to disrupt miRNA biogenesis. QRT-PCR was performed to check down-regulation of Dicer and Drosha after transfection. All measurements were done in triplicate. Data means were compared using one-way ANOVAs. MicroRNA prediction was done by the online microRNA prediction tools. No significant difference was shown in luciferase activity of CD36⁺ EPCs after co-transfection with shRNAs, while it was significant in non-permissive cells. Our study revealed that miRNAs may be involved in inhibition of VP expression in non-permissive cells, although further studies are required to demonstrate which miRNAs exactly are involved in regulation of PVB19 replication.     

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) improved osteogenic differentiation of the human induced pluripotent stem cells while considered as an artificial extracellular matrix

Cocell polymers can be the best implants for replacing bone defects in patients. The pluripotent stem cells produced from the patient and the nanofibrous polymeric scaffold that can be completely degraded in the body and its produced monomers could be also usable are the best options for this implant. In this study, electrospun poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers were fabricated and characterized and then osteogenic differentiation of the human-induced pluripotent stem cells (iPSCs) was investigated while cultured on PHBV scaffold. MTT results showed that cultured iPSCs on PHBV proliferation were increased compared to those cultured on tissue culture polystyrene (TCPS) as the control. Alkaline phosphatase (ALP) activity and calcium content were also significantly increased in iPSCs cultured on PHBV compared to the cultured on TCPS under osteogenic medium. Gene expression evaluation demonstrated that Runx2, collagen type I, ALP, osteonectin, and osteocalcin were upregulated in iPSCs cultured on PHBV scaffold in comparison with those cultured on TCPS for 2 weeks. Western blot analysis have shown that osteocalcin and osteopontin expression as two major osteogenic markers were increased in iPSCs cultured on PHBV scaffold. According to the results, nanofiber-based PHBV has a promising potential to increase osteogenic differentiation of the stem cells and iPSCs-PHBV as a cell-co-polymer construct demonstrated that has a great efficiency for use as a bone tissue engineered bioimplant.     

Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius)

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.     

PINK1-parkin-mediated neuronal mitophagy deficiency in prion disease

A persistent accumulation of damaged mitochondria is part of prion disease pathogenesis. Normally, damaged mitochondria are cleared via a major pathway that involves the E3 ubiquitin ligase parkin and PTEN-induced kinase 1 (PINK1) that together initiate mitophagy, recognize and eliminate damaged mitochondria. However, the precise mechanisms underlying mitophagy in prion disease remain largely unknown. Using prion disease cell models, we observed PINK1-parkin-mediated mitophagy deficiency in which parkin depletion aggravated blocked mitochondrial colocalization with LC3-II-labeled autophagosomes, and significantly increased mitochondrial protein levels, which led to inhibited mitophagy. Parkin overexpression directly induced LC3-II colocalization with mitochondria and alleviated defective mitophagy. Moreover, parkin-mediated mitophagy was dependent on PINK1, since PINK1 depletion blocked mitochondrial Parkin recruitment and reduced optineurin and LC3-II proteins levels, thus inhibiting mitophagy. PINK1 overexpression induced parkin recruitment to the mitochondria, which then stimulated mitophagy. In addition, overexpressed parkin and PINK1 also protected neurons from apoptosis. Furthermore, we found that supplementation with two mitophagy-inducing agents, nicotinamide mononucleotide (NMN) and urolithin A (UA), significantly stimulated PINK1-parkin-mediated mitophagy. However, compared with NMN, UA could not alleviate prion-induced mitochondrial fragmentation and dysfunction, and neuronal apoptosis. These findings show that PINK1-parkin-mediated mitophagy defects lead to an accumulation of damaged mitochondria, thus suggesting that interventions that stimulate mitophagy may be potential therapeutic targets for prion diseases.Subject terms: Targeted gene repair, Target validation, Neurodegeneration, Neurodegeneration, Prion diseases     

Rumen Microbiome Composition Determined Using Two Nutritional Models of Subacute Ruminal Acidosis

Subacute ruminal acidosis (SARA) is a metabolic disease in dairy cattle that occurs during early and mid-lactation and has traditionally been characterized by low rumen pH, but lactic acid does not accumulate as in acute lactic acid acidosis. It is hypothesized that factors such as increased gut permeability, bacterial lipopolysaccharides, and inflammatory responses may have a role in the etiology of SARA. However, little is known about the nature of the rumen microbiome during SARA. In this study, we analyzed the microbiome of 64 rumen samples taken from eight lactating Holstein dairy cattle using terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA genes and real-time PCR. We used rumen samples from two published experiments in which SARA had been induced with either grain or alfalfa pellets. The results of TRFLP analysis indicated that the most predominant shift during SARA was a decline in gram-negative Bacteroidetes organisms. However, the proportion of Bacteroidetes organisms was greater in alfalfa pellet-induced SARA than in mild or severe grain-induced SARA (35.4% versus 26.0% and 16.6%, respectively). This shift was also evident from the real-time PCR data for Prevotella albensis, Prevotella brevis, and Prevotella ruminicola, which are members of the Bacteroidetes. The real-time PCR data also indicated that severe grain-induced SARA was dominated by Streptococcus bovis and Escherichia coli, whereas mild grain-induced SARA was dominated by Megasphaera elsdenii and alfalfa pellet-induced SARA was dominated by P. albensis. Using discriminant analysis, the severity of SARA and degree of inflammation were highly correlated with the abundance of E. coli and not with lipopolysaccharide in the rumen. We thus suspect that E. coli may be a contributing factor in disease onset.The bovine rumen is a classical host-microbe symbiotic system, and disturbances in this exquisitely balanced ecosystem may lead to disease in the host. An example is subacute ruminal acidosis (SARA), or non-lactic acid acidosis, which has a disease etiology distinct from that of acute lactic acid acidosis because there is no accumulation of lactic acid (35). Field studies in the United States estimated that 19% of early lactating cows and 26% of mid-lactation cows suffered from SARA (11). In Germany and The Netherlands, approximately 11% of early lactation and 18% of mid-lactation cows suffered from this disease (22). In the acute form, lactic acid accumulates in the rumen, causing metabolic acidosis, and it usually occurs when animals are abruptly transitioned to a high-grain diet from a predominantly forage diet (38). If, however, the adaptation is gradual, slower-growing lactic acid-consuming bacteria, like Megasphaera elsdenii, convert the lactic acid to propionic acid (29). In SARA, lactic acid does not accumulate during low-pH conditions and other factors, like microbial population shifts and immune responses, appear to be associated with the disease etiology (35).In both acute and subacute acidosis, there is an increase in lipopolysaccharide (LPS) concentrations in the rumen (8, 14, 16). LPS and/or the low-pH rumen conditions may increase the permeability of the gut to LPS, which could trigger systemic inflammation (4). We previously developed two animal models of SARA, one based on grain and one based on alfalfa pellets (20, 21). Even though both models resulted in substantial reductions in rumen pH and an accumulation of LPS, only the grain induction model resulted in inflammation and the appearance of LPS in the peripheral blood (20, 21).In contrast to the rumen microbiome during lactic acid acidosis, the rumen microbiome during SARA has not been evaluated (13, 28). Even in acute acidosis, studies are largely culture based, and the uncultured members of the community have not been extensively assessed (31, 46, 49). In this article, we describe the rumen microbiome when two SARA induction models were used. The shifts in microbial community structure were assessed using terminal restriction fragment length polymorphism (TRFLP) analysis and real-time PCR of key microbial populations.     

Novel regulatory mechanism of cardiomyocyte contractility involving ICAM-1 and the cytoskeleton

ICAM-1 mediates interaction of cardiomyocytes with the extracellular matrix and leukocytes and may play a role in altering contractility. To investigate this possibility, rat ventricular cardiomyocytes were activated using TNF-alpha, IL-1beta, or LPS, washed, cultured with quiescent rat polymorphonuclear leukocytes (PMNs) for 4 h, and electrically stimulated to determine fractional shortening. PMNs cultured with activated cardiomyocytes reduced control fractional shortening of 20.5 +/- 0.7% by -2.8 +/- 0.3% per adherent PMN (P < 0.001). Fixing PMNs with paraformaldehyde or glutaraldehyde did not prevent PMN-mediated decreases in cardiomyocyte fractional shortening. However, PMN adherence and decreased fractional shortening were prevented by anti-ICAM-1 and anti-CD18 antibodies. Reduced fractional shortening was reproduced in the absence of PMNs by ICAM-1 binding using cross-linking antibodies (reduced by 36 +/- 3% from control, P < 0.01). Immunofluorescent staining demonstrated increased cortical cytoskeleton-associated focal adhesion kinase expression after ICAM-1 cross-linking, suggesting involvement of the actin cytoskeleton. Indeed, disruption of F-actin filament assembly using cytochalasin D or latrunculin A did not prevent PMN adherence but prevented decreased fractional shortening. Inhibition of the cytoskeleton-associated Rho-kinase pathway with HA-1077 prevented ICAM-1-mediated decreases in cardiomyocyte contractility, further suggesting a central role of the actin cytoskeleton. Importantly, ICAM-1 cross-linking did not alter the total intracellular Ca2+ transient during cardiomyocyte contraction but greatly increased heterogeneity of intracellular Ca2+ release. Thus we have identified a novel regulatory mechanism of cardiomyocyte contractility involving the actin cytoskeleton as a central regulator of the normally highly coordinated pattern of sarcoplasmic Ca2+ release. Cardiomyocyte ICAM-1 binding, by PMNs or other ligands, induces decreased cardiomyocyte contractility via this pathway.     

Dosimetric sensitivity of leaf width on volumetric modulated arc therapy plan quality: an objective approach

BackgroundSeveral authors investigated a dosimetric impact of leaf width on radiotherapy plan quality subjectively, and concluded that thinner leaf-width multileaf collimators (MLC) are beneficial because of their better coverage of clinically relevant structures. Study aimed to investigate the dosimetric effect of MLC leaf width on volumetric modulated arc therapy plan quality by objective approach.Materials and methodsTwelve of each prostate and head-and-neck patients were planned for volumetric modulated arc therapy (VMAT) treatments for MLC leaf widths of 4 mm and 10 mm. Three different VMAT schemes single-arc, dual-arc and two combined independent single-arcs were optimized. Dose volume histogram and Isodose distribution were used for quantitative and qualitative comparison of the treatment plan, respectively. Dose-volume-indices of the planning target volume, organs at risk and number of delivered monitor units were compared. The 4 mm leaf width being reference over 10 mm and results were noted as statistically significant if p ≤ 0.05 using student t-test.ResultsAll VMAT schemes for both tumor sites showed a gain in target coverage, similar organs at risk doses and higher monitor units to be delivered, when changing leaf width from 10 mm to 4 mm. The p-values were significant for majority of head-and-neck dose indices.ConclusionThe thinner-leaf MLCs, owing to their better spatial resolution, result in an overall gain for target coverage, while maintaining permissible doses to the organs at risk.