The effect of PCSK9 immunization on the hepatic level of microRNAs associated with the PCSK9/LDLR pathway

IntroductionMicroRNAs (miRNAs) are a class of gene expression epigenetic regulators that play roles in regulating genes involved in cholesterol homeostasis, including low-density lipoprotein receptor (LDLR) and PCSK9; therefore, miRNAs have been suggested as potential therapeutic targets for treating cardiometabolic disorders. Thus, the present study aimed to assess the effect of immunotherapy with the PCSK9 peptide vaccine on the hepatic expression levels of microRNAs associated with the LDLR pathway, including miRNA-27a, miRNA-30c, and miRNA-191, in normal vaccinated mice.Material and methodsPCSK9 immunogenic peptide and 0.4% alum adjuvant were mixed at a 1 : 1 ratio and used as a vaccine formulation. Male albino mice were randomly assigned to the vaccine or control group. Mice in the vaccine group were injected four times at two-week intervals with a PCSK9 peptide vaccine, and mice in the control group were injected with phosphate-buffered saline (PBS). Animal livers were sampled 2 weeks after the last injection to assess miRNA expression levels. The hepatic expression levels of miRNA-27a, miRNA-30c, and miRNA-191 were evaluated by SYBR Green real-time PCR, quantified by a comparative (2^–ΔΔCT) method (fold change (FC)) and normalized to U6 small nuclear RNA (U6snRNA) expression as an internal control.ResultsThe hepatic expression level of miRNA-27a was significantly lower in mice following immunotherapy with the PCSK9 peptide vaccine compared to the control group (FC: 0.731 ±0.1, p = 0.027). Also, there was a borderline significantly lower hepatic expression level of miRNA-30c in the vaccinated group compared to the control (FC: 0.569 ±0.1, p = 0.078). However, no significant differences were found in the hepatic expression level of miRNA-191 between the two studied groups (FC: 0.852 ±0.1, p = 0.343).ConclusionsAccording to the findings, the PCSK9 peptide vaccine could effectively reduce the hepatic expression level of miRNA-27a and may be helpful in the management of LDL-C level and atherosclerosis, which may be mediated through the LDLR pathway.     

In vivo regeneration of bladder muscular wall using decellularized colon matrix: an experimental study

Background

Finding a proper scaffold for augmentation is a serious challenge in bladder tissue engineering. We hereby aimed to determine the histological aspects of a decellularized colon graft for bladder augmentation in healthy rats.

Methods

Rat colon tissues were decellularized using perfusion-based method. After partial cystectomy, bladders were grafted with a patch of decellularized colon. Bladder specimens were investigated in 12 rats at 1, 3, and 9 months postoperatively for further histological changes and immunohistochemistry analyses were also performed.

Results

One month after implantation, partial seeding of new cells was observed. After 3 months continuity of transitional epithelium of natural bladder on the decellularized grafted colon tissue was confirmed with histological and immunohistochemical examinations. All augmented bladders demonstrated a spherical shape without stone formation, necrosis or graft rejection. The presence of urothelium with similar morphology to the natural urothelium and visible blood vessels were found within 3 months of operation. All immunohistochemical markers (except markers of colonic stem cells) were expressed in biopsies obtained 3 months after surgery demonstrating a progressive vascular and smooth muscle cell infiltration into the graft after implantation.

Conclusion

This study suggests that decellularized colon may provide a viable material for bladder augmentation in rats to pave the road for future applications of this natural collagen scaffold.

     

Safety and protective efficacy of intramuscular vaccination with a live aroA derivative of Pasteurella multocida B:2 against experimental hemorrhagic septicemia in calves

Three groups of five calves, namely, V1, V2, and V3, were immunized intramuscularly at 4 and 8 weeks of age with ca. 10⁹, 10⁸, and 10⁷ CFU, respectively, of a derivative of Pasteurella multocida B:2 wild-type strain 85020 containing a deletion in the aroA gene (strain JRMT12). The first and second vaccinations resulted in significantly (P < 0.01) higher rectal temperature responses in groups V1 and V2 than in group V3. Serum immunoglobulin M (IgM) and IgG titers did not increase in any group until after the second vaccination and were then significantly higher in groups V1 and V2 than in group V3 (P = 0.001 for both IgM and IgG). All vaccinated groups and three unvaccinated challenge control calves (group CC) were injected subcutaneously at 10 weeks of age with ca. 10⁷ CFU of strain 85020. Vaccinated calves survived the challenge, but two CC animals developed clinical disease and were killed for humane reasons. After challenge, mean serum amyloid A concentrations were significantly higher (P < 0.001) in the CC group than in the vaccinated groups. Postmortem examination revealed that calves in the CC group showed the most extensive range of bacteriologically positive tissues and gross and histopathological lesions. Overall, a clear dose-dependent response was present, with those receiving a higher vaccine dose being less affected clinically, bacteriologically, and pathologically by the wild-type challenge. The V2 treatment appeared to give the best combination of high immune response, protection, and safety.     

Early detection of acute kidney injury by serum cystatin C in critically ill children

Background

We prospectively evaluated whether serum cystatin C (CysC) detected acute kidney injury (AKI) earlier than basal serum creatinine (Cr).

Methods

In 107 pediatric patients at high risk of developing AKI, serum Cr and serum CysC were measured upon admission. Baseline estimated creatinine clearance (eCCl) was calculated using a CysC-based glomerular filtration rate (GFR) equation from a serum Cr measured at the pediatric intensive care unit (PICU) entrance.

Results

The median age was 10 months (interquartile range, 3–36 months). Serum Cr, serum CysC, and eCCl (mean ± standard deviation [range]) were 0.5?±?0.18 mg/dl (0.2–1.1 mg/dl), 0.53?±?0.78 (0.01–3.7 mg/l), and 72.55 ± 28.72 (20.6–176.2) ml/min per 1.73 m², respectively. The serum CysC level in patients with AKI was significantly higher than children with normal renal function (p?<?0.001). The values for the cut-off point, sensitivity, specificity, and the area under curve (AUC) were determined for CysC as 0.6 mg/l, 73.9 %, 78.9 %, and 0.92 [95 % confidence interval (0.82–1)], respectively, and for Cr the values were 0.4 mg/dl, 68 %, 46.2 %, and 0.39, [95 % confidence interval (0.24–0.54)], respectively. The receiver operating characteristics (ROC) curve analysis revealed that CysC had a significantly higher diagnostic accuracy than eCCl (p?<?0.001).

Conclusions

Our results identify that the sensitivity of serum CysC for detecting AKI is higher than that of serum Cr in a heterogeneous pediatric intensive care unit (PICU) population.     

Hepatitis D virus infection in Isfahan, central Iran: Prevalence and risk factors among chronic HBV infection cases

Background

Hepatitis D virus (HDV) is dependent on hepatitis B virus (HBV) infection. Acute infection with HDV can occur simultaneously with acute HBV infection or be superimposed onto a chronic HBV infection.

Objectives

This study aimed to identify cases of HDV and determine its prevalence in patients with chronic HBV infection for the first time study in Isfahan, central Iran.

Patients and Methods

In a cross-sectional study in 2009, 346 who had been diagnosed for at least 6 months with chronic HBV were enrolled consecutively. Anti-HDV was measured by ELISA in the serum of these patients.

Results

The study included 245 males (70.8%) and 101 (29.2%) females with a mean age of 39 ± 12.4 years. Anti-HDV was present in 8 (3.5%) HBe antibody-positive patients (p = 0.36) and in 2 (2.3%) HBe antigen-positive cases (p = 0.68). No association was found between hepatitis D and probable risk factors.

Conclusions

This study demonstrates that the prevalence of HDV infection is higher in patients who are positive for HBeAb compared those who are HBeAg-positive. Therefore, most HDV antibody-positive cases in Isfahan are HBV/HDV superinfections but not coinfections.     

MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells

Background

Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis.

Methods

Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays.

Results

We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC-derived exosomes on the expression of VEGF in breast cancer-derived cells. In addition, we found that down-regulation of VEGF mediated by MSC-derived exosomes can affect the vascular behavior of endothelial cells in vitro.

Conclusions

Overall, our findings suggest that exosomal transfer of miR-100 may be a novel mechanism underlying the paracrine effects of MSC-derived exosomes and may provide a means by which these vesicles can modulate vascular responses within the microenvironment of breast cancer cells.

     

Efficacy of vaccination of calves against hemorrhagic septicemia with a live aroA derivative of Pasteurella multocida B:2 by two different routes of administration

Two groups of four calves each were immunized either intramuscularly (i.m. vaccinated) or intranasally (i.n. vaccinated) at 2 and 6 weeks of age with ca. 10(9) CFU of a derivative of P. multocida serotype B:2 strain 85020 containing a deletion in the aroA gene (strain JRMT12). Both groups of calves and three unvaccinated control calves were challenged subcutaneously at 8 weeks of age with ca. 10(7) CFU of the wild-type 85020 strain. The first and second vaccinations caused a significant pyrexia and increase in the mean demeanor score (P <0.05) in i.m. but not i.n. vaccinated calves. Serum agglutinating activity against whole cells of P. multocida strain 85020 and immunoglobulin G antibody concentrations increased after the second vaccination in i.m. but not in i.n. vaccinated animals, and this difference was statistically significant (P <0.05). Concentrations of serum amyloid A (SAA) increased significantly 3 h after both the primary (P <0.05) and booster (P <0.001) i.m. vaccinations, but not in i.n. vaccinated calves. All four i.m. vaccinated calves were solidly immune to challenge with wild-type P. multocida B:2. However, the mean rectal temperatures, demeanor scores, and serum SAA concentrations of i.n. vaccinated and control calves increased significantly (P <0.01). Three i.n. vaccinated and two control calves were killed for humane reasons within 14 h postchallenge, and postmortem examination revealed pathological lesions consistent with hemorrhagic septicemia. These data showed that the aroA mutant strain, given i.m. as two doses 4 weeks apart, acted as an effective live-attenuated vaccine strain to protect calves against challenge with the virulent parent strain.