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Harsh gastric condition causes low bioavailability of probiotics when supplied orally. Polymeric encapsulation has successfully protected bacteria from harsh gastric condition and ultimately increased persistency and multiplication at the targeted region. In this study, we encapsulated LS29 into ACA microcapsules and characterized them. The survivability and release of LS29 from LS29-loaded ACA microcapsules in SGF and SIF were studied. Encapsulation efficiency of LS29 in ACA microcapsules was 99.9%. Approximately 70% of bacteria survived at pH 2 by 120 min after encapsulation. Although not much difference of the survivability of LS29 encapsulated into ACA and FDACA was observed, freeze-drying improved the controlled-release of LS29 in SIF and also showed better storage survivability at 4°C for 8 weeks. Furthermore, investigation of in vitro production of cytokines in RAW264.7 showed high level of induction of TNF-α and IL-10. These in vitro results support that the LS29 might have a balanced immunomodulatory effect. [ABSTRACT FROM AUTHOR]

Salmonellosis is one of the life threating diseases of goats in Bangladesh. Therefore, the present study was designed to study the prevalence of Salmonellosis, and the isolation and characterizations of the Salmonella spp. from apparently healthy and diarrheic goats. A total of 47 faecal samples was collected from selected places and cultured onto different prescribed media to isolate the Salmonella. In this study, 12.76% (6/47) of the samples were found to be positive for Salmonella spp. During culture, all of the Salmonella isolates produced round, smooth, opaque, translucent, and black colored colonies on SS agar medium. All of the isolated Salmonella spp. fermented dextrose, maltose, and mannitol with production of acid and gas but did not ferment sucrose and lactose. However, these isolates had shown negative for the Indole and Voges-Proskauer tests and positive for the Methyl-Red test. All of these isolates were subjected to a rapid plate agglutination test with polyvalent "O" (Poly 'O') and polyvalent "H" (poly 'H') antisera where positive agglutinations were observed. They were highly sensitive to ciprofloxacin, spiramycin, and gentamycin; moderately sensitive to oxytetracyline, streptomycin, and amoxicillin; less sensitive to sulphamethoxazole and resistant to penicillin-G. Based on the present findings, it may be concluded that the investigated Salmonella spp. from goats might be S. typhimurium, S. enteritidis, S. brandenburg, S. salford, S. newbrunswick, S. newport, or S. dublin. It was a preliminary study; therefore, further characterization is required using other serological and molecular techniques. [ABSTRACT FROM AUTHOR]

The enantiopure Schiff bases ( R or S )- N -1-(X-C 6 H 4 )ethyl-2-hydroxy-1-naphthaldimine {X = H [( R or S )-HL1], p -CH 3 O [( R or S )-HL2], and p -Br [( R - or S )-HL3]} react with cobalt(II) acetate to give bis[( R or S )- N -1-(X-C 6 H 4 )ethyl-2-oxo-1-naphthaldiminato-κ 2 N , O ]-Λ/Δ-cobalt(II) {X = H [Λ/Δ-Co-( R or S )-L1], p -CH 3 O [Λ/Δ-Co-( R or S )-L2], and p -Br [Λ/Δ-Co-( R or S )-L3]} ( 1 - 3 ), respectively. Induced Λ and Δ chirality originates at the metal center of the C 2 -symmetric molecule in pseudotetrahedral geometry. Differential scanning calorimetry analyses explored the thermal stability of the complexes, which undergo reversible phase transformation from crystalline solid to isotropic liquid phase for 1 and 3 but irreversible phase transformation for 2 . Like other cobalt(II) complexes, compounds 1 - 3 exhibit a continuous ensemble of absorption and circular dichroism bands, which span from the UV to IR region and can be collected into a superspectrum. Infrared vibrational circular dichroism (IR-VCD) spectra witness the coupling between Co 2+ -centered low-lying electronic states and ligand-centered vibrations. The coupling produces enhanced and almost monosignate VCD spectra, with both effects being mode-dependent in terms of the A or B symmetry (in the C 2 point group) and distance from the Co 2+ core.

Synthetic mRNA represents an exciting cancer vaccine technology for the implementation of effective cancer immunotherapy. However, inefficient in vivo mRNA delivery along with a requirement for immune co-stimulation present major hurdles to achieving anti-tumor therapeutic efficacy. Here, we demonstrate a proof-of-concept adjuvant-pulsed mRNA vaccine nanoparticle (NP) that is composed of an ovalbumin-coded mRNA and a palmitic acid-modified TLR7/8 agonist R848 (C16-R848), coated with a lipid-polyethylene glycol (lipid-PEG) shell. This mRNA vaccine NP formulation retained the adjuvant activity of encapsulated C16-R848 and markedly improved the transfection efficacy of the mRNA (>95%) and subsequent MHC class I presentation of OVA mRNA derived antigen in antigen-presenting cells. The C16-R848 adjuvant-pulsed mRNA vaccine NP approach induced an effective adaptive immune response by significantly improving the expansion of OVA-specific CD8 + T cells and infiltration of these cells into the tumor bed in vivo, relative to the mRNA vaccine NP without adjuvant. The approach led to an effective anti-tumor immunity against OVA expressing syngeneic allograft mouse models of lymphoma and prostate cancer, resulting in a significant prevention of tumor growth when the vaccine was given before tumor engraftment (84% reduction vs. control) and suppression of tumor growth when given post engraftment (60% reduction vs. control). Our findings indicate that C16-R848 adjuvant pulsation to mRNA vaccine NP is a rational design strategy to increase the effectiveness of synthetic mRNA vaccines for cancer immunotherapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Vaccines are one of the greatest medical interventions of all time and have been successful in controlling and eliminating a myriad of diseases over the past two centuries. Among several vaccination strategies, mucosal vaccines have wide clinical applications and attract considerable interest in research, showing potential as innovative and novel therapeutics. In mucosal vaccination, targeting (microfold) M cells is a frontline prerequisite for inducing effective antigen-specific immunostimulatory effects. In this review, we primarily focus on materials engineered for use as vaccine delivery platforms to target M cells. We also describe potential M cell targeting areas, methods to overcome current challenges and limitations of the field. Furthermore, we present the potential of biomaterials engineering as well as various natural and synthetic delivery technologies to overcome the challenges of M cell targeting, all of which are absent in current literature. Finally, we briefly discuss manufacturing and regulatory processes to bring a robust perspective on the feasibility and potential of this next-generation vaccine technology., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a well-characterized tumour-suppressor gene that is lost or mutated in about half of metastatic castration-resistant prostate cancers and in many other human cancers. The restoration of functional PTEN as a treatment for prostate cancer has, however, proven difficult. Here, we show that PTEN messenger RNA (mRNA) can be reintroduced into PTEN-null prostate cancer cells in vitro and in vivo via its encapsulation in polymer-lipid hybrid nanoparticles coated with a polyethylene glycol shell. The nanoparticles are stable in serum, elicit low toxicity and enable high PTEN mRNA transfection in prostate cancer cells. Moreover, significant inhibition of tumour growth is achieved when delivered systemically in multiple mouse models of prostate cancer. We also show that the restoration of PTEN function in PTEN-null prostate cancer cells inhibits the phosphatidylinositol 3-kinase (PI3K)-AKT pathway and enhances apoptosis. Our findings provide proof-of-principle evidence of the restoration of mRNA-based tumour suppression in vivo.

Influenza is a highly contagious viral infection associated with excessive hospitalizations and deaths throughout the world. Continuous antigenic shift and drift is not only responsible for this devastating effect of influenza but also causes ineffectiveness of antiviral drugs and vaccines. In this study, we investigated the effectiveness of ribavirin, oseltamivir, and amantadine drugs in vitro against nine influenza A isolates collected during June 2012-August 2013 from different slums in Dhaka city. The effectiveness of these drugs was determined by measuring the inhibition of virus-induced cytopathic effect on MDCK cells through MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Our data showed that all nine influenza isolates (6 H1N1 pdm09 and 3 H3N2 subtypes) were completely susceptible to ribavirin (The 50% effective concentrations, EC 50 3.0 µg/ml) and oseltamivir (EC 50 0.35 µg/ml). When influenza A infection was challenged with amantadine drug, eight out of nine isolates (88%) demonstrated susceptibility to amantadine drug (EC 50 0.30 µg/ml) while one H1N1 pdm09 isolate exhibited higher EC 50 value (>10 µg/ml) beyond the cell tolerance level of drug (>5 µg/ml). Genetic analysis of transmembrane matrix protein 2 (M2), which is a target for the amantadine drug and vital for viral replication, showed a substitution of amino acid at position 31(S31 N) of that amantadine-resistant isolate indicating the possible reason of amantadine drug resistance.

Gene therapy using recombinant DNA or gene silencing using siRNA have become a prominent area of research in cancer therapy. However, their use in clinical applications is limited due to overall safety concerns and suboptimal efficacy. Although non-viral vectors such as polycationic polymers do not offer the same level of transfection efficiency as their viral counterparts, they still demonstrate immense potential as alternatives to viral vectors, given their versatility, low immunogenicity, ease of large-scale production, and ability to accelerate gene transfer with well-designed delivery platforms. Among these polymers, polyethylenimine (PEI) is considered a state-of-the-art gene carrier, owing to its ability to improve gene transfer capacity and intracellular delivery. Nonetheless, PEI suffers from the critical shortcoming of non-degradability that can lead to severe cytotoxic effects, despite the fact that the level of this toxicity decreases with molecular weight (MW). As a result, a considerable amount of effort has been devoted to designing low-MW PEI derivatives with degradable linkages. This review will categorize the recent advances in these degradable PEI derivatives based on their degradable chemistries, including ester, disulfide, imine, carbamate, amide, and ketal linkages, and summarize their application in gene therapies against various major cancer malignancies.

Aside from kidney transplantation - a procedure which is exceedingly dependent on donor-match and availability leading to excessive costs - there are currently no permanent treatments available which reverse kidney injury and failure. However, kidney-specific targeted gene therapy has outstanding potential to treat kidney-related dysfunction. Herein we report a novel kidney-specific targeted gene delivery system developed through the conjugation of chitobionic acid (CBA) to a polysorbitol gene transporter (PSGT) synthesized from sorbitol diacrylate and low molecular weight polyethylenimine (PEI) carrying hepatocyte growth factor (HGF) gene to alleviate unilateral ureteral obstruction (UUO) in rats. CBA-PSGT performed exceptionally well for targeted delivery of HGF to kidney tissues compared to its non-targeted counterparts (P < 0.001) after systemic tail-vein injection and significantly reduced the UUO symptoms, returning the UUO rats to a normal health status. The kidney-targeted CBA-PSGT-delivered HGF also strikingly reduced various pathologic and molecular markers in vivo such as the level of collagens (type I and II), blood urea nitrogen (BUN), creatinine, and the expressions of ICAM-1, TIMP-1 and α-SMA which play a critical role in obstructive kidney functions. Therefore, CBA-PSGT should be further investigated because of its potential to alleviate UUO and kidney-related diseases using high affinity kidney targeting., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Small interfering RNA (siRNA)-mediated gene silencing represents a promising strategy for treating diseases such as cancer; however, specific gene silencing requires an effective delivery system to overcome the instability and low transfection efficiency of siRNAs. To address this issue, a polysorbitol-based transporter (PSOT) was prepared by low molecular weight branched polyethylenimine (bPEI) crosslinked with sorbitol diacrylate (SDA). Osteopontin (OPN) gene, which is highly associated with non-small cell lung cancer (NSCLC) was targeted by siRNA therapy using siRNA targeting OPN (siOPN). Characterization study confirmed that PSOT formed compact complexes with siOPN and protected siOPN against degradation by RNase. PSOT/siOPN complexes demonstrated low cytotoxicity and enhanced transfection efficiency in vitro, suggesting that this carrier may be suitable for gene silencing. In the A549 and H460 lung cancer cell lines, PSOT/siOPN complexes demonstrated significant silencing efficiency at both RNA and protein levels. To study in vivo tumor growth suppression, two lung cancer cell-xenograft mouse models were prepared and PSOT/siOPN complexes were delivered into the mice through intravenous injection. The siOPN-treated groups demonstrated significantly reduced OPN expression at both the RNA and protein levels as well as suppression of tumor volume and weight. Taken together, siOPN delivery using PSOT may present an effective and novel therapeutic system for lung cancer treatment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNA) are short oligonucleotides of endogenous origin involved in post-transcriptional regulation and are altered in disease, making them potential therapeutic targets. miRNA replacement is necessary in cells with downregulated miRNAs levels in response to disease. miRNA 145 is a novel tumor suppressor gene involved in cell suppression, invasion and migration of cancer cells; it is downregulated in most cancers. Delivery of therapeutic miRNA using nanoparticles enhances the chances of successful delivery and expression of genes at the target site. We evaluated polysorbitol-mediated transporter (PSMT) in the cellular delivery of miRNA 145. The polysorbitol backbone possesses osmotic properties and leads to enhanced cellular uptake. PSMT delivers genes into cells by a caveolae-mediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Physicochemical characterization, and the transfection efficiency and transgene expression capability of PSMT/reporter plasmid DNA nanoparticles, were determined. GFP-tagged miRNA 145 delivery with PSMT was confirmed by confocal microscopy and Western blotting. The functional effects of miRNA 145 delivered with PSMT were analyzed by confocal microscopy, as well as in apoptosis, proliferation and wound healing assays. Finally, the expression of an miRNA 145 target protein, c-myc, was determined by Western blotting after intracellular delivery of PSMT/miRNA 145 nanoparticle (NP)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chitosan on its own is a well-established natural polymer and is widely regarded as a biodegradable, biocompatible and nontoxic material for drug delivery applications. Although unmodified chitosan has some mucoadhesive properties on its own, its bioavailability is limited due to its short retention time in the body. Moreover, the high solubility of chitosan at acidic pH levels limits its use for mucosal drug delivery (especially through the oral route). Chemically-modified mucoadhesive chitosan, especially thiolated chitosan, has arisen as an alternative to create novel mucosal drug delivery systems. The mucoadhesive properties that are conferred to the thiolated chitosan certainly set this novel class of second or third-generation thiomers apart. To understand the significance of mucoadhesive chitosan, we first present the mechanism of mucoadhesion and provide comprehensive coverage of description of a variety of chemical modifications to prepare mucoadhesive thiolated chitosan derivatives. We then present the plethora of applications of these modified chitosan variants in a wide range of drug delivery fields, including the delivery of antigens, proteins and genes through a variety of routes, including oral, nasal, pulmonary, vaginal and others. By presenting the range of applications for mucoadhesive chitosan drug carriers we herein demonstrate that chemically-modified thiolated chitosan is a versatile and effective material for a new class of drug delivery vehicles.

Non-viral gene delivery systems are one of the most potential alternatives to viral vectors because of their less immunogenicity, less toxicity and easy productivity in spite of their low capacity of gene transfection using DNA or silencing using siRNA compared to that of viral vectors. Among non-viral systems, the polycationic derivatives are the most popular gene carriers since they can effectively condense nucleic acids to transfer into the cells, especially the polyethylenimine (PEI) which has been used as a golden standard polymer owing to its high buffering ability for endosomal escape of gene to be expressed. However, PEI has severe problems for its toxicity due to the high positive charge density and non-degradability although the toxicity of PEI depends on its molecular weight (MW) and structure. Therefore, a considerable attention has been paid on synthesis of degradable PEI derivatives using low MW one because low MW PEI is much less toxic than high MW PEI. Other degradable polycationic gene carriers such as polyamidoamines (PAA) and cyclodextrin (CD)-based polycations are also in a significant interest because of their high transfection efficiency with low toxicity. This review in detail explains the recent developments on these three major degradable polycations as promising carriers for deoxyribonucleic acid (DNA) and small interfering RNA (siRNA)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Respiratory syncytial virus (RSV) is one of the most common causes of viral deaths in infants worldwide, yet no effective vaccines are available. Here, we report an osmotically active polysaccharide-based polysorbitol transporter (PST) prepared from sorbitol diacrylate and low-molecular-weight polyethylenimine (PEI) showing a potent, yet safe, adjuvant activity and acting as an effective delivery tool for RSV glycoprotein (RGp) antigen. PST showed no toxicity in vitro or in vivo, unlike PEI and the well-known experimental mucosal adjuvant cholera toxin (CT). PST formed nano-sized complexes with RGp by simple mixing, without affecting antigenic stability. The complexes exhibited negative surface charges that made them highly efficient in the selective activation of phagocytic cells and enhancement of phagocytic uptake. This resulted in an improved cytokine production and in the significant augmentation of RGp-specific antibody production, which persisted for over 200 days. Interestingly, PST/RGp enhanced phagocytic uptake owing to the osmotic property of PST and its negative zeta potential, suggesting that PST could selectively stimulate phagocytic cells, thereby facilitating a long-lived antigen-specific immune response, which was presumably further enhanced by the polysaccharide properties of PST., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Polysorbitol-mediated transporter (PSMT) has been previously shown to achieve high transfection efficiency with minimal cytotoxicity. Polysorbitol backbone possesses osmotic properties and leads to enhanced cellular uptake. The PSMT/pDNA nanoparticles were prepared and the particle size, surface charge of the nanoparticles was determined for the study. PSMT delivers genes into cells by the caveolae mediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Transfection through the caveolae may help PSMT to selectively transfect cancer cells rather than normal cells. Transfection of the luciferase gene by PSMT was tested in various cell types including cancer cell lines, primary cells, and immortalized cells. Luciferase transgene expression mediated by PSMT was remarkably increased in HeLa cells compared to expression using the control carrier Lipofectamine. Moreover, the toxicity of PSMT was comparable to the control carrier (Lipofectamine) in the same cells. Selective transfection of cancer cells using PSMT was further confirmed by co-culture of cancer and normal cells, which showed that transgene expression was pre-dominantly achieved in cancer cells. A functional p53 gene was also delivered into HeLa cells using PSMT and the selective transgene expression of p53 protein in cancer cells was analyzed through western blotting and confocal microscopy. HeLa cells transfected with PSMT/p53 plasmid nanoparticles showed cellular damage and apoptosis, which was confirmed through propidium iodide staining., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Treating cancer patients by conventional chemotherapy to achieve prolonged survival still remains complicated. Autophagy is a topic of considerable interest in recent times, as it may contribute greatly to tumor suppression. Recent studies indicate that autophagy-deficient cells accumulate high levels of p62, an ubiquitin-binding scaffold protein, involved greatly in tumorigenesis. Here, we synthesized an osmotically active polysorbitol-mediated transporter (PSMT) to downregulate p62 using an RNAi strategy and described the mechanism of how p62 silencing using PSMT/siRNA p62 system activates autophagy and contributes to tumor suppression in the lungs of K-ras(LA1) mice. Downregulation of p62 by PSMT/siRNA p62 activated autophagy confirmed by the formation of autophagosomes and swelling of Golgi apparatus with a decreasing level of GM130, a cis-Golgi matrix protein. Activation of osmotic PSMT-mediated autophagy remarkably reduced the size and number of tumors by suppressing proliferative cell nuclear antigen, cluster of differentiation 31, and vascular endothelial growth factor levels. Furthermore, an increase in apoptosis was observed in the lungs of PSMT/siRNA p62-delivered K-ras(LA1) mice., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Non-viral carriers, able to regulate cellular uptake pathways are becoming promising vectors for efficient gene transfer into cells because the intracellular processing of gene is strongly determined by the internalization pathways and subsequent intracellular routes. The intracellular processing of gene can be directed either to digestive or non-digestive way depending on the endocytosis mechanism. Enormous attempts have been made to bypass or avoid the digestive way of lysosomal degradation for meaningful delivery of therapeutic genes at the target site. This review describes an updated information regarding regulation of endocytosis pathways in consequence with the cellular trafficking and the determinant factors of the initial mode of internalization route for rational design of non-viral gene carriers. Firstly, we describe various nonviral gene vectors and the key barriers to non-viral gene transfer. Secondly, various major endocytosis pathways and their significance in developing non-viral gene carriers are discussed. Thirdly, various factors that potentially regulate endocytosis pathways of gene carriers to improve gene delivery efficacy are elaborately described. Lastly, the perspectives of future studies to control endocytosis mechanisms are discussed to design potential non-viral gene vectors for active application in future.

The aim of the present study was to investigate the age-related production variation of T helper (Th)-type cytokines (IL-2, IL-4, IFN-γ and IL-10), granulocyte macrophage-colony stimulating factor (GM-CSF) and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated porcine alveolar macrophages (AMs) in a time-dependent manner. For this purpose, AMs were isolated from 5-days (newborn), 40-days (post-weaned) and 120-days (young) old pigs. Cells were incubated for 24h in the absence or presence of increasing concentrations of LPS (0.0, 0.01, 1.0, 5.0 and 10.0 μg/mL). IL-10, IFN-γ and GM-CSF mRNA expression was upregulated in a dose-dependent manner for all age groups (P<0.05). Age-related differences included a significantly increased IL-10 mRNA and protein production in newborn piglets compared to post-weaned and young pigs. IL-10 production pattern was similar with a higher peak between 12 and 36 h post-induction in all age groups. In contrast, IFN-γ mRNA and protein level was significantly elevated in young pigs 12h and 24h post-induction, respectively, while the time course production of IFN-γ was mostly consistent in newborn and post-weaned piglets. GM-CSF mRNA expression was significantly lower in newborn piglets than in post-weaned and young pigs. The kinetic of GM-CSF expression peaked at 12h in young and post-weaned pigs and at 24h in newborn piglets. IL-4 mRNA levels were very low and no apparent change of IL-2 expression was observed following LPS stimulation in all age groups. Only very low levels of NO were detected in the cell supernatants of young pigs. Collectively, these studies suggest age-related differences in time-dependent production of IL-10, IFN-γ and GM-CSF by porcine AMs with potential immunoregulatory consequences to be explored further., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs., Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study., Conclusion: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

The aim of the present study was to determine the age-related changes of phagocytic capacity and the kinetic production of cytokines in lipopolysaccharide-stimulated porcine alveolar macrophages. For this purpose, AMs were isolated from 5 (newborn), 40 (post-weaned) and 120 (young) day old pigs. Results of phagocytosis assay showed that AMs from newborn piglets had less phagocytic capacity than those of young pigs (P<0.05). For the kinetics study, cells and supernatant were collected at 1, 6, 12, 24, 36 and 48 h after LPS stimulation for quantification of cytokine mRNA and protein by quantitative real-time PCR and ELISA, respectively. The kinetics results showed that AMs from newborn piglets were significantly less capable of producing IL1β, IL6, IL12β, TNFα and IL8 than post-weaned piglets or young pigs. IL18 mRNA did not show significant differences between ages. MIP2 and MCP1 mRNA was higher in young pigs. Hence, higher production of cytokines by AMs may be the surfactant factors in the pulmonary host defense system. These results indicate that AMs from newborn piglets might be functionally immature, which may lead to increased susceptibility to lung infections. Future studies of cytokine kinetics in more animals are clearly needed to confirm these results across a wider age range., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Polymeric diversity allows us to design gene carriers as an alternative to viral vectors, control cellular uptake, target intracellular molecules, and improve transfection and silencing capacity. Recently, we developed a polysorbitol-based osmotically active transporter (PSOAT), which exhibits several interesting mechanisms to accelerate gene delivery into cells. Herein, we report the efficacy of using the PSOAT system for small interfering RNA (siRNA) delivery and its specific mechanism for cellular uptake to accelerate targeted gene silencing. We found that PSOAT functioned via a caveolae-mediated uptake mechanism due to hyperosmotic activity of the transporter. Moreover, this selective caveolae-mediated endocytosis of the polyplexes (PSOAT/siRNA) was regulated coincidently with the expression of caveolin (Cav)-1 and cyclooxygenase (COX)-2. Interestingly, COX-2 expression decreased dramatically over time due to degradation by the constant expression of Cav-1, as confirmed by high COX-2 expression after the inhibition of Cav-1, suggesting that PSOAT-mediated induction of Cav-1 directly influenced the selective caveolae-mediated endocytosis of the polyplexes. Furthermore, COX-2 expression was involved in the initial phase for rapid caveolae endocytic uptake of the particles synergistically with Cav-1, resulting in accelerated PSOAT-mediated target gene silencing., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Controlling the cellular uptake mechanism and consequent intracellular route of polyplexes is important to improve the transfection efficiency of the non-viral gene delivery. Here, we report a new non-viral vector, polymannitol-based gene transporter (PMT), generated by crosslinking low molecular weight polyethylenimine with mannitol diacrylate, which has low cytotoxicity and good transfection efficiency. Interestingly, the uptake pathway of PMT/DNA complexes was shifted into caveolae-mediated endocytosis, avoiding lysosomal degradation. The mechanism of increased caveolae-mediated endocytosis of PMT/DNA complexes was found to be correlated with mechanosensing signal transduction by the hyperosmotic polymannitol part. Our results suggested that PMT, polymannitol-based gene transporter, is a safe and efficient gene delivery system with a well-modulated uptake pathway and intracellular route for gene therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Toll-like receptor (TLR)4 is critical for the recognition of Gram-negative bacterial lipopolysaccharide (LPS) but in porcine peripheral blood mononuclear cells (PBMCs) it may cooperate with other TLRs and lead to the production of inflammatory cytokines. Therefore, we analyzed TLR1-10 mRNA expression in porcine PBMCs stimulated with LPS over time (1-48 h) by using quantitative real-time PCR and cytokine proteins level by ELISA in culture supernatant. TLR1-10 mRNA was detectable in porcine PBMCs. When compared with the control (non-stimulated), TLR1 mRNA were increased (p<0.05) at 3 h after challenge with 1 μg/ml LPS, whereas TLR1 and TLR2 mRNA were increased (p<0.01) at 6 h after challenge with 10 μg/ml LPS. TLR4 increased (p<0.001) at 3h after challenge with LPS and remained constant. TLR5 and TLR6 mRNA increased (p<0.05) at 9 h and 1 h after of LPS stimulation, respectively. The mRNA of CD14 and MD2 were increased (p<0.001) at 1h after LPS stimulation. Additionally, at most of the time analyzed, the mRNA expression increased with the dose of LPS. The LPS concentration had influence (p<0.05) on all the TLRs expression except TLR10; whereas time had effect (p<0.05) on all TLRs expression except TLR2, 3, 6 and 10. When compared to the control, the cytokines IL1b, IL8 and TNFα proteins were increased (p<0.001) immediately at 1 h after LPS stimulation and remained constant till 48 h. IL12b was increased (p<0.001) 12 h after challenge with 10 μg/ml of LPS. Although IL8 level was the highest, the higher (p<0.05) expression of all these inflammatory cytokines indicate that upon interacting with TLRs, LPS exerted inflammatory response in PBMCs through the production of Th1 type cytokines. The production of cytokines was influenced (p<0.001) by both the dose of LPS and the stimulation time. Hence, the porcine PBMCs are likely able to express all members of TLRs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A vaccine delivery system based on thiolated eudragit microsphere (TEMS) was studied in vivo for its ability to elicit mucosal immunity against enterotoxigenic Escherichia coli (ETEC). Groups of mice were orally immunized with F4 or F18 fimbriae of ETEC and F4 or F18 loaded in TEMS. Mice that were orally administered with F4 or F18 loaded TEMS showed higher antigen-specific IgG antibody responses in serum and antigen-specific IgA in saliva and feces than mice that were immunized with antigens only. In addition, oral vaccination of F4 or F18 loaded TEMS resulted in higher numbers of IgG and IgA antigen-specific antibody secreting cells in the spleen, lamina propria, and Peyer's patches of immunized mice than other groups. Moreover, TEMS administration loaded with F4 or F18 induced mixed Th1 and Th2 type responses based on similarly increased levels of IgG1 and IgG2a. These results suggest that F4 or F18 loaded TEMS may be a promising candidate for an oral vaccine delivery system to elicit systemic and mucosal immunity against ETEC., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range., (Copyright © 2012 Elsevier B.V. All rights reserved.)