An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult-they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

Sustained release of recombinant surface antigen 2 (rSAG2) from poly(lactide-co-glycolide) microparticles extends protective cell-mediated immunity against Toxoplasma gondii in mice.

SUMMARY Current development efforts of subunit vaccines against Toxoplasma gondii, the aetiological agent of toxoplasmosis, have been focused mainly on tachyzoite surface antigens (SAGs) such as SAG2, due to their attachment roles in the process of host-cell invasion. In the present study, we aimed to produce poly(lactide-co-glycolide) (PLG) microparticles (MPs) containing recombinant SAG2 (rSAG2) to induce improved immunity against T. gondii. The resulting PLG-encapsulated rSAG2 (PLG-rSAG2) MPs, 2·14-3·63 µm in diameter, showed 74-80% entrapment efficiency and gradually released antigenic rSAG2 protein (88·3% of the total protein load) for a long 33-day period. Peritoneal immunization with PLG-rSAG2 MPs in BALB/c mice resulted in not only sustained (10 weeks) lymphocyte proliferation and IFN-γ production but also an improved protective capacity (87%) against a lethal subcutaneous challenge of 1×104 live tachyzoites of T. gondii (RH strain). In conclusion, the sustained release of rSAG2 protein from PLG-rSAG2 MPs extends Th1 cell-mediated immunity (lymphocyte proliferation and IFN-γ production) and induces improved protection against T. gondii tachyzoite infection in mice.

Encapsulation of chimeric protein rSAG1/2 into poly(lactide-co-glycolide) microparticles induces long-term protective immunity against Toxoplasma gondii in mice.

In the present study, the poly(lactide-co-glycolide) (PLG) polymer was used as an adjuvant to encapsulate the chimeric protein rSAG1/2 for preparing a microparticle vaccine against Toxoplasma gondii. The resulting PLG-encapsulated rSAG1/2 (PLG-rSAG1/2) microparticles, 1.27-1.65µm in diameter, showed 72-83% entrapment efficiency. The amount of released rSAG1/2 protein from microparticles gradually increased over a long 56-day period and the protein still retained its antigenicity. Intraperitoneal immunization with the microparticles in BALB/c mice elicited significant long-term (10weeks) humoral and cell-mediated immune responses, accompanied by secretion of a large amount of IFN-γ, to achieve strong protection (83%) against a lethal subcutaneous tachyzoite challenge. In conclusion, we have successfully encapsulated rSAG1/2 with the PLG polymer to produce stable microparticles that can effectively induce not only long-term immunity but also high protection against T. gondii. These crucial data would be advantageous for developing long-term Toxoplasma vaccines for future use in humans and animals.

The Role of Mitochondrial Metabolism, AMPK-SIRT Mediated Pathway, LncRNA and MicroRNA in Osteoarthritis.

Osteoarthritis (OA) is the most common joint disease characterized by degeneration of articular cartilage and causes severe joint pain, physical disability, and impaired quality of life. Recently, it was found that mitochondria not only act as a powerhouse of cells that provide energy for cellular metabolism, but are also involved in crucial pathways responsible for maintaining chondrocyte physiology. Therefore, a growing amount of evidence emphasizes that impairment of mitochondrial function is associated with OA pathogenesis; however, the exact mechanism is not well known. Moreover, the AMP-activated protein kinase (AMPK)-Sirtuin (SIRT) signaling pathway, long non-coding RNA (lncRNA), and microRNA (miRNA) are important for regulating the physiological and pathological processes of chondrocytes, indicating that these may be targets for OA treatment. In this review, we first focus on the importance of mitochondria metabolic dysregulation related to OA. Then, we show recent evidence on the AMPK-SIRT mediated pathway associated with OA pathogenesis and potential treatment options. Finally, we discuss current research into the effects of lncRNA and miRNA on OA progression or inhibition.

The Role of Autophagy in Osteoarthritic Cartilage.

Osteoarthritis (OA) is one of the most common diseases leading to physical disability, with age being the main risk factor, and degeneration of articular cartilage is the main focus for the pathogenesis of OA. Autophagy is a crucial intracellular homeostasis system recycling flawed macromolecules and cellular organelles to sustain the metabolism of cells. Growing evidences have revealed that autophagy is chondroprotective by regulating apoptosis and repairing the function of damaged chondrocytes. Then, OA is related to autophagy depending on different stages and models. In this review, we discuss the character of autophagy in OA and the process of the autophagy pathway, which can be modulated by some drugs, key molecules and non-coding RNAs (microRNAs, long non-coding RNAs and circular RNAs). More in-depth investigations of autophagy are needed to find therapeutic targets or diagnostic biomarkers through in vitro and in vivo situations, making autophagy a more effective way for OA treatment in the future. The aim of this review is to introduce the concept of autophagy and make readers realize its impact on OA. The database we searched in is PubMed and we used the keywords listed below to find appropriate article resources.

Intra-Articular Injection of (-)-Epigallocatechin 3-Gallate (EGCG) Ameliorates Cartilage Degeneration in Guinea Pigs with Spontaneous Osteoarthritis.

Osteoarthritis (OA) is the most prevalent joint disease that causes an enormous burden of disease worldwide. (-)-Epigallocatechin 3-gallate (EGCG) has been reported to reduce post-traumatic OA progression through its anti-inflammatory property. Aging is the most crucial risk factor of OA, and the majority of OA incidences are related to age and not trauma. In this study, we assess whether EGCG can ameliorate cartilage degradation in primary OA. In an in-vitro study, real-time PCR was performed to assess the expression of genes associated with human articular chondrocyte homeostasis. A spontaneously occurring OA model in guinea pigs was used to investigate the effect of EGCG in vivo. OA severity was evaluated using Safranin O staining and Osteoarthritis Research Society International (OARSI) scores, as well as by immunohistochemical (IHC) analysis to determine the protein level of type II collagen (Col II), matrix metalloproteinase 13 (MMP-13), and p16 ink4a in articular cartilage. In the in-vitro study, EGCG increased the gene expression of aggrecan and Col II and decreased the expression of interleukin-1, cyclooxygenase 2, MMP-13, alkaline phosphatase, Col X, and p16 Ink4a; EGCG treatment also attenuated the degraded cartilage with a lower OARSI score. Meanwhile, IHC results showed that EGCG exerted an anti-OA effect by reducing ECM degradation, cartilage inflammation, and cell senescence with a less-immunostained Col II, MMP-13, and p16 Ink4a. In conclusion, these findings suggest that EGCG may be a potential disease-modifying OA drug for the treatment of primary OA.

PCR Detection and Phylogenetic Analysis of Megalocytivirus Isolates in Farmed Giant Sea Perch Lates calcarifer in Southern Taiwan.

The Megalocytivirus genus includes three genotypes, red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV), and has caused mass mortalities in various marine and freshwater fish species in East and Southeast Asia. Of the three genotypes, TRBIV-like megalocytivirus is not included in the World Organization for Animal Health (OIE)-reportable virus list because of its geographic restriction and narrow host range. In 2017, 39 cases of suspected iridovirus infection were isolated from fingerlings of giant sea perch (Lates calcarifer) cultured in southern Taiwan during megalocytivirus epizootics. Polymerase chain reaction (PCR) with different specific primer sets was undertaken to identify the causative agent. Our results revealed that 35 out of the 39 giant sea perch iridovirus (GSPIV) isolates were TRBIV-like megalocytiviruses. To further evaluate the genetic variation, the nucleotide sequences of major capsid protein (MCP) gene (1348 bp) from 12 of the 35 TRBIV-like megalocytivirus isolates were compared to those of other known. High nucleotide sequence identity showed that these 12 TRBIV-like GSPIV isolates are the same species. Phylogenetic analysis based on the MCP gene demonstrated that these 12 isolates belong to the clade II of TRBIV megalocytiviruses, and are distinct from RSIV and ISKNV. In conclusion, the GSPIV isolates belonging to TRBIV clade II megalocytiviruses have been introduced into Taiwan and caused a severe impact on the giant sea perch aquaculture industry.

Protective Immunity against Vibrio harveyi in Grouper Induced by Single Vaccination with Poly (Lactide-co-glycolide) Microparticles Releasing Pleurocidin Peptide and Recombinant Glyceraldehyde-3-phosphate Dehydrogenase.

The peptide adjuvant, pleurocidin (PLE), and the Vibrio harveyi antigen, recombinant glyceraldehyde-3-phosphate dehydrogenase (rGAPDH) protein, were encapsulated with poly (lactide-co-glycolide) (PLG) polymers in our previous study to produce PLG-encapsulated PLE plus rGAPDH microparticles (PLG-PLE/rGAPDH MPs) that sustained stable release of both PLE and rGAPDH as well as, after two-time vaccination with MPs, generated long-term protective immunity against V. harveyi in grouper. Stable controlled-release of PLE plus rGAPDH from PLG-PLE/rGAPDH MPs is an attractive feature for developing an effective single-dose vaccine. In the present study, therefore, we aim to evaluate whether single administration with PLG-PLE/rGAPDH MPs in grouper would result in protective immunity against V. harveyi. Peritoneal vaccination of grouper with one dose of PLG-PLE/rGAPDH MPs raised serum titers over a long 12-week period. Moreover, twelve weeks after vaccination, significant lymphocyte proliferation and maximum TNF-α production were found in grouper immunized with a single dose of PLG-PLE/rGAPDH MPs. More importantly, immune responses elicited by single vaccination with PLG-PLE/rGAPDH MPs protected 80% of fish against a lethal peritoneal challenge of the highly virulent V. harveyi (Vh MML-1). In conclusion, our data truly reveal the feasibility of the development of a single-dose vaccine against V. harveyi based on PLG-PLE/rGAPDH MPs.

Anti-Idiotype Vaccine Provides Protective Immunity Against Vibrio Harveyi in Grouper (Epinephelus Coioides).

Since anti-idiotype antibodies (anti-Id Abs) can display internal images similar to the epitopes of the original antigens, we aimed to produce an effective vaccine based on anti-Id Abs to protect grouper from Vibrio harveyi. Anti-Id IgG showing V. harveyi-like internal images was produced from rabbits immunized with the Id portion of grouper anti-V. harveyi antibodies and its Fab portion, anti-Id IgG (Fab), was then prepared to use as the anti-Id vaccine. The resulting anti-Id IgG (Fab) was intraperitoneally injected twice at a 21-day interval into grouper to evaluate its ability to induce effective anti-V. harveyi immunity and protection, in comparison with inactivated V. harveyi bacteria. We found that administration of grouper with anti-Id IgG (Fab) resulted in enhanced V. harveyi-specific serum titers, as well as lymphocyte proliferation. In addition, three weeks after boosting, 90% (18/20) of fish immunized with anti-Id IgG (Fab) survived at least 28 days after a lethal challenge of the heterologous, virulent strain of V. harveyi. The capability of this anti-Id IgG (Fab) to imitate the epitopes of V. harveyi antigens and effectively induce protective immunity would be advantageous for its application in developing an efficacious vaccine against V. harveyi for future farm use in fish.

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes.

BACKGROUND: Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. METHODS: We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164) of FMDV VP1. RESULTS: The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-gamma. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. CONCLUSION: Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

Protective immunity against toxoplasmosis in mice induced by single-dose immunization with rSAG1/2 protein released from poly(lactide-co-glycolide) microparticles.

Triphasic sustained release of tachyzoite chimeric protein, rSAG1/2, from poly(lactide-co-glycolide) (PLG)-encapsulated rSAG1/2 (PLG-rSAG1/2) microparticles (MPs) is a promising characteristic for developing a single-dose vaccine against Toxoplasma gondii in domestic animals. In the present study, we aimed to evaluate whether single immunization with PLG-rSAG1/2 MPs in BALB/c mice would achieve effective immunity and protection against T. gondii. Peritoneal immunization of mice with a single dose of PLG-rSAG1/2 MPs enhanced serum IgG titers and lymphocyte proliferation in a triphasic model over a long 12-week period. In addition, 12 weeks after immunization, significant production of IFN-γ was also monitored in mice vaccinated with one dose of PLG-rSAG1/2 MPs. More importantly, the immunity induced by one dose of PLG-rSAG1/2 MPs protected 70% of mice (14/20) against a lethal subcutaneous challenge of 1 × 104 live tachyzoites of T. gondii (RH strain). In conclusion, a single dose of PLG-rSAG1/2 MPs capable of sustaining triphasic release of rSAG1/2 protein induces long-lasting triphasic immunity against T. gondii in mice. Our data indicate the feasibility of PLG-rSAG1/2 MPs to be developed as a single-dose vaccine against T. gondii for potential use in domestic animals.

Pleurocidin Peptide Enhances Grouper Anti-Vibrio harveyi Immunity Elicited by Poly(lactide-co-glycolide)-Encapsulated Recombinant Glyceraldehyde-3-phosphate Dehydrogenase.

Outer membrane proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are considered immunodominant antigens for eliciting protective immunity against Vibrio harveyi, the main etiological agent of vibriosis in fish. Cationic antimicrobial peptides (AMPs), such as pleurocidin (PLE), play important roles in activating and recruiting immune cells, thereby contributing to subsequent innate and adaptive immune responses. In the present study, we aimed to use PLE peptide as a potent adjuvant to improve the immunogenicity of V. harveyi recombinant GAPDH (rGAPDH). In order to prepare a controlled-release vaccine, PLE peptide and rGAPDH protein were simultaneously encapsulated into polymeric microparticles made from the biodegradable poly(lactide-co-glycolide) (PLG) polymer. The resulting PLG-encapsulated PLE plus rGAPDH (PLG-PLE/rGAPDH) microparticles, 3.21-6.27 µm in diameter, showed 72%-83% entrapment efficiency and durably released both PLE and rGAPDH for a long 30-day period. Following peritoneal immunization in grouper (Epinephelus coioides), PLG-PLE/rGAPDH microparticles resulted in significantly higher (p < 0.05, nested design) long-lasting GAPDH-specific immunity (serum titers and lymphocyte proliferation) than PLG-encapsulated rGAPDH (PLG-rGAPDH) microparticles. After an experimental challenge of V. harveyi, PLG-PLE/rGAPDH microparticles conferred a high survival rate (85%), which was significantly higher (p < 0.05, chi-square test) than that induced by PLG-rGAPDH microparticles (67%). In conclusion, PLE peptide exhibits an efficacious adjuvant effect to elicit not only improved immunity, but also enhanced protection against V. harveyi in grouper induced by rGAPDH protein encapsulated in PLG microparticles.

Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), derived from the outer-membrane protein (OMP) fraction, has been used as a potential candidate for vaccine development. The gene-encoding 37 kDa GAPDH outer membrane protein (OMP) from Edwardsiella ictaluri was amplified using polymerase chain reaction (PCR) and was cloned and expressed in Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and nucleotide and amino acid sequencing were used to analyze the expressed antigenic protein and gene encoding this protein. Comparative DNA and protein sequence analysis of GAPDH from E. ictaluri GAPDHs from several Gram-negative bacterial species within the Enterobacteriaceae family revealed that the GAPDHs within this group are highly conserved and share a sequence similarity of 75-100% with E. ictaluri GDPDH. Rabbit antiserum raised against the E. ictaluri recombinant GAPDH (rGAPDH) protein recognized purified GADPH, indicating that it has a strong immunogenicity. Tilapia fish were intraperitoneally immunized with formalin-killed E. ictaluri whole cells, and rGAPDH (30 µg fish(-1)) from E. ictaluri, both of which were emulsified in ISA 763A adjuvant. At 3 months after immunization, fish were challenged with the E. tarda strain to assess vaccine efficacy; the relative percent survival (RPS) values were found to exceed 71.4%. The specific mean antibody titer log2 level of groups vaccinated with rGAPDH at 3 months was significantly higher than that of non-vaccinated fish (control group). Therefore, this recombinant protein can be considered a multi-purpose candidate vaccine against several pathogenic bacteria.

Induction of long-lasting protective immunity against Toxoplasma gondii in BALB/c mice by recombinant surface antigen 1 protein encapsulated in poly (lactide-co-glycolide) microparticles.

BACKGROUND: Current development efforts of subunit vaccines against Toxoplasma gondii, the etiological agent of toxoplasmosis, have been focused mainly on tachyzoite surface antigen 1 (SAG1). Since microparticles made from poly (lactide-co-glycolide) (PLG) polymers have been developed as safe, potent adjuvants or delivery systems, we aimed to encapsulate recombinant SAG1 (rSAG1) with the PLG polymers to prepare PLG-encapsulated rSAG1 (PLG-rSAG1) microparticles that would sustain rSAG1 release and generate long-lasting protective immunity against T. gondii in BALB/c mice. METHODS: In the present study, rSAG1 was encapsulated into PLG microparticles by the double emulsion method. PLG-rSAG1 microparticles were then intraperitoneally injected twice at a 14-day interval into BALB/c mice. We examined the ability of PLG-rSAG1 microparticles to induce and prolong effective anti-Toxoplasma immune responses, in comparison with rSAG1 formulated with a Vet L-10 adjuvant (rSAG1 (Vet L-10)). Eight weeks after the last immunization, protective activities were also evaluated after a lethal subcutaneous challenge of 1 x 10(4) live T. gondii tachyzoites. RESULTS: PLG-rSAG1 microparticles, 4.25~6.58 micrometers in diameter, showed 69%~81% entrapment efficiency. The amount of released rSAG1 protein from microparticles increased gradually over a 35-day period and the protein still retained native SAG1 antigenicity. Intraperitoneal vaccination of mice with the microparticles resulted in enhanced SAG1-specific IgG titers as well as lymphocyte proliferation and, more importantly, these enhanced activities were maintained for 10 weeks. In addition, eight weeks after the last immunization, maximum production of gamma interferon was detected in mice immunized with PLG-rSAG1 microparticles. Furthermore, 80% (8/10) of mice immunized with PLG-rSAG1 microparticles survived at least 28 days after a lethal subcutaneous tachyzoite challenge. CONCLUSIONS: Encapsulation of rSAG1 into PLG microparticles preserves the native SAG1 antigenicity and sustains the release of rSAG1 from microparticles. PLG-rSAG1 microparticles can effectively induce not only significant long-lasting SAG1-specific humoral and cell-mediated immune responses but also high protection against T. gondii tachyzoite infection. Our study provides a valuable basis for developing long-lasting vaccines against T. gondii for future use in humans and animals.