Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling.

Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.

Fusion of Dendritic Cells Activating Rv2299c Protein Enhances the Protective Immunity of Ag85B-ESAT6 Vaccine Candidate against Tuberculosis.

In Mycobacterium tuberculosis infection, naïve T cells that encounter mycobacterial antigens through dendritic cells (DCs) induce various CD4+ T-cell responses; therefore, appropriate DC activation is the key for protective immunity against tuberculosis. We previously found that Rv2299c-matured DCs induce Th1 differentiation with bactericidal activity. In this study, to prove that Rv2299c could enhance the protective immunity of other vaccine candidates comprising T-cell-stimulating antigens, Ag85B-ESAT6, a well-known vaccine candidate, was selected as a fusion partner of Rv2299c. Recombinant Rv2299c-Ag85B-ESAT6 protein induced DC maturation and activation. Furthermore, fusion of Rv2299c enhanced the protective efficacy of the Ag85B-ESAT6 vaccine in a mouse model and significantly higher production of TNF-α and IL-2 was detected in the lungs, spleen, and lymph nodes of the group immunized with the Rv2299c-fused protein than with Ag85B-ESAT6. In addition, fusion of Rv2299c enhanced the Ag85B-ESAT6-mediated expansion of multifunctional CD4+ T cells. These data suggested that the DC-activating protein Rv2299c may potentiate the protective immunity of the vaccine candidate comprising T cell antigens.

Mycobacterium avium subsp. paratuberculosis MAP1889c Protein Induces Maturation of Dendritic Cells and Drives Th2-biased Immune Responses.

:Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of chronic granulomatous bowel disease in animals and is associated with various autoimmune diseases in humans including Crohn's disease. A good understanding of the host-protective immune response and antibacterial immunity controlled by MAP and its components may contribute to the development of effective control strategies. MAP1889c was identified as a seroreactive antigen in Crohn's disease patients. In this study, we investigated the immunological function of MAP1889c in dendritic cells (DCs). MAP1889c stimulated DCs to increase expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) class molecules and to secret higher interleukin (IL)-10 and moderate IL-6, tumor necrosis factor (TNF)-α, and IL-12p70 levels through the Toll-like receptor (TLR) 4 pathway. MAP1889c-induced DC activation was mediated by mitogen-activated protein kinases (MAPKs), cAMPp-response element binding protein (CREB), and nuclear factor kappa B (NF-κB). In particular, the CREB signal was essential for MAP1889c-mediated IL-10 production but not TNF-α and IL-12p70. In addition, MAP1889c-matured DCs induced T cell proliferation and drove the Th2 response. Production of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokines and anti-inflammatory cytokines was suppressed and enhanced respectively by MAP1889c pretreatment in DCs and T cells. Furthermore, treatment of MAP1889c in M. avium-infected macrophages promoted intracellular bacterial growth and IL-10 production. These findings suggest that MAP1889c modulates the host antimycobacterial response and may be a potential virulence factor during MAP infection.

Cell wall skeleton of Mycobacterium bovis BCG enhances the vaccine potential of antigen 85B against tuberculosis by inducing Th1 and Th17 responses.

A safe and effective adjuvant is necessary to induce reliable protective efficacy of the protein-based vaccines against tuberculosis (TB). Mycobacterial components, such as synthetic cord factor and arabinogalactan, have been used as one of the adjuvant components. Mycobacterium bovis bacillus Calmette- Guérin cell-wall skeleton (BCG-CWS) has been used as an effective immune-stimulator. However, it is not proven whether BCG-CWS can be an effective adjuvant for the subunit protein vaccine of TB. In this study, we demonstrated that the BCG-CWS effectively coupled with Ag85B and enhanced the conjugated Ag85B activity on the maturation of dendritic cells (DCs). Ag85B-BCG-CWS-matured DCs induced significant Th1 and Th17 responses when compared to BCG-CWS or Ag85B alone. In addition, significant Ag85B-specific Th1 and Th17 responses were induced in Ag85B-BCG-CWS-immunized mice before infection with M. tuberculosis and maintained after infection. Moreover, Ag85B-BCG-CWS showed significant protective effect comparable to live BCG at 6 weeks after infection and maintained its protective efficacy at 32 weeks post-challenge, whereas live BCG did not. These results suggest that the BCG-CWS may be an effective adjuvant candidate for a protein-based vaccine against TB.

Mycobacterium tuberculosis acyl carrier protein inhibits macrophage apoptotic death by modulating the reactive oxygen species/c-Jun N-terminal kinase pathway.

Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host-mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms_AcpM) and a strain transformed with an empty vector (Ms_Vec). Ms_AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms_AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms_Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection.

Sinus floor elevation in sites with a perforated schneiderian membrane: What is the effect of placing a collagen membrane in a rabbit model?

OBJECTIVE: To investigate the healing following sinus grafting in sites with a perforated schneiderian membrane repaired using a collagen membrane, compared to control sites without membrane perforation. MATERIALS & METHODS: Following elevation of the sinus membrane in 16 rabbits, each sinus was assigned to one of the following groups: (a) intentional schneiderian membrane perforation, followed by the placement of a collagen membrane and bone grafting (group SMP) and (b) bone grafting without a perforation of the schneiderian membrane and without a collagen membrane placement (control group). At 2 and 4 weeks (n = 8 for each time-point), microcomputed tomographic (micro-CT) and histomorphometric analyses were performed. RESULTS: Overall new bone formation in group SMP was significantly delayed compared to the control group at 2 and 4 weeks (1.58 ± 1.25% vs. 9.23 ± 2.69% at 2 weeks, 10.43 ± 3.55 vs. 17.86 ± 4.11% at 4 weeks, p < 0.05). At 2 weeks, new bone formation for the areas close to lateral (1.19 ± 2.02%) and medial sinus bone walls (3.17 ± 1.98%) was markedly delayed in group SMP compared to the control group (13.08 ± 6.13% and 12.75 ± 5.63%, respectively, p < 0.05), but there was no statistical difference in those areas at 4 weeks (p > 0.05). The augmented volumes at 2 and 4 weeks were not statistically significantly different in both groups. CONCLUSION: The perforation of the schneiderian membrane and the repair using a collagen membrane delayed new bone formation in the augmented sinuses. However, the extension of the collagen membrane on the sinus bone walls was also attributable to this delayed bone formation.

Virulence properties of uropathogenic Escherichia coli isolated from children with urinary tract infection in Korea.

Urinary tract infections (UTIs) are one of the most common types of bacterial infection in humans in various parts of the world and are caused mainly by uropathogenic Escherichia coli (UPEC). A total of 58 UPEC isolates from urine were characterized by serotyping and pulsed-field gel electrophoresis (PFGE). The majority of the UPEC strains belonged to serogroups O2 and O6. The UPEC strains were grouped under different pulsotypes and majority of them belonged to serogroups O2 and O6. Among the 14 virulence factors considered, 13 were present in various serogroups. The virulence genes fimH and sfa were present in all the isolates while none of the isolates carried lt-1. The strains exhibited 36 different virulence patterns, of which 11, referred to as UP (UPEC pattern) 1 to UP 11 were most common. Antibiotic resistance profiling of the UPEC isolates revealed that the serogroups O2 and O6 contain the highest number of resistant strains. The data from the current study depicting the distribution of UPEC strains among various serogroups and pulsotypes, and the occurrence of virulence genes and antibiotics resistance offer useful information on the epidemiological features of UPEC in Korea for the enhanced surveillance of potential emergence of UPEC.

Fibroblast Growth Factor 10 Enhances the Developmental Efficiency of Somatic Cell Nuclear Transfer Embryos by Accelerating the Kinetics of Cleavage During In Vitro Maturation.

Somatic cell nuclear transfer (SCNT) is required for the generation of transgenic animals as disease models. During the in vitro development of SCNT embryos, the quality of matured oocytes is one of the major factors regulating the developmental potential of embryos. Time-lapse monitoring systems are new tools that assess the developmental capacity of embryos for use in embryo transfer. In this study, we investigated the effect of fibroblast growth factor 10 (FGF 10) on the developmental potential of SCNT embryos. After the in vitro maturation (IVM) of oocytes in IVM medium containing 10 ng/mL FGF 10 (10 F), the polar body extrusion rate was significantly higher than in the control. However, there was no difference in the percentage of fused embryos between the groups. The cleavage and blastocyst formation rates of embryos were significantly increased in the 10 F compared with the control. In addition, the total cell number was higher and the apoptotic index was lower in the 10 F than control at day 7. The messenger RNA (mRNA) expression of genes involved in apoptosis (baculoviral inhibitor of apoptosis repeat containing 5 [BIRC5] and caspase 3 [CASP3]) and development (octamer-binding transcription factor 4 [POU5F1] and sex determining region Y box 2 [SOX2]) increased after 10 F treatment. Furthermore, the kinetics of the first cleavage was faster and the percentage of embryos at cell block was significantly lower in the 10 F group than in the control. These results demonstrate that exposure of oocytes to FGF 10 during IVM promotes developmental competence.

Antioxidant ß-cryptoxanthin enhances porcine oocyte maturation and subsequent embryo development in vitro.

Oxidative stress is partly responsible for the poor quality of IVM oocytes. The present study investigated the effects of the antioxidant ß-cryptoxanthin on the IVM of porcine oocytes and the in vitro development of the ensuing embryos. Oocytes were matured in IVM medium containing different concentrations of ß-cryptoxanthin (0, 0.1, 1, 10 or 100µM). Treatment with 1µM ß-cryptoxanthin (Group 1B) improved polar body extrusion and the expression of maturation-related genes in cumulus cells and oocytes compared with control. In addition, levels of reactive oxygen species decreased significantly in Group 1B, whereas there were significant increases in glutathione levels and expression of the antioxidant genes superoxide dismutase 1 and peroxiredoxin 5 in this group. After parthenogenetic activation, although the cleavage rate did not differ between the control and 1B groups, the blastocyst formation rate was higher in the latter. Moreover, the total number of cells per blastocyst and relative mRNA levels of pluripotency marker and antioxidant genes were significantly higher in the 1B compared with control group. These results demonstrate that ß-cryptoxanthin decreases oxidative stress in porcine oocytes and improves their quality and developmental potential.

Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel.

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 µM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.

Treatment of allicin improves maturation of immature oocytes and subsequent developmental ability of preimplantation embryos.

Allicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study investigated the effects of allicin treatment on porcine oocyte maturation and developmental competence. Porcine oocytes were cultured in medium supplemented with 0 (control), 0.01, 0.1, 1, 10 or 100 µM AL, respectively, during in vitro maturation (IVM). The rate of polar body emission was higher in the 0.1 AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%) (P < 0.1). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 AL-treated group than in the control (P < 0.05). The reactive oxygen species level at metaphase II did not significantly differ among all groups. In matured oocytes, the expression of both BAK and CASP3, and BIRC5 was significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Similarly, the expression of BMP15 and CCNB1, and the activity of phospho-p44/42 mitogen-activated protein kinase (MAPK), significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.

Production of transgenic pig as an Alzheimer's disease model using a multi-cistronic vector system.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2-1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aß-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

Intermediate Reprogramming of Mouse Skin Fibroblasts into Stem-Like Cells by Bone Morphogenetic Protein 4.

Specific transcription factors are sufficient to reprogram fully induced pluripotent stem cells or other types of cells. These findings raise the question of whether chemical molecules or proteins can replace transcription factors to alter the defined cell fate. In this study, we treated mouse skin fibroblasts (MSFs) with bone morphogenetic protein 4 (BMP4) and examined intermediate reprogramming of MSFs into stem-like cells. Putative epidermal stem cells isolated from the ventral skin epidermis of an adult mouse were used to confirm the reprogramming activity of BMP4, which increased the proliferation of these cells. After these cells formed spheroids, they were treated with BMP4 and cultured for 5 days. Following BMP4 treatment, the characteristics of these cells changed, and they expressed Oct-4 and its target transcripts Nanog, Sox2, and alkaline phosphatase. To confirm the stem cell potency of these cells, we induced their differentiation into cardiomyocytes. Stem-like cell-derived cardiomyocytes exhibited mRNA expression of cardiac mesoderm markers such as Nk2 transcription factor-related locus 5 and connexin 40, and the cardiomyocyte marker troponin T. These differentiated cells exhibited contracting masses. These results suggest that BMP4-mediated somatic stem cell reprogramming may become an alternative approach for cell therapy.

Fibroblast growth factor 10 markedly improves in vitro maturation of porcine cumulus-oocyte complexes.

Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus-oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocyte's metaphase-II (MII) stage was significantly higher in the 10F-treated group than in the control. Transcript abundance of the cumulus cell expansion-related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F- and 50F-treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F-treated group. The percentage of oocytes with normal spindles was greater in the 10F- and 50F-treated group at GVBD than in the other groups; the 5F-, 10F-, and 100F-treated groups were higher than the control; and the 50F-treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F-treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F- to 50F-treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67-75, 2017. © 2016 Wiley Periodicals, Inc.

Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth.

Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.

Cell Synchronization by Rapamycin Improves the Developmental Competence of Porcine SCNT Embryos.

The cell cycle stage of donor cells influences the success of somatic cell nuclear transfer (SCNT). This study investigated the effects of rapamycin treatment on synchronization of porcine fibroblasts in comparison with control and serum-starved cells, SCNT donor cell viability, and SCNT-derived embryo development. Porcine fibroblasts were treated with 0.1, 1, 10, and 100 µM rapamycin for 1 or 3 days. The proportion of cells in G0/G1 phase was significantly higher among cells treated with 1 µM rapamycin for 3 days (D3-1R) than among control and serum-starved cells (p < 0.05). In comparison with control cells, rapamycin-treated cells exhibited reduced proliferation, similar to serum-starved cells. The viability (as assessed by the MTT assay) of D3-1R-treated cells was good, similar to control cells, showing their quality was maintained. To confirm nutrient regulation by rapamycin treatment, we checked the transcript levels of nutrient transporter genes (SLC2A2, SLC2A4, SLC6A14, and SLC7A1). These levels were significantly lower in D3-1R-treated cells than in control cells (p < 0.01). We performed SCNT with D3-1R-treated cells (SCNT(D3-1R)) to confirm the effect of cell cycle synchronization by rapamycin treatment. Although SCNT(D3-1R) embryos did not have an increased fusion rate, their cleavage and blastocyst formation rates were significantly higher than those of control embryos (p < 0.05). Regarding embryo quality, the numbers of total and apoptotic cells per blastocyst were increased and decreased, respectively, in SCNT(D3-1R) blastocysts. The mRNA levels of developmental (CDX2 and CDH1) and proapoptotic (FAS and CASP3) genes were significantly higher and lower, respectively, in SCNT(D3-1R) blastocysts than in control blastocysts (p < 0.05). These results demonstrate that rapamycin treatment affects the cell cycle synchronization of donor cells and enhances the developmental potential of porcine SCNT embryos.

Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

An evaluation of the use of immunoglobulin A antibody response against mycobacterial antigens for the diagnosis of Mycobacterium bovis infection in cattle.

Antibody responses are useful indicators of Mycobacterium bovis infection in cattle. Many studies have evaluated the ability of immunoglobulin G (IgG) to serodiagnose bovine tuberculosis (TB). In the current study, immunoglobulin A (IgA) and IgG responses against the MPB70 and MPB83 antigens of M. bovis, the 38 kDa phosphate-binding lipoprotein (PstS1) that is a well-known serodiagnostic M. tuberculosis antigen, and a newly identified protein, termed Rv1483c, were compared in M. bovis-infected and noninfected cattle as well as in field samples. The diagnostic utility of the IgA antibody to MPB70 and MPB83 for bovine TB was superior or comparable to that of the IgG antibody, and the sensitivity of serodiagnosis increased when the results of antigen binding by IgA and IgG were combined. The sensitivities of the IgG and IgA antibodies to the Rv1483c and PstS1 proteins were significantly lower than those to MPB70 and MPB83, and no diagnostic utility for Rv1483c was observed in field samples. Importantly, the IgA antibody reacted strongly to the MPB70 and MPB83 antigens and differentiated cattle with TB from healthy cattle in a multiantigen printed immunoassay. The results of this study support the feasibility of using IgA antibody against the MPB70 and MPB83 antigens to detect bovine TB. In addition, approaches using assays for both IgA and IgG antibodies may increase detection accuracy.

Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/- (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development.

Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT-MSC bioactive material (hAT-MSC-BM), and examined the effect of hAT-MSC-BM on porcine embryo development. hAT-MSC-BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT-MSC-BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT-MSC culture medium group, the best results were from the group cultured with 10% hAT-MSC-BM. Mitochondrial activity was also higher in the 10% hAT-MSC-BM-treated group. Moreover, the relative mRNA expression levels of development and anti-apoptosis genes were significantly higher in the 10% hAT-MSC-BM-treated group than in control, hAT-MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT-MSC-BM starting on Day 4 also improved the development rate and the total cell number of in vitro-fertilized embryos. This is the first report on the benefits of hAT-MSC-BM in a porcine embryo in vitro culture system. We conclude that hAT-MSC-BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro.