Doxycycline cotherapy with albendazole relieves neural function damage in C57BL/6 and BALB/c mice infected with Angiostrongylus cantonensis.

BACKGROUND: We investigated the effects of combination therapy albendazole and doxycycline in Angiostrongylus cantonensis-infected mice during early and late treatment. MATERIALS AND METHODS: C57BL/6 and BALB/c mice were divided into five groups: (i) uninfected, (ii) infected with A. cantonensis, (iii) infected + 10 mg/kg albendazole, (iv) infected + 25mg/kg doxycycline, and (v) infected + 10 mg/kg albendazole + 25 mg/kg doxycycline. We administered drugs in both early treatments started at 7-day post infections (dpi) and late treatments (14 dpi) to A. cantonensis-infected C57BL/6 and BALB/c mice. To assess the impact of these treatments, we employed the Morris water maze test to evaluate spatial learning and memory abilities, and the rotarod test to measure motor coordination and balance in C57BL/6 mice. Additionally, we monitored the expression of the cytokine IL-33 and GFAP in the brain of these mice using western blot analysis. RESULTS: In this study, A. cantonensis infection was observed to cause extensive cerebral angiostrongyliasis in C57BL/6 mice. This condition significantly affected their spatial learning and memory abilities, as assessed by the Morris water maze test, as well as their motor coordination, which was evaluated using the rotarod test. Early treatment with albendazole led to favorable recovery outcomes. Both C57BL/6 and BALB/c mice express IL-33 and GFAP after co-therapy. The differences of levels and patterns of IL-33 and GFAP expression in mice may be influenced by the balance between pro-inflammatory and anti-inflammatory signals within the immune system. CONCLUSIONS: Combination therapy with anthelmintics and antibiotics in the early stage of A. cantonensis infection, in C57BL/6 and BALB/c mice resulted in the death of parasites in the brain and reduced the subsequent neural function damage and slowed brain damage and neurobehavior impairment. This study suggests a more effective and novel treatment, and drug delivery method for brain lesions that can decrease the neurological damage of angiostrongyliasis patients.

The Exploration of miRNAs From Porcine Fallopian Tube Stem Cells on Porcine Oocytes.

Fallopian tube is essential to fertilization and embryonic development. Extracellular vesicles (EVs) from Fallopian tube containing biological regulatory factors, such as lipids, proteins and microRNAs (miRNAs) serve as the key role. At present, studies on oocytes from porcine oviduct and components from EVs remain limited. We aim to explore the effect of EVs secreted by porcine fallopian tube stem cells (PFTSCs) on oocyte. When the fifth-generation PFTSCs reached 80-90% of confluency, the pig in vitro maturation medium was utilized, and the conditioned medium collected for oocyte incubations. To realize the functions of EVs, several proteins were used to determine whether extracted EVs were cell-free. Field emission scanning electron microscope and nanoparticle tracking analyzer were used to observe the morphology. By next generation sequencing, 267 miRNAs were identified, and those with higher expression were selected to analyze the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment maps. The selected miR-152-3p, miR-148a-3p, miR-320a-3p, let-7f-5p, and miR-22-3p, were predicted to target Cepb1 gene affecting MAPK pathway. Of the five miRNAs, miR-320a-3p showed significant difference in maturation rate in vitro maturation. The blastocyst rate of pig embryos was also significantly enhanced by adding 50 nM miR-320a-3p. In vitro culture with miR-320a-3p, the blastocyst rate was significantly higher, but the cleavage rate and cell numbers were not. The CM of PFTSCs effectively improves porcine oocyte development. The miRNAs in EVs are sequenced and identified. miR-320a-3p not only helps the maturation, but also increases the blastocyst rates.

Improvements of cognitive functions in mice heavily infected by Angiostrongylus cantonensis after treatment with albendazole, dexamethasone, or co-therapy.

Angiostrongylus cantonensis, the causative agent of human eosinophilic meningitis and eosinophilic meningoencepalitis, has been reported to cause cognitive impairments in the host. To determine whether drug treatment improves the cognitive functions, BALB/c mice infected with 50 third-stage larvae were treated with albendazole, dexamethasone, or co-therapy since day 7 or 14 post-infection for one or two weeks. Abilities of spatial memory and learning of these animals were assessed with the Morris water maze. Our results showed that body weight was significant higher then infected group in the albendazole and combined therapy groups. Significantly lower worm recovery rates were found in mice treated with the same groups. The mice treated with dexamethasone since day 7 for 14 day had significant longer time in the remaining groups were found in forced swimming test. The animals treated with albendazole and combined therapy since day 7 for 14 days was demonstrated to have significantly shorter latencies to the platform in learning memory on day 3 and 4. Mice in these two groups were demonstrated to have significantly higher sores in spatial memory tests. These results indicate that treatment with albendazole or combined therapy may be more efficient in preventing brain damages and depression as well as preserving their capabilities in learning and memory. Therefore, administration of albendazole alone or combined with dexamethasone should have higher efficacies than dexamethasone alone in treatment of BALB/c mice infected with a heavy dose of 50 third-stage larvae of A. cantonensis.

Fallopian tube stem cell medium of porcine and bovine: In vitro regenerative effect on maturation and parthenogenesis of porcine oocytes.

Assisted reproductive technology (ART) has been widely developed over the decades. This advanced technology has shown efficacy in the conception and breeding of an animal. However, several issues such as polyspermy, low maturation rate, and low development rate in vitro remain unresolved. Fallopian tube derived cells are proposed to promote the maturation and development of oocyte. This study aims to characterize porcine (PFTSC) and bovine fallopian tube stem cell (BFTSC) while comparing allogeneic and xenogeneic paracrine effects on porcine oocyte. FTSC of Taiwan yellow cattle (B. indicus) and porcine (Landrace x Yorkshire dam x Duroc) were isolated and identified. Conditioned media (Medium 199 or PZM-3) from porcine and bovine was collected and added to porcine cells during in vitro maturation (IVM) and in vitro culture (IVC). Both PFTSC and BFTSC expressed little CD44, CD105, and CD4. Both cells were induced to transform into chondrocytes, very few cells gave rise to osteocytes and adipocytes. IVM test showed a significant elevation of maturation rate in both groups (Porcine: 66.5 ± 3.5% > 55.9 ± 1.7%, p < .05; Bovine: 68.9 ± 2.3% > 55.9 ± 1.7%, p < .05). IVC test demonstrated markedly reduction of blastocyst in both groups. In a diluted conditioned medium with different concentration, 25% and 50% PFTSC showed a decrease in blastocyst rate which is significantly different, but BFTSC demonstrated no significant difference. PFTSC and BFTSC possessed properties of stem cells. Conditioned media from both PFTSC and BFTSC could improve maturation rate but not blastocyst rate in vitro.

Infusion of Porcine-Derived Amniotic Fluid Stem Cells for Treatment of Experimental Colitis in Mice.

Recently, stem cells have offered an alternative treatment for inflammatory bowel disease (IBD) or colitis to overcome the poor outcomes associated with current therapies. Amniotic fluid-derived stem cells (AFSCs) have the potential for the regeneration of impaired organs and the recovery of normal physiologic functions of damaged tissues without ethical concerns or risk of tumor formation. In this work, we aimed to examine the therapeutic effects of infusion of porcine AFSCs (pAFSCs) in dextran sulfate sodium (DSS)-induced colitis in mice. Treatment with pAFSCs was shown to inhibit the shortening of the colon after induction of colitis and dramatically ameliorated the body weightloss induced by the DSS treatment. In addition, pAFSCs could also reduce the extent of the inflamed area represented by epithelial mesenchymal transformation in the colitis mice. The levels of the inflammatory cytokines interleukin 6 (IL-6) and interferon gamma (IFN-γ) were also reduced in colitis mice transplanted with pAFSCs. In conclusion, pAFSCs can ameliorate experimental colitis in mice, suggesting that they may be a potential treatment for IBD or colitis.

Artificial insemination of Holstein heifers with sex-sorted semen during the hot season in a subtropical region.

Our aim was to investigate insemination techniques in order to improve pregnancy rates of artificial insemination (AI) using sex-sorted semen (sexed AI) in cattle in tropical and subtropical (T/ST) regions. In T/ST regions, the pregnancy rates by sexed AI are reportedly the lowest in the hottest months of the year, with less than 15% in cows and 35-40% in heifers (PMID 24048822). We compared sexed AI by depositing the semen into the uterine body (UB-AI, n = 12) versus the unilateral uterine horn (UUH-AI, n = 14) of pre-ovulation heifers. The ovary and follicle were assessed by rectal ultrasound before AI. After insemination, pregnancy was determined by ultrasound at approximately 40 days and approximately 70 days. In the present study, we demonstrated that high pregnancy rates (>70%) by sexed AI in the hottest season in a subtropical region such as Taiwan can be achieved when heifers with pre-ovulation follicles are used. The overall pregnancy rates were 54% higher in the UUH-AI (71%) group than in the UB-AI (42%) group (P = 0.06), examined on approximately 40 days post-sexed AI. Surprisingly, however, the pregnancy outcome appeared to be higher in the hot season (62%) than in the cool season (46%) although this difference was not statistically significant. Based on the present study, we recommend that cattle breeders perform UUH-AI using sex-sorted semen for heifers with pre-ovulation follicles in order to achieve satisfactory pregnancy outcome in the hot seasons in T/ST regions.

Developmental origin of postnatal cardiomyogenic progenitor cells.

AIM: To trace the cell origin of the cells involved in postnatal cardiomyogenesis. MATERIALS & METHODS: Nkx2.5 enhancer-eGFP (Nkx2.5 enh-eGFP) mice were used to test the cardiomyogenic potential of Nkx2.5 enhancer-expressing cells. By analyzing Cre excision of activated Nkx2.5-eGFP+ cells from different lineage-Cre/Nkx2.5 enh-eGFP/ROSA26 reporter mice, we traced the developmental origin of Nkx2.5 enhancer-expressing cells. RESULTS: Nkx2.5 enhancer-expressing cells could differentiate into striated cardiomyocytes both in vitro and in vivo. Nkx2.5-eGFP+ cells increased remarkably after experimental myocardial infarction (MI). The post-MI Nkx2.5-eGFP+ cells originated from the embryonic epicardial cells, not from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells or perinatal/postnatal epicardial cells. CONCLUSION: Postnatal Nkx2.5 enhancer-expressing cells are cardiomyogenic progenitor cells and originate from embryonic epicardium-derived cells.

Establishment of a DsRed-monomer-harboring ICR transgenic mouse model and effects of the transgene on tissue development.

DsRed-monomer is an enhanced red fluorescent protein that may serve as a marker for studies in biotechnology and cell biology. Since the ICR mouse strain is a widely utilized outbred strain for oncology, toxicology, vaccine development and for aging studies, the objective of this study was to produce a DsRed-monomer transgenic mouse by means of pronuclear micro-injection of a vector driven by the cytomegalovirus (CMV) enhancer/chicken beta-actin promoter. Four transgenic mice were successfully produced, one of which expressed the DsRed-monomer protein in every tissue, although at varying levels. High expression levels were observed in the heart, pancreas and muscle. Moreover, amniotic fluid-derived progenitor cells, which also expressed the DsRed-monomer protein, could be collected from the DsRed-monomer- harboring ICR mice. As compared to wild-type mice, a few biochemical and histological dissimilarities were found in the DsRed-monomer transgenic mice, including the presence of intra-cytoplasmic eosinophilic threadlike materials in the acinar cells. Taken together, transgenic mice stably expressing DsRed-monomer can be produced using pronuclear micro-injection; however, expression of the DsRed-monomer gene or its insertion position may lead to minor influences.

Differentiation of Enhanced Green Fluorescent Protein-Labeled Mouse Amniotic Fluid-Derived Stem Cells into Cardiomyocyte-Like Beating Cells.

BACKGROUND: Amniotic fluid-derived stem cells (AFSCs) possess optimal differentiation potential and are a promising resource for cell therapy and tissue engineering. Mouse is a good model to be studied for pre-clinical research. METHODS: In this study, we successfully established enhanced green fluorescent protein mouse-derived amniotic fluid stem cells (EGFP-mAFSCs) and investigated whether EGFP-mAFSCs possess the ability to differentiate into cardiomyocytes by in vitro culture. We evaluated stem-cell differentiation using immunofluorescence. RESULTS: This study showed that EGFP-mAFSCs can give rise to spontaneously beating cardiomyocyte-like cells expressing the specific markers c-kit, myosin heavy chain, and cardiac troponin I. CONCLUSIONS: We demonstrated that mAFSCs have the in vitro propensity to acquire a cardiomyogenic phenotype and to a certain extent cardiomyocytes; however the process efficiency which gives rise to cardiomyocyte-like cells remains quite low (2 out of 10 were found). KEY WORDS: Amniotic fluid; Cardiomyocytes; In vitro differentiation; Stem cells.

Engraftment of mouse amniotic fluid-derived progenitor cells after in utero transplantation in mice.

BACKGROUND/PURPOSE: Amniotic fluid-derived progenitor cells (AFPCs) are oligopotent and shed from the fetus into the amniotic fluid. It was reported that AFPCs express stem cell-like markers and are capable of differentiating into specific cell type in in vitro experiments. However, no study has fully investigated the potentiality and destiny of these cells in in vivo experiments. METHODS: Ds-red transgenic mice (on Day 13.5 of pregnancy) were transplanted in utero with enhanced green fluorescent protein-labeled mouse AFPC (EGFP-mAFPCs). After birth, baby mice were euthanized at 3-week intervals beginning 3 weeks postnatally, and the specimens were examined by polymerase chain reaction, histology, and flow cytometry. RESULTS: Our results demonstrate the transplantability of mAFPCs into all three germ layers and the potential of mAFPCs in the study of progenitor cell homing, differentiation, and function. Engraftment of EGFP-mAFPCs was detected in the intestine, kidney, muscle, skin, bladder, heart, stomach, etc., at 3 weeks after delivery. CONCLUSION: This model using EGFP-mAFPCs injected in utero may provide an ideal method for determining the fate of transplanted cells in recipients and these findings may justify a clinical trial of in utero transplantation during gestation for patients who have inherited genetic disorders.

Intramuscular Transplantation of Pig Amniotic Fluid-Derived Progenitor Cells Has Therapeutic Potential in a Mouse Model of Myocardial Infarction.

Acute myocardial infarction (MI) is a fatal event that causes a large number of deaths worldwide. MI results in pathological remodeling and decreased cardiac function, which could lead to heart failure and fatal arrhythmia. Cell therapy is a potential strategy to repair the damage through enhanced angiogenesis or by modulation of the inflammatory process via paracrine signaling. Amniotic fluid-derived progenitor cells (AFPCs) have been reported to differentiate into several lineages and can be used without ethical concerns or risk of teratoma formation. Since pigs are anatomically, physiologically, and genetically similar to humans, and pregnant pigs can be an abundant source of AFPCs, we used porcine AFPCs (pAFPCs) as our target cells. Intramyocardial injection of AFPCs has been shown to cure MI in animal models. However, intramuscular transplantation of cells has not been extensively investigated. In this study, we investigated the therapeutic potential of intramuscular injection of pAFPCs on acute MI. MI mice were divided into 1) PBS control, 2) medium cell dose (1 × 10(6) cells per leg; cell-M), and 3) high cell dose (4 × 10(6) cells per leg; cell-H) groups. Cells or PBS were directly injected into the hamstring muscle 20 min after MI surgery. Four weeks after MI surgery, the cell-M and cell-H groups exhibited significantly better ejection fraction, significantly greater wall thickness, smaller infarct scar sizes, and lower LV expansion index compared to the PBS group. Using in vivo imaging, we showed that the hamstring muscles from animals in the cell-M and cell-H groups had RFP-positive signals. In summary, intramuscular injection of porcine AFPCs reduced scar size, reduced pathological remodeling, and preserved heart function after MI.

Generation and characterization of a transgenic pig carrying a DsRed-monomer reporter gene.

BACKGROUND: Pigs are an optimal animal for conducting biomedical research because of their anatomical and physiological resemblance to humans. In contrast to the abundant resources available in the study of mice, few fluorescent protein-harboring porcine models are available for preclinical studies. In this paper, we report the successful generation and characterization of a transgenic DsRed-Monomer porcine model. METHODS: The transgene comprised a CMV enhancer/chicken-beta actin promoter and DsRed monomeric cDNA. Transgenic pigs were produced by using pronuclear microinjection. PCR and Southern blot analyses were applied for identification of the transgene. Histology, blood examinations and computed tomography were performed to study the health conditions. The pig amniotic fluid progenitor/stem cells were also isolated to examine the existence of red fluorescence and differentiation ability. RESULTS: Transgenic pigs were successfully generated and transmitted to offspring at a germ-line transmission rate of 43.59% (17/39). Ubiquitous expression of red fluorescence was detected in the brain, eye, tongue, heart, lung, liver, pancreas, spleen, stomach, small intestine, large intestine, kidney, testis, and muscle; this was confirmed by histology and western blot analyses. In addition, we confirmed the differentiation potential of amniotic fluid progenitor stem cells isolated from the transgenic pig. CONCLUSIONS: This red fluorescent pig can serve as a host for other fluorescent-labeled cells in order to study cell-microenvironment interactions, and can provide optimal red-fluorescent-labeled cells and tissues for research in developmental biology, regenerative medicine, and xenotransplantation.

Therapeutic potential of amniotic-fluid-derived stem cells on liver fibrosis model in mice.

OBJECTIVE: Liver fibrosis results from the wound healing response to chronic liver damage. Advanced liver fibrosis results in cirrhosis and liver failure, and liver transplantation is often the only option for effective therapy; however, the shortage of available donor livers limits this treatment. Thus, new therapies for advanced liver fibrosis are essential. MATERIALS AND METHODS: Amniotic fluid contains an abundance of stem cells, which are derived from all three germ layers of the developing fetus. These cells do not induce teratomas in vivo and do not pose any ethical concerns. To generate liver fibrosis models, male ICR mice were treated with CCl4 via oral gavage for 4 weeks, and the serum levels of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and albumin were higher than in the control group following chemical induction. To assess the potential of amniotic-fluid-derived stem cells (mAFSCs) to ameliorate liver fibrosis in vivo, mAFSCs were isolated from amniotic fluid of 13.5-day-old transgenic mice, which globally express the fluorescent protein, enhanced green fluorescent protein (EGFP), for tracing purposes (EGFP-mAFSCs). Single cells were injected via the mesentery (1 × 10(6) cells/mouse) of transplanted mice with liver fibrosis. RESULTS: Four weeks after EGFP-mAFSC transplantation, the serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and albumin levels of recipient mice in the EGFP-mAFSC-injected group were significantly decreased when compared with mice in the saline-injected group. Additionally, fibrotic tissues were evaluated using Masson's trichrome staining 4 weeks after cell transplantation. Shrinkage of the fibrotic area was observed in the EGFP-mAFSC-injected group. The tissue-repair effects were also confirmed by hydroxyproline content analysis. CONCLUSION: The possible repair mechanism from our data revealed that EGFP-mAFSCs may fuse with the recipient liver cells. Overall, EGFP-mAFSCs can ameliorate liver fibrosis in mice, thus providing insight into the future development of regenerative medicine.

Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice.

OBJECTIVES: Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. METHODS: The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. RESULTS: Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. CONCLUSION: In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue.