Establishment of a diabetes susceptible family in the Bama minipig (BMP) by N-ethyl-N-nitrosourea (ENU) induction.

BACKGROUND: Diabetes mellitus (DM), characterized by hyperglycemia, is one of the leading causes of death worldwide. An appropriate DM animal model to explore the underlying mechanism of DM and develop new antidiabetic drugs is still desirable. Here, we aim to provide alternatives of DM animal models for medical researches. OBJECTIVE: To establish a diabetes susceptible family in the Bama minipig (BMP) by N-ethyl-N-nitrosourea (ENU) induction. METHODS: Male BMPs with hyperglycemia were selected from G1 and bred by the inbreeding strategy. After 5 generations, parameters such as fasting plasma glucose (FPG), 2-hour plasma glucose (2hPG), intravenous glucose-tolerance test (IVGTT), and insulin resistance were determined to evaluate susceptible family members. RESULTS: The male BMP 2907 (FPG = 6.1 mmol/L, IGVTT 2hPG = 11.9 mmol/L) with hyperglycemia was selected from G1 to generate the 2907 hyperglycemic family. With the number of breeding generations, average FPG levels in BMPs increased significantly (p < 0.05). G5 displayed the characteristics of elevated FPG, insulin resistance, dyslipidemia and abnormal glucose tolerance (p < 0.05). CONCLUSION: A diabetes susceptible family has been successfully established, which might be used for further inbreeding or induced to mimic the phenotype of diabetes.

Plasmodium infection inhibits tumor angiogenesis through effects on tumor-associated macrophages in a murine implanted hepatoma model.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death in China. The lack of an effective treatment for this disease results in a high recurrence rate in patients who undergo radical tumor resection, and the 5-year survival rate of these patients remains low. Our previous studies demonstrated that Plasmodium infection provides a potent antitumor effect by inducing innate and adaptive immunity in a murine Lewis lung carcinoma (LLC) model. METHODS: This study aimed to investigate the inhibitory effect of Plasmodium infection on hepatocellular carcinoma in mice, and various techniques for gene expression analysis were used to identify possible signal regulation mechanisms. RESULTS: We found that Plasmodium infection efficiently inhibited tumor progression and prolonged survival in tumor-bearing mice, which served as a murine implanted hepatoma model. The inhibition of tumor progression by Plasmodium infection was related to suppression of tumor angiogenesis within the tumor tissue and decreased infiltration of tumor-associated macrophages (TAMs). Further study demonstrated that matrix metalloprotease 9 (MMP-9) produced by TAMs contributed to tumor angiogenesis in the tumor tissue and that the parasite-induced reduction in MMP-9 expression in TAMs resulted in the suppression of tumor angiogenesis. A mechanistic study revealed that the Plasmodium-derived hemozoin (HZ) that accumulated in TAMs inhibited IGF-1 signaling through the PI3-K and MAPK signaling pathways and thereby decreased the expression of MMP-9 in TAMs. CONCLUSIONS: Our study suggests that this novel approach of inhibiting tumor angiogenesis by Plasmodium infection is of high importance for the development of new therapies for cancer patients. Video abstract.

The diagnostic value of [18F]-FDG-PET/CT in assessment of radiation renal injury in Tibet minipigs model.

BACKGROUND: Radiation-induced kidney damage can severely affect renal function, and have a serious impact on glucose reabsorption. Fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is routinely utilized for metabolic imaging of glucose utilization. In this study, we are trying to assess the diagnostic value of 18F-FDG-PET/CT on measuring hyperacute effect of total body irradiation (TBI) on the kidneys. METHODS: Forty-eight Tibet minipigs were treated by TBI of different dosages using an 8-MV X-ray linear accelerator. Whole-body 18F-FDG-PET/CT was performed at 6, 24 and 72 h followed by histologic examination, blood samples' and renal function analysis. RESULTS: The uptake of 18F-FDG was significantly different between 11/14 Gy dose groups and control group, the standard Uptake Values reached a maximal level at 72 h after 14-Gy TBI treatment. At doses over 8 Gy, histological observation showed formation of tube casts, degeneration, necrosis of tubular cells, inflammatory cell infiltration and dilatation of the mitochondria of tubule cells. Renal function analysis confirmed the changes in blood urea nitrogen and creatinine levels at various dosages and time intervals. Immunohistochemistry and western blot results indicate that the expression levels of IL-10 and TNF-α proteins were positively correlated with radiation dose up to 8 Gy. CONCLUSIONS: 18F-FDG PET/CT can reflect pathological changes in kidneys and it may be a useful tool for rapid and non-invasive assessment in cases of suspected radiation-induced kidney damage.

Granuloma formation in the liver is relatively delayed, although sustained, in BCG­infected mice co­infected with Plasmodium.

The purpose of the present study was to examine the effects of Plasmodium on the process of granuloma formation in Bacille Calmette­Guerin (BCG)­infected mice. Female six­week­old BALB/c mice were co­infected with BCG and Plasmodium. The liver index, pathological alterations and quantity of granulomas in the mice were observed when the mice were co­injected with BCG and Plasmodium. The expression of inducible nitric oxide synthase (iNOS) was assessed by immunohistochemistry and reverse transcription­polymerase chain reaction (RT­PCR) analysis. In addition, the expression of interleukin (IL)­10 in liver tissues was observed by RT­PCR. Following co­infection with BCG and Plasmodium, the swelling of the liver had been slowly restored to normal, and the time required to allow granulomas to subside had prolonged. In addition, the expression of iNOS increased, while the expression of IL­10 gradually decreased in Plasmodium­infected mice. It was concluded that the use of Plasmodium relatively delayed granuloma formation in livers of BCG­infected mice. In addition, iNOS and IL­10 are involved in this pathogenesis.

Comparison of naturally aging and D-galactose induced aging model in beagle dogs.

Animal models have been used to study aging for decades. In numerous aging studies, beagles are the most commonly used breed of dog. However, few studies have compared between naturally aging models and experimentally induced aging models in beagle dogs. In the present study, a D-galactose induced aging model was compared with a naturally aging model, and young adult dogs were considered as the young control group. The level of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum, and brain tissue were measured. Histopathological comparisons of the liver, kidneys, heart, lungs and spleen were evaluated using hematoxylin and eosin (H&E) staining, in addition, the brain was evaluated by H&E staining, and Nissl staining. The expression levels of aging-associated factors in the hippocampus, including proliferating cell nuclear antigen (PCNA), P16 and P21 were also determined through reverse transcription quantitative-polymerase chain reaction, and western blot analysis. The results indicated that D-galactose induced aging significantly increased the MDA level, while the levels of SOD and GSH-Px were diminished when compared with the young control group, which was similar to the naturally aging group. Parallel histopathological features were observed in the D-galactose induced aging and naturally aging groups compared with the young control group. However, a reduced expression level of PCNA, and increased expression levels of P16 and P21 were observed in the naturally ageing and induced aging groups compared with the young control group. The results of the current study demonstrated that the beagle dogs in D-galactose induced aging model exhibited significant similarities with the naturally aging model, providing evidence to support that the D-galactose induced aging model may be applied to aging studies.

Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer (SCNT).

Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.

Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells.

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However, Δ32 donors are scarce, heterologous bone marrow transplantation is not exempt of risks, and genetic engineering of autologous hHSCs is not trivial. Here, we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS.

Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer.

Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.