Effect of coordinator-based osteoporosis intervention on quality of life in patients with fragility fractures: a prospective randomized trial.

We examined the effects of the coordinator-based intervention on quality of life (QOL) in the aftermath of a fragility fracture, as well as factors predictive of post-fracture QOL. The coordinator-based interventions mitigated the decrease in QOL. Secondary fracture after primary fracture, however, was a significant predictor of lower QOL. PURPOSE: This study aimed to determine the effects of the coordinator-based intervention on QOL in the aftermath of a fragility fracture, as well as factors predictive of post-fracture QOL, in an Asian population. METHODS: Patients with new fractures in the intervention group received the coordinator-based intervention by a designated nurse certified as a coordinator, within 3 months of injury. QOL was evaluated using the Japanese version of the EuroQol 5 Dimension 5 Level (EQ-5D-5L) scale  before the fracture (through patient recollections) and at 0.5, 1, and 2 years after the primary fracture. RESULTS: Data for 141 patients were analyzed: 70 in the liaison intervention (LI) group and 71 in the non-LI group. Significant intervention effects on QOL were observed at 6 months after the fracture; the QOL score was 0.079 points higher in the LI group than in the non-LI group (p=0.019). Further, the LI group reported significantly less pain/discomfort at 2 years after the fracture, compared to the non-LI group (p=0.037). In addition, secondary fractures were found to significantly prevent improvement and maintenance of QOL during the recovery period (p=0.015). CONCLUSION: Short-term intervention effects were observable 6 months after the primary fracture, with the LI group mitigated the decrease in QOL. Few patients in the LI group reported pain/discomfort 2 years after the fracture, but there is uncertainty regarding its clinical significance. Secondary fracture after initial injury was a significant predictor of lower QOL after a fracture.

Efficiency of coordinator-based osteoporosis intervention in fragility fracture patients: a prospective randomized trial.

We examined the effectiveness of coordinators' interventions to prevent secondary fractures in patients with fragility fractures. These coordinator-based interventions improved bone density assessment implementation and treatment rates, and enhanced treatment persistence rates in the early stages following fractures. INTRODUCTION: This study aimed to determine the efficiency of coordinator-based osteoporosis intervention in fragility fracture patients during a 2-year period. METHODS: A prospective intervention randomized control study was conducted at seven medical facilities from January 2015 to March 2017. Postmenopausal women and men over 50 years old with fragility fractures were randomly divided into the coordinator intervention (LI; 70 patients) and without intervention (non-LI; 71 patients) groups. The osteoporosis treatment rate, osteoporosis treatment persistence rate, fall rate, fracture incidence rate, and bone density measurement rate 3 months, 6 months, 1 year, and 2 years after registration were compared between the two groups. Non-parametric tests were used to analyze data at each inspection period. RESULTS: The osteoporosis treatment initiation rate was significantly higher in the LI group than in the non-LI group (85.7% vs. 71.8%; p = 0.04). The LI group had significantly higher bone density assessment implementation rates than the non-LI group at the time of registration (90.0% vs. 69.0%; p = 0.00) and 6 months after registration (50.0% vs. 29.6%; p = 0.01), but not 1 or 2 years after registration. In addition, no significant differences in fall or fracture incidence rates were found between the two groups. CONCLUSION: The coordinator-based interventions for fragility fractures improved bone density assessment implementation and treatment rates and enhanced treatment persistence rates in the early stages following bone fractures. The findings suggest that liaison intervention may help both fracture and osteoporosis physicians for the evaluation of osteoporosis and initiation and continuation of osteoporosis medication.

DWI for Monitoring the Acute Response of Malignant Gliomas to Photodynamic Therapy.

BACKGROUND AND PURPOSE: Photodynamic therapy is a novel treatment that provides effective local control, but little is known about photodynamic therapy-induced changes on MR imaging. The aim of this study was to assess the utility of DWI and ADC in monitoring the response of malignant gliomas to photodynamic therapy. MATERIALS AND METHODS: Time-dependent changes in DWI and ADC values after photodynamic therapy were analyzed in a group that received photodynamic therapy in comparison with a group that did not. RESULTS: Twenty-four patients were enrolled (photodynamic therapy, n = 14; non-photodynamic therapy, n = 10). In all patients who received photodynamic therapy, linear high signals on DWI in the irradiated area were detected adjacent to the resection cavity and were 5-7 mm in depth from 1 day posttreatment and disappeared in about 30 days without any neurologic deterioration. The non-photodynamic therapy group did not show this change. The photodynamic therapy group had significantly lower ADC values from 1 day posttreatment (P < .001), which increased steadily and disappeared by 30 days. There was no decline or time-dependent change in ADC values in the non-photodynamic therapy group. CONCLUSIONS: The acute response of malignant gliomas to photodynamic therapy was detected as linear high signals on DWI and as a decrease in ADC values. These findings were asymptomatic and transient. Although the photodynamic therapy-induced acute response on MR imaging disappeared after approximately 30 days, it may be helpful for confirming the photodynamic therapy-irradiated area.

Immunological Response of Pigs to Human Cells, Including Issues Such as the Production of Natural Antibodies in Newborns.

Pigs have recently become very popular for use not only in xenotransplantation field, but in regeneration studies as well, sometimes with pigs being used as the scaffold. We have already presented our findings related to the pig immune system against human cells, including the complement systems, natural antibodies (NAs), and NK cells. In this study, we investigated the pig innate immunological reaction against human cells further. Our investigations included issues such as the production of NAs in newborns, day 0 and day 1, and sow colostrum. The alternative pathway for pig complement reacted with human cells, and pig NK cells and macrophages directly injured human aortic endothelial cells. Pig serum clearly contains the natural antibodies IgG and IgM to human peripheral blood mononuclear cells (PBMCs). Pig plasma from day 1 newborns contained almost the same levels of these natural antibodies to human PBMCs as those of sow plasma. On the other hand, pig plasma from day 0 newborns did not contain IgG and IgM to human PBMCs. In addition, sow colostrum clearly contained both IgG and IgM to human PBMCs. As expected, the pig innate immunity system reacted to human cells, including natural antibodies. However, the NAs of pigs, both IgM and IgG, against human cells do not exist in pig serum at day 0, but at day 1 and in mother's milk, indicating that NAs in newborns did not come from the placenta but from sow colostrum.

A molecular dynamics study of nuclear quantum effect on diffusivity of hydrogen molecule.

In this paper, the nuclear quantum effect of the hydrogen molecule on its diffusivity was analyzed using the molecular dynamics (MD) method. The centroid MD (CMD) method was applied to reproduce the time evolution of the molecules. The diffusion coefficient of hydrogen was calculated using the Green-Kubo method over a wide temperature region, and the temperature dependence of the quantum effect of the hydrogen molecule on its diffusivity was addressed. The calculated results were compared with classical MD results based on the principle of corresponding state (PCS). It was confirmed that the difference in the diffusion coefficient calculated in the CMD and classical MD methods was small, and the PCS appears to be satisfied on the temperature dependence of the diffusion coefficient, even though the quantum effect of the hydrogen molecules was taken into account. It was clarified that this result did not suggest that the quantum effect on the diffusivity of the hydrogen molecule was small but that the two changes in the intermolecular interaction of hydrogen due to the quantum effect offset each other. Moreover, it was found that this tendency was related to the temperature dependence of the ratio of the kinetic energy of the quantum fluctuational motion to the classical kinetic energy.

Oral CD103-CD11b+ classical dendritic cells present sublingual antigen and induce Foxp3+ regulatory T cells in draining lymph nodes.

Sublingual immunotherapy (SLIT) is a safe and efficient treatment for type 1 allergies; however, the underlying immunological mechanisms, particularly the phenotype of oral antigen-presenting cells (APCs) responsible for the induction of regulatory T (Treg) cells, remain unclear. We show here that the sublingual application of ovalbumin (OVA) induced antigen-specific Foxp3+ Treg cells in draining submandibular lymph nodes (ManLNs). Oral APCs were classified into macrophages, classical dendritic cells (cDCs), and Langerhans cells by flow cytometry. A major subset of oral cDCs with the CD103-CD11b+ phenotype showed retinoic acid (RA)-producing activity and converted naive CD4+ T cells to Foxp3+ Treg cells in a transforming growth factor-ß- and RA-dependent manner in vitro. In the ManLNs, migratory CD103-CD11b+ cDCs also showed RA-producing activity. After the sublingual application of fluorescent OVA, fluorescence was detected in oral macrophages in tissues, followed by migratory CD103-CD11b+ cDCs in ManLNs and migratory CD103-CD11b+ cDCs were the main APCs responsible for the induction of sublingual antigen-specific Treg cells. The transfer of OVA-SLIT-induced Treg cells suppressed the OVA-induced hypersensitivity response. These results suggest that oral CD103-CD11b+ cDCs transport sublingual antigens to draining ManLNs and induce antigen-specific Foxp3+ Treg cells, and, thus, provide a rationale for developing cDC-based therapeutic approaches in SLIT.

Maternal Separation Induces Orofacial Mechanical Allodynia in Adulthood.

It is well known that exposure to maternal separation (MS) in early life causes plastic changes in the nervous system in adulthood, occasionally resulting in ubiquitous chronic pain. However, the pathogenic mechanisms of pain hypersensitivity remain unclear. Here, the authors examined the involvement of corticosterone in orofacial mechanical hypersensitivity induced by MS. To establish a rat model of MS, pups were placed in isolated cages 180 min/d and kept in a temperature-controlled environment at 22 ± 2 °C for 14 d. Mechanical allodynia in the whisker pad skin in adulthood was induced by MS and was significantly suppressed by successive postnatal subcutaneous administration of the glucocorticoid receptor antagonist mifepristone. Corticosterone levels were increased in the serum of MS rats, and successive postnatal administration of subcutaneous corticosterone to naive rats induced mechanical allodynia in the whisker pad skin. The number of P2X3 receptor-immunoreactive (P2X3R-IR) trigeminal ganglion (TG) neurons innervating the whisker pad skin was significantly increased in MS rats and decreased following subcutaneous administration of mifepristone. The number of P2X3R-IR TG neurons innervating the whisker pad skin was also significantly increased following successive postnatal administration of subcutaneous corticosterone in naive rats. Moreover, the mechanical allodynia was suppressed 30 min after administration of the P2X3R antagonist A317491 to the whisker pad skin in MS rats. These findings suggest that the increase in P2X3R-IR TG neurons innervating the whisker pad skin via enhanced neonatal corticosterone signaling by MS plays an important role in orofacial mechanical allodynia in adulthood.

Studies of Pig Complement: Measurement of Pig CH50, ACH50, and Components.

BACKGROUND: On the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. METHODS: Hemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate-labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg(++)EGTA. RESULTS: Both the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. CONCLUSIONS: As a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.

An Effective New Cryopreservation Procedure for Pancreatic Islets Using Hollow Fiber Vitrification.

The present study aimed at establishing a new cryopreservation method for mouse pancreatic islets by vitrification using hollow fibers as a container. A unique feature of the hollow fiber vitrification (HFV) method is that this method achieves stable vitrification using a minimum volume of cryoprotectant (CPA) solution, thereby ensuring high viability of the islets. The cytotoxicity, optimum composition, and concentration of the CPAs for vitrifying islets were examined. The viability, functional-integrity of vitrified islets were evaluated in comparison with those vitrified by conventional methods. Insulin secretion was measured in vitro by a static incubation assay and the metabolic functions was tested after transplantation into Streptozotocin-induced diabetic mice. The combination of 15% dimethyl sulfoxide+15% ethylene glycol resulted in the best CPA solution for the HFV of islets. HFV showed the highest viability in comparison to 2 vitrification methods, open pulled straws and vitrification with EDT324 solution. The vitrified islets stably expressed ß-cells markers NeuroD, Pancreatic and duodenal homeobox-1, and MafA. Transplantation of the vitrified islets achieved euglycemia of the host diabetic mice and response to an intraperitoneal glucose tolerance test to a similar extent as non-vitrified transplanted islets. The HFV method allows for efficient long-term cryopreservation of islets.

Involvement of Microglial P2Y12 Signaling in Tongue Cancer Pain.

To elucidate if microglial P2Y12 receptor (P2Y12R) mechanisms are involved in the trigeminal spinal subnucleus caudalis (Vc; also known as the medullary dorsal horn) in intraoral cancer pain, we developed a rat model of tongue cancer pain. Squamous cell carcinoma (SCC) cells were inoculated into the tongue of rats; sham control rats received the vehicle instead. Nociceptive behavior was measured as the head-withdrawal reflex threshold (HWRT) to mechanical or heat stimulation applied to the tongue under light anesthesia. On day 14 after the SCC inoculation, activated microglia and P2Y12R expression were examined immunohistochemically in the Vc. The HWRT was also studied in SCC-inoculated rats with successive intra-cisterna magna (i.c.m.) administration of specific P2Y12R antagonist (MRS2395) or intraperitoneal administration of minocycline, a microglial activation inhibitor. Tongue cancer was histologically verified in SCC-inoculated rats, within which the HWRT to mechanical stimulation of the tongue was significantly decreased, as compared with that of vehicle-inoculated rats, although the HWRT to heat stimulation was not. Microglia was strongly activated on day 14, and the administration of MRS2395 or minocycline reversed associated nocifensive behavior and microglial activation in SCC-inoculated rats for 14 d. The activity of Vc wide dynamic range nociceptive neurons was also recorded electrophysiologically in SCC-inoculated and sham rats. Background activity and noxious mechanically evoked responses of wide dynamic range neurons were significantly increased in SCC-inoculated rats versus sham rats, and background activity and mechanically evoked responses were significantly suppressed following i.c.m. administration of MRS2395 in SCC-inoculated rats as compared with sham. The present findings suggest that SCC inoculation that produces tongue cancer results in strong activation of microglia via P2Y12 signaling in the Vc, in association with increased excitability of Vc nociceptive neurons, reflecting central sensitization and resulting in tongue mechanical allodynia.

RAD51 G135C genetic polymorphism and their potential role in gastric cancer induced by Helicobacter pylori infection in Bhutan.

In order to evaluate the role of the RAD51 G135C genetic polymorphism on the risk of gastric cancer induced by Helicobacter pylori infection, we determined allele frequency and genotype distribution of this polymorphism in Bhutan--a population documented with high prevalence of gastric cancer and extremely high prevalence of H. pylori infection. The status of RAD51 G135C was examined by restriction fragment length polymorphism analysis of PCR amplified fragments and sequencing. Histological scores were evaluated according to the updated Sydney system. G135C carriers showed significantly higher scores for intestinal metaplasia in the antrum than G135G carriers [mean (median) 0·33 (0) vs. 0·08 (0), P = 0·008]. Higher scores for intestinal metaplasia of G135C carriers compared to those of G135G carriers were also observed in H. pylori-positive patients [0·3 (0) vs. 0·1 (0), P = 0·002] and H. pylori-positive patients with gastritis [0·4 (0) vs. 0·1 (0), P = 0·002] but were not found in H. pylori-negative patients. Our findings revealed that a combination of H. pylori infection and RAD51 G135C genotype of the host showed an increasing score for intestinal metaplasia. Therefore, RAD51 G135C might be the important predictor for gastric cancer of H. pylori-infected patients.

Epidemiology of fragility fractures in Sakaiminato, Japan: incidence, secular trends, and prognosis.

UNLABELLED: We investigated the incidence of fragility fractures from 2010 to 2012 in Sakaiminato, Japan. The incidence rates of limb fractures in Sakaiminato were lower than in Caucasian populations but had increased relative to data obtained in Japan in the 1990s. Clinical vertebral fractures occurred at higher rates in Sakaiminato than in Caucasian populations. INTRODUCTION: To elucidate the incidence and prognosis of fragility fractures in Sakaiminato, Japan. METHODS: A survey of all hip, distal radius, proximal humerus, and clinical vertebral fractures was performed from 2010 to 2012 in patients aged 50 or older in Sakaiminato city, Tottori prefecture, Japan. The age- and gender-specific incidence rates (per 100,000 person-years) were calculated based on the population of Sakaiminato city each year. The incidence rates of hip, distal radius, and proximal humerus fractures were compared with previous reports. We conducted a follow-up study assessing patients within 1 year following their initial treatment at two Sakaiminato hospitals. RESULTS: The age-adjusted incidence rates in population aged 50 years or older (per 100,000 person-years) of hip, distal radius, proximal humerus, and clinical vertebral fractures were, respectively, 217, 82, 26, and 412 in males and 567, 432, 96, and 1229 in females. Age-specific incidence rates of hip, distal radius, and proximal humerus fractures all increased since the 1990s. Our study also revealed that anti-osteoporotic pharmacotherapy was prescribed 1 year post-fracture at rates of 29, 20, 30, and 50 % for patients with hip, distal radius, proximal humerus, and clinical vertebral fractures, respectively. CONCLUSIONS: The incidence rates of limb fractures in Sakaiminato were substantially lower than Caucasian populations in northern Europe but had increased relative to data obtained in Japan in the 1990s. Unlike upper and lower limb fractures, clinical vertebral fractures occurred at higher rates in our study population than in other Asian and North European countries.

An analysis of quantum effects on the thermodynamic properties of cryogenic hydrogen using the path integral method.

In this paper, we describe the analysis of the thermodynamic properties of cryogenic hydrogen using classical molecular dynamics (MD) and path integral MD (PIMD) method to understand the effects of the quantum nature of hydrogen molecules. We performed constant NVE MD simulations across a wide density-temperature region to establish an equation of state (EOS). Moreover, the quantum effect on the difference of molecular mechanism of pressure-volume-temperature relationship was addressed. The EOS was derived based on the classical mechanism idea only using the MD simulation results. Simulation results were compared with each MD method and experimental data. As a result, it was confirmed that although the EOS on the basis of classical MD cannot reproduce the experimental data of saturation property of hydrogen in the high-density region, the EOS on the basis of PIMD well reproduces those thermodynamic properties of hydrogen. Moreover, it was clarified that taking quantum effects into account makes the repulsion force larger and the potential well shallower. Because of this mechanism, the intermolecular interaction of hydrogen molecules diminishes and the virial pressure increases.

Detection of masked mycotoxins derived from type A trichothecenes in corn by high-resolution LC-Orbitrap mass spectrometer.

Masked mycotoxins (mycotoxin glucosides) derived from type A trichothecenes were detected in commercially available corn powder reference material. These new glucosides were identified as neosolaniol-glucoside (NESGlc) and diacetoxyscirpenol-glucoside (DASGlc) on the basis of accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometric (LC-Orbitrap MS) analysis. Although the absolute structure was not clarified, 3-OH glucosylation appeared to be the most probable when considering the structures of neosolaniol and diacetoxyscirpenol and the fragmentation profiles of these masked mycotoxins. Concomitant detection of deoxynivalenol-3-glucoside, the most well-known masked mycotoxin derived from the type B trichothecene, deoxynivalenol, in the identical material further supports this probability.

Transgenic pig expressing the red fluorescent protein kusabira-orange as a novel tool for preclinical studies on hepatocyte transplantation.

INTRODUCTION: Research on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as pigs. We used transgenic pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells. MATERIALS AND METHODS: Donor hepatocytes were isolated from transgenic Kusabira-Orange (KO)-expressing pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a transgenic-cloned KO pig by Matsunari et al. and ova from a domestic pig. The hepatocyte transplant recipients were the nontransgenic, KO-negative littermates. In these recipient pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the transgenic male pig were isolated using collagenase perfusion. Hepatocytes (1 × 10(9) cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys. RESULTS: Strong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the transgenic male donor pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7. CONCLUSION: Transgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.

Emerging applications of sperm, embryo and somatic cell cryopreservation in maintenance, relocation and rederivation of swine genetics.

Advances in porcine assisted reproductive technology (ART) make it possible to use cryopreserved sperm, embryos and somatic cells in the maintenance, relocation and regeneration of swine genetics. In this review, development of key application-limiting technology is discussed in each cell type, focusing on the efficiencies, ease of storage and transportation, and minimization of pathogen transmission. Methods to regenerate swine genetics and/or models using frozen sperm, embryos and somatic cells in combination with other porcine ARTs, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transplantation (SCNT), are also discussed. The applications of these ARTs utilizing cryopreserved cells will greatly increase the efficiency as well as biosecurity for maintenance, relocation and rederivation of swine genetics/models.

Advancing pig cloning technologies towards application in regenerative medicine.

Regenerative medicine is expected to make a significant contribution by development of novel therapeutic treatments for intractable diseases and for improving the quality of life of patients. Many advances in regenerative medicine, including basic and translational research, have been developed and tested in experimental animals; pigs have played an important role in various aspects of this work. The value of pigs as a model species is being enhanced by the generation of specially designed animals through cloning and genetic modifications, enabling more sophisticated research to be performed and thus accelerating the clinical application of regenerative medicine. This article reviews the significant aspects of the creation and application of cloned and genetically modified pigs in regenerative medicine research and considers the possible future directions of the technology. We also discuss the importance of reproductive biology as an interface between basic science and clinical medicine.

A study of the glycoantigens of neonatal porcine islet-like cell clusters using a lectin microarray.

BACKGROUND: The pig pancreas is considered to be the most suitable source of islets for clinical xenotransplantation. Two types of islet transplantation are: adult pig islets and neonatal porcine islet-like cell clusters (NPCC). However, besides a-Gal expression, differences in glycosylation and xenoantigenicity between both types were not clear so fat to date. In this study, we performed lectin microarray analyses of NPCCs cultured for 1, 5, or 9 days. METHODS: We studied differences in gycoantigens among several kinds of wild-type NPCCs isolated from 1- to 3-day-old neonatal wild-type pigs (Large White/Landrace × Duroc) and cultured for 1, 5 and 9 days in Ham's 10 in the presence of nicotinamide, using a previously published technique. After sonication and centrifugation, supernatant proteins from each islet were labeled with Cy3, applied to a lectin array and scanned with an SC-Profiler for evaluation using an Array Pro Analyzer. RESULTS: The overall signals of NPCC at days 5 and 9, showed almost the same values to most lectins, whereas those on day 1 showed differences, suggesting that the NPCC on day 1 contain immature cells that gradually turn to mature NPCCs in culture.

A cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase from porcine endothelial cells.

INTRODUCTION: The Hanganutziu-Deicher (H-D) antigen with terminal N-glycolyl neuraminic acid-(NeuGc) is widely distributed in mammalian species including monkeys and apes, but is not found in humans and birds. After the knock out of α1, 3galactosyltransfease, the H-D antigen became a major antigen of the "non-Gal antigen." The expression of NeuGc is controlled by the activity of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In this study, molecular cloning of pig CMAH was performed, as the first step in producing H-D knockout pigs. METHODS: A pig endothelial cell line, MYP30, was used. The DNA sequence of pig CMAH was queried in dbEST (NCBI) using the BLAST program to search for cDNA fragments of pig CMAH, based on an alignment analysis of the mouse CMAH sequence. A polymerase chain reaction experiment was performed and candidate cDNA clones were isolated. To obtain the 5'-end and 3'-end of the open reading frame sequence, a 5'-full RACE Core Set and 3'-full RACE Core Set were used. RESULTS: We cloned and characterized the pig CMAH gene. The ATG is located in exon 4, which corresponds to the mouse gene, and the stop codon is in exon 17. In the case of the 5' site of the gene, exon 3 was identified but exons 1 and 2 are still being investigated. On the other hand, exon 18 was newly identified in the 3' site of the gene. CONCLUSION: The results represent useful information for future clinical xenotransplantation studies.

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.