Investigation of SLA class I and II haplotypes in the NIH miniature pigs

Xenotransplantation involves the transplantation of organs, tissues and cells from one species to another. A major barrier to successful xenotransplantation is the rejection of the donor tissue by the recipient immune system. Swine leukocyte antigens (SLA) are important molecules within the immune system and play an essential role in fighting infectious diseases and viruses. The present study investigated three SLA class I (SLA-1, SLA-3 and SLA-2) and three SLA class II (DRB1, DQB1 and DQA) alleles in 60 NIH miniature pigs using PCR with sequence-specific primers (PCR-SSP). As the results, nine combinations of SLA class I and II haplotypes, comprising of three homozygous and six heterozygous haplotypes, were examined. The SLA homozygous haplotype Lr-2.4/2.4 was the most prevalent, with an overall frequency of 28.3% (17/60) and heterozygous haplotype Lr-2.2/4.4 was the second most common (20.0%; 12/60), followed by haplotype Lr-4.2/4.2 (16.7%; 10/60), Lr-2.2/2.4 (15.0%; 9/60), Lr-2.2/2.2 (5.0%; 3/60), Lr-2.2/4.2 (5.0%; 3/60), Lr-2.4/4.4 (5.0%; 3/60) and Lr-2.2/3.3 (3.3%; 2/60), Lr-4.2/4.4 (1.7%; 1/60), respectively. These results provide useful information that can be used to establish highly inbred pig lines with fixed SLA homozygous alleles and haplotypes.     

Physiology of feeding in miniature pigs

The anatomy and physiology of feeding in miniature swine were studied, using gross dissection, electromyography, cinematography, and cineradiography. Small particles are preferred for ingestion, and large items are usually broken down outside of the oral cavity. The particles are initially picked up with the lower lip and then retrieved by the tongue; the tongue very rarely leaves the oral cavity. Geniohyoid, mylohyoid and digastric are the most active muscles during food collection. Mastication is fairly rapid (3 cycles/sec) and involves a transverse component which may be either medially or laterally directed. The direction of motion is generally reversed with every chew. Electromyography indicates that the transverse rotation is caused by a force couple consisting of protrusors (masseter, medial and lateral pterygoids) on one side and retrusors (zygomaticomandibularis and temporalis) on the other. The direction of the rotation is not necessarily related to the side containing the bolus. Mandibular depression is electromyographically biphasic. Concurrent tongue and hyoid movements complicate the interpretation of activity patterns in the oral floor. Both masticated food and liquid are stored between the tongue and the soft palate and epiglottis before being swallowed. Swallowing involves very strong activity in all of the hyoid and tongue muscles and very low activity in the adductors. Liquid is taken in by suction rather than lapping. Comparisons with other mammals are presented.     

Sleep apnea in obese miniature pigs

Lonergan, Robert P., III, J. Catsby Ware, Richard L. Atkinson, W. Christopher Winter, and Paul M. Suratt. Sleep apnea in obese miniature pigs. J. Appl.Physiol. 84(2): 531-536, 1998.We postulated thatthree extremely obese Yucatan miniature pigs would have more sleepapnea than three nonobese Yucatan miniature pigs. Pigs were studiedwith the use of electroencephalograms, inductance plethysmography,oximetry, expired nasal CO₂, orthermistors. All of the obese pigs, but none of the nonobese pigs, hadboth sleep apnea (8.5, 10.3, and 97.0 in obese pigs vs. 0 apnea + hypopnea/h in all nonobese pigs; P < 0.05) and oxyhemoglobin desaturation episodes during sleep [9.4 ± 3.0 vs. 0 + 0.53 (SD) mean desaturation episodes/h in obese pigsvs. nonobese pigs, respectively; P < 0.05]. Two of the extremely obese pigs had obstructive sleepapnea, whereas the third obese pig had central sleep apnea. We conclude that sleep apnea occurs in extremely obese Yucatan minipigs and suggestthat this animal can be used as a model for sleep apnea in obesity.

     

Hereditary melanoblastoma in miniature pigs and its successful therapy by devitalization technique.

Applying selective breeding for 10 years we established the MeLiM (Melanoblastoma-bearing Libechov Minipigs) strain. Melanoblastoma (MB) in this strain shows a hereditary occurrence. Cutaneous tumours are usually nodular, multiple and distributed on various parts of body. They appear in darkly pigmented animals already at the birth or during two months thereafter (57% of all animals). Numerous organ metastases mainly into the spleen, lymph nodes and lungs are regularly ascertained in animals with cutaneous MB. Tumour cells were surprisingly found also in the inner organs of phenotypically healthy minipigs in which no cutaneous MBs were observed visually (27% of all animals). About 34% of all affected piglets die during the first 2 months of age. These features document a malignancy of this tumour in the MeLiM strain. Original surgical technique was applied in more than 40 affected minipigs at 1-2 months of age. It consists in a devitalization (ischemization) of one of cutaneous tumours by the mattress sutures conducted around the tumour base without any excision of tumour tissue. This simple procedure causes a total destruction of MB cells in all cutaneous tumours as well as in all organ metastases during 4-6 months. Animals treated by this technique were fully healed of tumour cells and no relapses were observed. This technique could bring similar positive results also in therapy of human MB.     

巴马小型猪与贵州小型香猪遗传多样性的RAPD分析

RAPD analysis was performed with 31 selected single primers to study genetic diversity of two strains of miniature pigs, The percentages of polymorphisic loic in or between two strains of miniature pigs were 30.9%, 29.2% and 25.7% respectively, and the average genetic distances in and between two strains of miniature pigs were 0.120, 0.072, and 0.067 respectively. These results suggested that genetic diversity and genetic variability were poorer in and between two strains of miniature pigs than ordinary breeds' pigs.     

Repair and regeneration of osteochondral defects in the articular joints

People suffering from pain due to osteoarthritic or rheumatoidal changes in the joints are still waiting for a better treatment. Although some studies have achieved success in repairing small cartilage defects, there is no widely accepted method for complete repair of osteochondral defects. Also joint replacements have not yet succeeded in replacing of natural cartilage without complications. Therefore, there is room for a new medical approach, which outperforms currently used methods. The aim of this study is to show potential of using a tissue engineering approach for regeneration of osteochondral defects. The critical review of currently used methods for treatment of osteochondral defects is also provided. In this study, two kinds of hybrid scaffolds developed in Hutmacher's group have been analysed. The first biphasic scaffold consists of fibrin and PCL. The fibrin serves as a cartilage phase while the porous PCL scaffold acts as the subchondral phase. The second system comprises of PCL and PCL-TCP. The scaffolds were fabricated via fused deposition modeling which is a rapid prototyping system. Bone marrow-derived mesenchymal cells were isolated from New Zealand White rabbits, cultured in vitro and seeded into the scaffolds. Bone regenerations of the subchondral phases were quantified via micro CT analysis and the results demonstrated the potential of the porous PCL and PCL-TCP scaffolds in promoting bone healing. Fibrin was found to be lacking in this aspect as it degrades rapidly. On the other hand, the porous PCL scaffold degrades slowly hence it provides an effective mechanical support. This study shows that in the field of cartilage repair or replacement, tissue engineering may have big impact in the future. In vivo bone and cartilage engineering via combining a novel composite, biphasic scaffold technology with a MSC has been shown a high potential in the knee defect regeneration in the animal models. However, the clinical application of tissue engineering requires the future research work due to several problems, such as scaffold design, cellular delivery and implantation strategies.     

小型猪在肾脏疾病研究中的应用

随着对小型猪研究的不断深入,其作为人类疾病模型的优势日益明显,在生物医学研究中的应用也日趋增多。比较解剖学和生理学研究表明,小型猪的诸多生物学特性均与人类极为相似,尤其在肾脏的解剖和功能方面几乎是人类的复制品,使其在复制肾脏疾病模型,研究疾病发病机制和评估治疗策略等中具有无可替代的作用。本文将综述小型猪作为疾病动物模型在肾脏疾病研究中的应用。     

Serum biochemistry of healthy Yucatan miniature pigs

Sera collected from 24 (12 boars and 12 gilts) healthy, fasted 28-43 week-old Yucatan miniature pigs were analyzed for 21 clinical chemistry parameters. The mean, standard deviation, median, and observed range for each parameter are presented as reference values for the normal blood chemistry for this breed. Comparison with published values reveals that the Yucatan miniature pig has a blood chemistry profile comparable to that of domestic pigs and other breeds of miniature swine.     

Assessment of the in vivo biofunctionality of a biomimetic hybrid scaffold for osteochondral tissue regeneration

Chondral and osteochondral lesions represent one of the most challenging problems in the orthopedic field, as these types of injuries lead to disability and worsened quality of life for patients and have an economic impact on the healthcare system. The aim of this in vivo study was to develop a new tissue engineering approach through a hybrid scaffold for osteochondral tissue regeneration made of porous polyurethane foam (PU) coated under vacuum with calcium phosphates (PU/VAC). Scaffold characterization showed a highly porous and interconnected structure. Human amniotic mesenchymal stromal cells (hAMSCs) were loaded into scaffolds using pectin (PECT) as a carrier. Osteochondral defects in medial femoral condyles of rabbits were created and randomly allocated in one of the following groups: plain scaffold (PU/VAC), scaffold with hAMSCs injected in the implant site (PU/VAC/hAMSC), scaffold with hAMSCs loaded in pectin (PU/VAC/PECT/hAMSC), and no treated defects (untreated). The therapeutic efficacy was assessed by macroscopic, histological, histomorphometric, microtomographic, and ultrastructural analyses at 3, 6, 12, and 24 weeks. Histological results showed that the scaffold was permissive to tissue growth and penetration, an immature osteocartilaginous tissue was observed at early experimental times, with a more accentuated bone regeneration in comparison with the cartilage layer in the absence of any inflammatory reaction.     

Immunological state of adult germfree miniature minnesota pigs

Fundamental hematological and immunological data were obtained on sexually mature germfree miniature pigs fed, after the milk diet period, with cereal-type diet sterilized by γ-radiation, and were compared with data of control conventional animals. Germfree adult pigs had a lower count of peripheral blood leukocytes with a lower percentage of neutrophil granulocytes and without any younger forms, a lower total serum protein level with a negligible amount of γ and α₂ globulin fractions and a higher serum albumin and β globulin level. In the mesenteric lymph nodes and in spleen, surface IgA-bearing cells predominated over surface IgG-bearing cells. Also a large amount of IgA-containing cells was found in the intestinal lamina propria, where the IgG cells were present in a negligible amount. IgM cells were the most frequent surface isotype in peripheral blood. The count of blood T lymphocytes was more than doubled.     

Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Cartilage graft is considered to be useful in repairing chondral or osteochondral defects. One method of the cartilage graft is achieved by autologous chondrocyte transplantation following cell culture. However, chondrocytes change their phenotype during culture. We used costal chondrocytes cultured over agarose (suspension culture) as a source of graft materials. The suspension-cultured chondrocytes formed aggregate in culture. We first examined the expressions of cartilage-specific matrices of cultured chondrocytes after two weeks in culture. The chondrocytes cultured over agarose expressed more type II collagen mRNA than those cultured on plastic dishes did after two weeks in culture. Safranin O staining showed the presence of glycosaminoglycans in the chondrocyte culture over agarose, while glycosaminoglycans were not observed in the culture on plastic dishes. We then examined the changes of rat articular osteochondral defects after transplantation of suspension-cultured chondrocytes. The aggregate of suspension-cultured chondrocytes was easily picked up with forceps and transplanted in the osteochondral defects. The defects were filled with safranin O-stained hyaline cartilage tissue two weeks after chondrocyte transplantation. On the contrary, the fibrous materials, which were not stained with safranin O, were observed in the control defects. These results suggest that the suspension-cultured chondrocytes are useful for autologous cartilage grafts by preserving chondrocyte phenotype.     

Production of transgenic miniature pigs by pronuclear microinjection

Miniature pig is an attractive animal for a wide range of research fields, such as medicine and pharmacology, because of its small size, the possibility of breeding it under minimum environmental controls and the physiology that is potentially similar to that of human. Although transgenic technology is useful for the analysis of gene function and for the development of model animals for various diseases, there have not yet been any reports on producing transgenic miniature pig. This study is the first successful report concerning the production of transgenic miniature pig by pronuclear microinjection. The huntingtin gene cloned from miniature pig, which is a homologue of candidate gene for Huntington's disease, connected with rat neuron-specific enolase promoter region, was injected into a pronucleus of fertilized eggs with micromanipulator. The eggs were transferred into the oviduct of recipient miniature pigs, whose estrus cycles were previously synchronized with a progesterone analogue. A total of 402 injected eggs from 171 donors were transferred to 23 synchronized recipients. Sixteen of them maintained pregnancy and delivered 65 young, and one resulted in abortion. Five of the 68 offspring (three of which were aborted) were determined to have transgene by PCR and Southern analysis. The overall rate of transgenic production was 1.24% (transgenic/injected eggs). This study provides the first success and useful information regarding production of transgenic miniature pig for biomedical research.     

Mechano-regulation of stem cell differentiation and tissue regeneration in osteochondral defects

Cartilage defects that penetrate the subchondral bone can undergo spontaneous repair through the formation of a fibrous or cartilaginous tissue mediated primarily by mesenchymal stem cells from the bone marrow. This tissue is biomechanically inferior to normal articular cartilage, and is often observed to degrade over time. Whether or not biomechanical factors control the type and quality of the repair tissue, and its subsequent degradation, have yet to be elucidated. In this paper, we hypothesise a relationship between the mechanical environment of mesenchymal stem cells and their subsequent dispersal, proliferation, differentiation and death. The mechano-regulation stimulus is hypothesised to be a function of strain and fluid flow; these quantities are calculated using biphasic poroelastic finite element analysis. A finite element model of an osteochondral defect in the knee was created, and used to simulate the spontaneous repair process. The model predicts bone formation through both endochondral and direct intramembranous ossification in the base of the defect, cartilage formation in the centre of the defect and fibrous tissue formation superficially. Greater amounts of fibrous tissue formation are predicted as the size of the defect is increased. Large strains are predicted within the fibrous tissue at the articular surface, resulting in significant cell apoptosis. This result leads to the conclusion that repair tissue degradation is initiated in the fibrous tissue that forms at the articular surface. The success of the mechano-regulation model in predicting many of the cellular events that occur during osteochondral defect healing suggest that in the future it could be used as a tool for optimising scaffolds for tissue engineering.     

Anaphylactoid reactions mediated by autoantibodies to cholesterol in miniature pigs

Antoantibodies to cholesterol were detected and purified from normal (nonimmunized) pig serum. The antibodies were assayed by ELISA with crystalline cholesterol as an Ag and by C-dependent damage to cholesterol-laden liposomes. Intravenous injection of liposomes containing cholesterol into anesthetized animals caused decreased hemolytic complement titers, and induced a reaction consisting of transient neutropenia, thrombocytopenia, respiratory distress, cyanosis, pulmonary and systemic hypertension, and decreased cardiac output. Plasma levels of thromboxane B2 and 6-keto-prostaglandin F1 alpha increased 1300 and 200%, respectively, and leukocyte and platelet counts decreased by 36 and 38%, respectively. Injection of cholesterol-free liposomes did not induce the reaction. These results show that naturally occurring autoantibodies to cholesterol can initiate C activation and can be associated with anaphylactoid reaction to exogenously administered cholesterol in pigs.     

The time of appearance of blood group antigens in miniature pigs

Erythrocytes of foetuses and piglets of miniature pigs of different age were tested with 50 to 55 blood group reagents of 15 genetic systems. Out of 49 blood factors found to be present in parent animals, 40 were present in 34–46-day-old foetuses. Factor Kb was detected in 66-day-old foetuses, and other factors of the K system (Ka, Kc, Kd and Ke) at 77 days of age. Factor A was demonstrated in one day old piglets, factor 0 not earlier than in piglets aged 13 to 24 days.