Cardiac function and morphology of Hanford miniature swine and Yucatan miniature and micro swine

The use of miniature swine in biomedical research is increasing; however, a comparison of cardiac function and morphology between strains has yet to be characterized. The purpose of this project was to examine comprehensive hemodynamics and cardiac morphology of three groups of ten normal, 4 months old, age-matched Yucatan miniature (MINI) pigs, Yucatan micropigs (MICRO) and Hanford (HAN) miniature pigs, 5 males and five females per group. Closed chest cardiac catheterization under equivalent conditions was performed followed by post mortem cardiac morphometry. Mean arterial pressure was significantly greater in the Hanford group when compared to both the minipig and micropig pigs (HAN: 89 +/- 4; MINI: 48 +/- 3; MICRO: 53 +/- 2 mmHg). Pulmonary vascular resistance was significantly different between the three groups (HAN: 9 +/- 1; MINI: 60 +/- 12; MICRO: 111 +/- 29 dyne x sec/cm x m2). The Hanford strain had a significantly smaller heart weight to body weight ratio than the other two groups (HAN: 4.6 +/- 1.0; MINI: 5.7 +/- 0.1; MICRO: 5.5 +/- 1.0). Variations in cardiovascular parameters occur among these strains and should be considered when constructing experimental designs.     

Cardiovascular function in anesthetized miniature swine.

Miniature swine anesthetized with pentobarbital were studied with respect to their cardiovascular function under control conditions and in response to catecholamines, baroreceptor inhibition, bilateral vagotomy and vagal nerve stimulation. Measurements included aortic pressure, heart rate, intraventricular pressure and its maximum rate of rise during contraction, carotid blood flow and resistance, femoral blood flow and resistance, and renal blood flow and resistance. The cardiovascular actions of norepinephrine, epiniphrine and isoproterenol were similar to those in other mammals, and the adrenergic receptor mechanisms also were susceptible to blockade with phentolamine or propranolol. Inhibition of the carotid baroreceptors was accompanied by elevation of aortic pressure, reflex bradycardia and increased femoral and renal resistances. Bileteral vagotomy was followed by hypertension, tachycardia and increased renal resistance. Changes in femoral resistance to these procedures differed between the two strains of miniature swine studied. Stimulation of the peripheral end of either vagus nerve was accompanied by bradycardia without hypotension.     

Post-ischemia immunosuppression in a miniature swine model

Yucatan miniature swine were the experimental model used to examine the effect of ischemia-injury on post-ischemic monocyte (MO) and immune function. Monocyte plasminogen activator (PA) was depressed while MO tissue factor activity was increased. The ability of porcine monocytes to generate a primary in vitro antibody forming cell (AFC) response to sheep red blood cells (SRBC) also was depressed by ischemic injury. The mechanism by which ischemic injury modulated immunosuppression appeared to be through generation of immunosuppressive serum substances.     

Class II genes of miniature swine

Genomic clones corresponding to class II genes of theSLA ^c haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding theDRB andDQB genes were identified on the basis of hybridization with locus-specific 3 untranslated cDNA probes. Cluster 4 contained exons of bothDOB andDQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containingDP, DR, andDO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the association number M29944. Address correspondence and offprint requests to: C. LeGuern.     

Class II genes of miniature swine

Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.     

Class II genes of miniature swine

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLA ^c haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique genes were obtained which showed no evidence of cross-hybridization, while genes showed extensive cross-hybridization and were frequently detected in the library by more than one human gene probe. These data are consistent with early evolutionary divergence of a genes, prior to mammalian speciation, and with continuing evolution of genes, with possible shared usage of these genes by different a loci. The data also imply that genes can readily be assigned to loci homologous to their human counterparts, but that genes will require further mapping and/or sequence analysis to confirm assignments.     

Heritable ventricular septal defect in Yucatan miniature swine

A heritable ventricular septal defect (VSD) was found in a strain of Yucatan miniature swine. The defect was determined to be a high membranous VSD analogous in anatomic location to the most common from of VSD in humans. Eighteen animals were studied clinically, hemodynamically and at necropsy to characterize the defect. Three mature animals developed pulmonary hypertension. Three animals were found to have a patent foramen ovale (PFO) in addition to the VSD. VSD is heritable probably due to polygenic factors. VSD in Yucatan miniature swine may be a suitable model of the human disease syndrome.     

A model of postprandial hyperinsulinemia in miniature swine

This report describes a new animal model of postprandial hyperinsulinemia (PPH) in adult miniature swine that consume a diet simulating that of affluent Western societies. Two progressive levels of PPH were induced experimentally by injecting subcutaneously low and high doses of purified porcine insulin without causing acute detrimental clinical effects or significant biological effects on total serum cholesterol, sodium and potassium concentrations, mean arterial blood pressure, or heart rate. Physiologic postprandial increments in total serum triglyceride concentrations were inhibited by experimentally-induced PPH. With this model, the in vivo effects of homologous PPH can be studied in a dose-responsive manner. Areas of potential research use of this model include study of the chronic effects of PPH on lipoprotein metabolism, the development of atherosclerosis and diabetes mellitus, and the association with regional body fat distribution and metabolism.     

Vascularized composite islet-kidney transplantation in a miniature swine model

Previous work from this laboratory has demonstrated that transplantation of allogeneic thymic tissue as part of a composite vascularized graft is far more successful in terms of both engraftment and long-term survival than transplantation of thymic tissue or cells alone. We have subsequently extended this concept to transplantation of allogeneic islets, comparing survival of islet cell suspensions to that of vascularized composite islet-kidneys (IK), prepared by injection of autologous islets underneath the renal capsule 2-3 months prior to allogeneic transplantation of the composite organ. We have utilized partially inbred miniature swine with defined MHC loci as the experimental large animals for this study, permitting reproducible transplantation across specific MHC barriers. Composite IK have been transplanted successfully across minor and full MHC mismatch barriers, using treatment regimens previously demonstrated to induce long-term tolerance of kidney allografts across these barriers. IK allografts containing ≥5000 islet equivalents (IE)/kg recipient body weight were found capable of reversing surgically induced diabetes, while injection of comparable numbers of purified islets via the portal vein or under the renal capsule did not. Studies are also being directed toward preparation of autologous “thymo-islet-kidneys” (TIK), for potential use as xenografts, in which the thymic component is intended to induce tolerance and the islets to reverse diabetic hyperglycemia. The use of both types of composite organ transplants may eventually be applicable to the treatment of type I diabetic patients suffering from end-stage diabetic nephropathy.     

Physiological levels and action of dehydroepiandrosterone in Yucatan miniature swine

The biological role of dehydroepiandrosterone (DHEA) and its less active sulphated conjugate DHEAS was investigated in two experiments using Yucatan miniature swine. In experiment 1, plasma levels of both DHEA(S) among males were greater than female pigs that ranged in age from 0.3 to 84 mo old (P < 0.0001). In males, DHEA(S) were related inversely to serum triglycerides; DHEA was positively related to triglycerides in females (P < 0.01). In experiment 2, four 2-yr old male pigs, used as their own control, showed a 5% decrease in body weight, 11% increase in energy expenditure, 88% increase in lipid, and 100% decrease in glucose utilization (P < 0.0001) in response to DHEA vs. placebo treatments when adjusted for body weight. Plasma DHEA(S) were not different between treatment conditions. Glucose tolerance and plasma insulin levels were not different from controls. In vivo response to norepinephrine indicated beta-adrenergic sensitivity was altered by DHEA. Present findings suggest DHEA and/or its hormone products are important in modulating energy expenditure and lipid utilization for energy in male animals. The role of DHEA in energy metabolism and the difference between sexes warrant further investigation.     

Acute renal function in chronically sympathectomized deoxycorticosterone acetate-treated miniature swine

We recently validated a swine model in which chronic treatment with 6-hydroxydopamine (6-OHDA) produced an effective sympathectomy. These sympathectomized swine demonstrated a significantly attenuated hypertensive response when treated with deoxycorticosterone acetate (DOCA). Because renal nerve activity is elevated and important in controlling renal function and blood pressure in the DOCA swine model, we wanted to study the effect of chronic sympathectomy on acute renal hemodynamics and tubular function. Kidney function was assessed in 14 DOCA-treated miniature swine, 8 of which were sympathectomized by chronic treatment with 6-OHDA, while 6 served as controls. Effective renal sympathectomy in this model has been previously confirmed by a significant reduction (97%) of norepinephrine in renal cortical tissue. When anesthetized, mean arterial pressure and renal blood flow were similar between the two groups. Glomerular filtration rate was lower by 43%, urine flow rate by 71%, sodium excretion by 66%, and potassium excretion by 48% in the 6-OHDA DOCA animals. All of these parameters were significantly different from the intact DOCA controls. These results indicate that anesthetized, chronically sympathectomized swine exhibit decreased renal excretory function. The changes in renal function may have been due to the development of a tubular or glomerular supersensitivity to circulating antinatriuretic factors, since the 6-OHDA group had a 28% greater pressor response to the alpha-agonist phenylephrine and a significantly greater fall in mean arterial pressure in response to alpha-blockade with prazosin when compared with the controls. These changes in renal function may also explain why the 6-OHDA animals demonstrated a slight increase in mean arterial pressure in response to DOCA. Because acute renal denervation in DOCA-treated swine produces a diuresis and natriuresis, this study affirms that there may be important functional differences in acutely versus chronically denervated kidneys for which the implications under normal physiologic conditions are unknown.     

Uterine-specific proteins in luminal secretions of swine leukocyte antigen-inbred miniature swine with cystic endometrial hyperplasia

Uterine-specific proteins were evaluated in luminal secretions of Swine Leukocyte Antigen (SLA)-inbred miniature swine with cystic endometrial hyperplasia (CEH) by polyacrylamide gel electrophoresis (PAGE) and Sephacryl S-200 chromatography. CEH and non-CEH (NCEH) pigs (n = 23) were killed on Days 4, 9, and 15 of the estrous cycle (estrus = Day 0) and reproductive tracts were excised for collection of serum and uterine luminal protein. Uterine luminal protein was greater (p less than 0.05) on Day 9 than on Days 4 and 15 (42.9 vs. 6.1 and 29.4 mg, respectively) for CEH pigs and Days 4, 9, and 15 (8.5, 10.1, and 25.6 mg, respectively) for NCEH pigs. The presence of the uterine-specific acidic and basic proteins, as revealed by PAGE, was affected (p less than 0.025) by day of the cycle and CEH condition. All Day 15 NCEH pigs (4 of 4) produced the complete profile of these proteins, whereas none of the uterine protein samples representing other treatment groups contained them. Some minor acidic protein components were present in cystic fluids from CEH pigs, but these fluids lacked the typical uterine-specific proteins. PAGE analysis of Sephacryl S-200 fractions from uterine fluids of Day 15 NCEH pigs revealed the uterine-specific proteins in fractions IV (Mr 40,000) and V (Mr 15,000). The results of the investigation demonstrate an impairment in the secretion of uterine-specific proteins in cyclic SLA miniature swine with cystic endometrial hyperplasia.     

Identification of novel porcine endogenous betaretrovirus sequences in miniature swine

PCR amplification of genomic DNA from miniature swine peripheral blood lymphocytes, using primers corresponding to highly conserved regions of the polymerase (pol) gene, allowed the identification of two novel porcine endogenous retrovirus (PERV) sequences, PMSN-1 and PMSN-4. Phylogenetic analyses of the nucleotide sequences of PMSN-1 and PMSN-4 revealed them to be most closely related to betaretroviruses. The identification of PERVs belonging to the Betaretrovirus genus shows that endogenous retroviruses of this family are more broadly represented in mammalian species than previously appreciated. Both sequences contained inactivating mutations, implying that these particular loci are defective. However, Southern blot analysis showed additional copies of closely related proviruses in the miniature swine genome. Analyses of fetal and adult miniature swine tissues revealed a broad mRNA expression pattern of both PMSN-1 and PMSN-4. The most abundant expression was detected in whole bone marrow c-kit(+) (CD117(+)) progenitor bone marrow cells, fetal liver, salivary gland, and thymus. It appears unlikely that functional loci encoding these novel PERV sequences exist, but this remains to be established. The betaretrovirus sequences described in this report will allow such investigations to be actively pursued.     

Developmental changes of myelin-related lipids in brain of miniature swine

The biochemical development of whole brains from male and female miniature swine aged 2 weeks to 1 years was studied. The data were similar for both sexes. The brain-body weight ratio declined rapidly for the first 10–12 weeks after birth, then decreased at a slower rate up to 1 year. Total brain lipid weight and lipid phosphorus changed rapidly during the first 8–10 weeks of life, but thereafter changed very little. The glycolipid content rapidly increased during the first 12–14 weeks of life and then increased at a slower rate. Total brain cholesterol increased continuously over the time period studied, although the rate of increase appeared to decline with age. Monogalactosyl diacylglycerol concentration remained constant up to about 8 weeks of age, but then decreased continuously up to 1 year. The alkali-labile fatty acid composition of pig brain remained relatively constant except for increases in 181 and 226(n – 3) and a decrease in 160. The increase in percentage of 181 was most rapid during the first 10 weeks of age. These data suggest that the growth spurt or active myelination phase of miniature pig brain development ends at 8–10 weeks post partum.     

Teaching cases: Heterochromia

Abstract: A patient was noted to have 2 different eye colours and miosis in her left eye. She ultimately received a diagnosis of congenital Horner syndrome. Determinants of eye colour and possible clinical significance are discussed.The case: A 35-year-old woman with a hypertensive emergency and confusion presented to the emergency department. Incidentally, we noted that she had 2 different coloured eyes (heterochomia) and miosis of her left eye (Figure 1). The patient reported that her eyes had been different colours since very early in her childhood.Open in a separate windowFigure 1: This 35-year-old woman had different coloured eyes since birth. The entire iris of her right eye is brown, and the iris of the left eye is greenish brown. Her left pupil is smaller than the right, which is consistent with the diagnosis of congenital Horner syndrome.Although some patients have pigment changes involving only 1 segment of the iris (segmental heterochromia or heterochromia iridium),¹ our patient''s entire iris was involved (complete heterochromia or heterochromia iridis). Heterochromia iridis is rare, affecting fewer than 200 000 people in the United States.² Although uncommon in humans, it is common in some breeds of cats, dogs and horses.Eye colour is determined by the concentration and distribution of melanin in the iris, with both genetic and physiologic factors affecting determination and maintenance of iris colour. Most human cases of heterochromia are sporadic and benign, and they occur without any detectable underlying abnormality. Congenital heterochromia occurs in a variety of syndromes, including Sturge–Weber syndrome, Waardenburg syndrome and Parry–Romberg syndrome (3Table 1Open in a separate windowDisruption of the sympathetic stimulation of the melanocytes in the superficial stroma of the iris (especially as a child) can lead to heterochromia. Horner syndrome from the unilateral impairment of sympathetic nerves leads to ptosis, miosis, a lag in pupil dilation, enopthalmos (the impression of a sunken eye) and facial anhidrosis (decreased sweating on 1 side of the face). Acquired heterochromia can occur in adults in rare cases as a result of acquired Horner syndrome. In contrast to patients with acquired Horner syndrome, patients with congenital Horner syndrome, such as our patient, often lack several features of the syndrome.In adults with acquired heterochromia and miosis, Fuchs heterochromic cyclitis and sympathetic heterochromia must be considered. Unilateral sympathetic nerve lesions such as paravertebral neurilemmoma and neuroblastoma should also be considered. Our patient''s clinical presentation was inconsistent with any of these causes. Sympathetic heterochromia was suspected but investigations, including urinary catecholamines and an MIBG (iodine-131-meta-iodobenzylguanidine) scan, did not reveal excess catecholamine secretion or a sympathetic tumour.The patient''s blood pressure was managed with appropriate medication, and she was ultimately discharged from our care with a reversal of her confusion. There was no further follow-up with regard to her eye colour.Habib Ur Rehman MBBS Department of Internal Medicine Regina General Hospital Qu''Appelle Health Region Regina, Sask.     

Hematologic and serum biochemical values for Yucatan miniature swine

Hematologic and serum biochemical values were determined for healthy, mature Yucatan miniature swine, Sus scrofa. These values were similar to those reported for other breeds of swine. There was no effect on erythrocyte count, hematocrit, MCV, MCH, MCHC, RDW, platelet count, or leukocyte count attributable to sex (p greater than 0.05). Differential leukocyte counts generated on an automated multichannel blood cell counter, having a three part leukocyte differential capability, were compared to 100-cell manual leukocyte differentials. Determination of lymphocyte and non-lymphocyte fractions on this system were not significantly different from microscopic differentials (p less than 0.001). However, the mononuclear cell count did not correlate well with the percentage of monocytes determined manually (r = 0.084, p greater than 0.5). Leukocytes, erythrocytes, and platelets behaved properly with respect to counting thresholds as modified for counting cells of other common domestic species on this automated cell counter.     

Characterization of a polymorphism of CD4 in miniature swine.

A polymorphism of CD4 in miniature swine has been identified by failure of cells from some animals to react with mAb 74-12-4. The phenotypic, molecular genetic, and functional characteristics of these animals have been defined. Cells from heterozygous animals bear approximately 50% the number of 74-12-4-reactive molecules on their surface as do cells from animals homozygous for the wild type. Animals of both phenotypes demonstrate similar flow cytometric profiles for CD8+ T cells. Northern blot analysis confirms the presence of mRNA for CD4 among PBL of animals failing to stain with 74-12-4. CD4 allelism is confirmed by Southern blot analysis, revealing RFLP. Function of the CD4 subset in vivo, as demonstrated by antibody production against a T cell-dependent Ag, is similar between animals of both phenotypes. Proliferative responses to PHA and alloantigen stimulation by a full haplotype mismatch or a class II mismatch alone are equivalent for animals of both phenotypes. These data suggest that the allelic form of CD4, designated CD4.2 in contrast to the wild-type CD4.1, is capable of performing normally as an accessory molecule in the generation of immune responses. Furthermore, antixenogeneic responses to C57B10.BR were equivalent, suggesting that both CD4 molecular types may be capable of interacting with xenogeneic class II molecules. Although the polymorphism includes differences in exons 3 and 4, regions thought to encode portions of the molecule interacting with MHC class II, these results imply that this naturally occurring CD4 polymorphism does not affect the interaction with class II molecules.     

Genomic presence of recombinant porcine endogenous retrovirus in transmitting miniature swine

Background

The Coccolithoviridae is a recently discovered family of viruses that infect the marine coccolithophorid Emiliania huxleyi. Following on from the sequencing of the type strain EhV-86, we have sequenced a second strain, EhV-163.

Results

We have sequenced approximately 80% of the EhV-163 genome, equating to more than 200 full length CDSs. Conserved and variable CDSs and a gene replacement have been identified in the EhV-86 and EhV-163 genomes.

Conclusion

The sequencing of EhV-163 has provided a wealth of information which will aid the re-annotating of the EhV-86 genome and identified a gene insertion in EhV-163.