Sinclair Swine Research Articles

Performance of Molecular Lymphosonography for Detection and Quantification of Metastatic Involvement in Sentinel Lymph Nodes.

To assess the performance of molecular lymphosonography with dual-targeted microbubbles in detecting and quantifying the metastatic involvement in sentinel lymph nodes (SLNs) using a swine melanoma model. Targeted microbubbles were labeled with P-selectin and αV β3 -integrin antibodies. Control microbubbles were labeled with immunoglobulin G antibodies. First lymphosonography with Sonazoid (GE Healthcare, Oslo, Norway) was used to identify SLNs. Then dual-targeted and control microbubbles were injected intravenously to detect and quantify metastatic disease in the SLNs. Distant non-SLNs were imaged as benign controls. All evaluated lymph nodes (LNs) were surgically removed, and metastatic involvement was characterized by a histopathologic analysis. Two radiologists blinded to histopathologic results assessed the baseline B-mode images of LNs, and the results were compared to the histologic reference standard. The mean intensities of targeted and control microbubbles within the examined LNs were measured and compared to the LN histologic results. Thirty-five SLNs and 34 non-SLNs from 13 Sinclair swine were included in this study. Twenty-one SLNs (62%) were malignant, whereas 100% of non-SLNs were benign. The sensitivity of B-mode imaging for metastatic LN diagnosis for both readers was relatively high (90% and 71%), but the specificity was very poor (50% and 58%). The sensitivity and specificity of molecular lymphosonography for metastatic LN detection were 91% and 67%, respectively. The mean intensities from dual-targeted microbubbles correlated well with the degree of metastatic LN involvement (r = 0.6; P < 0.001). Molecular lymphosonography can increase the specificity of metastatic LN detection and provide a measure to quantify the degree of metastatic involvement.

Treatment with Modified Heat Shock Protein Repigments Vitiligo Lesions in Sinclair Swine

HSP70i is secreted by stressed melanocytes, is associated with human vitiligo lesions, and functionally contributes to a mouse model of vitiligo. Henning etal. report that treatment with a modified version of the protein reversed depigmentation in Sinclair swine, a useful animal model of vitiligo. These studies provide the rationale for testing in human studies.

Informing efficient pilot development of animal trauma models through quality improvement strategies

Poor quality data in preclinical trials can result from inconsistent and unstandardized experimental processes. Unpredictable pre-intervention variability generates unreliable data, biases outcomes and results in needless waste of animals and resources. We applied Define-Measure-Analyse-Improve-Control (DMAIC) quality improvement processes to pilot development of a swine model of trauma, haemorrhagic shock and coagulopathy. The goal was to reduce variability through protocol standardization and error reduction. Six male Sinclair swine were sequentially anesthetized, intubated, mechanically ventilated and instrumented, then subjected to multiple-hit injury, followed by fluid resuscitation monitoring and coagulation testing. Experimental tasks were defined and mapped. Performance measures were task performance times, subject stabilization time and number of task execution errors. Process improvement was assessed by reduced times and errors, and subject stability at target physiological values. Previously-overlooked performance errors and deficiencies were identified. 'Mistake-proofing' actions included personnel retraining, revisions of standard operating procedures and use of checklists. The quality improvement pilot trial produced a stable model with reduced protocol deviations. Data quality can be improved and animal waste minimized, if experimental planning incorporates strategies to ensure protocol adherence and reduced operator performance variation and errors. Properly designed pilot trials can be essential components of refinement and reduction strategies in animal-based research.

HSP70iQ435A-Encoding DNA Repigments Vitiligo Lesions in Sinclair Swine

Human HSP70iQ435A carries a single amino-acid modification within the dendritic cell activating region and tolerizes dendritic cells invitro. The underlying DNA was used to prevent and treat disease in vitiligo mouse models through reduced dendritic cell activation and diminished skin T-cell infiltration, suggesting the same may be useful for patients. Physiologic differences between mouse and human skin then called for studies in large animals with human-like skin. We established the efficiency of DNA jet injection into swine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Vitiligo lesions in Sinclair swine were treated with plasmid DNA to measure changes in depigmentation, T-cell infiltration, expression of HSP70i in skin, serum HSP70i, and anti-HSP70i serum titers. Remarkable repigmentation following HSP70iQ435A-encoding DNA treatment persisted throughout the 6-month follow-up period. Repigmentation was accompanied by an initial influx of T cells accompanied by increased CD4/CD8 ratios, waning by week 15. Melanocytes spanned the border of repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. Serum titer fluctuations were not treatment-associated. Importantly, treatment did not interfere with melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.

Interventional vitamin C: A strategy for attenuation of coagulopathy and inflammation in a swine multiple injuries model.

Coagulopathy and inflammation induced by hemorrhagic shock and traumatic injury are associated with increased mortality and morbidity. Vitamin C (VitC) is an antioxidant with potential protective effects on the proinflammatory and procoagulant pathways. We hypothesized that high-dose VitC administered as a supplement to fluid resuscitation would attenuate inflammation, coagulation dysfunction, and end-organ tissue damage in a swine model of multiple injuries and hemorrhage. Male Sinclair swine (n = 24; mean body weight, 27 kg) were anesthetized, intubated, mechanically ventilated, and instrumented for physiologic monitoring. Following stabilization, swine were subjected to shock/traumatic injury (hypothermia, liver ischemia and reperfusion, comminuted femur fracture, hemorrhagic hypotension), resuscitated with 500 mL of hydroxyethyl starch, and randomized to receive either intravenous normal saline (NS), low-dose VitC (50 mg/kg; LO), or high-dose VitC (200 mg/kg; HI). Hemodynamics, blood chemistry, hematology, and coagulation function (ROTEM) were monitored to 4 hours postresuscitation. Histological and molecular analyses were obtained for liver, kidney, and lung. Compared with VitC animals, NS swine showed significant histological end-organ damage, elevated acute lung injury scores, and increased mRNA expression of tissue proinflammatory mediators (IL-1β, IL-8, TNFα), plasminogen activation inhibitor-1 and tissue factor. There were no statistically significant differences between treatment groups on mean arterial pressure or univariate measures of coagulation function; however, NS showed impaired multivariate clotting function at 4 hours. Although correction of coagulation dysfunction was modest, intravenous high-dose VitC may mitigate the proinflammatory/procoagulant response that contributes to multiple organ failure following acute severe multiple injuries. Prospective randomized controlled blinded trial study, Preclinical (animal-based).

1262 HSP70iQ435A-encoding DNA repigments vitiligo lesions in Sinclair swine

HSP70iQ435A carries an amino acid modification within the substrate binding domain, resulting in a stress protein that can tolerize dendritic cells in vitro. Introduced by gene gun, an expression plasmid encoding HSP70iQ435A can both prevent and treat autoimmune disease in mouse models of vitiligo. Here we tested the efficacy and safety of human HSP70iQ435A encoding DNA in human-like skin. We compared available methods of introducing DNA to porcine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Next, vitiligo lesions in a substrain of Sinclair swine were treated with up to 5 mg plasmid DNA (N=6 lesions) or vehicle (N=4) weekly for 4 weeks to measure changes in lesional area, CD4 and CD8 T cell infiltration and serum HSP70 content and HSP70 antiserum titers in monthly biopsies and blood samples. After the gene gun, jet injection demonstrated greatest plasmid-encoded gene expression. Whereas untreated lesions continued to enlarge, significant repigmentation in response to HSP70iQ435A-encoding DNA lasted throughout the 6 months follow-up. Thus, serial samples of repigmenting, human-like skin and associated serum samples are now available to study the repigmentation process. Repigmentation was accompanied by an initial influx of CD4 T cells, followed by a significant reduction in perilesional T cells 3 months after treatment. Ta99+ melanocytes were 50% less plentiful but not absent from repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. No treatment-related changes in HSP70 or anti-HSP70 titers were observed. Importantly, treatment did not interfere with but rather supported melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.

Sentinel Lymph Node Characterization with a Dual-Targeted Molecular Ultrasound Contrast Agent.

The purpose of this study was to assess the performance of molecular ultrasound with dual-targeted microbubbles to detect metastatic disease in the sentinel lymph nodes (SLNs) in swine model of naturally occurring melanoma. The SLN is the first lymph node in the lymphatic chain draining primary tumor, and early detection of metastatic SLN involvement is critical in the appropriate management of melanoma. Nine Sinclair swine (weight 3-7kg; Sinclair BioResources, Columbia, MO, USA) with naturally occurring melanoma were examined. Siemens S3000 scanner with a 9L4 probe was used for imaging (Siemens Healthineers, Mountain View, CA). Dual-targeted contrast agent was created using Targestar SA microbubbles (Targeson, San Diego, CA, USA) labeled with ανβ3-integrin and P-selectin antibodies. Targestar SA microbubbles labeled with IgG-labeled were used as control. First, peritumoral injection of Sonazoid contrast agent (GE Healthcare, Oslo, Norway) was performed to detect SLNs. After that, dual-targeted and IGG control Targestar SA microbubbles were injected intravenously with a 30-min interval between injections. Labeled Targestar SA microbubbles were allowed to circulate for 4min to enable binding. After that, two sets of image clips were acquired several seconds before and after a high-power destruction sequence. The mean intensity difference pre- to post-bubble destruction within the region of interest placed over SLN was calculated as a relative measure of targeted microbubble contrast agent retention. This process was repeated for non-SLNs as controls. All lymph nodes evaluated on imaging were surgically removed and histologically examined for presence of metastatic involvement. A total of 43 lymph nodes (25 SLNs and 18 non-SLNs) were included in the analysis with 18 SLNs demonstrating metastatic involvement greater than 5% on histology. All non-SLNs were benign. The mean intensity (± SD) of the dual-targeted microbubbles for metastatic SLNs was significantly higher than that of benign LNs (18.05±19.11 vs. 3.30±6.65AU; p=0.0008), while IgG-labeled control microbubbles demonstrated no difference in retained contrast intensity between metastatic and benign lymph nodes (0.39±1.14 vs. 0.03±0.24AU; p=0.14). The results indicate that dual-targeted microbubbles labeled with P-selectin and ανβ3-integrin antibodies may aid in detecting metastatic involvement in SLNs of melanoma.

Novel Small Molecule Probes for Metastatic Melanoma.

Actively targeting probe 1b, an unsymmetrical bivalent dipeptide mimic, selectively bound melanoma over healthy skin tissue in histological samples from patients and Sinclair swine. Modifications to 1b gave agents 2-4 that contain a near-IR aza-BODIPY fluor. Contrary to our expectations, symmetrical probe 3 gave the highest melanoma-to-healthy skin selectivity in histochemistry and experiments with live cells; this was surprising because 2, not 3, is unsymmetrical like the original lead 1. Optical imaging of 3 in a mouse melanoma model failed to show tumor accumulation in vivo, but the probe did selectively accumulate in the tumor (some in lung and less in the liver) as proven by analysis of the organs post mortem.

Lack of TERT promoter mutations in melanomas with extensive regression

Lack of TERT promoter mutations in melanomas with extensive regression

Contrast-enhanced sonography for detection of secondary lymph nodes in a melanoma tumor animal model.

To investigate the use of contrast-enhanced ultrasound imaging (US) for detection of secondary lymph nodes (LNs) in a naturally occurring melanoma swine model compared to surgery and pathologic assessment. Twenty-seven Sinclair swine were studied. The perfluorobutane microbubble contrast agent Sonazoid (GE Healthcare, Oslo, Norway) was administered (1.0 mL total dose) around the melanoma, and contrast-enhanced US was used to localize contrast-enhanced sentinel lymph nodes (SLNs). Then Sonazoid (dose, 0.25-1.0 mL) was injected into the SLNs to detect contrast-enhanced efferent lymphatic channels and secondary LNs. After peritumoral injection of blue dye, a surgeon (blinded to the contrast-enhanced US results) performed a radical LN dissection. Contrast-enhanced US was used to guide removal of any enhanced secondary LNs left after radical LN dissection. Clustered conditional logistic regression analyzed the benefit of contrast-enhanced US-directed secondary LN dissection over radical LN dissection using pathologic findings as the reference standard. A total of 268 secondary LNs were resected, with 59 (22%) containing metastases. Contrast-enhanced US detected 92 secondary LNs; 248 were identified by radical LN dissection; and 68 were identified by both methods. Metastases were detected in 20% (51 of 248) and 40% (37 of 92) of the secondary LNs identified by radical LN dissection and contrast-enhanced US, respectively. Thus, secondary LNs detected by contrast-enhanced US were nearly 5 times more likely to contain metastases than secondary LNs removed by radical LN dissection (odds ratio, 4.8; P < .0001). Twenty-two of the 180 secondary LNs (12%) identified only by radical LN dissection contained metastases, whereas contrast-enhanced US identified 20 secondary LNs after the surgeon completed the radical LN dissection, of which 8 (40%) contained metastases. Secondary LNs can be detected by using contrast-enhanced US after injection of Sonazoid into SLNs. Secondary LNs detected with contrast-enhanced US are significantly more likely to contain metastases than those removed by radical LN dissection.

Abstract 203: Sodium Hydrogen Exchanger Isoform Switching And Kchip2 Upregulation In Elderly Porcine Atria

Aging-associated changes in the heart contribute to a wide variety of cardiovascular diseases. The aging atrium becomes increasingly more susceptible to development of atrial fibrillation, and even its pharmacological response to therapeutic drugs alters over time. Changes in structure, function, and pharmacology of the heart can potentially be tied to specific changes in gene expression with aging. Here, seeking molecular correlates of myocardial aging processes, we performed global “whole transcript” analysis of 25,388 genes using 572,667 probes to compare the left atrium (LA) transcriptomes of young adult (9 months old) versus elderly (10 years old) female Sinclair swine. The two genes exhibiting the largest increase in LA expression with aging were NHE2 (9.2-fold; n = 3-4; P = 0.00001) and KCNIP2 (3.8-fold; n = 3-4; P = 0.0002). Real-time qPCR recapitulated the NHE2 results and revealed strongest upregulation with aging in LA, and age-independent expression in the ventricles. NHE2 encodes sodium hydrogen exchanger isoform 2, which was previously considered to not be expressed in mammalian heart, the favored cardiac isoform being NHE1. NHE1 is considered to be important for reperfusion injury after ischemia but, surprisingly, has been a relatively disappointing therapeutic target in clinical trials. Here, we found NHE1 transcript expression in all chambers, but no upregulation in aging heart, raising the possibility that NHE2 upregulation could negatively impact the pharmacological responsiveness of aging tissue to NHE1-specific inhibitors. KCNIP2 encodes potassium channel beta subunit KChIP2, which tightly regulates Ito density. Real-time qPCR indicated equal KCNIP2 upregulation with aging in the LA and RA, and aging-independent expression in the ventricles. In silico modeling predicted the increased KCNIP2 observed here would increase Ito sufficiently to shorten the atrial refractory period, an established substrate for atrial fibrillation. In sum, the findings uncover potential molecular substrates for aging-associated changes in cardiac function, and suggest possible novel therapeutic avenues for aging-related cardiovascular diseases.

Abstract 199: Whole-Transcript Genomics Reveals Atrial KCNE1 Downregulation in a Model of Postoperative Atrial Fibrillation Susceptibility

Lone atrial fibrillation (AF) is associated with various ion channel gene sequence variants, notably the common S38G loss-of-function polymorphism in the KCNE1 K + channel ancillary subunit gene. New-onset postoperative AF (PAF) generally occurs 48-72 hours after major surgery, particularly following procedures within the chest, but its molecular bases remain poorly understood. Here, to begin to address this gap in knowledge, we analyzed molecular changes in the left atrium (LA) three days following left upper lung lobectomy in Sinclair swine in relation to simultaneous changes in hemodynamics, LA effective refractory period (ERP), and the capacity to sustain electrically-induced AF. Relative to control pigs (no previous surgery), lobectomy increased mean pulmonary artery pressure (16 ± 3 vs 22 ± 5 mm Hg; P = 0.045) and pulmonary vascular resistance (273 ± 93 vs 481 ± 142 dyn¶s/cm 5 ; P = 0.025), whereas other hemodynamic parameters were unchanged. Left atrial ERP measured from a 300 ms cycle length trended toward reduction in lobectomy animals (187 ± 37 vs 170 ± 44 ms, P &gt;0.05). None of the lobectomy pigs developed spontaneous postoperative AF as assessed by telemetric ECG. However, all lobectomy pigs, but none of the controls, were able to sustain AF induced by a 10 s burst of rapid pacing for ≥30 s ( P =0.0079), independent of atrial ERP; AF was sustained ≥60 s in 3/5 postoperative pigs versus 0/5 controls and correlated with a shorter ERP overall ( P =0.023). Whole-transcript genomics of left atrial tissue using 25 probes per transcript revealed 13 genes with expression changes ≥2-fold in postoperative pigs. Strikingly, of these, atrial KCNE1 showed the statistically strongest link to surgery, and was the most downregulated gene in the postoperative group compared to controls (2.2-fold reduced, P =0.0027), recapitulated at the protein level with western blotting ( P =0.039). The data suggest KCNE1 remodeling as a potential marker or influencing factor in the early progression of, or susceptibility to, PAF [[Unable to Display Character: –]] uncovering a possible common predisposing genetic factor in PAF and lone AF.

Gene expression in Sinclair swine with malignant melanoma

Sinclair swine develop an aggressive form of melanoma, which, in many cases, spontaneously regresses after a complete metastatic phase. We used Affymetrix GeneChip® Porcine Genome Arrays consisting of 24 123 probe sets to compare gene expression in white blood cells (WBCs) and various tissues including the liver, lungs, inguinal lymph nodes and spleen harvested from a Sinclair piglet afflicted by melanoma at birth and exhibiting metastatic lesions at weaning (6 weeks) with those from a full-sibling piglet that showed no incidence of melanoma at birth and weaning. The highest number (3489; ∼14%) of significantly upregulated transcripts (fold change in gene expression ⩾2.0 and t-test P-value ≤0.05) was observed in the liver, while the spleen exhibited the lowest number of upregulated transcripts (528; ∼2%). Among significantly downregulated genes, the highest numbers were observed in the inguinal lymph nodes (3651; ∼15%) and the least in WBCs (730; ∼3%). Differentially expressed transcripts included genes involved in melanoma pathogenesis including SILV, TYR and RAB28. SILV was over-expressed 784-, 430- and 164-fold, while TYR was over-expressed 138-, 81- and 28-fold in the liver, lungs and inguinal lymph nodes, respectively. Quantitative real-time RT-PCR (qRT-PCR) confirmed the microarray data of 12 selected differentially expressed sequences. These results suggest that significant changes in gene expression occur during metastasis of malignant melanoma in the Sinclair swine model. In addition, qRT-PCR analysis of the above 12 differentially expressed sequences was carried out on liver samples collected from 22 pigs (12 of which had melanoma during the first 6 weeks of life), and an ANOVA test contrasting absolute RNA expression between pigs with regressing, progressing and without tumors was significant for TYR, TACSTD1, MATP, GPNMB and CYP4A22, with P-values of 0.034, 0.015, 0.007, 0.050 and 0.022, respectively.

Swine leukocyte antigen (SLA) diversity in Sinclair and Hanford swine

The swine leukocyte antigen (SLA) haplotype B is associated with increased penetrance of the tumor traits in Sinclair swine cutaneous melanoma (SSCM). We established a series of Sinclair × Hanford swine crosses to facilitate genetic mapping of the tumor-associated loci. In this study, the SLA diversity in the founding animals was characterized for effective selection of maximum tumor penetrance in the pedigrees. Using the sequence-based typing (SBT) method we identified a total of 29 alleles at five polymorphic SLA loci ( SLA-1, SLA-3, SLA-2, DRB1 and DQB1) representing six class I and five class II haplotypes. We subsequently developed a rapid PCR-based typing assay using sequence-specific primers (PCR-SSP) to efficiently follow the SLA types of the crossbred progeny. In a total of 469 animals we identified three crossovers within the class I region and three between the class I and class II regions, which corresponded to recombination frequencies of 0.39% and 0.56%, respectively. We also confirmed the presence of two expressed SLA-1 loci in three of the class I haplotypes and were able to determine the relative chromosomal arrangement of the duplicated loci in two haplotypes. This study furthers our understanding of the allelic architecture and polymorphism of the SLA system and will facilitate the mapping of loci associated with the expression of SSCM.

Role of selection and inbreeding on the incidence of cutaneous malignant melanoma in Sinclair swine

This paper reports the quantitative analysis of the historical database of a herd of Sinclair swine affected by cutaneous malignant melanoma. The herd was under partial and non-systematic selection for melanoma susceptibility (animals having at least one tumour during the first 6 weeks of life). Weighted selection differentials for the number of tumours at birth and the number of tumours at 6 weeks were generally positive and between -0.43 and 4.76 tumours for the number of tumours at 6 weeks. Estimates of the heritability for number of tumours at birth and at 6 weeks using 1934 animals were 0.27 (+/-0.03) and 0.25 (+/-0.03), respectively. The estimate of the genetic correlation between these two traits was 0.95 (+/-0.03). Genetic trends were positive for the number of tumours at birth and at 6 weeks. In spite of positive selection differentials and a moderate heritability, there was a negative phenotypic trend in the number of tumours. Natural selection might be acting in a direction opposite to artificial selection in the Sinclair herd. The slopes of the regression of the number of tumours at birth, at 6 weeks, and melanoma susceptibility on individual inbreeding coefficients were non-significant, indicating no evidence of dominance. The number of live-born pigs was lower in litters from parents susceptible to the disease (p < 0.01).

Modeling Inheritance of Malignant Melanoma With DNA Markers in Sinclair Swine

Cutaneous malignant melanoma in Sinclair swine is a hereditary disease that develops in utero or during the first 6 weeks of life. In many cases, the tumors regress and piglets survive the disease. Two different sets of gene(s) might be involved in the disease: tumor initiator (suppressor) locus or loci and loci affecting the aggressiveness of the disease (number and stage of tumors). We develop maximum-likelihood methods for interval mapping for both types of loci. The experimental design consisted of a boar mated to tumor-bearing sows with recording of tumor status and number of tumors in the 6 weeks of life of the offspring. The model to search for the tumor initiator locus (with alleles T and t) was tested by computer simulation. Estimates of penetrances (Psi(TT) and Psi(Tt) for genotypes TT and Tt, respectively) were accurate even for small family sizes. Statistical power was >99% for a family size of 70 with Psi(TT) = 1 and Psi(Tt) = 0. The models to test for number of tumors incorporated genotype information for the tumor initiator locus. All models were tested with data from a single boar family of 72 piglets over swine chromosomes 6 and 8 (SSC6 and SSC8). No tumor evidence for initiator loci was found associated with these chromosomes. However, association of a QTL affecting number of tumors at birth near microsatellite SW1953 on SSC8 was chromosomewise significant (P<0.0124).

Image-Guided Oncologic Surgery Using Invisible Light: Completed Pre-Clinical Development for Sentinel Lymph Node Mapping

Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. In this study, we complete pre-clinical development of the technology for sentinel lymph node (SLN) mapping using a large animal model of spontaneous melanoma. Sinclair swine with spontaneous melanoma metastatic to regional lymph nodes were used because of their similarity to human melanoma. Organic lymphatic tracers tested included FDA-approved indocyanine green adsorbed non-covalently to human serum albumin (HSA), and NIR fluorophore CW800 conjugated covalently to HSA (HSA800). The inorganic/organic hybrid tracer tested was type II NIR quantum dots with an anionic coating. Primary tumors received four peri-tumoral injections of each tracer, with a fluorophore dose of 100 pmol to 1 nmol per injection. SLN mapping and image-guided resection were performed in real-time. Each of the 3 lymphatic tracers was injected into n = 4 separate primary melanomas in a total of 6 animals. All 12 injections resulted in identification of the SLN(s) and their associated lymphatic channels within 1 minute in 100% of cases, despite highly pigmented skin and black fur. Hydrodynamic diameter had a profound impact on tracer behavior in vivo. This study completes the pre-clinical development of NIR fluorescence-guided SLN mapping and provides insight into imaging system optimization and tracer choice for future human clinical trials. The technology is likely to eliminate the need for radioactive and colored tracers, permits real-time image guidance throughout the procedure, and assists the pathologist in tissue analysis.

Retinal venous oxygen saturation and cardiac output during controlled hemorrhage and resuscitation.

The objective was to test calibration of an eye oximeter (EOX) in a vitiligo swine eye and correlate retinal venous oxygen saturation (Srv(O(2))), mixed venous oxygen saturation (Sv(O(2))), and cardiac output (CO) during robust changes in blood volume. Ten anesthetized adult Sinclair swine with retinal vitiligo were placed on stepwise decreasing amounts of oxygen. At each oxygen level, femoral artery oxygen saturation (Sa(O(2))) and retinal artery oxygen saturation (Sra(O(2))) were obtained. After equilibration on 100% O(2), subjects were bled at 1.4 ml. kg(-1). min(-1) for 20 min. Subsequently, anticoagulated shed blood was reinfused at the same rate. During graded hypoxia, exsanguination, and reinfusion, Sra(O(2)) and Srv(O(2)) were measured by using the EOX, and CO and Sv(O(2)) were measured by using a pulmonary artery catheter. During graded hypoxia, Sra(O(2)) correlated with Sa(O(2)) (r = 0.92). Srv(O(2)) correlated with Sv(O(2)) (r = 0.89) during exsanguination and reinfusion. Sv(O(2)) and Srv(O(2)) correlated with CO during blood removal and resuscitation (r = 0.92). Use of vitiligo retinas improved the calibration of EOX measurements. In this robust hemorrhage model, Srv(O(2)) correlates with CO and Sv(O(2)) across the range of exsanguination and resuscitation.

An intra-oral hydraulic system for controlled loading of dental implants

This study reports a method for controlling loads on an in vivo dental implant and its application for the investigation of early loading versus delayed loading of dental implants. The method was developed for the purpose of studying an ongoing hypothesis that amounts to bone loss around dental implants are related to mechanical-mediated adaptation of the alveolar bone. Using a customized intra-oral hydraulic system, the daily loading over a dental implant has been completed and recorded for six Sinclair swine. Each pig had a 5-month duration implant loading. During the experiments (loading), no analgesic treatment was supplied. The mean of the in vivo daily loadings was confirmed through an in vitro bench test after each animal was euthanized. Variations of the averaged loading input among the six animals were smaller than 10%. Preliminary data produced by the model suggests that cervical bone loss is less for early loading than for delayed loading. The current system is expected to provide a useful load control model for the study of alveolar bone adaptation around dental implants in relation to various loadings.

Spontaneous regression of cutaneous melanoma in sinclair swine is associated with defective telomerase activity and extensive telomere erosion.

Recently we proposed the hypothesis that extensive telomeric association of chromosomes is an early manifestation of cell death and asked whether there are extensive telomeric associations present in metaphases of the spontaneously regressing Sinclair swine cutaneous melanoma (SSCM). Our results indicate that early passage SSCMs, in the accelerated growth phase, do not show telomeric associations but do have numerical and other specific structural abnormalities. However, the same melanoma cell lines at late passages or melanomas obtained from middle- and old-aged Sinclair swine show extensive telomeric associations in the form of dicentric, multicentric, and ring configurations. Such abnormal structures are present mostly in metaphases that are hyperploids. Increasing frequencies of apoptotic bodies were also observed in higher passage tumor cell lines obtained from younger animals or in melanomas obtained from older animals. The polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay shows no detectable telomerase activity in any of these regressing swine melanoma cell lines, neither in normal swine skin fibroblasts nor in nevi. However, the fetal swine (i.e., non-regressing) melanoma cells show telomerase activity. Fluorescence in situ hybridization (FISH) results using the commercially available human telomeric repeat DNA probe indicate a reduction of telomeric signals in metaphase and interphase cells of regressing melanomas. From these observations we conclude that spontaneous regression of SSCM is associated with the loss of telomerase activity and a reduction of telomeric repeats that results in the formation of multicentric and ring configurations. Such abnormal chromosome configurations are lost, following the breakage-fusion-bridge-cycles, and result in extensive DNA fragmentation, as shown by laddering experiments, and, finally, cell death.