Effect of rapamycin on renal allograft survival in canine recipients treated with antilymphocyte serum, donor bone marrow, and cyclosporine

Rapamycin (Rapa) monotherapy can promote renal allograft survival in dogs, but it is very toxic. To attempt to augment the effectiveness of Rapa and reduce its toxicity in a tolerance induction protocol, canine renal allograft recipients were treated briefly with antilymphocyte serum (ALS), donor bone marrow cells (BMC), and a limited course of cyclosporine (CsA). Rapa had little effect when CsA-treated recipients were given ALS on days -5 to -1 and BMC on day +1. When combined with CsA given days +13 to +42, ALS on days -5 to +7, and BMC on day +10, Rapa at 0.3 mg/kg on day +8 plus alternate days +15 to +39 significantly increased overall survival and was compatible with long-term survival after immunosuppression (6 grafts, 1 graft > 212 days, 1 graft > 470 days). Rapa appeared to prevent early rejections that can occur during treatment with these ALS/BMC/CsA protocols. Little toxicity of Rapa was observed with any treatment.     

Analysis of failure times for multiple infections following bone marrow transplantation: an application of the multiple failure time proportional hazards model

We report an application of the proportional hazards model for multiple failure times in a study arising from the Bone Marrow Transplant Database at the University of Minnesota. The study compared the risk of infections after transplantation for patients who received allogeneic bone marrow transplants from unrelated donors (URD) versus related donors (RD). In 249 patients there was a total of 365 infections in 2.5 years of follow-up. The multiple failure time model uses all the data and provides empirical estimates of standard errors that incorporate the within-person dependencies in the data. The estimate of relative risk associated with URD was 1.4 (naive 95 per cent confidence interval 1.14 to 1.73, empirical 1.08 to 1.79), compared to the estimate 1.6 (naive or empirical, 1.1 to 2.1) from the proportional hazards model on 165 first infections only. The multivariate model gives great flexibility in modelling, for example, in accommodating a separate base hazard function for each type of failure and in allowing analysis of intervals between infections as an alternative to analysis of time from a marker event, here transplantation.     

A comparison of patellar tendon autograft and allograft used for anterior cruciate ligament reconstruction in the goat model

Similar-sized patellar tendon autografts and fresh-frozen allografts were used to reconstruct the anterior cruciate ligament of one knee in 40 female goats. Evaluations of the reconstructions and contralateral controls at the 6-week and 6-month postoperative periods included anterior-posterior translation, mechanical properties determined during tensile failure tests, measurement of cross-sectional area, histology, collagen fibril size and area distribution, and associated articular cartilage degenerative changes. Six months after anterior cruciate ligament reconstruction, the autografts demonstrated a smaller increase in anterior-posterior displacement, values of maximum force to failure two times greater, a significant increase in cross-sectional area, a more rapid loss of large-diameter collagen fibrils, and an increased density and number of small-diameter collagen fibrils compared to the allografts. Clinical significance. More surgeons are allowing their patients to return to running and sports 6 months after anterior cruciate ligament reconstruction. While the structural and material properties of autografts and allografts at time zero are similar, in the goat model during the first 6 months they differ. The allografts demonstrate a greater decrease in their implantation structural properties, a slower rate of biologic incorporation, and the prolonged presence of an inflammatory response. At 6 months the autograft demonstrates a more robust biologic response, improved stability, and increased strength to failure values.     

A prognostic model of survival in surgically resected squamous cell carcinoma of the lung using clinical, pathologic, and biologic markers

The biologic behavior of tumoral cells plays a significant role in the progression of the neoplasia, because 30 to 35% of patients with Stage I squamous cell carcinoma relapse. The present study was designed to determine whether age, pathologic parameters, DNA ploidy, and a cell proliferation index (the area of nucleolar organizer regions, AgNOR), could be used to predict survival in patients who undergo resection for limited squamous cell carcinoma of the lung. For histopathologic analysis, the parameters of histologic grading, pleural involvement, vascular invasion, and residual disease were considered. The cell proliferation index was evaluated by mitotic index, AgNOR quantification, and DNA ploidy by means of digital image analysis. Fifty-two patients (median age, 60 yr +/- 8.6 yr) were staged according to the TNM staging system. Cox univariate analysis showed that stage, residual disease, vascular invasion, histologic grading, DNA ploidy, and AgNOR were significant predictors of survival. Many of the univariate predictors of cancer death, however were eliminated when Cox multivariate models were computed. The variable that exhibited the most robust predictive value for overall survival was AgNOR. We conclude that measurement of cell proliferation might serve as a prognostic marker in squamous cell carcinoma of the lung.     

Growth of macrophage colonies from normal canine peripheral blood: morphological, cytochemical and functional parameters

Canine macrophage colonies were grown at high colony-forming efficiencies (average of 4.5 macrophage colonies/10(4) mononuclear cells plated) from gradient-separated peripheral blood mononuclear cells. The colonies were first observed on day 5 of the culture period, reached maximum numbers between days 10-14, and differed kinetically from CFU-GM colonies. Colony cells had typical macrophage morphology at the cellular and ultrastructural levels, were nonspecific-esterase positive, specific-esterase negative, and were actively phagocytic. Colony growth in semisolid cultures and 3H-TdR incorporation in liquid cultures occurred following stimulation with rabbit anti-canine immunoglobulin antisera (anti-Ig), anti-bovine serum albumin and normal rabbit serum. Addition of 2-mercaptoethanol (2-Me) to stimulated cultures significantly enhanced the proliferative response. A maximal response was obtained using anti-IgM and 2-ME. Preincubation of anti-Ig with goat anti-rabbit IgG or purified canine immunoglobulin in the presence or absence of 2-ME significantly reduced the proliferative response, suggesting the presence of both specific and nonspecific components of stimulation. The growth of canine macrophage colonies from peripheral blood provides a method for non-invasive, sequential and kinetic studies of macrophage progenitor cells in large animals.     

The evaluation of the lung transplantation program at Groningen: survival rate, quality of life, and cost-effectiveness

OBJECTIVE: Assessment of the effectiveness and cost-effectiveness of lung transplantation, and the supply and need of donor lungs in the Netherlands. DESIGN: Prospective and comparative. SETTING: University Hospital Groningen, the Netherlands. METHOD: Costs and effects were compared of the situations with and without a lung transplantation programme. Costs and effects were registered during all phases of this programme (November 1990 to February 1996; 76 patients reached the transplantation phase), and the situation without a programme was judged on pre-transplant evidence. The effect of lung transplantation on survival was assessed by means of a Cox regression model, and in the quality of life study of a longitudinal analysis was applied. The supply and the need of donor lungs were based on several registrations. RESULTS: Lung transplantation led to a significant improvement in survival and quality of life. The average gain in life years and quality adjusted life years (Qalys) were estimated at 4.4 and 5.2, respectively. The average costs per transplanted patient were estimated at Hfl. 670,000 (Hfl. 1 is about 0.5 dollar). The average costs per life year and Qaly gained were estimated at Hfl 153,000 and Hfl. 120,000 (both discounted), respectively. The annual need of donor lungs was estimated at 50-75, the annual supply at 17-27. CONCLUSIONS: Lung transplantation leads to improvement of survival and quality of life. However, it involves considerable costs and the cost-effectiveness is unfavourable compared with other Dutch transplant programmes. Moreover, there is a great discrepancy between the need and supply of donor lungs in the Netherlands.     

Expression of endothelin-1 and effects of an endothelin receptor antagonist, TAK-044, at reperfusion after cold preservation in a canine lung transplantation model

BACKGROUND: Rapid increase of pulmonary vascular resistance (PVR) early after reperfusion remains a major issue in clinical lung transplantation. A potent vasoconstrictor peptide, endothelin- plays an important role in various pulmonary pathophysiologic conditions and might induce increased PVR. We investigated the expression and influence of endothelin-1, and the effects of an ETA and ETB nonselective endothelin receptor antagonist, TAK-044, at reperfusion after cold preservation in a canine lung transplantation model. METHODS: Left single lung allotransplantation procedures were performed in three groups of animals. In group I (n=5) lungs were preserved for 12 hours; in group II (n=5) lungs were preserved for 18 hours; and in group III (n=6) lungs were also preserved for 18 hours, and TAK-044 (5 mg/kg) was administered just before reperfusion. All donor lungs were flushed and preserved with low-potassium dextran glucose solution at 4 degrees C. RESULTS: Six hours after reperfusion, arterial oxygen tension (mm Hg, inspired oxygen fraction=1.0) was 512.9+/-34.7 in group I, 152.4+/-46.7 in group II, and 509.6+/-29.0 in group III; PVR index (dyne x sec x cm(-5) x m2) was 1130+/-142 in group I, 1820+/-142 in group II, and 1287+/-191 in group III. Plasma endothelin-1 level was elevated significantly, and endothelin-1-like immunoreactivity was found in a variety of pulmonary vascular tissue and was seen less with immunohistochemical evaluation in group II in bronchial tissue. Conclusions: These results suggest that endothelin-1 is expressed as a result of ischemia-reperfusion injury and may worsen early graft function. TAK-044 is beneficial in protecting the graft from high pulmonary vascular resistance and pulmonary edema during the early posttransplantation stage.     

Feeding mice palm carotene prevents DNA damage in bone marrow and reduction of peripheral leukocyte counts, and enhances survival following X-ray irradiation

This study examined the effects of palm carotene feeding on DNA damage of bone marrow, recovery of peripheral leukocyte counts, and the survival of mice that received whole-body X-ray irradiation. The palm carotene was composed of alpha- and beta-carotene in a ratio of 1:3. Mice were fed either a basal diet or a carotene diet (50 mg carotene/100 g diet) for 2 weeks, then irradiated. The carotene diet was prepared by the dietary protocol that markedly enhanced the accumulation of carotene in tissues (J. Nutr. 125, 3081, 1995). DNA damage in bone marrow was evaluated by micronucleus assay using peripheral blood. When mice received X-ray (1.5 Gy), marked DNA damage in bone marrow and reduction of peripheral leukocyte count were observed. These changes were significantly attenuated in mice fed the carotene diet. In addition, X-ray (6.5 Gy)-induced survival of mice fed the carotene diet was higher than those fed the basal diet. In mice fed the carotene diet, alpha- and beta-carotene were detected in bone marrow and liver, and concentration of vitamin A in liver was about four times higher compared with that in mice fed the basal diet. These findings suggest that feeding mice palm carotene prevents radiation-induced damages by way of its antioxidant activity and/or vitamin A activity.     

Spectral analysis of electrograms during ventricular tachycardia in a canine model: relation with epicardial isochronal maps

The purpose of this study was to assess the capability of magnitude-squared coherence and bicoherence to differentiate monomorphic ventricular tachycardia (MVT) and polymorphic ventricular tachycardia (PVT) in a canine model and to relate these results to the epicardial isochronal maps on a beat-to-beat basis. Unipolar electrograms were simultaneously recorded from the surface of both ventricles with a 127-lead sock electrode array in 12 open-chest anesthetized dogs. The sampling frequency was 500 Hz. Atrioventricular block was induced by formaldehyde injection into the atrioventricular node. The left anterior descending coronary artery was occluded for 60 minutes under ventricular pacing (140 beats/min). During reperfusion, 12 MVT episodes lasting more than 42 seconds were recorded. Left stellate ganglion stimulation induced five PVT episodes lasting more than 42 seconds. Each of these recordings was divided into seven segments of 3,072 points (6.144 seconds). After visual selection, 104 segments were extracted and classified as 73 MVT and 31 PVT segments. Magnitude-squared coherence was estimated as the cross-spectrum from two epicardial signals (on the right and left ventricles, respectively), normalized with the respective autopower spectrum. Bicoherence was estimated as the bispectrum normalized with the autopower spectrum. Magnitude-squared coherence correctly identified 96% of MVT and 81% of PVT segments for a total accuracy of 91%. Bicoherence estimated with the left ventricular lead correctly identified 100% of MVT and 77% of PVT segments with an accuracy of 93%. Beat-to-beat epicardial maps of MVT displayed a cluster of sites of origin close to the reperfusion area, while the sites of origin from beats during PVT were much more dispersed over both ventricles. A strong and significant correlation was found between the number of electrodes with the earliest epicardial activation and coherence (r = .76, P < .0001) and bicoherence (r = .68, P < .0001), respectively. A high and significant correlation was also found between both spectral estimators (r = .74, P < .0001). Coherence and bicoherence discriminated accurately between MVT and PVT. Coherence achieved better results compared with bicoherence. Coherence and bicoherence measurements showed a quantitative relation with the spatial dispersion of the sites of origin. Both spectral techniques seemed powerful enough to be used in the development of implantable devices.     

Use of randomly amplified polymorphic DNA markers for the genetic analysis of relatedness and diversity in chickens and turkeys

A study involving the use of random amplified polymorphic DNA (RAPD) was conducted to evaluate genetic polymorphism and relatedness within and among four chicken breeds: Araucona, Rhode Island Red, White Leghorn, and White Plymouth Rock, and two turkey populations, a long-term randombred and a commercial strain. A total of 60 random primers were used in the RAPD analyses. Forty-two of the 60 primers tested amplified patterns with at least one polymorphic fragment in one or more of the populations. Six of these 42 primers amplified polymorphic fragments in each of the six strains with a within- and between-population average band-sharing frequency of less than one but above zero (P < 0.05). Differences among the six primers for genetic distance (D) among populations were significant (P < 0.05). A consensus dendogram was therefore developed to show the phylogenetic relationships among the populations. As expected, estimates of D between populations were lowest within species and highest between species. The results provide evidence of the applicability of RAPD to determining genetic relatedness within and among different poultry populations and in developing reproducible markers useful in evaluating individual variation in chickens and turkeys.     

Simultaneous assessment of the hemodynamic, cardiomechanical, and electrophysiological effects of terfenadine on the in vivo canine model

The G2 block is a major response of cells to DNA damage and seem to be induced independently of p53 status. It is thought that the G2 block has a protective function and allows cells to repair their DNA. The molecular events involved in the formation of the G2 block therefore are of great interest. We have used pentoxifylline, a potent G2 delay abrogator, to study the expression of an essential component of the mitosis promoting complex (MPF), cyclin B1. Cyclin B1/G2 ratios are used to show that irradiation induces a decrease in cyclin B1 expression and that pentoxifylline restores cyclin B1 expression to control level. This confirms that suppression of cyclin B1 plays a role in the formation of the G2 cell cycle delay, and that elevating cyclin B1 expression is part of the mechanism of action of pentoxifylline on G2 blocked cells.     

Inhibition of lipid peroxidation with the lazaroid U74500A attenuates ischemia-reperfusion injury in a canine orthotopic heart transplantation model

BACKGROUND: The lazaroid U74500A is a 21-aminosteroid that inhibits lipid peroxidation and attenuates ischemia-reperfusion injury. We examined the effect of U74500A on heart preservation with the use of a clinically relevant canine orthotopic heart transplantation model. METHODS AND RESULTS: Six donor dogs (group L) were pretreated intravenously with U74500A (10 mg/kg), and the dogs without pretreatment served as a control (group C, n = 6). The donor heart was preserved in cold University of Wisconsin solution for 24 hours. The heart was then transplanted orthotopically. Myocardial biopsy was performed to measure the adenosine triphosphate level at the end of ischemia. Before reperfusion, recipients in group L received another dose of U74500A (10 mg/kg) intravenously. After 3 hours of reperfusion, left ventricular function was evaluated by left ventricular pressure-volume relations with the use of a Millar catheter and conductance catheter, thereby deriving the slope of the end-systolic pressure-volume relation, the slope of the stroke work-- end-diastolic volume relation, and the slope of the maximum dP/dt--end-diastolic volume relation. At the same time, serum creatine kinase MB isoenzyme and lipid peroxide levels were measured. The slopes of the end-systolic pressure-volume relation, the stroke work--end-diastolic volume relation, and the maximum dP/dt--end-diastolic volume relation for group L were significantly higher than those for group C. The adenosine triphosphate levels for group L were significantly higher than those for group C. Serum creatine kinase MB isoenzyme and lipid peroxide levels for group L were significantly lower than those for group C. CONCLUSIONS: Inhibition of lipid peroxidation by the administration of U74500A was effective for 24-hour canine cardiac preservation. These results indicate that U74500A is a promising agent for heart allograft preservation.     

Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae

Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein. These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.     

Single cell multiple biomarker analysis in archival breast fine-needle aspiration specimens: quantitative fluorescence image analysis of DNA content, p53, and G-actin as breast cancer biomarkers

Fine-needle aspiration (FNA) is a sensitive and cost-effective method for evaluating breast lesions. However, the diagnosis of early premalignant lesions is less reliable by FNA because of a lack of distinctive cytological features. Accurately defining the risk of such lesions at the individual level may have significant impact in breast cancer prevention and management. The main objective of this preliminary study was to develop a method to study multiple biomarkers on archival FNA slides using quantitative fluorescence image analysis (QFIA). Biomarkers p53, G-actin, and DNA content were labeled with an immunofluorescence technique and measured by QFIA simultaneously on a single cell basis. QFIA allows the labeling and measurement procedures to be carried out in situ, without the need to remove cells from the slide while preserving the morphology of the cells. FNA slides from 72 incident patients were obtained for this study. Fifty-six cases had an adequate number of cells for the actual analysis (25 benign breast lesions, 14 proliferative breast diseases with nuclear atypia, and 17 malignant lesions). The DNA content (> or = 5c) and G-actin (average gray mean, > 90) were positive in 81% and 88% of malignant lesions, respectively. These were significantly higher than the corresponding positive rates in benign lesions (7% and 15%, respectively; P <0.01 for both). None of the benign cases were positive for G-actin and DNA simultaneously, and none of the malignant cases were negative for G-actin and DNA together. p53 was positive in 33% of malignant lesions and 8% of benign lesions (P >0.05). Our study demonstrates the feasibility of evaluating multiple biomarkers by QFIA on archival FNA-fixed specimens. The G-actin and DNA content assayed by QFIA may be potential intermediate end point markers for breast cancer individual risk assessment.     

Auditory scene analysis in European starlings (Sturnus vulgaris): Discrimination of song segments, their segregation from multiple and reversed conspecific songs, and evidence for conspecific song categorization.

The ability of male European starlings (Sturnus vulgaris) to perceive conspecific song amid a variety of background distractor conditions was investigated. In Experiment 1, test starlings rapidly discriminated 10 song segments produced by conspecific Starling A from 10 song segments produced by Starling B. Experiment 2 demonstrated that birds maintained the A vs. B discrimination against a background of song from a novel singer, Starling C. Experiment 3 demonstrated that birds could discriminate song segments from Starling A and Starling B amid superimposed distractor songs from 4 conspecifics. Experiment 4 demonstrated that the Starling A vs. Starling B discrimination could be maintained in the presence of starling song played backward. Experiments 1 and 5 suggested that test starlings were treating the A and B songs as open-ended, possibly abstract, auditory categories. Thus, starlings identify and discriminate conspecific songs amid many types of conspecific distractors. The results are well described by principles of auditory scene analysis. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Longitudinal analysis of deciduous tooth emergence: II. Parametric survival analysis in Bangladeshi, Guatemalan, Japanese, and Javanese children

We present a form of parametric survival analysis that incorporates exact, interval-censored, and right-censored times to deciduous tooth emergence. The method is an extension of common cross-sectional procedures such as logit and probit analysis, so that data arising from mixed longitudinal and cross-sectional studies can be properly combined. We extended the method to incorporate and estimate a proportion of agenic teeth. While we concentrate on deciduous tooth emergence, the method is relevant to studies of permanent tooth emergence and other developmental events. Deciduous tooth emergence data were analyzed from four longitudinal studies. The samples are 1,271 rural Guatemalan children examined every three months up to age two and every six months thereafter as part of the INCAP study; 397 rural Bangladeshi children examined monthly to age one and quarterly thereafter as part of the Meheran Growth and Development Study; 468 rural Indonesian children examined monthly as part of the Ngaglik study; and 114 urban Japanese children examined monthly in studies from 1910 and 1920. Although all four studies were longitudinal, many observations from the Guatemala and Bangladesh studies were effectively cross-sectionally observed. Three different parametric forms were used to model the eruption process: a normal distribution, a lognormal distribution, and a lognormal distribution with age shifted to shortly after conception. All three distributions produced reliable estimates of central tendencies, but the shifted lognormal distribution produced the best overall estimates of shape (variance) parameters. Estimates of emergence were compared to other studies that used similar methods. Japanese children showed relatively fast emergence times for all teeth. Bangladeshi and Javanese children showed emergence times that were slower than are found in most previous studies. Estimates of agenesis were not significantly different from zero for most teeth. One or two central incisors showed significant agenesis that ranged from 0.1 to 0.8% in three of the samples; even so, failure to model the agenic proportion did not seriously bias the estimates.