Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Precision diagnostics in transplantation: from bench to bedside.

The Canadian and American Societies of Transplantation held a symposium on February 22, 2012 in Quebec City focused on discovery, validation and translation of new diagnostic tools into clinical transplantation. The symposium focused on antibody testing, transplantation pathology, molecular diagnostics and laboratory support for the incompatible patient. There is an unmet need for more precise diagnostic approaches in transplantation. Significant potential for increasing the diagnostic precision in transplantation was recognized through the integration of conventional histopathology, molecular technologies and sensitive antibody testing into one enhanced diagnostic system.

Banff 2011 Meeting report: new concepts in antibody-mediated rejection.

The 11th Banff meeting was held in Paris, France, from June 5 to 10, 2011, with a focus on refining diagnostic criteria for antibody-mediated rejection (ABMR). The major outcome was the acknowledgment of C4d-negative ABMR in kidney transplants. Diagnostic criteria for ABMR have also been revisited in other types of transplants. It was recognized that ABMR is associated with heterogeneous phenotypes even within the same type of transplant. This highlights the necessity of further refining the respective diagnostic criteria, and is of particular significance for the design of randomized clinical trials. A reliable phenotyping will allow for definition of robust end-points. To address this unmet need and to allow for an evidence-based refinement of the Banff classification, Banff Working Groups presented multicenter data regarding the reproducibility of features relevant to the diagnosis of ABMR. However, the consensus was that more data are necessary and further Banff Working Group activities were initiated. A new Banff working group was created to define diagnostic criteria for ABMR in kidneys independent of C4d. Results are expected to be presented at the 12th Banff meeting to be held in 2013 in Brazil. No change to the Banff classification occurred in 2011.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

Correlation of donor-specific antibodies, complement and its regulators with graft dysfunction in cardiac antibody-mediated rejection.

Antibody-mediated rejection (AMR) is an immunopathologic process in which activation of complement often results in allograft injury. This study correlates C4d and C3d with HLA serology and graft function as diagnostic criteria for AMR. Immunofluorescence staining for C4d and C3d was performed on 1511 biopsies from 330 patients as part of routine diagnostic work-up of rejection. Donor-specific antibodies were detected in 95% of those with C4d+C3d+ biopsies versus 35% in the C4d+C3d- group (p = 0.002). Allograft dysfunction was present in 84% in the C4d+ C3d+ group versus 5% in the C4d+C3d- group (p < 0.0001). Combined C4d and C3d positivity had a sensitivity of 100% and specificity of 99% for the pathologic diagnosis of AMR and a mortality of 37%. Since activation of complement does not always result in allograft dysfunction, we correlated the expression pattern of the complement regulators CD55 and CD59 in patients with and without complement deposition. The proportion of patients with CD55 and/or CD59 staining was highest in C4d+C3d- patients without allograft dysfunction (p = 0.03). We conclude that a panel of C4d and C3d is diagnostically more useful than C4d alone in the evaluation of AMR. CD55 and CD59 may play a protective role in patients with evidence of complement activation.

Technical, Economical, and Microbiological Aspects of the Microaerobic Process on H2S Removal for Low Sulfate Concentration Wastewaters.

We studied the feasibility of the microaerobic process, in comparison with the traditional chemical absorption process (NaOH), on H2S removal in order to improve the biogas quality. The experiment consisted of two systems: R1, biogas from an anaerobic reactor was washed in a NaOH solution, and R2, headspace microaeration with atmospheric air in a former anaerobic reactor. The microaeration used for low sulfate concentration wastewater did not affect the anaerobic digestion, but even increased system stability. Methane production in the R2 was 14 % lower compared to R1, due to biogas dilution by the atmospheric air used. The presence of oxygen in the biogas reveals that not all the oxygen was consumed for sulfide oxidation in the liquid phase indicating mass transfer limitations. The reactor was able to rapidly recover its capacity on H2S removal after an operational failure. Bacterial and archaeal richness shifted due to changes in operational parameters, which match with the system functioning. Finally, the microaerobic system seems to be more advantageous for both technical and economical reasons, in which the payback of microaerobic process for H2S removal was 4.7 months.

Effect of serum albumin on vascular smooth muscle metabolism.

In studies on metabolism of vascular smooth muscle, it was observed that incubation of intact porcine carotid artery strips with 3% bovine or porcine serum albumin had profound effects on the oxidation of substrates and O2 consumption. Arteries incubated over 180 min with charcoal-treated and dialyzed albumin demonstrated time-dependent stimulation of glucose oxidation (145%; P < 0.0001, n=6) and O2 consumption (116%; P< 0.001, n=6). These results were not mimicked by incubation with 3% solutions of ovalbumin or porcine skin gelatin. However, the oxidation of the medium chain fatty acid octanoate was inhibited in the presence of albumin over a broad range of octanoate concentrations (0.5-5.0 mM). Short chain fatty acid oxidation (acetate, 5 mM), in contrast, was not inhibited by albumin. Wash-out of albumin only partially reversed the stimulation of O2 consumption and incubation of arteries with a polyanionic compound, polyethylene sulfonate (5 mg/ml), blunted the stimulatory effect of albumin on O2 consumption. Albumin also produced anaplerosis of the Krebs cycle, and an increase in the content of glutamate and alanine (P < 0.005, n=8). The metabolic effects of albumin were associated with time-dependent uptake of albumin (30.9 +/- 1.5 nmol/g per 210 min; P<0.01, n=15). ATP-dependent proteolysis of the albumin taken up was also observed. These results demonstrate novel and important intracellular effects of serum albumin on energy metabolism of vascular smooth muscle.

Inducible nitric oxide synthase inhibition of weibel-palade body release in cardiac transplant rejection.

BACKGROUND: Inducible nitric oxide synthase (iNOS, or NOS2) reduces the severity of accelerated graft arteriosclerosis (AGA) in transplanted organs, although the precise mechanism is unclear. METHODS AND RESULTS: We transplanted wild-type murine hearts into either wild-type or NOS2-null recipient mice; we then measured cardiac allograft survival and analyzed tissue sections by immunohistochemistry. We have confirmed that NOS2 increases cardiac allograft survival. We now show that there is less inflammation of cardiac allografts in wild-type hosts than in NOS2-null hosts. Furthermore, staining for von Willebrand factor reveals that the presence of NOS2 is correlated with the presence of Weibel-Palade bodies inside endothelial cells, whereas the absence of NOS2 is correlated with the release of Weibel-Palade bodies. CONCLUSIONS: Weibel-Palade bodies contain mediators that promote thrombosis and inflammation. Therefore, nitric oxide (NO) may stabilize the vessel wall and prevent endothelial activation in part by inhibiting the release of the contents of Weibel-Palade bodies. Prevention of Weibel-Palade body release might be a mechanism by which NO protects the vessel wall from inflammatory disorders such as atherosclerosis or graft arteriosclerosis.

Thermal effects of laser and electrical discharge on cardiovascular tissue: implications for coronary artery recanalization and endocardial ablation.

To determine the thermal responses of cardiovascular tissues to laser and electrical ablation, and to characterize the effects of different superfusing media and temperatures on target tissue temperatures and resulting extent of tissue injury, 184 laser and 15 electrical discharges were delivered to segments of human and canine aorta and canine ventricular endocardium. Tissue temperatures were measured 2 mm from the point of contact of laser fiber tip and tissue. When superfusing media consisted of whole blood or plasma at room temperature, a standard 40 J laser discharge caused peak arterial temperatures to rise 29.2 +/- 1.6 degrees C and 30 +/- 1.4 degrees C, respectively; however, tissue cooling was significantly slower in blood than in plasma. When saline solution was superfused, tissue temperatures rose by 11.4 +/- 2.2 degrees C, and tissue cooling occurred significantly faster than with either plasma or blood. The dimensions of the resulting aortic lesions were larger when blood (1.69 +/- 0.26 mm) was superfused than when plasma (1.39 +/- 0.04 mm) or saline (0.77 +/- 0.13 mm) was superfused (p less than 0.0001). Similar findings were observed with ventricular endocardium using blood or saline as the superfusing medium. In arterial tissue, superfusion with cold blood or saline solution resulted in lower peak temperature elevations (22 +/- 3.8 degrees C and 13.5 +/- 1.3 degrees C, respectively) and faster tissue cooling after laser discharge. Corresponding aortic lesion sizes were significantly smaller (1.4 +/- 0.03 and 0.5 +/- 0.02 mm, respectively) than when blood or saline medium was superfused at room temperature (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study.

In this study we demonstrated that the vaccine candidate against avian influenza virus H5N1 based on the hemagglutinin H5 (HA) fused to the chicken CD154 (HACD) can also be used for differentiating infected from vaccinated animals (DIVA). As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli. After its purification by IMAC, the competence of the proteins NP49-375 and HACD as coating antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the proteins HA and HACD and the reference sera from several avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian viral diseases were analyzed by competition ELISA assays coated with the proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the protein NP49-375. Moreover, only the antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases. This study supported the fact that the ELISA assays using the proteins NP49-375 and HACD could be valuable tools for avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic avian influenza virus H5N1 outbreaks.

Cardiac myocytes and dendritic cells harbor human immunodeficiency virus in infected patients with and without cardiac dysfunction: detection by multiplex, nested, polymerase chain reaction in individually microdissected cells from right ventricular endomyocardial biopsy tissue.

Two hundred fifteen patients infected with human immunodeficiency virus (HIV) participated in a prospective longitudinal study of HIV-related heart disease. Evaluation included signal-averaged electrocardiography and echocardiography. Fifteen patients underwent endomyocardial biopsy, 5 had cardiovascular symptoms and 10 did not. Cardiac myocytes or dendritic cells were prepared by individual cell microdissection to sort them from other cell types such as interstitial cells or circulating blood elements. HIV proviral sequences were amplified in samples of 15 to 20 cells of each type by multiplex, nested, polymerase chain reaction and hybridized to 32P-labeled probes specific for regions within the gag and pol genes of HIV-1. The results showed the presence of HIV sequences in myocytes of 2 of 5 patients with cardiac symptoms and in 6 of 10 without. Thus, symptomatic HIV cardiomyopathy did not appear to be a direct consequence of the virus on myocardial cells. In dendritic cells, HIV sequences were detected in 5 of 5 patients with cardiac symptoms and in 8 of 10 with apparently normal ventricular function. Furthermore, dendritic cells were somewhat more numerous in the myocardium of symptomatic than asymptomatic patients. Our studies are the first to directly detect the HIV genome in purified cardiac myocytes from patients with and without cardiac dysfunction. Our findings do not support a direct role of the virus in myocardial dysfunction. However, the results do suggest that the interstitial dendritic cells may be involved in some manner in the development of cardiac dysfunction observed in HIV-infected patients.

Effect of ibuprofen on the healing phase of experimental myocardial infarction in the rat.

The healing phase of acute myocardial infarction (AMI) is initiated by proteolysis of necrotic myocardium, followed by infiltration of fibroblasts and deposition of collagen. To assess whether ibuprofen, a potent antiinflammatory agent, preserves existing collagen and enhances deposition of new collagen during infarct healing, biochemical and morphologic studies were made of experimentally induced myocardial infarcts in untreated rats and in rats treated with ibuprofen. All treated rats received 12.5 mg/kg of ibuprofen at 1, 6 and 18 hours after AMI. Group 1 rats underwent measurement of myocardial hydroxyproline (HP) content at 24 hours after AMI. Group 2 rats received ibuprofen, 12.5 mg/kg, twice a day for 2 additional days, with measurement of myocardial HP at 3 days (group 2a) or 21 days (group 2b) after AMI. Group 3 rats received ibuprofen, 12.5 mg/kg, twice a day for 6 additional days with measurement of HP content, or infarct size and degree of thinning at 21 days after AMI. Compared with untreated rats, ibuprofen-treated rats had significantly greater amounts of HP in the infarct at 24 hours (group 1, 8.9 +/- 2.2 nmol/mg dry weight vs untreated, 7.1 +/- 2.8 nmol/mg dry weight, p less than 0.04) and at 21 days (group 2b, 112 +/- 37 nmol/mg dry weight vs untreated, 91 +/- 39 nmol/mg dry weight, p less than 0.05, and group 3, 125 +/- 51 nmol/mg dry weight vs untreated, 91 +/- 39 nmol/mg dry weight, p less than 0.003). Substantial scar thinning was noted in all rats; no difference in scar thinning was noted between treated and untreated rats at 21 days after AMI.(ABSTRACT TRUNCATED AT 250 WORDS)

Cuspal perforations caused by long suture ends in implanted bioprosthetic valves.

A description is presented of the gross anatomic, histologic, and scanning electron microscopic features of cuspal abrasions, perforations, and tears caused by excessively long ends of braided sutures in bioprosthetic cardiac valves implanted in the mitral position in sheep. These lesions are produced as consequences of contact between the ends of the sutures and the inflow surfaces of the bioprosthetic cusps, leading to a process of surface erosion that progresses to actual perforation of the cusps. The perforation has the appearance of a crater, the wider end of which faces the inflow surface and the walls of which are formed by broken ends of collagen fibrils. Suture perforations can extend to form tears that involve the free edge of the cusp and result in hemodynamically important regurgitation. Therefore, care must be taken to avoid leaving excessively long suture ends during the implantation of bioprosthetic cardiac valves.

Ventricular aneurysms and other lesions produced by the struts of bioprosthetic valves implanted in sheep.

Damage to the posterolateral wall of the left ventricle was found in eight of approximately 700 sheep undergoing mitral valvular replacement as part of animal model studies of bioprosthetic valves. The damage consisted of left ventricular aneurysms in five animals, subacute rupture of the left ventricle in one, acute left ventricular laceration in one, and endocardial scarring in one. Six of the eight bioprostheses were bovine pericardial valves, including five low-profile valves and one standard valve; of the two porcine bioprostheses, one was intentionally oversized and the other was a low-profile supra-annular valve. In each of these animals the damage appeared to have been caused by contact between the most posterior strut of the bioprosthesis and the left ventricular wall.

Atherogenic effect of barotrauma on in situ saphenous veins in monkeys.

The objectives of this study were to determine whether veins subjected to barotrauma in situ undergo lipid uptake and morphologic changes to the same extent as veins grafted into the arterial circulation. Saphenous veins in seven stump-tailed macaque monkeys were exposed bilaterally and were circumferentially dissected free from surrounding tissue only at isolated sites. Segments of the veins were distended for 1 minute at hydrostatic pressures of 125 or 350 mm Hg. An undistended segment served as control. A cephalic vein graft was interposed in the femoral artery for comparison with in situ veins. The animals were fed a diet that sustains plasma cholesterol levels of approximately 225 mg/dl. Saphenous veins and the cephalic vein grafts were explanted at 3 months for biochemical and histologic analyses. Cholesterol content in undistended saphenous veins was similar to that in veins distended at 125 or 350 mm Hg--105 +/- 15, 122 +/- 14, and 109 +/- 30 micrograms/100 mg wet tissue weight, respectively. Cholesterol content in cephalic vein grafts, 473 +/- 122 micrograms/100 mg, was greater (p less than 0.001) than in saphenous veins at all distention pressures studied. There was no difference among the distention pressures in the intimal fraction of saphenous vein wall, with the pooled value being 20% +/- 12%. This contrasted with the value of 59% +/- 11% in cephalic vein grafts (p less than 0.01). Endothelial coverage of the luminal surface in saphenous veins was similar among the levels of barotrauma, with the pooled value being 83% +/- 15%. Less of the lumen was covered with endothelium in cephalic vein grafts, 46% +/- 18% (p less than 0.01). Slightly more medial fibrosis was observed in cephalic vein grafts as compared with saphenous veins (p less than 0.05). These data demonstrate that barotrauma alone does not cause veins that remain in the venous system to undergo the lipid uptake or morphologic changes that occur in veins grafted into the arterial circulation in nonhuman primates.

The relative influence of arterial pressure versus intraoperative distention on lipid accumulation in primate vein bypass grafts.

Atherosclerotic degeneration has been well documented to be the limiting factor for long-term function of aortacoronary vein bypass grafts. Injury, including that induced by pressure distention in preparation for grafting, is thought to play a role in this degeneration. Injury can be minimized by limiting the distending pressure, but vein grafts are chronically subjected to arterial pressures that far exceed native venous pressure. We evaluated the relative influence of arterial pressure and of higher pressure of distention on cholesterol and apolipoprotein-B accumulation by grafts in our established animal model of graft atherogenesis. Grafts were interposed in the femoral arteries of eight normolipemic stump-tailed macaque monkeys. Before insertion, each vein was distended at 125 mm Hg (arterial pressure) for 1 minute with autologous blood, followed by distention of one half of the vein at 350 mm Hg for 1 additional minute. Grafts and ungrafted control vein were removed 3 months later. Cholesterol concentration in grafts distended at 125 mm Hg was 213% (p less than 0.01) and apolipoprotein-B concentration was 430% (p less than 0.001) of that in ungrafted control veins, whereas in grafts distended at 350 mm Hg cholesterol was 250% (p less than 0.01) and apolipoprotein-B was 925% (p less than 0.001) of the control concentrations. Although morphologic differences between the two groups of grafts were less profound than biochemical differences, foam cells were observed more frequently in grafts distended at 350 mm Hg than in those distended at 125 mm Hg. These data demonstrate that chronic exposure to arterial pressure has a significant effect on graft cholesterol that is proportionally greater than that caused by intraoperative distention at moderate pressure. Nevertheless, the detrimental effects of excessive distending pressures should not be ignored.

Histologic, morphometric, and biochemical evolution of vein bypass grafts in a nonhuman primate model. III. Long-term changes and their modification by platelet inhibition with aspirin and dipyridamole.

The objectives of this study were to elucidate the long-term influence on vein bypass grafts of platelet inhibition and its late discontinuation. Cephalic vein grafts were interposed bilaterally in the femoral arteries of stump-tailed macaque monkeys fed a diet that sustains plasma cholesterol levels of approximately 225 mg/dl. Fifteen animals were divided into three groups of five animals each. Group I received no medications and served as a control group. Group II received for the full duration of the study a combination of aspirin, 80 mg/day, and dipyridamole, 50 mg/day. Group III received the same regimen of platelet inhibition as in group II during the first 9 months, but were not treated during the subsequent 9-month interval. Grafts were excised for analysis from groups I and II at both 9 and 18 months and from group III at 18 months. Cholesterol content in group I grafts was 470 +/- 89 micrograms/100 mg at 9 months and 388 +/- 127 micrograms/100 mg at 18 months. In group II grafts, cholesterol content was 208 +/- 72 micrograms/100 mg at 9 months (p less than 0.001 compared with group I) and 266 +/- 84 micrograms/100 mg at 18 months. In group III grafts, cholesterol content was 249 +/- 71 micrograms/100 mg at 18 months. Differences in cholesterol content among the three groups of grafts at 18 months were not found to be statistically significant. Stepwise regression analysis at 18 months showed that cholesterol content was best predicted by medial fibrosis (r2 = 0.66) followed by abundance of foam cells (increase in r2 = 0.26) in group I, by fibrin in group II (r2 = 0.63), and by prevalence of macrophages in group III (r2 = 0.74). In all groups, platelets, fibrin, and polymorphonuclear leukocytes were less abundant than they had been at 3 months. Cross-sectional area occupied by the intima was not influenced by platelet inhibition.

Histologic, morphometric, and biochemical evolution of vein bypass grafts in a nonhuman primate model. II. Modification of early changes by platelet inhibition with aspirin and dipyridamole.

The objective of this study was to determine the early influence of platelet inhibition on the histologic, morphometric, and biochemical evolution of vein bypass grafts in a nonhuman primate model. Cephalic vein grafts were interposed bilaterally in the femoral arteries of 15 stump-tailed macaque monkeys fed a diet that sustains plasma cholesterol levels of approximately 225 mg/dl. All animals received in combination aspirin, 80 mg/day, and dipyridamole, 50 mg/day. Grafts were excised from five animals for analysis on each of postoperative days 3, 7, 14, 30, 60, and 90. In animals subjected to platelet inhibition, cholesterol content in the graft was 170 +/- 52 micrograms/100 mg at 90 days, 205% of the level in ungrafted vein (p less than 0.01). This change was small in comparison with the increase to 686% of ungrafted vein observed in our study of control animals. In stepwise regression analysis, cholesterol content of grafts was best predicted by prevalence of foam cells (r2 = 0.82), and the proportion of intima as a fraction of total wall area was best predicted by the presence of macrophages (r2 = 0.69). Platelet inhibition did not decrease the extent of intimal hyperplasia. The prevalence of adherent platelets (r = -0.72) and the amount of fibrin (r = -0.78) correlated inversely with the amount of endothelium present during the first 14 days. The strength of these correlations declined with time, despite persistent lack of endothelium in some areas. Medial fibrosis occurred to the same extent as in control grafts, as did the early appearance of platelet factor VIII and fibronectin and the lack of vasa vasorum at 3 days followed by reappearance at 7 days. These data demonstrate that platelet inhibition dramatically reduces lipid uptake by grafts in the first 90 days but has less influence over histologic or morphometric changes.

Histologic, morphometric, and biochemical evolution of vein bypass grafts in a nonhuman primate model. I. Sequential changes within the first three months.

The objective of this study was to define the histologic and morphometric evolution that accompanies the increase in cholesterol content of vein bypass grafts in a nonhuman primate model. Cephalic vein grafts were interposed bilaterally in the femoral arteries of 15 stump-tailed macaque monkeys (Macaca arctoides), which were fed a diet that sustains plasma cholesterol levels of approximately 225 mg/dl. Grafts were excised from five animals for analysis on each of postoperative days 3, 7, 14, 30, 60, and 90. Cholesterol content increased from 69 +/- 24 micrograms/100 mg (mean +/- standard deviation) in ungrafted vein to 473 +/- 122 micrograms/100 mg in grafts 90 days after implantation (p less than 0.05). By stepwise regression analysis, cholesterol content was best predicted by abundance of foam cells (r2 = 0.82). Intima comprised 13% +/- 5% of the total cross-sectional area of the wall in ungrafted vein and 59% +/- 11% at day 90 (p less than 0.001). With cholesterol content excluded from the stepwise regression, intimal area was best predicted by the presence of foam cells (r2 = 0.39). There was consistently an increase in the prevalence of polymorphonuclear leukocytes on the luminal surface and in both the intima and media during the first 14 days after grafting. Vasa vasorum, which were always present in ungrafted vein, were sparse at 3 days but reappeared by day 7. Medial fibrosis occurred in grafts, and in the 30- to 90-day interval it was directly correlated with the number of adventitial vasa vasorum present (r = 0.64, p less than 0.05). Immunohistochemistry revealed prominent staining for both platelet factor VIII and fibronectin during the first month, with a gradual decline in staining intensity thereafter. The evolution of changes in vein bypass grafts documented in this report are in general agreement with graft changes observed in humans and support the validity of our model in evaluating the histologic correlates of increased graft cholesterol content.

Acute rejection after cardiac transplantation: detection by interstitial myocardial pH.

Intramyocardial pH was assessed as a potential marker for clinical evaluation and treatment of acute rejection following cardiac transplantation. Fifteen cats underwent forty operative procedures. Following intra-abdominal heterotopic heart transplantation, serial laparotomies were performed in the early (days 0 to 2), intermediate (days 5 to 7), and late (days 7 to 16) postoperative periods. Rejection was assessed by serial clinical examinations, ECG analyses, B-mode echocardiography, histological and ultrastructural analyses, and measurements of interstitial myocardial pH. Intramyocardial pH was measured by a new miniature (0.6 X 3.0 mm) fiberoptic pH transducer. At confirmed rejection, concomitant laparotomy and thoracotomy were performed and pH sensors were implanted in both native (anatomical) and graft hearts. Nine animals at rejection were given methylprednisolone and changes in graft and native heart pH were measured. The pH during absence of rejection, mild acute rejection, and severe acute rejection averaged 7.430 +/- 0.019, 7.233 +/- 0.040 (p less than .02), and 6.860 +/- 0.066 (p less than .02), respectively (mean +/- standard error of the mean). A progressive decline in pH was noted in each heart. In animals receiving steroids, graft heart pH increased over 90 minutes from 6.852 +/- 0.065 to 7.043 +/- 0.077 (p less than .05). Although pH decline may be secondary to either inflammatory or ischemic etiology, histological and ultrastructural analyses demonstrate a predominant inflammatory response with progressive mononuclear cell infiltration, interstitial edema, vascular wall edema, infiltration by polymorphonuclear neutrophil leukocytes, vacuolation of sarcoplasmic reticulum, and disarray of myocytes associated with falling pH. Degree of pH change correlated closely with degree of histological rejection, presence of ECG voltage decline, and change in wall thickness by ultrasound.