Localization of inducible nitric oxide synthase in acute renal allograft rejection in the rat

There is increasing evidence for a role for nitric oxide (NO) in the alloimmune response and induction of NO synthesis occurs during allograft rejection. The aim of this study was to investigate the source of NO synthesis in rejecting allografts. Localization of inducible nitric oxide synthase (iNOS) was studied by immunohistochemistry, in a rat model of acute renal allograft rejection, in unmodified Lewis recipients in which rejection is complete 7 days after transplantation of F1 hybrid Lewis-Brown Norway kidneys. High levels of iNOS expression were found in infiltrating mononuclear cells in glomeruli and interstitium of rejecting kidneys; there was no expression in parenchymal renal cells, or in control isografts of either rat strain. Expression of iNOS in the cortex was present from 4 to 6 days posttransplantation, and had declined by the 7th day, where expression was principally in the medulla. The pattern of iNOS staining was similar to ED1 staining, a marker for rat macrophages. These findings suggest that infiltrating macrophages in the graft reaction are a prominent source of NO; this iNOS expression supports a role for NO in the modulation of local allogeneic responses, and possibly as a mediator of cytotoxic graft damage.     

The co-culture of dermal fibroblasts with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts

BACKGROUND: We recently demonstrated that inhibition of nitric oxide (NO) production ameliorated acute pulmonary allograft rejection. This study examined whether inducible NO synthase (iNOS) was expressed in the transplanted lung during acute rejection. METHODS: With a rat left lung transplant model, tissue from syngeneic (Fischer 344 to Fischer 344) and allogeneic (Brown Norway to Fischer 344) transplants were harvested on postoperative day 4 and analyzed for iNOS mRNA expression (ribonuclease protection assay), iNOS enzyme activity (conversion of L-[3H]-arginine to NO and L-[3H]-citrulline), and serum nitrite/nitrate levels. RESULTS: The iNOS mRNA was expressed in allograft lungs but was not detected in isografts or controls. The iNOS protein was present in allograft lungs, as demonstrated by high levels of L-[3H]-citrulline production compared with minimal iNOS enzyme activity in isograft and control lungs (10.1 +/- 2.4 vs 0.6 +/- 0.2 and 0.7 +/- 0.2 pmol L-[3H]-citrulline.mg-1.min-1, respectively; n = 6, p < 0.001). Allografts had significantly elevated systemic serum nitrite/nitrate levels compared with isografts and controls (38 +/- 6 vs 18 +/- 2 and 16 +/- 1 mumol/L, respectively; n = 6; p < 0.005). CONCLUSIONS: These results, together with our previous demonstration that iNOS inhibition ameliorated lung allograft rejection, suggest that (1) iNOS expression and increased NO production contributed to acute rejection of the transplanted lung, (2) iNOS inhibition may offer an alternative in management of acute lung allograft rejection, and (3) increased NO production, detected by the presence of iNOS mRNA or protein or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.     

The tetravalent guanylhydrazone CNI-1493 blocks the toxic effects of interleukin-2 without diminishing antitumor efficacy

The use of interleukin 2 (IL-2) as an antineoplastic agent has been limited by the serious toxicities that accompany the doses necessary for a tumor response. Elevation of nitric oxide (NO) and tumor necrosis factor (TNF) both have been implicated in IL-2 toxicities. CNI-1493, a tetravalent guanylhydrazone, is an inhibitor of macrophage activation including the synthesis of TNF and other cytokines. Doses of CNI-1493 as low as 1 mg/kg/day conferred complete protection against fatal toxicity of IL-2 with IL-2 doses tenfold higher than the safely tolerated level in Sprague-Dawley rats. Moreover, typical pathologic changes in the lungs, kidneys, and the liver caused by IL-2 infusion were blocked by cotreatment with CNI-1493. When animals bearing established hepatomas were given IL-2 and CNI-1493 combination therapy, 10 of 10 hepatomas regressed from 1 cm3 to <1 mm3. Intracytoplasmic TNF levels were increased in normal tissues from IL-2 treated animals, and treatment with CNI-1493 maintained TNF at control levels. The degree of apoptosis measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining of tumors following IL-2 therapy was not reduced compared with IL-2 cotreated with CNI-1493. In contrast, apoptosis in the liver and lung parenchyma following IL-2 therapy was blocked completely by cotreatment with CNI-1493. Taken together, these data showed that low and infrequent doses of CNI-1493 markedly protected animals from IL-2 systemic toxicities whereas not affecting tumor response to IL-2 therapy. With the protection afforded by CNI-1493 treatment, IL-2 therapy dose levels could be increased to provide significant antitumor effects in animals with established hepatomas.     

Abdominal findings in AIDS-related pulmonary tuberculosis correlated with associated CD4 levels

BACKGROUND: There is evidence that inducible nitric oxide (NO) may be directly related to the process of allograft rejection. Because of its strong pulmonary vasodilatory activity, inhaled NO (INO) has recently been used as a therapeutic option for allograft dysfunction after lung transplantation. The action of inducible NO and inhaled NO seems contradictory for preserving posttransplantation pulmonary allograft function. INO used for lung transplant recipients may actually enhance acute allograft rejection. We studied the effect of INO on acute allograft rejection with a rat pulmonary allograft model. METHOD: A total of 24 left lung allotransplantations were performed from Lewis donors into F344 recipients. Animals were divided into two groups and inhaled either room air alone or 20 ppm NO with room air in a closed chamber immediately after transplantation until rats were killed on days 7 and 14. During observation, NO uptake was monitored by measuring serum NO2-/NO3- level. Acute rejection was evaluated by use of a semiquantitative radiographic scoring method (aeration score: 0 to 6, opaque to normal appearance) and rejection score (0 to 4, no sign of rejection to diffuse mononuclear infiltration). RESULTS: Markedly elevated serum NO2-/NO3- levels were observed in the NO inhalation group compared with levels in the normal air inhalation control group (110.8 +/- 25.3 vs 16.3 +/- 4.0 micromol/L/ml on day 7, p < 0.01; 107.0 +/- 30.9 vs 16.8 +/- 4.8 micromol/L/ml on day 14, p < 0.01). However, no positive effect of INO on acute rejection was found histologically or radiographically. CONCLUSION: The effect of INO on acute rejection is likely so minimal as not to be clinically relevant.     

Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development

The switch from myocyte hyperplasia to hypertrophy occurs during the early postnatal period. The exact temporal sequence when cardiac myocytes cease dividing and become terminally differentiated is not certain, although it is currently believed that the transition takes place gradually over a 1-2-week period. The present investigation has characterized the growth pattern of cardiac myocytes during the early postnatal period. Cardiac myocytes were enzymatically isolated from the hearts of 1, 2, 3, 4, 6, 8, 10, and 12-day-old rats for the measurements of binucleation, cell volume and myocyte number. Almost all myocytes were mononucleated and cell volume remained relatively constant during the first 3 days of age. Increases in cell volume and binucleation of myocytes were first detected at day 4. Myocyte volume increased 2.5-fold from day 3 to day 12 (1416 +/- 320 compared to 3533 +/- 339 microns 3). The percentage of binucleated myocytes began to increase at day 4 and proceeded at a high rate, reaching the adult level of approximately 90% at day 12. Myocyte number increased 68% during the first 3 days (from 13.6 +/- 3.5 x 10(6) at day 1 to 22.9 +/- 5.6 x 10(10) at day 3) and remained constant thereafter. To confirm that no further myocyte division exists after 4 days, bromodeoxyuridine (Brdu) was administered to 4-day-old rats and the fate of DNA-synthesizing myocytes was examined 2 h and 2, 4, 6 and 8 days after Brdu injection. About 12% of myocytes were labeled with Brdu at 2 h and all were mononucleated at that time. Gradually, these Brdu-labeled myocytes became binucleated. However, the percentage of labeled myocytes in all groups was identical, indicating that DNA-synthesizing myocytes were becoming binucleated without further cell division after 4 days of age. Within 8 days after injection, approximately 82% of total labeled myocytes were binucleated, while the others remained mononucleated. Sarcomeric alpha-actinin was fully disassembled in dividing myocytes of 2-day-old rats, while typical alpha-actinin striations were present in dividing myocytes of 4-day-old rats. The results from this study suggest that a rapid switch from myocyte hyperplasia to hypertrophy occurs between postnatal day 3 and 4 in rat hearts.     

Regulation of cardiac myocyte contractile function by inducible nitric oxide synthase (iNOS): mechanisms of contractile depression by nitric oxide

Inflammatory cytokines have been implicated in the reversible depression of cardiac contractile function accompanying local or systemic immune stimulation. Incubation of cardiac myocytes with soluble components in the supernatant from cultured rat lung macrophages activated with endotoxin decreases their contractile response to beta-adrenergic stimulation through the induction of iNOS and the subsequent production of nitric oxide by these cells. In the present study, we characterize the mechanisms underlying NO's attenuation of adrenergic responsiveness in cardiac myocytes. iNOS was induced in cultured ventricular myocytes from adult rats by incubation for 20 h with conditioned medium from lipopolysaccharide (LPS)-activated macrophages. iNOS induction did not induce any alteration in beta-adrenergic receptor density or affinity, Galphai protein abundance, or adenylyl cyclase activity in cultured myocytes. Myocyte exposure to activated macrophage-conditioned medium markedly attenuated the elevation of cAMP in response to isoproterenol (Iso, 2 nM). Induction of iNOS with the macrophage-conditioned medium also potentiated the Iso-induced increase in myocyte cGMP. This cGMP increase was totally abolished by NOS inhibitors. NOS inhibition also returned the attenuated cAMP response to 2 nM Iso to levels observed in control cells. Pre-incubation of the cells in isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, also partly reversed the attenuation of cAMP increase with 2 nM Iso in cells expressing iNOS. Brief (15 min) exposure of myocytes to the NO donor, S-nitrosoacetylcysteine (SNAC, 100 micro M) which produced a three-fold increase in intracellular cGMP, also decreased by half the contractile response of cardiac myocytes to Iso (2 nM). We conclude that NO endogenously produced by iNOS decreases the intracellular levels of cAMP in response to beta-adrenergic stimulation in isolated cardiac myocytes, in part through a cGMP-mediated mechanism. This effect may participate in the NO-dependent depression of cardiac function following cytokine exposure.     

NOS2 mediates opposing effects in models of acute and chronic cardiac rejection: insights from NOS2-knockout mice

To compare regulatory effects of NOS2 in acute and chronic cardiac allograft rejection, we used NOS2 knockout mice as recipients in a cardiac transplant model. To study acute and chronic rejection separately but within the same genetic strain combination, we compared allografts placed into recipients without or with immunosuppression (anti-CD4/8 for 28 days). NOS2 mRNA and protein expression were compared using 32P-RT-PCR and immunohistochemistry. In our acute rejection model, NOS2 was predominately localized to graft-infiltrating immune cells. At day 7, grafts in NOS2-deficient recipients (n = 7) showed reduced inflammatory infiltrates and myocyte damage resulting in significantly lower rejection scores (1.6 +/- 0.4) compared to wild-type controls (n = 18; 2.8 +/- 0.2, P = 0.002). In contrast, in our chronic rejection model, additional NOS2 expression was localized to graft-parenchymal cells. At day 55, grafts in NOS2-deficient recipients (n = 12) showed more parenchymal infiltration and parenchymal destruction (rejection score 3.8 +/- 0.1) than wild-type controls (n = 15; 1.6 +/- 0.2, P < 0.0001). This was associated with a significant decrease in ventricular contractility (palpation score 0.3 +/- 0.1 compared to 2.3 +/- 0.3 in wild-type, P < 0.0001). Hence, NOS2 promotes acute but prevents chronic rejection. These opposing effects during acute and chronic cardiac allograft rejection are dependent on the temporal and spatial expression pattern of NOS2 during both forms of rejection.     

Fetuin (alpha2-HS-glycoprotein) opsonizes cationic macrophagedeactivating molecules

Macrophages become activated by bacterial endotoxin (lipopolysaccharide) and other stimuli to release proinflammatory cytokines and NO. To prevent release of toxic or potentially lethal quantities of these factors, the state of macrophage activation is counter-regulated by anti-inflammatory mediators (e.g., glucocorticoid hormones, interleukin 10, and transforming growth factor type beta). Fetuin, a negative acute-phase protein, recently was implicated as an anti-inflammatory mediator, because it is required for macrophage deactivation by spermine. In the present studies, we found that fetuin is necessary for macrophages to respond to CNI-1493, a tetravalent guanylhydrazone inhibitor of p38 mitogen-activated protein kinase phosphorylation. Fetuin dose-dependently increases macrophage uptake of CNI-1493, which can be specifically inhibited by anti-human fetuin antibodies. Anti-human fetuin antibodies render primary human peripheral blood mononuclear cells insensitive to deactivation by CNI-1493. Thus, macrophages use fetuin as an opsonin for cationic-deactivating molecules, both endogenous (e.g., spermine) and pharmacologic (e.g., CNI-1493). This role of fetuin as an opsonic participant in macrophage-deactivating mechanisms has implications for understanding and manipulating the innate immune response.     

Early cardiac allograft rejection is independent of regional myocardial blood flow

Noninvasive telemetric monitoring of canine heterotopic cardiac allograft unipolar peak-to-peak amplitude (UPPA) has permitted prospective surveillance for rejection; moreover, this technique is able to reliably detect rejection before the development of histologic evidence of myocyte necrosis. This study was performed to determine whether early cardiac allograft rejection and the accompanying decline in allograft UPPA were associated with alterations in regional myocardial blood flow (RMBF). Seven heterotopic, intrathoracic canine cardiac transplantations were performed using triple-drug immunosuppression. Native hearts and allografts were instrumented with right ventricular and left ventricular epicardial screw-in electrodes connected to subcutaneous telemeters. Daily measurement of native and graft UPPA was performed; using radioactive microspheres, native and graft RMBF were determined during the control period and when UPPA had declined by 15%, 30%, and 45%. Graft histologic status was determined by endomyocardial biopsy at the time of RMBF determination. Mean duration of the study was 19.7 +/- 3.9 days. Rejection was documented in all animals. The UPPA was stable in native hearts; UPPA declined in the allografts after the onset of rejection. A biphasic change in allograft blood flow was seen. Initially RMBF increased as UPPA declined; a 30% to 45% reduction in UPPA was associated with a 41% increase in RMBF (p = 0.028 versus allograft control). Subsequently, a significant decline in blood flow was observed for reductions in UPPA greater than 45% (0.68 +/- 0.44 versus 1.07 +/- 0.47 mL.g-1 x min-1 for a 30% to 45% decline in UPPA; p = 0.007).(ABSTRACT TRUNCATED AT 250 WORDS)     

Surgical resection for hepatocellular carcinoma in Cape Town--a clinical and histopathological study

BACKGROUND: The effect of mycophenolate mofetil (MMF) and sirolimus (rapamycin, RAPA) mono- and combination-therapy was examined in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat. METHODS: Both drugs were administered orally for up to 30 days. Eleven groups (n=6) were involved in the first part of the heart allografting model. Brown Norway (RT1n) to Lewis (RT1(1)) combination was used in the heart and pancreas transplantation models, whereas Buffalo (RT1b) to Wistar Furth (RT1u) was used in the kidney transplantation model. RESULTS: The naive control group showed a mean survival time of 6.5+/-0.6 days. There were graded dose-responses to monotherapy of MMF 10 and 20 mg(kg/ day (12.5+/-2.6 days; 19.3+/-9.0 days) and RAPA 0.2, 0.4, 0.8, and 1.8 mg/kg/day (19.2+/-2.0 days; 30.0+/-7.3 days; 50.8+/-12.5 days; 51.2+/-2.6 days), respectively (P=0.001). Results with the combined use of drugs indicate that a synergistic or very strong synergistic interaction was produced when compared with monotherapy of MMF or RAPA: MMF 10 mg(kg/day+RAPA 0.2 mg/kg(day (52.7+/-5.7 days, combination index [CI] =0.189), MMF 20 mg(kg/day+RAPA 0.2 mg/kg/day (57.7+/-5.7 days, CI=0.084), MMF 10 mg/kg/day+RAPA 0.4 mg(kg/day (50.2+/-13.5 days, CI=0.453), and MMF 20 mg/kg(day+ RAPA 0.4 mg/kg(day (51.5+/-6.8 days, CI=0.439), respectively. These results were repeatable in the prevention of acute pancreas and kidney allograft rejection in the rat. In the second part of the study of reversal of ongoing acute heart allograft rejection model, the combined treatment of MMF 10 mg/kg(day+RAPA 0.2 mg/ kg(day (35.5+/-16.0 days, CI=0.794) and MMF 20 mg/kg day+RAPA 0.2 mg(kg/day (57.2+/-4.7 days, CI=0.310) represented synergistic interaction compared with monotherapy of MMF or RAPA. CONCLUSIONS: Concomitant therapy of MMF and RAPA produces a synergistic effect in prevention of heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat.     

Free radical-mediated tissue injury in acute lung allograft rejection and the effect of superoxide dismutase

BACKGROUND: The role of monocytes and neutrophils is crucial during acute allograft rejection. They have the capacity to generate toxic reactive oxygen intermediates in response to specific agonists that may act as tissue destructive molecules. We examined the possibility of reactive intermediate-mediated tissue injury in acute lung allograft rejection, as well as the effect of superoxide dismutase. METHODS: Allogenic (Brown Norway to F344) or syngenic (F344 to F344) rat left-lung transplantation was performed. Generation of reactive oxygen intermediates in peripheral blood was evaluated by the method of luminol-dependent chemiluminescence. Cell membrane phospholipid peroxidation in the graft was measured as malondialdehyde concentration. The third group of animals having allografts received bovine erythrocyte superoxide dismutase (5,000 U/kg intravenously every 12 hours after transplantation). RESULTS: Relative chemiluminescence response in the allograft recipient to normal F344 was elevated on postoperative day 1 (257%), then decreased slightly on day 3 (156%) and was elevated again on day 7 (560%) as the process of rejection progressed. Allograft tissue malondialdehyde levels (248.37 +/- 112.35 nM/whole lung, n = 6; p < 0.05 by Student's t test) were higher than isograft levels (139.29 +/- 35.93 nM/whole lung, n = 6) on day 7. Superoxide dismutase treatment significantly ameliorated the histologic degree of rejection on day 7. CONCLUSIONS: These results demonstrate the tissue destructive activity of reactive oxygen intermediates during lung allograft rejection. To scavenge free radicals may be a useful therapeutic modality in the management of acute lung allograft rejection.     

Ionic mechanisms mediating the differential effects of methohexital and thiopental on action potential duration in guinea pig and rabbit isolated ventricular myocytes

BACKGROUND: The stress response to injury concept has been proposed as a mechanism of chronic rejection. This hypothesis has been tested with a rat cardiac allograft model in recipients pretreated with donor bone marrow (BM) cells. Chronic rejection is manifested in this BM group by obliterative arteriopathy and the epicardium and endocardium contains lymphocytic infiltrates resembling Quilty lesions. Pretreatment with a liver allograft (the orthotopic liver transplant [OLTx] group) is associated with an absence of chronic rejection in the transplanted heart. METHODS AND RESULTS:. Stress responses in the allograft were assessed by determining heat shock protein (hsp) expression by immunohistology of graft tissues and immunoblot analysis of stromal tissue lysates with monoclonal antibodies (mAb) to mammalian hsp60, the inducible hsp72, the constitutively expressed hsc73, and the grp78 C-terminal sequence KSEKDEL (grp78seq). Immunostaining showed clusters of grp78seq-positive cells in the inflammatory infiltrates of obliterated blood vessels and Quilty lesions in the BM group of cardiac allografts. Such grp78seq-positive cells were not seen in the OLTx group of heart allografts nor in syngrafts. Neither group showed significantly different graft myocyte staining of grp78 or hsp72, whereas hsp60 and hsc73 showed higher expression in the BM group and, to a lesser extent, the OLTx group. The increased expression of hsc73 was seen especially in the obliterated arteries and in myocytes nearby cellular infiltrates. Immunoblot analysis of graft stromal tissue lysates showed additional bands with mAb to hsp60 and hsc73 for the OLTx and especially the BM group. No significant bands were seen for hsp72 and grp78. Lymphocytes isolated from chronically rejecting allografts reacted with irradiated autologous spleen cells in the presence of mycobacterial hsp65 and interleukin-2. Culturing of graft-infiltrating cells with mycobacterial hsp71 and interleukin-2 yielded lymphocyte clones without alloreactivity, but with strong proliferative responsiveness to self-antigen-presenting cells and, only in the presence of mycobacterial hsp71 or murine grp78. This T-cell reactivity seemed to require intact hsp molecules because treatment of hsp71 with proteolytic enzymes, polymyxin, or ATP abrogated this induction of the stimulatory effect of self-antigen-presenting cells. These T cells are similar to the hsp-dependent, autoreactive lymphocytes cloned from acutely rejecting rat allografts. CONCLUSIONS: These findings support the concept that the pathogenesis of chronic rejection involves a stress response and the participation of graft-infiltrating autoreactive T cells that operate under hsp-dependent mechanisms.     

Influence of gravity and light on the developmental polarity of Ceratopteris richardii fern spores

Starburst dendrimer, a structurally defined, spherical macromolecule composed of repeating polyamidoamino subunits, was investigated to augment plasmid-mediated gene transfer efficiency in a murine cardiac transplantation model. The grafts were directly injected with naked pCH110, a plasmid encoding beta-galactosidase (beta-Gal), or pCH110-dendrimer complex, and reporter gene expression determined by X-Gal staining. The grafts injected with pCH110-dendrimer demonstrated widespread and extended beta-Gal expression in both myocytes and the graft infiltrating cells from 7 to 28 days, compared to the grafts injected with naked pCH110 that expressed beta-Gal only in myocytes for less than 14 days. p alphaMHC-vIL-10, as plasmid encoding viral interleukin-10 (vIL-10) under the control of alpha-myosin heavy chain promoter, was able to prolong allograft survival from 13.9 +/- 0.9 days to 21.4 +/- 2.3 days (p < 0.005). When dendrimer G5EDA was used with p alphaMHC-vIL-10, 60-fold less DNA resulted in significant prolongation of graft survival to 38.6 +/- 4.7 days (p < 0.0005). The dose of DNA, the charge ratio of DNA to dendrimer, and the size generation of the dendrimers were all determined to be critical variables for prolongation of allograft survival in this model system. Thus, the use of the Starburst dendrimer dramatically increased the efficiency of plasmid-mediated gene transfer and expression. Production of immunosuppressive cytokines at higher amounts for longer periods of time in a greater expanse of tissue enhanced the immunosuppressive effect and prolonged graft survival further.     

Ramiprilat attenuates hypoxia/reoxygenation injury to cardiac myocytes via a bradykinin-dependent mechanism

Isolated rat neonatal cardiac myocytes were subjected to immersion in hypoxic (PO2 < 2 mm Hg), glucose-free Tyrode's solution for 5 h followed by concomitant reoxygenation and staining with the membrane-impermeant fluorophore, propidium iodide, in normoxic (PO2 > 150 mm Hg), serum-free culture media for 15 min in order to assess sarcolemmal damage indicative of myocyte viability due to hypoxia/reoxygenation injury. Prior to hypoxic exposure, cells were pretreated for 90 min with the angiotensin-converting enzyme inhibitor cyclopenta[b]pyrrole-2-carboxylic acid, 1-[2-[(1-carboxy-3-phenylpropyl)amino]-l-oxopropyl]octahydro-[2S-[1[R* (R*)]2 alpha, 3a beta, 6a beta]] (ramiprilat), concomitantly with ramiprilat and H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH (bradykinin B2 receptor antagonist HOE 140), the bioactive peptide Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin) or concomitantly with bradykinin and HOE 140. Hypoxia/reoxygenation injury to untreated control cardiac myocytes was characterized by a significant loss of sarcolemmal integrity measured at 75 +/- 4% of total cell fluorescence (mean +/- S.E., n = 42 cultures). Compared to propidium iodide staining of the above untreated control myocytes, those pretreated with 30 or 100 microM ramiprilat showed a significant reduction of propidium iodide staining to 45 +/- 9% and 40 +/- 8% (n = 9, P < 0.05) of untreated controls, respectively. Pretreatment with the protective concentrations of ramiprilat concomitant with 10 microM HOE 140 abolished the significant reduction in propidium iodide staining observed with ramiprilat alone. Similarly, pretreatment with 10 or 100 nM bradykinin significantly reduced propidium iodide staining to 35 +/- 5% and 60 +/- 10% (n = 6, P < 0.05) of the untreated hypoxic controls, respectively. In addition, concomitant pretreatment with protective concentrations of bradykinin and 10 microM HOE 140 also abolished the significant reduction in propidium iodide staining observed with bradykinin alone. The results indicate that the angiotensin-converting enzyme inhibitor ramiprilat has a protective effect on isolated cardiac myocytes exposed to hypoxia/reoxygenation and that this effect is most likely related to a local action of bradykinin on the cardiac myocyte via the activation of the kinin B2 receptor.     

The expression of constitutively active isotypes of protein kinase C to investigate preconditioning

The role of protein kinase C (PKC) in ischemic preconditioning remains controversial because of difficulties with both its measurement and pharmacological manipulation. We investigated preconditioning in isolated neonatal rat cardiocytes by expressing constitutively active isotypes of PKC. Observations at differing durations of simulated ischemia suggested beta-galactosidase (beta-gal) activity reflected viability within transfected myocytes. Preconditioning with 90 min of ischemia significantly increased beta-gal activity and myocyte survival after 6 h of ischemia; an effect abolished by PKC inhibitors. After co-transfection with plasmids encoding beta-gal and either constitutively active mutants of PKC-delta, PKC-alpha, wild type PKC-delta, or empty vector, cardiocytes were subjected to 6 h of ischemia. Only PKC-delta, rendered constitutively active by a limited deletion within the pseudosubstrate domain, consistently increased resistance to simulated ischemia (beta-gal activity was 85.6 +/- 11.9% versus 53.7 +/- 6.5% (p 相似文献   

Genetics of kidney tumours

BACKGROUND: Allograft rejection in heart transplant recipients is associated with lymphocytic extracellular infiltration and edema resulting in increased myocardial stiffness and abnormal relaxation. We hypothesize that these abnormalities will result in reduced myocardial relaxation velocities. Doppler tissue imaging is a novel noninvasive imaging modality that is capable of quantifying myocardial tissue velocities and may therefore be useful to identify allograft rejection. METHODS: In this observational study, 121 heart transplant recipients underwent pulsed-wave Doppler tissue imaging at the time of their surveillance endomyocardial biopsies. Peak relaxation and systolic velocities were measured from the inferior wall blinded to clinical biopsy. Biopsy results were classified as rejecting (3a, 3b, 4) or nonrejecting (0, 1a, 1b). RESULTS: The peak relaxation velocity in nonrejecting allograft recipients (n = 98) was 0.21 m/sec +/- 0.01. During moderate allograft rejection (n = 16), peak relaxation velocities decreased to 0.14 m/sec +/- 0.01 (p < 0.0001), and subsequently increased to 0.23 m/sec +/- 0.0 after successful treatment (p = 0.0001). Peak systolic velocities did not change during rejection, 0.08 m/sec +/- 0.02 when compared with nonrejecting recipients 0.09 +/- 0.02 (p = NS). With a cutoff value of less than 0.16 m/sec, the sensitivity of peak myocardial relaxation velocities for detection of rejection was 76%. The specificity and negative predictive values were 88% and 92%, respectively. CONCLUSION: Moderate allograft rejection results in reduced myocardial relaxation velocities, which can be detected noninvasively with pulsed-wave Doppler tissue imaging. Hence, Doppler tissue imaging is a useful noninvasive tool to exclude allograft rejection.