Negative selection by apoptosis enriches progenitors in naïve and expanded human umbilical cord blood grafts.

The influence of TNF-α and Fas-ligand (FasL) on viability and function was evaluated in fresh- and expanded-umbilical cord blood (UCB) cells. CD34(+) progenitors and T cells display outstanding survival, whereas ~30% and >50% B lymphocytes and myeloid cells undergo spontaneous apoptosis within 24 and 48 h, respectively. Although the impact of exposure to toxic doses of FasL and TNF-α was undetectable in measurements of apoptosis; removal of dead cells after 2 days of incubation with the ligands revealed a twofold increase in frequency of colony-forming cells (CFU). The sensitivity of progenitors to apoptosis was also unaffected by Fas cross-linking following TNF-induced upregulation of the receptor, increasing CFU frequency without impairing SCID repopulating cell (SRC) activity. Most significant enrichment in CD34(+) progenitors and corresponding increase in CFU frequency were observed when FasL was applied during the final week of ex vivo expansion under the influence of nicotinamide, without impairing SRC activity. These data emphasize differential sensitivities of UCB progenitors and lineage-positive cells to apoptotic signaling mediated by the Fas and TNF receptors, which might be useful in improving the efficiency of ex vivo expansion and improving UCB cell engraftment.

Apoptotic signaling through Fas and TNF receptors ameliorates GVHD in mobilized peripheral blood grafts.

Mobilized peripheral blood (mPB) is a prevalent source of hematopoietic progenitors for transplantation; however, allogeneic and haploidentical transplants are often accompanied by severe GVHD. Following the observation that murine GVHD is ameliorated by pretransplant donor cell exposure to Fas-ligand (FasL) without host-specific sensitization, we assessed the susceptibility of mPB cells to spontaneous and receptor-induced apoptosis as a possible approach to GVHD prophylaxis. Short incubation for 4 h resulted in spontaneous apoptosis of 50% of the T and B lymphocytes and 60% myeloid cells. Although expression of Fas and TNF-R1 was proportionate to fractional apoptosis, cell death was dominated by spontaneous apoptosis. Functional assays revealed that the death receptors modulated mPB graft composition as compared with incubation in medium, without detectable quantitative variations. Removal of dead cells increased the frequency of mPB myeloid progenitors (P<0.001 vs medium), and recipients of mPB exposed to death ligands displayed reduced GVHD (P<0.01 vs medium) and improved survival following lipopolysacharide stimulation. mPB grafts exposed to the apoptotic challenge retained SCID reconstituting potential and graft versus tumor activity. These data emphasize that short-term exposure of mPB grafts to an apoptotic challenge is effective in reduction of GVHD effector activity.

Early immunisation with dendritic cells after allogeneic bone marrow transplantation elicits graft vs tumour reactivity.

BACKGROUND: Perspectives of immunotherapy to cancer mediated by bone marrow transplantation (BMT) in conjunction with dendritic cell (DC)-mediated immune sensitisation have yielded modest success so far. In this study, we assessed the impact of DC on graft vs tumour (GvT) reactions triggered by allogeneic BMT. METHODS: H2K(a) mice implanted with congenic subcutaneous Neuro-2a neuroblastoma (NB, H2K(a)) tumours were irradiated and grafted with allogeneic H2K(b) bone marrow cells (BMC) followed by immunisation with tumour-inexperienced or tumour-pulsed DC. RESULTS: Immunisation with tumour-pulsed donor DC after allogeneic BMT suppressed tumour growth through induction of T cell-mediated NB cell lysis. Early post-transplant administration of DC was more effective than delayed immunisation, with similar efficacy of DC inoculated into the tumour and intravenously. In addition, tumour inexperienced DC were equally effective as tumour-pulsed DC in suppression of tumour growth. Immunisation of DC did not impact quantitative immune reconstitution, however, it enhanced T-cell maturation as evident from interferon-γ (IFN-γ) secretion, proliferation in response to mitogenic stimulation and tumour cell lysis in vitro. Dendritic cells potentiate GvT reactivity both through activation of T cells and specific sensitisation against tumour antigens. We found that during pulsing with tumour lysate DC also elaborate a factor that selectively inhibits lymphocyte proliferation, which is however abolished by humoral and DC-mediated lymphocyte activation. CONCLUSION: These data reveal complex involvement of antigen-presenting cells in GvT reactions, suggesting that the limited success in clinical application is not a result of limited efficacy but suboptimal implementation. Although DC can amplify soluble signals from NB lysates that inhibit lymphocyte proliferation, early administration of DC is a dominant factor in suppression of tumour growth.

Engineering of bone marrow cells with fas-ligand protein-enhances donor-specific tolerance to solid organs.

Effective immunomodulation to induce tolerance to tissue/organ allografts is attained by infusion of donor lymphocytes endowed with killing capacity through ectopic expression of a short-lived Fas-ligand (FasL) protein. The same approach has proven effective in improving hematopoietic stem and progenitor cell engraftment. This study evaluates the possibility of substitution of immune cells for bone marrow cells (BMC) to induce FasL-mediated tolerance to solid organ grafts. Expression of FasL protein on BMC increased the survival of simultaneously grafted vascularized heterotopic cardiac grafts to 90%, as compared to 30% in recipients of naïve BMC. Similar results were obtained for skin allografts implanted into radiation chimeras at 1 week after bone marrow transplantation. Further reduction of preparative conditioning to busulfan resulted in acceptance of donor skin implanted at 2 weeks after transplantation of naïve and FasL-coated BMC, whereas third-party grafts were acutely rejected. The levels of donor chimerism were in the range of 0.7% to 12% at the time of skin grafting, with higher levels in recipients of FasL-coated BMC. It is concluded that FasL-mediated abrogation of alloimmune responses can be effectively attained with BMC. There is no threshold of donor chimerism, but tolerance to solid organs evolves during the process of donor-host mutual acceptance.

Immunomodulation with dendritic cells and donor lymphocyte infusion converge to induce graft vs neuroblastoma reactions without GVHD after allogeneic bone marrow transplantation.

BACKGROUND: Mounting evidence points to the efficacy of donor lymphocyte infusion (DLI) and immunisation with tumour-pulsed dendritic cells (DC) in generating graft vs leukaemia reactions after allogeneic bone marrow transplantation (BMT). We assessed the efficacy of DLI and DC in generating potent graft vs neuroblastoma tumour (GVT) reactions following allogeneic BMT. METHODS: Mice bearing congenic (H2K(a)) Neuro-2a tumours were grafted with allogeneic (H2K(b)) T-cell-depleted bone marrow cells. Tumour-pulsed donor DC (DC(Neuro2a)) were inoculated (on day +7) in conjunction with donor (H2K(b)) and haploidentical (H2K(a/b)) lymphocytes. RESULTS: Murine Neuro-2a cells elicit immune reactions as efficient as B lymphoma in major histocompatibility complex antigen-disparate mice. Lymphopenia induced by conditioning facilitates GVT, and transition to adaptive immunity is enhanced by simultaneous infusion of and DC(Neuro2a) and lymphocytes devoid of graft vs host (GVH) activity (H2K(a/b)). In variance, the efficacy of DC-mediated immunomodulation was diminished by severe graft vs host disease (GVHD), showing mechanistic dissociation and antagonising potential to GVT. CONCLUSIONS: The GVHD is not a prerequisite to induce GVT reactivity after allogeneic BMT, but is rather detrimental to induction of anti-tumour immunity by DC-mediated immunomodulation. Simultaneous inoculation of tumour-pulsed donor DC and DLI synergise in stimulation of potent GVT reactions to the extent of eradication of established NB tumours.

Regulatory functions of TRAIL in hematopoietic progenitors: human umbilical cord blood and murine bone marrow transplantation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling pathway has selective toxicity to malignant cells. The TRAIL receptors DR4 and DR5 are expressed at low levels in human umbilical cord blood cells (3-15%) and are upregulated by incubation with the cognate ligand, triggering apoptosis in 70-80% of receptor-positive cells (P<0.001). Apoptosis is not induced in hematopoietic progenitors, as determined from sustained severe combined immunodeficiency reconstituting potential and clonogenic activity. Furthermore, elimination of dead cells after incubation with TRAIL for 72 h results in a threefold enrichment in myeloid progenitors. Exposure to TRAIL in semisolid cultures showed synergistic activity of DR4 and granulocyte/macrophage colony-stimulating factor in recruiting lineage-negative (lin(-)) and CD34(+) progenitors and in promoting the formation of large colonies. In murine bone marrow, approximately 30% of lin(-) cells express TRAIL-R2 (the only murine receptor), and the receptor is upregulated after transplantation in cycling and differentiating donor cells that home to the host marrow. However, this receptor is almost ubiquitously expressed in the most primitive (lin(-)SCA-1(+)c-kit(+)) progenitors, and stimulates the clonogenic activity of lin(-) cells (P<0.001), suggesting a tropic function after transplantation. It is concluded that TRAIL does not trigger apoptosis in hematopoietic progenitors, and upregulation of its cognate receptors under stress conditions mediates tropic signaling that supports recovery from hypoplasia.

Targeting of IL-2 receptor with a caspase fusion protein disrupts autoimmunity in prediabetic and diabetic NOD mice.

AIMS/HYPOTHESIS: Interruption of IL-2 signalling is an attractive therapeutic target in autoimmune disorders. In this study we evaluated the effect of a fusion protein composed of IL-2 and caspase-3 (IL2-cas) on NOD mice, as compared with disease induction by cyclophosphamide. METHODS: IL2-cas was assessed in NOD mice at various ages and in conjunction with cyclophosphamide administration. The effect of IL2-cas on diabetogenic cells was evaluated in adoptive transfer experiments and in cell suspension in vitro. RESULTS: IL2-cas induced apoptosis in T cells expressing the alpha chain of the IL-2 receptor (cluster of differentiation [CD]25) in vitro, with superior survival of T cells expressing CD4 and forkhead box P3 (FOXP3). The fusion protein decreased mixed lymphocyte reactivity, and pretreatment with IL2-cas decreased the efficacy of adoptive transfer of diabetes into NOD severe combined immunodeficiency mice. Administration of one dose of IL2-cas decreased the incidence of diabetes in NOD mice, showing a superior beneficial effect when administered at young age, and effectively blocked induction of hyperglycaemia by cyclophosphamide, reducing the severity of islet inflammation. Administration of IL2-cas caused an acute increase in CD25(-)FOXP3(+) T cells in the lymph nodes, pancreas and thymus in NOD mice, with similar effects in wild-type mice. Administration of IL2-cas after onset of hyperglycaemia resulted in superior survival. CONCLUSIONS/INTERPRETATION: Targeted elimination of cells expressing the IL-2 receptor by this fusion protein disrupts the autoimmune pathogenesis in prediabetic and diabetic NOD mice, despite depletion of CD25(+) regulatory T cells. Furthermore, this particular fusion protein is permissive to the development of FOXP3(+) T cells that might contribute to protracted protection from the progression of insulitis and overt hyperglycaemia.

NMR spectroscopic characterization of sarcolemmal permeability during myocardial ischemia and reperfusion.

This study aims to characterize the pattern of membrane disintegration during myocardial ischemia and reperfusion. Intracellular volumes were measured by 1H and 59Co NMR in isolated rat hearts during 10, 30 and 60 min of total ischemia and 30 min of reperfusion at normothermia. Perfusion with hypo-osmotic medium (210 mosm/l) increased intracellular water from 2.50+/-0.06 to 3.07+/-0.07 ml/g dry weight (P<0.001) during pre-ischemia. Hypo-osmotic swelling decreased by 16+/-3, 32+/-6 and 44+/-11% of the pre-ischemic value after 10, 30 and 60 min of ischemia (n.s., P<0.005, P<0.001) respectively, indicating that membrane permeabilization facilitated efflux of osmolytes and counterbalanced the osmotic driving force for water influx. Hypo-osmotic swelling decreased during 30 min of reperfusion by 18+/-5% in all groups (P<0.0.005 v post-ischemia). The volume of distribution of the extracellular marker cobalticyanide increased by more than 3.2+/-0.4 and 5.8+/-0.5% of the intracellular space after 30 and 60 min of ischemia respectively (P<0.001), and by an additional 2% after reperfusion. During 30 min of reperfusion, hearts released 1.6+/-0.2 and 3.2+/-0.4% of the intracellular creatine kinase contents after 30 and 60 min of ischemia, respectively (P<0.001). In addition to the correlation between ischemia duration and membrane permeability, evident from the analysis of each probe, the data showed a progressive increase in severity of membrane injury over time and permeabilization to larger molecules. 23Na NMR spectroscopy in conjunction with an extracellular shift reagent (SR) showed formation of a resonance at an intermediate chemical shift in between the intra and extracellular Na+ peaks, suggesting penetration of SR into cells with disrupted membranes. The constant chemical shift and narrow line shape of this resonance, characteristic of a homogeneous chemical environment, suggested that the distribution of SR was contained within the cytosol of cardiomyocytes. We propose that sarcolemmal membranes are gradually permeabilized to larger molecules by ischemia, and the evolving chemical instability is spatially contained within the myocyte.

Is cytotoxic cellular edema real? The effect of calcium ion on water homeostasis in the rat heart.

The objective of this study was to determine the effect of calcium on water homeostasis in cardiac muscle and whether cytotoxic edema is involved in ischemic-reperfusion injury. Cellular water was quantified by multinuclear nuclear magnetic resonance (NMR) spectroscopy in isolated rat hearts during 30 min of ischemia and reperfusion. An increase in extracellular calcium from 0.75 to 2.25 mM increased ischemic swelling twofold, with a marginal effect on functional recovery at reperfusion. Inhibition of Ca2+ channels with verapamil and bepridil reduced ischemic swelling by 58-66% and improved cardiac viability and functional recovery. Inhibition of the Na/Ca exchange with amiloride resulted in reperfusion swelling resulting from the inhibition of calcium efflux via the Na/Ca exchange. This was confirmed by inhibition with amiloride of 63% of cellular swelling induced by calcium overload via the Na/Ca exchange after calcium-free perfusion (calcium paradox). The volume-related activity of amiloride was attributed to the inhibition of Na/Ca exchange because the inhibition of the Na/H antiport resulted in low cellular volumes. The consistent changes in cellular volumes induced by the various agents used to alter calcium fluxes provide evidence of a pro-edematous effect of intracellular calcium accumulation during myocardial ischemia. The administration of toxic doses of bepridil to the perfused and ischemic heart resulted in the shrinkage of cellular volumes and in functional failure. The toxicity of bepridil was mediated by unrestricted release of calcium from the sarcoplasmic reticulum because magnesium cardioplegia, which limits calcium release, lowered ischemic swelling without cellular shrinkage and improved functional recovery. In all experimental groups, cellular edema subsided during the early stages of reperfusion, indicating efficient dissipation of intracellular hyperosmolarity. There was no evidence that cytotoxic edema develops during ischemia or reperfusion, or both. On the contrary, calcium-mediated cardiac toxicity was associated with cellular volume shrinkage as a result of the efflux of osmolytes through permeabilized sarcolemmal membranes. It is concluded that calcium ion is involved in the regulation of cellular volumes, and its intracellular accumulation promotes swelling in ischemic myocytes with intact membranes.

Glycolysis protects sarcolemmal membrane integrity during total ischemia in the rat heart.

Experimental evidence indicates that ischemic glycolysis improves myocardial tolerance to low flow ischemia and anoxia, and cellular membrane disruption signals and/or causes transition to irreversible ischemic injury. The objective of this study was to determine the impact of ischemic glycolysis on membrane integrity and myocardial viability during total ischemia. Phosphorus metabolites were measured by 31P NMR spectroscopy and cellular volumes were determined by 1H and 59Co NMR in conjunction with the extracellular marker cobalticyanide. Isolated rat hearts were submitted to 30 min of total ischemia, followed by 30 min of reperfusion. Glycogen contents were modulated by pre-ischemic perfusion with various substrates. Increased glycolytic activities, as determined from lactate production, delayed onset of ischemic contracture (p < 0.05), induced cytosolic acidification (p < 0.005) and cellular swelling during ischemia (p < 0.05), reduced post-ischemic diastolic tone (p < 0.05), improved recovery of high energy phosphates and contraction force (p < 0.005). Inhibition of glycolysis with iodoacetate and glycogen depletion with 2-deoxyglycose resulted in early onset of ischemic contracture (p < 0.005), elevated post-ischemic diastolic pressures (p < 0.05), reduced coronary flow rates and mechanical activities (p < 0.05). Cellular viability was evaluated by creatine kinase efflux, and membrane integrity was determined from cellular swelling during perfusion with hypoosmotic medium. High activities of ischemic glycolysis correlated with improved cellular viability and preserved membrane integrity, while low glycolytic fluxes were associated with membrane permeabilization (p < 0.05). The protective effect of ischemic glycolysis over sarcolemmal integrity was attributed to continuous provision of energy, undetected by 31P NMR spectroscopy. There was no evidence that ischemic swelling caused by glycolytic end-metabolites accumulation had detrimental consequences, and of excessive swelling during reperfusion. It is concluded that one of the cardio-protective mechanisms of ischemic glycolysis is energy-dependent preservation of sarcolemmal integrity and cellular viability.

Sensitivity of mechanical and metabolic functions to changes in coronary perfusion: A metabolic basis of perfusion-contraction coupling.

Experimental evidence indicates a metabolic basis of contraction-perfusion coupling during an increase in cardiac work load. This study aims to characterize adjustment of myocardial energy metabolism in response to acute low flow ischemia (LFI), and to determine its involvement in perfusion-contraction coupling. Intracellular parameters were measured in isolated rat hearts by NMR spectroscopy and biochemical methods during 30 min of graded LFI and reperfusion as compared to continuous perfusion (control). Oxygen pressure was set to reach maximal oxygen extraction at 70% coronary flow rate (CFR), therefore oxygen limitation was proportional to coronary underperfusion. At 69, 38 and 10% CFR left ventricular pressures decreased to 71, 43 and 25% of pre-ischemic values respectively (P<0.005 v 97% in control) without an increase in diastolic tone, and recovered to 92+/-3% after 30 min of reperfusion. Despite hydrolysis of high energy phosphates and cellular acidification, ADP concentrations were stable in underperfused hearts. At 69, 38 and 10% CFR, cytosolic phosphorylation potentials (PP) decreased from 74+/-10 m M(-1)during pre-ischemia to 40+/-6, 25+/-4 and 14+/-4 m M(-1)respectively (P<0.05 v 63+/-9 m M(-1)in control), and lactate efflux increased to 256+/-18, 386+/-22 and 490+/-43 micromol /gdw respectively (P<0.005 v 186+/-22 micromol/gdw in control). Glycogen contents decreased (P<0.005 v control) and accounted for 27-30% of lactate efflux. These results indicate: (a) proportionate depression of contraction force and glycogen contents, and increased glucose uptake and anaerobic energy production in the underperfused myocardium. Coordinated modulation of these parameters attributes cytosolic PP a regulatory function; (b) resetting of cytosolic PP to lower levels mediates perfusion-contraction coupling during graded LFI. The data are consistent with the concept that glycolytic energy production improves myocardial tolerance to ischemia.

Efficient limitation of intracellular edema and sodium accumulation by cardioplegia is dissociated from recovery of rat hearts from cold ischemic storage.

Energy deficiency and disturbances of sodium and water homeostasis are considered as mechanisms of injury during hypothermic preservation of cardiac muscle. The present study attempts to characterize the effect of potassium (K+) and magnesium (Mg2+) cardioplegia on these mechanisms. Cellular parameters were measured by multinuclear NMR spectroscopy in isolated rat hearts during 12 h of ischemia at 4 degrees C and 2 h of normothermic reperfusion with an isoosmotic Krebs-Henseleit (KH) solution. Potassium and magnesium cardioplegia (a) reduced the rate of ATP hydrolysis and cellular acidification during early stages of ischemia; (b) caused an early cessation of the phase of fast sodium influx after 40 min (P<0.001 vs 120 min with KH); (c) reduced intracellular sodium accumulation to 148-165 micromol/gdw after 12 h (P<0.01 vs 268+/-15 micromol/gdw with KH); (d) decreased ischemic volumes to 2.7+/-0.1 and 2.8+/-0.1 ml/gdw after 8 and 12 h of storage, respectively (P<0.005 v 3.0 and 3.3 ml/gdw with KH). Quantitative analysis of these parameters showed that both hypothermia and cardioplegia increased the relative contribution of sodium to intracellular water accumulation by a factor of 2-2.5. In view of the marked reduction in absolute sodium and water contents, the data indicate that cold cardioplegia limits the increase in intracellular osmolarity. Myocardial mechanical and metabolic recoveries, and cellular viability deteriorated during prolongation of the ischemic period from 8 to 12 h in all experimental groups (P<0.005). Reperfusion was efficient in reversing intracellular sodium and water accumulation in hearts stored with cardioplegia, in contrast to hearts stored in KH. Magnesium, but not potassium cardioplegia, lowered interstitial water contents (P<0.01 v KH), increased intracellular magnesium concentrations (P<0.001), improved mechanical and metabolic recoveries (P<0.01) and cellular viability (P<0.001). These results indicate (a) cardioplegia reduces intracellular sodium (by approximately 46%) and water accumulation (by 66%) during cold ischemia; (b) both hypothermia and cardioplegia limit the rise in intracellular osmolarity and increase the contribution of sodium to cellular swelling; (c) intracellular sodium and water contents were dissociated from myocardial viability and recovery from cold ischemia in potassium and magnesium cardioplegic solutions. It is concluded that intracellular sodium and water accumulation are not dominant factors in determination of cardiac outcome from ischemia.

Creatine and cyclocreatine treatment of human colon adenocarcinoma xenografts: 31P and 1H magnetic resonance spectroscopic studies.

Creatine (Cr) and cyclocreatine (cyCr) have been shown to inhibit the growth of a variety of human and murine tumours. The purpose of this study was to evaluate the anti-tumour effect of these molecules in relation to drug accumulation, energy metabolism, tumour water accumulation and toxicity. Nude mice carrying a human colon adenocarcinoma (LS174T) with a creatine kinase (CK) activity of 2.12 units mg(-1) protein were fed Cr (2.5% or 5%) or cyCr (0.025%, 0.1% or 0.5%) for 2 weeks and compared with controls fed standard diet. Cr concentrations of 2.5% and 5% significantly inhibited tumour growth, as did 0.1% and 0.5% cyCr. In vivo 31P magnetic resonance spectroscopy (MRS) after 2 weeks of treatment showed an increase in [phosphocreatine (PCr)+phosphocyclocreatine (PcyCr)]/nucleoside triphosphate (NTP) with increasing concentrations of dietary Cr and cyCr, without changes in absolute NTP contents. The antiproliferative effect of the substrates of CK was not related to energy deficiency but was associated with acidosis. Intratumoral substrate concentrations (measured by 1H-MRS) of 4.8 micromol g(-1) wet weight Cr (mice fed 2.5% Cr) and 6.2 micromol g(-1) cyCr (mice fed 0.1% cyCr) induced a similar decrease in growth rate, indicating that both substrates were equally potent in tumour growth inhibition. The best correlant of growth inhibition was the total Cr or (cyCr+Cr) concentrations in the tissue. In vivo, these agents did not induce excessive water accumulation and had no systemic effects on the mice (weight loss, hypoglycaemia) that may have caused growth inhibition.

Intracellular volumes and membrane permeability in rat hearts during prolonged hypothermic preservation with St Thomas and University of Wisconsin solutions.

The study aims to determine a possible relationship between intracellular water, energy metabolism, functional recovery and membrane permeability, during and after hypothermic cardiac preservation. Isolated rat hearts were stored for 12 h at 4 degrees C with University of Wisconsin (UW), St Thomas Hospital (ST) and Krebs-Henseleit (KH) solutions, and were reperfused for 1 h. Cellular volumes were measured by 1H NMR of water and 59Co NMR of the extracellular marker cobalticyanide, and energetic profiles by 31P NMR spectroscopy. Storage in ST solution reduced ischemic swelling from 2.50 +/- 0.06 to 2.73 +/- 0.09 (P < 0.001 v 3.56 +/- 0.10 ml/g dry weight in KH), while UW solution caused cellular shrinkage to 2.12 +/- 0.08 ml/g dry weight. Intracellular ATP concentrations and pH values were higher in UW as compared to ST solution. At reperfusion, hearts stored in ST shrank while those stored in UW expanded, resulting in similar intracellular volumes. Storage with UW was superior to ST in post-ischemic function 65 +/- 5% (P < 0.01 v 49 +/- 4% with ST) and in recovery of ATP 46 +/- 3% (P < 0.001 v 25 +/- 4% with ST). Storage with both ST and UW solutions did not prevent interstitial edema. Sarcolemmal membrane integrity, as assessed by cellular swelling in response to a hypo-osmotic shock (210 mmol/l), was significantly improved by ST and UW solutions as compared to KH (P < 0.05). Creatine kinase efflux was reduced by ST and UW as compared to KH (P < 0.05), and by UW as compared to ST (P < 0.05). Coronary flow was higher following storage with UW than ST solutions. 66 +/- 6 and 45 +/- 4%, respectively (P < 0.01). According to these data, the beneficial effects of UW and ST solutions on hypothermic ischemic storage of rat hearts included prevention of cellular edema and preservation of sarcolemmal membrane integrity. It is concluded: (a) UW and ST solutions reduce ischemic and reperfusion cellular volumes: (b) both solutions, and UW in particular were efficient in preservation of membrane integrity: (c) prevention of cellular edema is not the single or main mechanism responsible for the improved preservation with UW and ST solutions.

Differential effects of creatine kinase isoenzymes and substrates on regeneration in livers of transgenic mice.

Creatine kinase (CK) has been implicated in affecting cell growth, and the CK substrates creatine (Cr) and cyclocreatine (CyCr) have been shown to have anti-tumor activity. The influence of Cr and CyCr on liver regeneration following major hepatectomy was evaluated in normal and transgenic mice expressing the human ubiquitous mitochondrial isoform of CK (CK-mit) or the brain isoform of CK (CK-B) or livers expressing both CK-mit and CK-B (CK-comb). Expression of CK isoenzymes had little effect on liver regeneration in the absence of dietary supplementation with Cr or CyCr as assayed by the increase in liver mass. Dietary supplementation with Cr and CyCr significantly reduced liver growth in normal mice. Liver regeneration was almost completely inhibited in mice expressing CK-mit in the presence of Cr. Livers expressing CK-mit regenerated better than normal livers in the presence of CyCr. In mice expressing CK-B, Cr and CyCr had opposite effects from those found in CK-mit mice. In the presence of CyCr, regeneration was inhibited in livers expressing CK-B, and, in the presence of Cr, CK-B-expressing livers regenerated better than normal livers. The amount of DNA synthesized 2 days after hepatectomy confirmed the results obtained from measurements of liver mass for all groups. Growth and DNA synthesis were completely abolished by Cr in CK-mit mice, whereas CyCr mainly affected growth 2 days after hepatectomy in CK-B-expressing mice. Coexpression of the CK isoforms in CK-comb mice ameliorated the effects detected with either isoform alone. Inhibition of growth by Cr and CyCr was not correlated to water accumulation. These results clearly demonstrate isoenzyme and substrate-specific effects of CK on cell growth.

Intermittent ischemia: energy metabolism, cellular volume regulation, adenosine and insights into preconditioning.

Interruption of ischemia by brief reperfusions (I/R) is better tolerated by the heart than continuous ischemia. The present study aims to determine the metabolic profiles of isolated rat hearts during intermittent ischemia, the possible cardioprotective role of adenosine and the influence of I/R on intracellular volumes, using multinuclear NMR spectroscopy. After five I/R (5/5 min) episodes, hearts paced at 5 Hz developed pressures comparable to those of hearts continuously perfused for 50 min at 37 degrees C (CP). Following the first 5 min episode of no-flow ischemia, [ADP] dropped from 72 +/- 9 to 43 +/- 5 microM (P < 0.001) and remained stable at the end of the following reperfusions, despite a 2.5-4-fold increase during each episode of 5 min ischemia. Intracellular volumes were stable during CP at a value of 2.50 +/- 0.06 ml/g dry weight, and decreased by 4, 8, and 12% after 1, 3, and 5 I/R episodes. The phosphorylation potentials decreased from 54 +/- 8 to 4 mM-1 during each period of 5 min ischemia and were 40 +/- 6 and 28 +/- 6 mM-1 after CP and I/R5, respectively. Cardiac glycogen had decreased during 50 min of CP from 103 +/- 13 to 81 +/- 9 mumol/g dry weight and lactate production was 116 +/- 15 mumol/heart. Five I/R episodes decreased glycogen to 46 +/- 7 mumol/g dry weight (P < 0.005 v CP) and increased lactate efflux to 262 +/- 31 mumol/ heart (P < 0.005 v CP). These findings suggest that a brief ischemia/reperfusion episode increases anaerobic metabolism of exogenous glucose, reduces [ADP] and induces cellular shrinkage. Administration of the adenosine receptor blocker 8-phenyl theophylline (8PT) during intermittent perfusion depressed the developed pressure to 78 +/- 7%, accentuated the decrease in phosphorylation potential (14 +/- 4 mM-1), abolished cellular shrinkage, reduced lactate efflux and blunted the decrease in ADP following the first I/R episode. In variance, no detectable changes were observed during intermittent ischemia when the ATP-sensitive potassium channel blocker glibenclamide was administered. These data demonstrate: (a) a brief episode of ischemia/reperfusion stimulates anaerobic metabolism of exogenous glucose and lowers intracellular ADP concentration: (b) adenosine receptors are partially responsible for the glycolytic stimulation during intermittent ischemia; (c) cellular shrinkage is related to the rate of glycolysis during intermittent ischemia/reperfusion.

Continuous monitoring of intracellular volumes in isolated rat hearts during normothermic perfusion and ischemia.

The present study describes an experimental setup that enables continuous measurement of cellular volumes in isolated organs. The procedure is a modification of a recently reported method that uses multinuclear NMR measured by 59Co NMR of cobalticyanide and 1H NMR of water in isolated rat hearts at normothermia. The new apparatus contains a background flow which is shown to improve the rate of exchange of the marker between the interstitium and the external solution and allows detection of cellular shrinkage during no-flow ischemia. A series of experiments of marker loading and wash-out were performed to validate the method. In the Langendorff preparation, intracellular volumes (in units of milliliters per gram dry weight) of hearts perfused with Krebs-Henseleit solution oscillated around a mean value of 2.50 +/- 0.06 ml/gdw. During 30 min of ischemia the cells swelled to 2.88 +/- 0.08 ml/gdw and residual edema was observed after 30 min of reperfusion (2.62 +/- 0.08 ml/gdw). A hypoosmotic shock was used to assess changes in membrane permeability at different time points of ischemia and reperfusion. Water influx induced by the hypoosmotic shock at the end of ischemia was similar to that elicited in perfused hearts. After 15 and 30 min of reperfusion, the magnitude of the response to hypoosmolarity decreased by 9 and 37%, respectively, indicating a gradual permeabilization of the membranes, presumably to ions. The experimental setup was also used to monitor intracellular volumes as a function of time in anisoosmotic conditions. Cellular swelling/shrinkage were delayed for periods of 5 and 8 min at osmolarities of +/-50 and +/-100 mosmol/liter, suggesting a limited capability of the heart to absorb an anisoosmotic shock. The variation in cellular volumes was proportional to the deviation of the conditions from isoosmolarity, and activation of volume-regulatory mechanisms was demonstrated. The noninvasive technique presented in this study is capable of providing quantitative evidence of changes in cellular volumes in isolated hearts at a temporal resolution of 1 min and a spatial resolution of 4% (of cellular volume). As demonstrated in the cases of global ischemia and anisoosmolar conditions, the technique is expected to provide new insights into the mechanism of cellular-volume regulation.

Inhibition of muscle sympathetic nerve activity during yawning.

Yawning is a complex event that depends largely on the autonomic nervous system. Microneurographic techniques were used to study the mechanism involved in yawning. A series of spontaneous yawns displayed by a healthy 39-year-old male offered us the opportunity to study the muscle sympathetic nerve activity (MSNA) during this phenomenon. It was found that 2 s of yawning inhibited the MSNA recorded at the right peroneal nerve in the lateral knee area, while 3 s of slow expiration succeeding a yawn provoked an MSNA discharge. Blood pressure decreased with each slow expiration by 5-6 mmHg, and increased again with the renewed MSNA discharge. We conclude that yawning is associated with a sympathetic suppression that favours a parasympathetic dominance, as indicated by the MSNA and the decrease in blood pressure. The slow expiration following a yawn is associated with a sympathetic activation marked by an MSNA discharge and an increase in blood pressure.