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A successful case of a hand-assisted laparoscopic splenectomy with low-pressure pneumoperitoneum for autoimmune thrombocytopenic purpura in a patient at 23 weeks' gestation is reported. Preoperative splenic arterial embolization was performed on the same day as the operation using painless contour embolic material and super-absorbent polymer microspheres. The abdominal wall retraction method first was applied to avoid the effects of pneumoperitoneum on systemic hemodynamic alterations. However, a sufficient surgical view could not be obtained, as the intra-abdominal organs were elevated because of the enlarged uterus. A surgical view with 4 to 6-mm Hg pneumoperitoneum was available for the hand-assisted splenectomy. The postoperative course was uneventful, and the patient vaginally delivered a healthy infant. A hand-assisted laparoscopic splenectomy with low-pressure pneumoperitoneum after splenic arterial embolization would be feasible for patients with autoimmune thrombocytopenic purpura during a relatively advanced pregnancy.

We recently published our work in this journal on the ef-fectsofrapamycinonpancreaticb-cellsdysfunctioninisletgrafts, including autophagy (1). The comment followed byPallet(2)providesfurtheropportunitytoemphasizeseveralpoints discussed in our paper.Autophagy is a tightly regulated degradation process thatdelivers cytoplasmic material to the lysosomes via theautophagosomes. We reported previously that rapamycinupregulates autophagy in islet b-cells and that this ef-fect results in cell death and impaired insulin potency.Autophagosomes contain microtubule-associated protein-light-chain-3(LC3),whichisusedasamarkerofautophagy.The rapamycin-induced autophagy in pancreatic islets wasconfirmed by two widely used methods. First, the uncon-jugated (LC3-I) and conjugated forms (LC3-II) of LC3 wereseparated by SDS-PAGE; then immunoblotting showed ac-cumulation of LC3-II, which correlates with the numberof autophagosomes relative to the amount of endoge-nous LC3-II protein in islets. Second, LC3 distribution wasmappedinGFP-LC3transgenicmice.ThismethodisbasedondetectionofLC3-positivestructuresbyfluorescencemi-croscopy, although it has certain limitations. Under certainconditions, e.g. LC3 overexpression, LC3 easily incorpo-rates into the protein aggregates, hindering accurate lo-calization. Although this limitation is independent of au-tophagy, any LC3 localization by this method should beinterpreted with caution. In our study (1), we did not per-form transient transfection with GFP-LC3 gene but usedstable GFP-LC3 transgenic mice. The data showed in-creased autophagosome production in islets, though thiswas not confirmed by electron microscopy.A major problem inautophagyresearch isthelack of highlyspecific autophagy inhibitors. PI3-kinase inhibitors such aswortmannin and 3-MA (3) are the most commonly usedautophagy inhibitors, but they inhibit the activities of bothclass I and III PI3-kinase. Indeed, 3-MA, which was used inour study (1), targets other kinases and affects other cel-lular processes, especially at high concentrations. Accord-ingly, we used this regent at 10 mM, which is the recom-mended concentration for inhibition of autophagy. While3-MA and wortmannin block the formation of autophago-somes at early stages of autophagy, other PI3-kinase in-hibitors (e.g. bafilomycin A1), which was recommended byPallet (2), block later stages of autophagy. Bafilomycin A1was reported to inhibit autophagosome-lysosome fusion,and also altered intralysosomal degradation by inhibitingacidification (4).Recent loss-of-function studies of Atg genes have ana-lyzedtheparadoxicalfunctionsofautophagyincellsurvivaland death. We also examined this issue in our rapamycin-treated islets (1). Another study demonstrated that in-duction of autophagy over 48 h resulted in cell death (5)whereas induction over 12 h had no effect on cell viabil-ity. These findings support the more rational belief that au-tophagyfacilitatestheremovalofdamagedproteinsandor-ganelles, thus providing temporary protection during tran-sient stress; but can also lead to “autophagic cell death”under prolonged stimulation. Perhaps this death-inducingfunctionofautophagyistriggeredbythedigestionofacriti-calamountofcytoplasmiccontentsnecessaryforsurvival,with subsequent activation of death-promoting molecules.Further research is required in this field including crosstalkbetween autophagic and apoptotic cell death.

The effect of CsA on hypoxia-reoxygenation injury was studied in perfused rat livers. CsA did not attenuate hypoxic injury, as assessed by the release of lactate dehydrogenase and mitochondrial aspartate aminotransferase. During reoxygenation, the release of lactate dehydrogenase was also not affected by CsA. However, the release of mitochondrial aspartate aminotransferase into the cytosol, which indicates mitochondrial injury, was significantly reduced by CsA. The effect of CsA on mitochondrial function during hypoxia-reoxygenation was also investigated. CsA administration increased both the respiratory control ratio and the adenine nucleotide content after reoxygenation in both isolated mitochondria and perfused livers. In addition, glucose production by perfused livers after reoxygenation was increased by CsA. We conclude that the beneficial effect of CsA on hypoxia-reoxygenation injury may be partly due to protection of the mitochondria against reoxygenation injury.

Human C4b-binding protein (C4bp) functions as a cofactor for factor I in the degradation of C4b and C3b and, in addition, accelerates the rate of decay of the C4b2a complex.In this study, we constructed a surface-bound form of human C4b-binding protein (C4bp-PI) consisting of a short consensus repeat 1-8 of the alpha-chain of C4bp and a glycosyl phosphatidylinositol (GPI) of the decay-accelerating factor (CD55) and established stable swine endothelial cell (SEC) lines expressing C4bp-PI by transfection of cDNA. Amelioration of complement-mediated lysis by the transfectant molecules was tested as an in vitro hyperacute rejection model of swine to human discordant xenograft, using the lactate dehydrogenase assay.Flow cytometric profiles of the stable SEC lines with C4bp-PI showed a high level of expression of this molecule. The cell lysate of the SEC line with C4bp-PI showed strong cofactor activity in not only C4b but also C3b, whereas the activity of plasma C4bp to bind to C3 was very weak. Approximately 150 x 10(4) molecules of C4bp-PI per SEC blocked human complement-mediated cell lysis by approximately 75%.The results suggest that the surface-bound form of C4bp will be very useful in clinical xenotransplantation.

Many strategies of tolerance induction by intrathymic (IT) injection of donor alloantigens have been reported to date; however, the timing of IT injection is usually 1-3 weeks before transplantation.To apply IT injection to cadaveric organ transplantation, 1 x 10(8) fully allogeneic bone marrow cells (BMC) of Buffalo (BUF; RT1b) rats were intrathymically injected into Wistar Furth (WF; RT1u) rats at the time of BUF cardiac allografting with short-course therapy of antilymphocyte serum (ALS) and FK506 in our experimental model.Allogeneic IT injection of BUF BMC with ALS and FK506 indefinitely prolonged graft survival (mean survival time210 days) in all WF rats. On day 130 after grafting, tolerant WF rats accepted donor BUF skin grafts (120 days) but not third-party Lewis skin grafts. In control groups, syngeneic IT injection of WF BMC or intravenous injection of donor BUF BMC in combination with ALS/FK506 therapy failed to induce tolerance. In vivo testing was performed during induction (1 month) or during maintenance (6 months of tolerance. In the mixed lymphocyte reaction (MLR), spleen T cells of tolerant rats at 1 month after grafting displayed hyporesponsiveness after stimulation with donor cells. The addition of interleukin (IL)-2 to MLR culture did not restore T-cell responsiveness. Tolerant rats had a significantly decreased frequency of T cytotoxic cell precursors (fTcp) of 1:4,926, and frequency of IL-2-producing T helper cell precursors (fThp) of 1:23,925, compared with naive rats (1: 2,158 and 1:4,266, respectively). By 6 months after grafting, however, the anti-donor MLR proliferative responses of tolerant rats had been restored to the levels of naive splenic T cells. These tolerant rats displayed restoration of the (fTcp) of 1:2,842 and of the (fThp) of 1:5,630, which were comparable frequencies of naive rats. Suppressor T cells did not contribute in this model. In cardiac grafts of tolerant rats induced by IT injection, expression of both Th1 (interferon-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokines was detected in the early phase; thereafter, expression was completely inhibited, except for interferon-gamma in the chronic phase.Perfect donor-specific tolerance was obtained by IT injection of donor BMC at the time of transplantation, while alloimmune responses were maintained at levels similar to those of naive rats.

We examined the use of an abdominal wall retraction method instead of pneumoperitoneum in laparoscopic cholecystectomy for patients with cardiac disease to prevent the hemodynamic deterioration associated with pneumoperitoneum. Eight patients with cardiac diseases, mainly valvular or coronary artery diseases, underwent laparoscopic cholecystectomy under hemodynamic monitoring. Five patients without cardiac disease served as controls. As hemodynamic parameters, heart rate, mean systemic arterial pressure (mAP), mean pulmonary arterial pressure (mPAP), central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP), and cardiac index (CI) were measured. The patients with cardiac disease showed significantly elevated mPAP and PCWP compared with the control group under pneumoperitoneum, and one patient showed critically decreased CI due to increased tricuspid regurgitation under pneumoperitoneum. These changes, however, were resolved on the abdominal wall retraction. There was no major perioperative complication. This abdominal wall retraction method is, therefore, favorable for patients with underlying cardiac disease to minimize the hemodynamic deterioration during laparoscopic cholecystectomy.

The proto-oncogene bcl-2 and a bcl-2-related gene bcl-x prevent apoptotic cell death induced by various treatments. Although a mechanism has been proposed that involves Bcl-2 activity on reactive oxygen species (ROS), we find that expression of Bcl-2 or Bcl-xL prevents cell death induced by withdrawal of oxygen (hypoxia) and that the cell death does not involve ROS, suggesting that Bcl-2 or Bcl-xL exerts an anti-cell death function by a mechanism other than through regulation of ROS activity. Using electron microscopy, and confocal and non-confocal fluorescence microscopy, we show that hypoxia induces both necrosis and apoptosis. Overexpression of Bcl-2 or Bcl-xL blocks hypoxia-induced apoptosis and, although to a lesser extent, necrosis. The anti-apoptotic proteins Bcl-2 and Bcl-xL effectively inhibit KCN-induced cell death which is characterized by necrotic features including apparently intact chromatin, remarkable mitochondrial swelling with loss of crista structure and loss of plasma membrane integrity. The necrotic cell death is also inhibited by inhibitors of ICE (interleukin-1 beta converting enzyme)(-like) proteases, the common mediators of apoptosis. These results indicate that Bcl-2/Bcl-xL and ICE(-like) proteases modulate both apoptotic and at least some forms of necrotic cell death, suggesting that both cell death pathways involve some common mediators.

Loss of fibronectin (FN) from the cell surface has been shown to be closely associated with malignant transformation of cells. To elucidate the role of the FN matrix in the modulation of malignant phenotypes, we overexpressed a full-length cDNA encoding plasma-type FN in HT1080 human fibrosarcoma cells. The cells overexpressing FN adopted a more flattened morphology and deposited a moderately developed FN matrix both in vitro and in vivo, although the level of expression of integrin alpha5beta1 remained unchanged. FN-overexpressing cells exhibited a reduced cell motility on the substratum and grew poorly when injected s.c. into nude mice. Overexpression of FN also suppressed the ability of the tumor cells to proliferate in soft agar, whereas the suppression was reversed by inclusion in soft agar of the Arg-Gly-Asp (RGD)-containing peptide and adhesion-blocking antibodies against the central cell-binding domain of FN. Neither cell motility nor growth potential was altered by overexpression of a truncated form of FN lacking the central cell-binding domain. These results, taken together, indicate that increased deposition of FN in the pericellular matrix per se can suppress the motility and growth potential of tumor cells through interaction with RGD-recognizing integrins, most likely alpha5beta1.

Bcl-2, Bcl-xL, CrmA and tetrapeptide ICE inhibitor reduce the extent of necrotic cell death induced by cyanide, which primarily damages mitochondria. Although none of them affects the drastic decrease in ATP levels induced by cyanide, Bcl-2 and Bcl-xL but not CrmA or ICE inhibitor inhibit the cyanide-induced decrease in mitochondrial membrane potential. A similar blocking effect is observed on necrotic cell death induced by other respiration inhibitors, rotenone and antimycin A, and on apoptotic cell death induced by etoposide or calcium ionophore. These results indicate that Bc1-2 and Bcl-xL protect mitochondria against the loss of function during both apoptosis and at least some forms of necrotic cell death. The ICE family proteases act at a different step other than the loss of mitochondrial membrane potential.

Diabetes-prone (DP) BB rats (RT1(u), RT6.1) spontaneously develop insulin-dependent diabetes mellitus (IDDM) and the disease manifestation resembles that in human IDDM. DP rats are immunodeficient with severe T lymphocytopenia due to the absence of T cells expressing the RT6 differential alloantigen, which have immunoregulatory functions. MHC- and non-MHC-compatible Wistar Furth (WF; RT1(u), RT6.2) pancreases were transplanted into DP rats. WF pancreas grafts were destroyed by IDDM recurrence (insulitis), but not by rejection, with a mean survival time of 65.3 +/- 21.7 days. To prevent the recurrence of IDDM in the grafts, monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 were administered. WF pancreas grafts were indefinitely accepted (108.0 +/- 26.8 days) in monoclonal antibody-treated DP recipients. The number of T cells was increased and cellular immune responses restored only in the DP rats that had accepted grafts. The increased number of T cells was due to the peripheral appearance of donor-type RT6.2+ T cells, which represented 34.3 +/- 7.0% of total splenic T cells. The cytotoxicity of splenic T cells to WF islet cells was suppressed in the presence of RT6+ T cells in vitro. These findings demonstrated that stable macrochimerism of donor-derived RT6+ T cells could restore the immune responses and prevent the recurrence of IDDM in the DP recipients.

The Bcl-2 family and the ICE family of cysteine proteases play important roles in regulating cell death. We show here that induction of cell death by a Ca2+ ionophore or hypoxia results in increased levels and activity of active ICE(-like) proteases and the subsequent activation of CPP32/Yama(-like) proteases, and that inhibition of these protease activities reduces the extent of cell death. Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-xL inhibits the cell death and the activation of ICE(-like) and CPP32/Yama(-like) proteases, indicating that Bcl-2 and Bcl-xL act upstream of these proteases. We also show that specific inhibition of ICE(-like) proteases in vivo prevents activation of CPP32/Yama(-like) proteases, whereas inhibition of CPP32/Yama(-like) proteases does not prevent activation of ICE(-like) proteases, suggesting the existence of a protease cascade in vivo that requires ICE(-like) proteases for activation of CPP32/Yama(-like) proteases. Induction of necrotic cell death by KCN also induces activation of ICE(-like) proteases but not of CPP32/Yama(-like) proteases, and Bcl-2 and Bcl-xL inhibit the activation and the cell death, suggesting that the functional site of Bcl-2 and Bcl-xL is also upstream of ICE(-like) proteases in at least some forms of necrosis.

Inhibition of the respiratory chain reaction by cyanide, rotenone or antimycin A (chemical hypoxia) induces necrotic cell death characterized by apparently intact chromatin, remarkable mitochondrial swelling with loss of crista structure, and loss of plasma membrane integrity. The treatments induce no apoptotic cell death, as defined by fragmented nuclei with condensed chromatin, fragmented or condensed cytoplasm. The anti-apoptotic proteins Bcl-2 and Bcl-xL effectively retard the chemical hypoxia-induced necrotic cell death. The necrotic cell death is also retarded by inhibitors of ICE(-like) proteases, including interleukin-1beta converting enzyme (ICE), which are common mediators of apoptosis. These results indicate that Bcl-2/Bcl-xL and ICE(-like) proteases modulate apoptotic and at least some forms of necrotic cell death. Both cell death pathways appear to involve some common mediators; however necrotic or apoptotic cell death signals might be transduced through multiple pathways, because Bcl-2/ Bcl-xL or inhibitors of ICE(-like) proteases are relatively less potent in blocking necrotic cell death than in preventing apoptosis.

The molecular mechanism of cell death due to hypoxia has not been elucidated. Our recent observations that overexpression of the anti-apoptotic proto-oncogene bcl-2 and a bcl-2-related gene, bcl-x, prevents hypoxic cell death suggest that hypoxia induces apoptosis. Using electron microscopy and confocal and nonconfocal fluorescence microscopy, we show here that hypoxia does, in fact, induce both necrosis and apoptosis, and that the proportion of these two modes is highly dependent on the cell type. Overexpression of Bcl-2 or Bcl-Xl blocks hypoxia-induced apoptosis in a dose-dependent manner.

Fas (Apo-1/CD95) belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits apoptotic signals by binding to its ligand. Interleukin-1beta-converting enzyme (ICE), which shows substantial homology to the product of the cell death gene, ced-3, of Caenorhabditis elegans, is reported to be involved in Fas-mediated apoptosis. Using two human carcinoma-derived cell lines with undetectable levels of ICE, we found that an agonistic antihuman Fas antibody induces the activation of CPP32/Yama(-like) proteases that are ICE(-like) protease family members, and that a tetrapeptide inhibitor of CPP32/Yama protease, DEVD-CHO, inhibits the Fas-mediated activation of the proteases, Fas-mediated apoptosis, and CPP32/Yama(-like) proteolytic activities in vitro. Fas-mediated apoptosis is inhibited by the CPP32/Yama inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO, suggesting a dominant role for the CPP32/Yama(-like) proteases and not ICE itself in Fas-mediated apoptosis of the human carcinoma cell lines.

A patient with severe ulcerative colitis developed portal vein thrombosis. Intraportal infusion of plasminogen activators through a percutaneous transhepatic catheter produced an effective thrombolysis without increasing rectal bleeding. This is the first report in which portal vein thrombosis associated with active ulcerative colitis was treated successfully.

The cellular levels of adenine nucleotides and their metabolites in ischemic rat liver were assayed by high pressure liquid chromatography with high theoretical plate numbers. The method was sensitive enough to measure all the metabolites in about 1 mg of tissue, and to examine changes in their levels in a single liver in ischemia. In ischemia the cellular level of ATP decreased rapidly. Concomitantly there was a transitory increase in AMP, followed by its degradation to allantoin via adenosine with accumulation of all species of purine catabolites. NAD was also degraded gradually with concomitant accumulation of nicotinamide. Thus, the level of total adenine nucleotides decreased during ischemia and the amount of this decrease ws equal to the sum of the amounts of catabolites produced. The ATP level was rapidly restored on recirculation after an ischemic period of less than 15 min. However, recovery of the ATP level was depressed by prolonged ischemia and was not observed after an ischemic period of 2 h. Intermediate purine catabolites that accumulated in ischemia were also cleared during recirculation either by their removal in the blood flow or by further oxidative degradation, but they were not salvaged for reuse until the cellular level of ATP was restored. Administration of allopurinol resulted in marked accumulation of hypoxanthine in ischemic liver, but neither this drug nor chlorpromazine had any appreciable effect on recovery of the ATP level during recirculation.

During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption of the diffusion barrier against protons. The external addition of ATP and its non-metabolizable analog, beta,gamma-methylene adenosine 5'-triphosphate, prevented both the release of Ca2+ and increase in the rate of state 4 respiration. Addition of EGTA, which did not prevent release of the ion, resulted in little increase in the respiration rate. Addition of an inhibitor of mitochondrial phospholipase A2, such as quinacrine, dibucaine, or chlorpromazine, also prevented increase in the respiration rate without affecting Ca2+ release from mitochondria during anoxic incubation. Non-esterified polyunsaturated fatty acids were also found to be liberated from anoxic mitochondria. External addition of the ATP-analog, EGTA, and inhibitors of phospholipase A2 suppressed the liberation of non-esterified polyunsaturated fatty acids. Melittin and Ca2+, which activate phospholipase A2, increased the rate of state 4 respiration and the liberation of fatty acids. These findings support the hypothesis proposed previously that the following sequence changes occurs in mitochondria during anoxia; depletion of ATP, liberation of free calcium from mitochondria, and disruption of the diffusion barrier against H+ of the inner membrane. The results also indicate another event; activation of phospholipase A2 by release Ca2+ which results in H+ leakiness of the inner membrane.

Biochemical markers of ischemic injury of rat liver were studied in an extracorporeal perfusion system. During anoxic perfusion, purine compounds appeared in the perfusate as soon as they were formed in the liver and their recovery in the perfusate balanced the loss of adenine nucleotides from the liver. In contrast, cytosolic aspartate aminotransferase did not appear in the perfusate at slow rates of liver perfusion or during hypothermic perfusion. The production of purine compounds was further investigated in hypothermically preserved liver in connection with the restoration of some metabolic functions of liver. The amount of purine compounds released into the perfusate was found to be closely related to the degrees of damage of the hepatic functions of gluconeogenesis, ureogenesis, and mitochondrial respiration on reperfusion. These results indicate that release of purine compounds into the perfusate is a good marker of ischemic damage.

We recently encountered a case of a huge vertebral aneurysm involving the entire right neck, extending from the clavicle to the mandibular area. The patient had undergone laminectomy of the cervical spine at age 45 for neuritis or spinal caries. This operative procedure apparently caused a small vertebral laceration and produced the giant aneurysm. The aneurysm was resected under hypothermia in order to protect the brain. One vertebral artery was ligated, but cerebral damage did not occur. Only a few cases of huge aneurysms extending from the clavicle to the mandible have been reported in the literature.

The process of mitochondrial dysfunction in ischemic rat liver was studied. A close correlation was found between decrease in the mitochondrial adenine nucleotide content and deterioration of oxidative phosphorylation capacity. The level of total adenine nucleotides, which was 15--20 nmol/mg protein in mitochondria isolated from normal liver, fell to 1--2 nmol/mg protein with concomitant loss of oxidative phosphorylation capacity after anoxic incubation in vitro or in vivo for 120 min. However, neither the permeability barrier to adenine nucleotides nor matrix enzymes were affected under these conditions. The loss of adenine nucleotides was ascribed to degradation of AMP to adenosine and then leakage of the latter. Conventional procedures for maintenance of oxidative phosphorylation capacity of isolated mitochondria, preservation in the cold and addition of ATP or a respiratory substrate under aerobic conditions, were very effective in maintaining the intramitochondrial levels of adenine nucleotides. Of the three species of adenine nucleotides, only AMP was ineffective in maintaining mitochondrial function; mitochondria containing more than 5 nmol of ATP plus ADP/mg protein exhibited normal activity of oxidative phosphorylation, but with less than 2 nmol they showed no activity.

The relationship between adenine nucleotide metabolism and ischemic damage was studied in human liver. Thirty transplanted grafts were divided into two groups according to their functional outcome. Cellular adenine nucleotide levels were assayed by high-performance liquid chromatography. During cold ischemia, the adenosine triphosphate (ATP) level was not correlated with graft function, but two grafts with low total adenine nucleotides (TAN) levels showed poor function after transplantation. After recirculation, the ATP level showed good recovery in grafts that functioned satisfactorily (n = 24, 5.47 +/- 1.51 mumol/g dry weight), but remained low in poorly functioning grafts (n = 6, 3.30 +/- 1.68 mumol/g dry weight) (P less than 0.01). The level of recovery of ATP was inversely related to the period of warm ischemia during implantation (P less than 0.01). Bile production, used as a parameter of initial function, was observed shortly after implantation in 17 of 24 grafts that functioned satisfactorily, but in only 1 of 6 poorly functioning grafts. It is concluded that loss of adenine nucleotides and lack of bile production during transplantation are good markers of damaged grafts in human liver transplantation.