Clinical significance of circulating hepatocyte growth factor, a new risk marker of carotid atherosclerosis in patients with Type 2 diabetes.

AIMS: Recent studies have provided increasing evidence that hepatocyte growth factor (HGF) has a pathophysiological role in the development of diabetic complications. We set out to determine the relationship between serum HGF and risk factors for macroangiopathy including carotid atherosclerosis. Carotid atherosclerosis is an established and important risk factor for both cerebral and coronary artery diseases. METHODS: We studied 89 patients (48 males, 41 females, mean age 62.5 +/- 10.3 years) with Type 2 diabetes (DM). RESULTS: Serum levels of HGF correlated positively with both intimal-media thickness (IMT) (r = 0.24, P = 0.0248) and plaque score (r = 0.27, P = 0.0126). In multiple regression analysis, serum HGF was associated independently with IMT (standardized beta = 0.28, P = 0.0499). We also found that both IMT and plaque score were higher in patients with ischaemic heart disease (IHD) than in patients without IHD, and that plaque score in patients with lacunar infarcts was higher than in patients without lacunar infarcts. CONCLUSIONS: Serum HGF concentration may be a new marker of atherosclerotic complications in patients with Type 2 DM.

Prognostic value of cyclic GMP in patients undergoing allogeneic bone marrow transplantation after conditioning with total body irradiation.

This study was performed to investigate whether measurement of cyclic GMP (cGMP), a marker for nitric oxide production, before and after allogeneic bone marrow transplantation (BMT) with total body irradiation (TBI) conditioning was of prognostic value. cGMP levels were monitored in 23 consecutive patients who received TBI as conditioning for BMT, and were compared with the outcome. cGMP became positive during the aplastic phase after BMT in 12 patients. In nine of these 12 patients, cGMP level decreased during the recovery phase. Eight of the nine patients survived, one dying after relapse. In three other patients, the cGMP level continued to increase even during the recovery phase and they died of severe complications. cGMP became positive on day 0 of BMT and during the leukocyte recovery phase after BMT in two and seven of the 23 patients, respectively. Subsequently, all patients died of severe complications. The two patients who were negative for cGMP both before and after BMT survived without complications. These results suggest that monitoring cGMP from early after BMT may be useful for predicting outcome and that it may be a useful prognostic marker.

Endothelial damage caused by cytomegalovirus and human herpesvirus-6.

Infection with cytomegalovirus (CMV) or human herpesvirus-6 (HHV-6) may have a role in vascular endothelial damage after bone marrow transplantation (BMT). In total, 41 patients who underwent BMT were classified into four groups (12, 10, 7, and 12 patients who were infected with both CMV and HHV-6, CMV alone, HHV-6, and neither virus, respectively). Levels of thrombomodulin, plasminogen activator inhibitor-1, and cyclic GMP were 7.5+/-1.7 FU/ml, 76.4+/-24.1 ng/ml, and 9.51+/-1.1 pmol/ml, respectively, in the patients with both viruses, while the respective values were 2.9+/-0.67 FU/ml, 33.8+/-8.09 ng/ml, and 2.90+/-1.4 pmol/ml in patients infected with CMV alone, 4.8+/-0.96 FU/ml, 47.7+/-9.21 ng/ml, and 5.48+/-0.55 pmol/ml in patients with HHV-6 alone, and 1.6+/-0.39, 17.5+/-7.88 ng/ml, and 0.45+/-0.3 in those with neither virus. All three markers were significantly higher in the three groups with at least one virus than in the uninfected patients (P<0.05), and were also higher in patients with HHV-6 alone than in those with CMV alone (P<0.05). These results suggest that infection by CMV or HHV-6 causes vascular endothelial injury, with HHV-6 having a stronger effect than CMV, and combined infection having a stronger effect than either virus alone. Such viral infection may be a cause of thrombotic microangiopathy after BMT.

Influence of Helicobacter pylori on platelets after bone marrow transplantation from unrelated donors.

This study was performed to clarify the influence of Helicobacter pylori on the platelet count in patients undergoing bone marrow transplantation (BMT) from unrelated donors. Of 23 consecutive patients undergoing BMT from unrelated donors, the H. pylori antibody test did not change from before conditioning until recovery of the platelet count in 15 patients. These patients were classified into H. pylori antibody-positive (n=8) and -negative (n=7) groups. In the H. pylori antibody-positive group, the platelet count exceeded 20 x 10(9)/l significantly faster after BMT, than in the H. pylori antibody-negative group. When myelosuppression was most severe, the interleukin-6 (IL-6) level was significantly higher in the positive group than in the negative group (67.0+/-10.6 vs 9.9+/-2.4 pg/ml, P<0.05). In addition, the thrombopoietin level was significantly lower in the positive group than in the negative (510.1+/-313.9 vs 3209.1+/-2006.7 pg/ml, P<0.01). These data suggest that H. pylori infection accelerates recovery of the platelet count after BMT from unrelated donors, possibly by stimulating IL-6 production.

Superior vena cava syndrome after bone marrow transplantation caused by aspergillosis: a case report.

Aspergillosis is known for the variety of unusual presentations in immuno-suppressed patients. We report a patient in whom aspergillosis caused the superior vena cava (SVC) syndrome. A 37-year-old woman became febrile soon after bone marrow transplantation (BMT). Chest radiography demonstrated a 5-cm mass extending from the right lung apex to the right supraclavicular fossa beside her Hickman catheter. She then developed SVC syndrome, which progressed despite treatment. Despite recovery of the white blood cell count, the patient continued to deteriorate, became comatose, suffered a cardiac arrest and died 31 days after BMT. Autopsy revealed Aspergillus infection at the apex of the right lung associated with innominate artery thrombosis.

Similarity between eruptions induced by sulfhydryl drugs and acute cutaneous graft-versus-host disease after bone marrow transplantation.

Cutaneous GVHD is histologically similar to eruptions induced by drugs containing a sulfhydryl group. The levels of interleukin-2 and interleukin-2 receptor were determined in a group of patients undergoing bone marrow transplantation (BMT) without graft-versus-host disease or any other complications and in a group with cutaneous graft-versus-host disease (GVHD) alone. In patients who only developed cutaneous GVHD, both interleukin-2 and inter-leukin-2 receptor levels were elevated when the disease was evident. As the elevation of these parameters became more marked, the grade of cutaneous graft versus-host disease also increased. In some patients, only one of the two parameters was elevated and the grade of graft-versus-host disease was low or no skin manifestations were seen. These findings suggest that interleukin-2 and interleukin-2 receptor act together in the development of cutaneous GVHD. This study also showed that the mechanism of cutaneous GVHD resembles that involved in the induction of eruptions by sulfhydryl-containing drugs.

The molecular mechanism in activation-induced cell death of an Ag-reactive B cell clone.

TPA-1 is a subclone of B cell hybridomas established by somatic hybridization using B cells of A/J mice immunized with TNP-LPS, and expresses a receptor for TNP on the cell membrane. The present study showed that TPA-1 was induced to apoptotic cell death upon treatment with TNP-BSA. Therefore, TPA-1 is considered to provide a good model for the study on activation-induced cell death of mature B cells induced by soluble antigen. TNP-BSA treatment caused the generation of a large amount of intracellular reactive oxygen species (ROS) of TPA-1, and the addition of the monovalent thiol-reactive compound: monochlorobimane (MCB) rescued it from apoptosis as well as the antioxidant reagent: N-acetyl-L-cysteine. Furthermore, MCB markedly inhibited the generation of ROS and prevented the disruption of mitochondrial membrane potential that was induced by TNP-BSA treatment. In addition, it counteracted the effect of TNP-BSA on the expression of the Bcl-2 family, resulting in down-regulation of Bax and Bad and up-regulation of Bcl-XL. Taken together, these results suggest strongly that oxidative stress of mitochondria may be involved directly in apoptotic cell death by engagement of antigen receptors on mature B cells with soluble antigen.

Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome following allogeneic bone marrow transplantation.

We monitored the levels of various cytokines and chemokines, as well as an adhesion molecule and factors related to vascular endothelial damage, in three patients with thrombotic thrombocytopenic purpura and hemolytic uremic syndrome after bone marrow transplantation. Measurements were done at the onset of this condition and during plasma exchange for treatment. At the onset of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, the levels of interleukin-8, thrombomodulin, and plasminogen activator inhibitor-1 were all markedly increased. A close relationship was observed between improvement in symptoms by plasma exchange and a decrease in interleukin-8 level, suggesting that this chemokine may be related to the development of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome after bone marrow transplantation.

Enhancement by cyclosporine A and tacrolimus of serotonin-induced formation of small platelet aggregation.

Cyclosporine A (CsA) may increase the incidence of thrombotic events, but whether tacrolimus (Tc) has such effects is still unclear. The serotonergic system has been linked to the thrombotic effects of CsA, but a direct effect of CsA on serotonin-induced platelet aggregation has not been demonstrated because of methodological difficulties. We measured the effects of CsA and Tc on serotonin-induced platelet aggregate formation by particle counting using light scattering. CsA and Tc both enhanced serotonin-induced formation of small platelet aggregates, however, neither CsA nor Tc affected aggregation induced by high or low concentrations of ADP, with or without addition of a serotonin receptor antagonist. Both CsA and Tc enhance platelet aggregation induced via the serotonin pathway.

Oral eicosapentaenoic acid for acute colonic graft-versus-host disease after bone marrow transplantation.

We investigated whether pretreatment with eicosapentaenoic acid, an inhibitor of leukotriene (LT) B4, could ameliorate acute colonic graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). Seventeen patients undergoing unrelated BMT were divided into two groups, with eight patients receiving eicosapentaenoic acid and nine not receiving it. The grade of GVHD after transplantation was compared with that estimated from the pretransplantation LTB4 level. The levels of LTB4 and several cytokines were also monitored. The actual grade of GVHD was lower than that estimated from LTB4 levels in three of the eight patients from the treated group, and there was a significant difference between the treated and untreated groups (p < 0.05, chi 2 test). The levels of LTB4, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) were all significantly lower in the treated group (p < 0.05, Student's t-test). These findings suggest that eicosapentaenoic acid may ameliorate acute colonic GVHD when administered from before BMT.

Extracellular pH affects platelet aggregation associated with modulation of store-operated Ca(2+) entry.

The pH dependence of store-operated Ca(2+) influx (SOCI) into human platelets, as well as its physiological consequence, aggregation, was studied. In Ca(2+)-free medium, thapsigargin (1 microM) induced a small increase in intracellular free-Ca(2+) ([Ca(2+)](i)), which was not affected by changes in extracellular pH. The addition of Ca(2+) (0.5-3 mM) after Ca(2+) store depletion caused by thapsigargin resulted in concentration-dependent increases in [Ca(2+)](i) (SOCI), which were strongly inhibited by SKF-96365 (100 microM), an inhibitor of receptor-mediated Ca(2+) entry. SOCI was inhibited by acidosis (pH 6.9) and augmented by alkalosis (pH 7.9). The addition of Ca(2+) (0.5-3 mM) to platelets, which were kept in Ca(2+)-free medium, slightly but significantly increased [Ca(2+)](i). This Ca(2+) leak entry was also decreased and increased by extracellular acidosis (pH 6.9) and alkalosis (pH 7.9), respectively, but not affected by SKF-96365. Neither thapsigargin (1 microM) stimulation in Ca(2+)-free solution nor elevation of extracellular Ca(2+) alone was sufficient to induce platelet aggregation. In contrast, the addition of Ca(2+) (1 mM) to platelets activated by thapsigargin resulted in aggregation, which was markedly inhibited by SKF-96365 (100 microM). Platelet aggregation associated with SOCI was also inhibited by extracellular acidosis (pH 6.9) and augmented by extracellular alkalosis (pH 7.9). These results suggest that acidosis-induced inhibition, as well as alkalosis-induced promotion of platelet aggregation, involve pH effects on SOCI.

Treatment of intestinal graft-versus-host disease using betamethasone enemas.

Intestinal graft-versus-host disease (GVHD) can readily easily induce generalized metabolic disturbance that influences morbidity and mortality after allogeneic bone marrow transplantation. Although adding a new drug or increasing the doses of immunosuppressive agents will probably be effective for controlling intestinal GVHD, the systemic side effects of such therapy cannot be ignored. In this study, we used betamethasone retention enemas as a local treatment for eight patients with refractory and/or severe intestinal GVHD. Six of the eight patients showed improvement of diarrhea and/or abdominal pain, with a reduction in the stage of GVHD. When treatment with betamethasone enemas was continued for 10 to 27 days in the 6 responding patients, no severe toxicity was observed. One patient failed to respond to treatment and another could not tolerate the enemas. Despite some uncertainty regarding the indications and duration of treatment, betamethasone enemas seem to be a potential alternative method for the management of intestinal GVHD.

Daunorubicin inhibits gene expression of cyclooxygenase-2 in vascular smooth muscle cells.

Daunorubicin (0.1-1 microM) concentration-dependently inhibited prostacyclin production induced by interleukin-1beta (IL-1beta, 2.5 ng/ml) in cultured aortic smooth muscle cells isolated from rats. IL-1beta stimulation caused activation of nuclear factor-kappaB (NF-kappaB) and expression of cyclooxygenase-2 (COX-2) mRNA and protein, which were inhibited by daunorubicin. However, COX activity, evaluated by conversion of exogenous arachidonic acid to prostacyclin, was not affected by daunorubicin (0.1-1 microM). Protein expression of COX-1 and NF-kappaB was not affected by daunorubicin. Daunorubicin also inhibited nitric oxide (NO) production induced by IL-1beta. These results suggest that daunorubicin attenuated prostacyclin synthesis through inhibiting expression of COX-2 mRNA, which could be explained by perturbation of NF-kappaB activation.

Increased hepatocyte growth factor in serum in acute graft-versus-host disease.

Hepatocyte growth factor (HGF) was reported to be effective in preventing acute graft-versus-host disease (GVHD) in a murine model. We examined serum HGF concentrations in 38 patients receiving allogeneic bone marrow transplants, and investigated the relationship of serum HGF concentrations to severity of acute GVHD. More HGF was present in sera from patients with than without acute GVHD. Serum HGF correlated significantly with grade of acute GVHD. Furthermore, serum HGF correlated with serum concentrations of C-reactive protein, gamma-glutamyltranspeptidase (GTP), and aspartate aminotransferase (AST). Serum concentrations of HGF in transplanted patients without GVHD were consistently low, while those in patients with acute GVHD increased with exacerbation. We conclude that HGF was produced during induction of the GVH reaction, and probably increased as a physiological response.

The role of donor T cells for target organ injuries in acute and chronic graft-versus-host disease.

Donor T cells are crucial for target organ injury in graft-versus-host disease (GVHD). We examined the effects of donor T cells on the target organs using a parent-into-F1 model of acute and chronic GVHD. Donor T cells showed engraftment in the spleen, small intestine and liver of mice with acute GVHD, causing typical GVHD pathology in these organs. Interferon-gamma and Fas ligand expression were up-regulated, and host lymphocytes were depleted in the target organs of these mice. In contrast, donor T cells did not show engraftment in the small intestine of mice with chronic GVHD, and no GVHD pathology was observed in this organ. However, both donor T-cell engraftment and GVHD pathology were observed in the spleen and liver of chronic GVHD mice, along with the up-regulation of interleukin-4 (IL-4) and IL-10 expression plus the expansion of host lymphocytes such as splenic B cells and hepatic natural killer (NK) 1.1+ T cells. Donor anti-host cytotoxic T-lymphocyte activity was observed in spleen cells from mice with acute GVHD, but not in spleen cells from mice with chronic GVHD. Transplantation of Fas ligand-deficient (gld) spleen cells did not induce host lymphocyte depletion in target organs. These results indicate that donor T cells augment type 1 T helper immune responses and deplete the host lymphocytes from target organs mainly by Fas-mediated pathways in acute GVHD, while donor T cells augment type 2 T helper immune responses and expand host splenic B cells and hepatic NK1.1+ T cells in chronic GVHD.

Fas ligand-induced caspase-1-dependent accumulation of interleukin-18 in mice with acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD), the fatal side effects of bone marrow transplantation, was shown to be accompanied by elevation of serum levels of interleukin 18 (IL-18). In this study, the mechanism underlying the accumulation of IL-18 in aGVHD in mice was investigated. Lethally irradiated recipients having transplantation with H-2 disparate donor splenocytes demonstrated aGVHD and contained markedly elevated serum levels of IL-18. In contrast, recipients having transplantation with gld/gld spleen cells, which lack functional Fas ligand (FasL), contained only normal ranges of IL-18, indicating FasL-mediated IL-18 release in aGVHD. The wild-type hosts engrafted with caspase-1-deficient cells revealed marked increases of IL-18 similar to those engrafted with wild-type cells, whereas caspase-1-deficient recipients engrafted with wild-type cells showed only a slight elevation of serum IL-18, indicating that IL-18 elevation is derived from host cells in a caspase-1-dependent manner. These results suggest FasL-mediated caspase-1-dependent IL-18 secretion in aGVHD in mice.

Hepatocyte growth factor ameliorates acute graft-versus-host disease and promotes hematopoietic function.

Acute graft-versus-host disease (GVHD) is a major complication of bone marrow transplantation (BMT) and is characterized by hematopoietic dysfunction, immunosuppression, and tissue injury in the skin, liver, and intestinal mucosa. Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, induces mitogenic and antiapoptotic activity in various epithelial cells and promotes hematopoiesis. Working in a murine model of acute GVHD, we performed repeated transfection of the human HGF cDNA into skeletal muscle and showed that this treatment inhibited apoptosis of intestinal epithelial cells and donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by acute GVHD. HGF also markedly suppressed IFN-gamma and TNF-alpha expression in the intestine and liver and decreased the serum IL-12. Furthermore, extramedullary hematopoiesis by donor cells was increased, and the survival rate was improved. These results suggest that HGF may be useful for controlling acute GVHD after allogeneic BMT.

Urinary trypsin inhibitor concentration can predict the immunological insult of chemotherapy and complications after bone marrow transplantation.

Urinary trypsin inhibitor has attracted attention as an index of the systemic inflammatory response syndrome. In this study, the urine concentration of trypsin inhibitor was measured to compare the immunological insult of conventional chemotherapy and conditioning chemotherapy for bone marrow transplantation. We also investigated whether urinary trypsin inhibitor was a useful index of the complications and outcome of bone marrow transplantation. Urinary trypsin inhibitor concentration was determined before chemotherapy, on the day after finishing chemotherapy (day 0 of transplantation), and during recovery of the white cell count, in 17 patients (seven receiving conventional chemotherapy and 10 receiving conditioning for bone marrow transplantation). Urinary trypsin inhibitor concentrations were significantly higher after conditioning for bone marrow transplantation than after conventional chemotherapy (P < 0.001), indicating that conditioning was more invasive. After bone marrow transplantation, the incidence of severe complications and the mortality rate were higher in patients whose urinary trypsin inhibitor concentrations rose during recovery of the white cell count. Comparison of urinary trypsin inhibitor concentrations suggested that conditioning for bone marrow transplantation was more invasive than conventional chemotherapy. This study also suggested that the urine concentration of trypsin inhibitor could be useful for predicting the risk of complications and outcome of bone marrow transplantation.

Oral eicosapentaenoic acid for complications of bone marrow transplantation.

The 'systemic inflammatory response syndrome' (SIRS) may represent the underlying cause of complications after bone marrow transplantation (BMT). This study was conducted to determine whether blocking the etiologic factors of SIRS could improve the complications of BMT. Sixteen consecutive patients with unrelated donors were allocated alternately to two groups. Seven patients received 1.8 g/day of eicosapentaenoic acid (EPA) orally from 3 weeks before to about 180 days after transplantation, while nine patients did not. These two groups were compared with respect to complications, survival, and various cytokines and factors causing vascular endothelial damage. All seven patients receiving EPA survived and only two had grade III graft-versus-host disease (GVHD). Among the nine patients not receiving EPA, three had grade III or IV GVHD. In addition, thrombotic microangiopathy developed in four patients and cytomegalovirus disease occurred in four. Five patients died in this group. The levels of leukotriene B(4), thromboxane A(2), and prostaglandin I(2) were significantly lower in patients receiving EPA than in those not receiving it (all P < 0.01). Cytokines such as tumor necrosis factor-alpha, interferon-gamma, and interleukin-10 were also significantly decreased by EPA (P < 0.05), as were factors causing vascular endothelial damage such as thrombomodulin and plasminogen activator inhibitor-1 (P < 0.05). The survival rate was significantly higher in the group given EPA (P < 0.01). EPA significantly reduced the complications of BMT, indicating that these complications may be manifestations of the systemic inflammatory response syndrome.