Eizo Kakishita

Relationship of CD4+CD25+ regulatory T cells to immune status in HIV-infected patients.

Objective:To determine whether the frequencies of CD4+CD25+ regulatory T cells (T Reg) were related to immune status in HIV-infected patients. Methods:Peripheral blood CD4 T-cell populations were examined for T-helper 1 cells (Th1), T-helper 2 cells (Th2), and T Reg by intracellular staining for interferon (IFN)-γ and interleukin (IL)-4, and surface staining for CD25, respectively. The immunoregulatory properties of T Reg were assessed by measurement of the inhibitory effects of isolated CD4+CD25+ T Reg on CD4+CD25− T-cell proliferation. Results:Isolated CD4+CD25+ T Reg from both HIV-infected patients and healthy controls strongly expressed CD45RO, HLA-DR, and FoxP3. HIV-infected patients with detectable plasma HIV-1 RNA showed a statistically significant increase in CD4+CD25high T Reg frequencies (P < 0.05) compared to healthy controls, with T Reg frequency inversely proportional to CD4 T-cell count (P < 0.01). However, in HIV-infected patients with undetectable plasma HIV-1 RNA, CD4+CD25high T Reg frequencies were not increased and were not related to CD4 T-cell counts. In both HIV-infected patient groups, T Reg frequency was inversely related to Th1 frequency (detectable HIV-1 RNA: P < 0.05; undetectable: P < 0.001), but positively related to Th2 frequency (detectable HIV-1 RNA: P < 0.01; undetectable: P < 0.001). T Reg activity was lower in patients with detectable plasma HIV-1 RNA than in patients with undetectable plasma HIV-1 RNA. Conclusions:Increased T Reg frequencies in peripheral blood were related to low peripheral blood CD4 T-cell counts and polarization toward Th2 immune responses in HIV-infected patients.

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.

Diminution of contractile response of the aorta from endotoxin-injected rats

The contractility of a helical strip of the thoracic aorta was studied in rats injected intraperitoneally with endotoxin. The contractile response to any of the agonistic agents, KCl, norepinephrine or 5-hydroxytryptamine was time dependently diminished in the endotoxin-injected rats compared to the controls. This diminution preceded the depression of blood pressure. When the external calcium concentration was increased from 2.5 to 7.5 mM after the KCl (80 mM)-induced contractile response reached a plateau, the diminished contractile response was reversed in the endotoxin-injected group. The strips from the endotoxin-injected rats showed a higher 45CaCl2 uptake into the vascular tissue with the KCl-stimulated contraction. These findings suggest that the blood pressure depression during endotoxic shock may be attributed partially to the diminished contractility of the blood vessels and that this diminution is induced by a disorder of calcium utilization within vascular smooth muscle during vascular contraction.

Complications after bone marrow transplantation are manifestations of systemic inflammatory response syndrome

Bone marrow transplantation has been established as a useful treatment for various hematological disorders and is now performed widely, but the mortality rate is still high due to various complications. A clear therapeutic policy for such complications has not yet been established because of their complex nature. We investigated whether the major complications occurring after bone marrow transplantation could be classified as aspects of the systemic inflammatory response syndrome. Subjects were 10 patients who developed severe complications after bone marrow transplantation (graft-versus-host disease, thrombotic microangiopathy, respiratory disorders, and cytomegalovirus interstitial pneumonitis) and 16 patients without complications. Their symptoms, serum cytokines, and factors related to vascular endothelial damage were compared before and after transplantation. Whereas all 10 patients who developed complications had fever in the aplastic phase after transplantation, 15 of the 16 patients without complications remained afebrile (P < 0.001, t-test). When compared with the patients who did not develop complications, the patients with complications also showed significantly higher cytokine levels during the recovery phase after transplantation (P < 0.0001, t-test). Thus, the patients with complications developed fever in the aplastic phase and showed an increase of cytokines during the recovery phase, which triggered the occurrence of vascular endothelial damage shown by factors such as the thrombomodulin and plasminogen activator inhibitor type 1. This sequence of events corresponds with that occurring during systemic inflammatory response syndrome, so many of the complications of bone marrow transplantation can be considered as manifestations of this syndrome. Bone Marrow Transplantation (2000) 26, 419–426.

Clinical significance of vascular endothelial growth factor and hepatocyte growth factor in multiple myeloma

Summary. Angiogenesis is a crucial process in the progression of multiple myeloma (MM). Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are multifunctional cytokines that potently stimulate angiogenesis including tumour neovascularization. Serum levels of VEGF and HGF were measured in 52 patients with MM by enzyme‐linked immunosorbent assay (ELISA). Serum levels of VEGF and HGF were elevated in MM patients compared with healthy controls (VEGF: mean 0·31 ng/ml and 0·08 ng/ml respectively, P < 0·01; HGF: mean 2·17 ng/ml and 0·45 ng/ml, respectively, P < 0·001). In serial samples taken after chemotherapy, serum VEGF and HGF levels were correlated with M‐protein levels. Serum levels of VEGF were higher in patients with extramedullary plasmacytomas than in patients without them (P < 0·05). They were also significantly higher in a group of patients who showed poor response to chemotherapy (P < 0·01). Serum levels of HGF were higher in patients with complications such as anaemia, hypercalcaemia and amyloidosis than in patients without these complications (P < 0·01, P < 0·05, P < 0·05 respectively). Both serum VEGF and HGF levels were significant predictors of mortality (P = 0·01, P = 0·02, respectively, log‐rank test). The present study demonstrated that serum levels of VEGF and HGF are significantly elevated and dependent on the severity of MM, suggesting that measurement of VEGF and HGF may be useful for assessing disease progression and for predicting the response to chemotherapy in MM patients.

Modulation of vascular tonus by the endothelium in experimental diabetes

The role of the vascular endothelium in the contractile response of aortas from streptozotocin-induced diabetic rats was investigated using selected agents. Contractile response to KCl was not affected by removal of the endothelium in both diabetic and control groups, but was diminished in the diabetic rats compared to the control rats. Contractile response to clonidine markedly increased after removal of the endothelium in the control group, with the increment being less in the diabetic group. After removal of the endothelium, contractile response to clonidine was poorer in the diabetic group than the control group. Vascular relaxation induced by acetylcholine disappeared when the endothelium was removed in both diabetic and control groups. The degree of reaction to acetylcholine did not significantly differ between the two groups. These results suggest that in diabetic rats, abnormality of the endothelium-dependent vascular relaxation is specific for alpha 2 receptor while that of the vascular smooth muscle reactivity is not receptor-specific.

Elevated interleukin (IL)-18 levels during acute graft-versus-host disease after allogeneic bone marrow transplantation.

Acute graft‐versus‐host disease (aGVHD) after allogeneic bone marrow transplantation (BMT) is mediated by grafted T lymphocytes after their polarization into type 1 T cells. Interleukin (IL)‐18, a novel immunoregulatory cytokine, strongly stimulates type 1 T cells, therefore we postulated that IL‐18 may be involved in the pathogenesis of aGVHD. Using an enzyme‐linked immunosorbent assay (ELISA), we serially measured serum levels of IL‐18 in 37 patients with haematological malignancy before and after allogeneic BMT. Patients with aGVHD had high levels of IL‐18 that strongly correlated with the severity of aGVHD. We also found that they showed reduced serum levels of IL‐18 after appropriate treatment or at a state of resolution. IL‐18 levels were not affected by the pretransplant regimen, engraftment or bacterial infection. Compared with circulating interferon (IFN)‐γ or IL‐12 levels, serum levels of IL‐18 showed a more sensitive and specific correlation with the disease status of aGVHD. These findings suggest that IL‐18 may play important roles in the pathogenesis of aGVHD and that measurement of serum IL‐18 levels can be useful indicator of aGVHD.

Impairment of endothelium-dependent relaxation in human basilar artery after subarachnoid hemorrhage.

Background and Purpose The goal of this study was to determine the alterations in vascular reactivity of human basilar artery after subarachnoid hemorrhage. Methods Human basilar arteries were obtained from subjects who died within 1 day after subarachnoid hemorrhage and control subjects who died from causes other than brain involvement. Basilar artery strips were suspended for isometric tension recording in Krebs-Ringer solution. Morphometric study was also carried out on paraffin-embedded sections stained with van Giesons elastica stain of preselected sites from the basilar arteries. The intimal and medial area and the intimal index ([intimal area/area circumscribed by internal elastic lamina] × 100) were evaluated. Results Contractile responses to KCl, norepinephrine, and 5-hydroxytryptamine did not differ between subarachnoid hemorrhage and control groups. The endothelium-dependent relaxation responses to thrombin, bradykinin, and calcium ionophore A23187 were less for the subarachnoid hemorrhage group than for the control group. However, the endothelium-independent response to sodium nitroprusside of the subarachnoid hemorrhage group did not differ from that of the control group. Morphometric measurements were comparable between the two groups. Conclusions These results suggest that the decreased relaxation responses to thrombin and bradykinin occur at the level of endothelial cells and not smooth muscle cells and that decreased relaxation may be involved in delayed vasospasm after subarachnoid hemorrhage. Although the decreased relaxation was observed within 1 day after subarachnoid hemorrhage, a period in which delayed spasm does not occur, this time difference may be dependent on the severity of bleeding after rupture of an aneurysm.

Effect of aging on endothelium-dependent vascular relaxation of isolated human basilar artery to thrombin and bradykinin.

Using strips of human basilar arteries mounted in organ chambers to record isometric tension, we investigated vascular reactivity to thrombin and bradykinin. Both agents produced endothelium-dependent relaxation of basilar artery strips precontracted with phenylephrine but had no effect on resting tension in strips with or without endothelium. The relaxations caused by thrombin were abolished by antithrombin III/heparin, hirudin, and MD805. Thrombin but not bradykinin caused complete tachyphylaxis toward a second exposure. Indomethacin did not inhibit the relaxations induced by thrombin or bradykinin, whereas bromophenacyl bromide and methylene blue did. Aging decreased the relaxation induced by thrombin but did not affect the concentration needed to reach 50% maximal relaxation, nor did it affect the maximal relaxation in response to bradykinin, calcium ionophore A23187, and sodium nitroprusside. Our results suggest that thrombin and bradykinin produce endothelium-dependent relaxations mediated by an endothelium-derived relaxing substance and that the relaxation caused by thrombin is mediated by a proteolytic action on endothelial cells. The decrease in relaxations in response to thrombin with increasing age might be due to a decrease in the number or sensitivity of thrombin receptors on endothelial cells.

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 B4, thromboxane A2, and prostaglandin I2 were significantly lower in patients receiving EPA than in those not receiving it (all P < 0.01). Cytokines such as tumor necrosis factor-α, interferon-γ, 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. Bone Marrow Transplantation (2001) 28, 769–774.