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A small proportion of individuals infected with HIV-1 known as long-term nonprogressors (LTNPs) remain healthy and immunologically normal, with stable numbers of CD4+ lymphocytes, for prolonged periods without the administration of antiretroviral agents. The long terminal repeat (LTR) of HTV-1 proviral DNA of an LTNP from whom virus was consistently not recoverable has now been isolated by a nested polymerase chain reaction (PCR) method and shown to contain a total of 38 point mutations, only four of which affect promoter and enhancer elements, compared with the IIIB strain of HIV-1. Almost the entire HIV-1 proviral DNA was then isolated from the proband by a long PCR approach. Restriction enzyme digestion of the proviral DNA revealed no large deletions in the gag, pol, or env genes, although the loss of an Nco I site was apparent. Amplification of the env gene by long PCR also yielded a product apparently identical in size to that obtained with HIV-1 strain IIIB. Analysis by long PCR of HIV-1 proviral DNA from LTNPs with nonrecoverable virus may clarify the mechanism of long-term nonprogression and contribute to the development of HIV-1 vaccines.

To investigate whether iloprost, a stable analog of prostacyclin, is useful for the prevention of posttraumatic spinal cord injury, we examined its effects on compression trauma-induced spinal cord injury in rats. Spinal cord injury was induced by applying a 20-g weight for 20 minutes to the spinal cord at the level of T-12, resulting in motor disturbances in the hindlimbs. These motor disturbances, evaluated using Tarlov's index, were markedly attenuated in rats with nitrogen mustard-induced leukocytopenia. Administration of iloprost also attenuated the motor deficits. Histological examination revealed that intramedullary hemorrhages observed 24 hours after trauma were significantly attenuated in leukocytopenic animals and in animals that received iloprost. The accumulation of leukocytes at the site of trauma, evaluated by measuring tissue myeloperoxidase activity, significantly increased with time following the trauma, peaking at 3 hours postinjury. Spinal cord myeloperoxidase activity in sham-operated animals did not increase postoperatively. Leukocyte depletion and administration of iloprost reduced the accumulation of leukocytes in the damaged spinal cord segment 3 hours posttrauma. These findings indicate that iloprost attenuates motor disturbances induced by spinal cord trauma and that its therapeutic efficacy can be partly explained by its inhibition of leukocyte accumulation at the traumatized site.

We investigated the novel role of prostacyclin (PGI2) in gastric mucosal lesion formation induced by stress in rats. Gastric 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) levels were significantly increased 30 minutes after water-immersion restraint stress (WIR). Subcutaneous indomethacin (IM) (5 mg/kg) inhibited this increase but significantly exacerbated gastric mucosal lesion formation in rats subjected to WIR. Although gastric myeloperoxidase (MPO) activity was not increased by WIR, it significantly increased with time after WIR in animals pretreated with IM. NS-398, a selective inhibitor of cyclooxygenase-2, did not inhibit the WIR-induced increase in gastric 6-keto-PGF1alpha. Neither the gastric lesion index nor gastric MPO activity were affected in animals pretreated with NS-398 and subjected to WIR. WIR-induced mucosal lesion formation was significantly inhibited in animals given iloprost, a stable analog of PGI2, and in those with nitrogen mustard-induced leukocytopenia. Iloprost prevented the gastric leukocyte accumulation and exacerbation of gastric mucosal lesions induced by IM in animals subjected to WIR. These IM-induced events also were prevented in animals subjected to WIR with nitrogen mustard-induced leukocytopenia. These observations implicate leukocytes in the process leading to gastric mucosal lesions induced by WIR. The increase in WIR-induced gastric PGI2 synthesis, mainly mediated by cyclooxygenase-1, appears important in preventing lesion formation, not only by maintaining gastric mucosal blood flow but also by inhibiting leukocyte activation.

To clarify the aspects affected by the PEBP2beta/MYH11 fusion gene involved in the inv(16), we analysed immunophenotypes in myelomonoblastic leukaemias. We found high expressions of CD34 and c-KIT antigens in myelomonoblastic cells from all patients carrying this fusion gene, including two with M4 and one CML blastic phase, in contrast to those with M4 without the fusion gene. These findings indicate that immunophenotyping is useful for detecting a leukaemia with the fusion gene in myelomonoblastic leukaemias and that the PEBP2beta/MYH11 gene is involved in immature cells expressing CD34 and c-KIT antigens.

The effects of interleukin-11 (IL-11) which stimulates megakaryopoiesis and thrombopoiesis and/or stem cell factor (SCF) which stimulates multiple lineage cells in the peripheral blood by an increase in marrow cellularity on hematopoietic suppression in carboplatin (CBDCA)-treated rats were determined. CBDCA was administered to rats at a dose of 35 mg/kg as a single intraperitoneal bolus on day 0. IL-11 (20 micrograms/day) was given subcutaneously for 10 days from day 5 after CBDCA treatment. IL-11 ameliorated the suppression of hematopoiesis in rats treated with CBDCA. Especially, the production of the platelets was stimulated by IL-11 more markedly than that of cells of other lineages. However, the combination of SCF and IL-11 intensified the CBDCA-induced suppression of hematopoiesis. These findings may be helpful in the design of future therapies to prevent or treat thrombocytopenia induced by chemotherapy.

Objective: Gabexate mesilate is a synthetic protease inhibitor capable of inhibiting both coagulation and cytokine production by monocytes. To investigate whether gabexate mesilate is useful for the prevention of posttraumatic spinal cord injury, we examined its effect on compression trauma-induced spinal cord injury in rats., Design: Prospective, randomized, blinded, controlled study., Setting: Research laboratory at a university medical center., Subjects: Male Wistar rats weighing 300 to 350 g., Interventions: Spinal cord injury was induced by applying a 20-g weight extradurally to the spinal cord at the level of the 12th thoracic vertebra for 20 mins. Spinal cord injury was evaluated by assessing the motor function of the rats 24 hrs posttrauma. The accumulation of leukocytes and histologic changes in the injured spinal cord tissue also were examined. Rats received gabexate mesilate (10 or 20 mg/kg i.p.) 30 mins before or after the compressive trauma. The effects of heparin or an inactive derivative of activated factor X (a selective inhibitor of thrombin generation) on compressive trauma-induced spinal cord injury also were examined. Leukocytopenia was induced by the administration of nitrogen mustard., Measurements and Main Results: The motor disturbances observed following traumatic spinal cord compression, evaluated by Tarlov's score, and the accumulation of leukocytes in the injured tissue, evaluated by measuring tissue myeloperoxidase activity, were markedly reduced by leukocyte depletion induced by nitrogen mustard and by pre- or posttreatment of animals with gabexate mesilate. Neither heparin nor the inactive derivative of activated factor X prevented the motor disturbances and the accumulation of leukocytes. Histologic examination demonstrated that intramedullary hemorrhages observed 24 hrs after trauma at the 12th thoracic vertebra were significantly attenuated by nitrogen mustard-induced leukocytopenia and the administration of gabexate mesilate., Conclusions: The compression trauma-induced spinal cord injury demonstrated by this model was mainly mediated by leukocytes. Gabexate mesilate prevented spinal cord injury not by inhibiting coagulation, but by inhibiting the activation of leukocytes.

Adult T cell leukemia (ATL) is an aggressive neoplasm of mature helper T cell, which is etiologically linked with human T-lymphotropic virus type 1 (HTLV-I). We studied HTLV-I provirus in 61 cases of ATL with Southern blot analyses and long PCR. These methods detected defective virus in 34 cases (56%). Furthermore, it found two types of defective virus. The first type (type 1) defective virus had both LTRs, but lacked internal sequences, such as gag and pol. Type 1 defective virus was seen in 50% of all defective virus. The second form (type 2) of defective virus had only one LTR, and 5'-LTR was preferentially deleted. This type of defective virus could be more frequently detected in aggressive types of ATL (16/44 cases) than chronic type (1/17 cases). This defective virus might be associated with clinical subtype.

The plasma level of soluble E-selectin (sE) reflects the activation of endothelial cells induced by cytokines such as tumor necrosis factor-alpha and interleukin-1 in vitro. These cytokines are important in the development of coagulation abnormalities in patients with sepsis. We compared the plasma levels of sE in patients with infections suspected of having disseminated intravascular coagulation (DIC) (n = 33) and in patients with underlying disorders other than infections, including solid tumors (n = 28), obstetric disorders (n = 13), hematologic malignancies (n = 13), and liver disease (n = 9), to clarify the involvement of cytokines in the development of coagulation abnormalities in patients with sepsis. Plasma levels of sE in patients with infection were significantly higher than in patients with the other underlying disorders. The plasma level of sE was also significantly higher in patients with infection with DIC (114.6 +/- 77.9 ng/ml, n = 21) than in patients with infection without DIC (54.5 +/- 53.1 ng/ml, n = 12, P < 0.02). There was no significant difference in sE level between patients with the other underlying disorders with and without DIC. The plasma level of sE was significantly correlated with the serum level of FDP(E) in patients with infection. The plasma level of sE was significantly higher in patients with infection with organ failure compared to patients without organ failure. There was no significant difference between patients with the other underlying disorders with and without organ failure. Plasma levels of tumor necrosis factor-alpha and interleukin-6 were detected in only 12.1% and 20.0% of patients with infections, respectively. These observations strongly suggest that plasma levels of sE reflect the activation of endothelial cells induced by cytokines, which may lead to DIC and organ failure in the presence of sepsis. Furthermore, determination of plasma level of sE may be useful for detecting the endothelial activation induced by cytokines in the pathologic conditions of sepsis, even when plasma levels of cytokines cannot be detected.

Partial overlapping cDNA sequences likely to encode a novel human CC chemokine were identified from the GenBank Expressed Sequence Tag data base. Using these sequences, we isolated full-length cDNA encoding a protein of 96 amino acid residues with 20-28% identity to other CC chemokines. By Northern blot, this chemokine was mainly expressed in liver among various tissues and strongly induced in several human cell lines by phorbol myristate acetate. We thus designated this chemokine as LARC from Liver and Activation-Regulated Chemokine. We mapped the LARC gene close to the chromosomal marker D2S159 at chromosome 2q33-q37 by somatic cell and radiation hybrid mappings and isolated two yeast artificial chromosome clones containing the LARC gene from this region. To prepare LARC, we subcloned the cDNA into a baculovirus vector and expressed it in insect cells. The secreted protein started at Ala-27 and was significantly chemotactic for lymphocytes. At a concentration of 1 microg/ml, it also showed a weak chemotactic activity for granulocytes. Unlike other CC chemokines, however, LARC was not chemotactic for monocytic THP-1 cells or blood monocytes. LARC tagged with secreted alkaline phosphatase-(His)6 bound specifically to lymphocytes, the binding being competed only by LARC and not by other CC or CXC chemokines. Scatchard analysis revealed a single class of receptors for LARC on lymphocytes with a Kd of 0.4 nM and 2100 sites/cell. Collectively, LARC is a novel CC chemokine, which may represent a new group of CC chemokines localized on chromosome 2.

To examine whether activated leukocytes may impair the endothelial production of prostaglandin (PG) I2, an important cytoprotective agent in gastric mucosa, we investigated the effects of leukocyte depletion and ONO-5046, a specific inhibitor of granulocyte elastase, on the gastric level of this prostaglandin and gastric mucosal injury in rats subjected to water-immersion restraint stress (WIR). Gastric 6-keto-PGF1 alpha was increased after 30 min of WIR, followed by a decrease to below baseline after 6 h of stress. Gastric levels of 6-keto-PGF1 alpha in leukopenic animals or animals pretreated with ONO-5046 after 1 h of stress were significantly higher than those of controls, levels after 6 h of stress were not lower than those preceding stress. Leukocytopenia or ONO-5046 significantly inhibited WIR-induced gastric mucosa lesion formation. Iloprost, a stable derivative of PGI2, prevented stress-induced lesions. These results suggest that activated leukocytes may play an important role in stress-induced gastric mucosal lesion formation by inhibiting production of PGI2.

Adult respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC) are serious complications of sepsis. Thrombomodulin, an important endothelial anticoagulant, binds thrombin to generate activated protein C (APC). To determine whether thrombomodulin purified from human urine (urinary thrombomodulin, UTM) is useful for the treatment of DIC and ARDS in sepsis, we examined the effect of UTM on endotoxin (ET)-induced coagulation abnormalities and pulmonary vascular injury in rats. Intravenous administration of UTM prevented the ET-induced pulmonary accumulation of leukocytes and the increase in pulmonary vascular permeability, as well as ET-induced histological changes such as leukocyte infiltration and pulmonary interstitial edema. On the other hand, dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, did not prevent these effects of ET. UTM did not prevent ET-induced pulmonary accumulation of leukocytes and pulmonary vascular injury in rats pretreated with DEGR-Xa. Our findings suggest that UTM attenuates ET-induced coagulation abnormalities and pulmonary vascular injury. Furthermore, the latter effect may be dependent on the capacity of UTM to activate protein C.

Although Bence Jones protein (BJP) is generally accepted to be critically involved in the pathogenic process of kidney impairment in patients with myeloma, patients with BJP do not always have kidney dysfunction. As proteins often undergo glycosylation and alter their molecular nature, it is expected that the heterogeneity in kidney dysfunction can be explained at least partly by the differential affinity to the kidneys of BJP dependent on its glycosylation. Accordingly, we analyzed the structures of carbohydrates of urine BJP biochemically to correlate the structure with kidney function. BJP was obtained from 16 patients with myeloma, 2 patients with light chain amyloidosis, a patient with plasma cell leukemia, and a patient with Waldenstrom's macroglobulinemia. All BJP had five forms of oligosaccharides: three forms of biantennary oligosaccharides and two forms of triantennaries. The three biantennaries correspond to previously reported oligosaccharides on only lambda-type BJP, whereas the triantennaries are novel oligosaccharides found on BJP. Among the five oligosaccharides, the triantennary oligosaccharide Gal(beta)1-4GlcNAc(beta)1-2Man(alpha)1-6 [Gal(beta)1-GlcNA(beta)1-4(Gal(beta)1-4GlcNAc(beta) 1-2)Man(alpha)1-3]Man(beta)1-4GlcNAc(beta)1-4GlcNAc showed a significant negative correlation with the serum creatinine level (p = 0.015 by Spearman's correlation test, R = 0.744). Thus determination of BJP glycosylation may be useful for the evaluation of kidney impairment in patients with BJP.

Granulocyte elastase released from activated leukocytes plays an important role in leukocyte infiltration. Since activated leukocytes have been shown to be involved in the pathogenesis of gastric mucosal lesion formation induced by nonsteroidal antiinflammatory drugs, inhibition of granulocyte elastase release from activated leukocytes may be useful in the prevention of these lesions. Rebamipide, a novel antiulcer agent, inhibited granulocyte elastase release from activated neutrophils in vitro. Rebamipide and ONO-5046, a granulocyte elastase inhibitor, markedly inhibited gastric mucosal lesion formation in rats. Gastric myeloperoxidase activity was significantly increased 3 hr after indomethacin administration. This increase was significantly inhibited by rebamipide and ONO-5046. Cimetidine did not inhibit granulocyte elastase release from activated neutrophils. Although cimetidine markedly prevented the indomethacin-induced gastric mucosal lesion formation, it did not reduce the gastric myeloperoxidase activity. Therefore, unlike cimetidine, rebamipide may prevent indomethacin-induced gastric mucosal lesion formation by inhibiting neutrophil activation.

We investigated the effect of activated protein C (APC) on pulmonary vascular injury and the increase in tumor necrosis factor (TNF) levels in lipopolysaccharide (LPS)-treated rats to determine whether APC reduces LPS-induced endothelial damage by inhibiting cytokine production. Intravenously administered LPS (5 mg/kg) induced pulmonary vascular injury, as indicated by an increase in the lung wet-to-dry weight ratio. LPS-induced pulmonary vascular injury was prevented by APC but not by active site-blocked factor Xa [dansyl glutamyl-glycyl-arginyl chloromethyl detone-treated activated factor X (DEGR-Xa)], a selective inhibitor of thrombin generation, or inactivated APC [diisopropyl fluorophosphate-treated APC (DIP-APC)]. APC, but not DEGR-Xa or DIP-APC, significantly inhibited the LPS-induced increase in the plasma level of TNF. APC significantly inhibited the production of TNF by LPS-stimulated monocytes in a dose-dependent fashion in vitro, but DIP-APC did not. APC did not inhibit the functions of activated neutrophils in vitro. These findings suggest that APC prevented LPS-induced pulmonary vascular injury by inhibiting TNF production by monocytes and not via its anticoagulant activity. The serine protease activity of APC appears to be essential for inhibition of TNF production.

Nafamostat mesilate (NM) is a synthetic protease inhibitor that is capable of inhibiting the various coagulation factors such as factor VIIa and thrombin. To determine whether NM may also be useful in treating adult respiratory distress syndrome (ARDS) related in sepsis, we investigated the effect of NM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intraperitoneal administration of NM prevented the pulmonary vascular injury and coagulation abnormalities induced by LPS. DEGR-factor VIIa, a selective inhibitor of factor VIIa, prevented the coagulation abnormalities, but not the pulmonary vascular injury, induced by LPS. NM did not reduce LPS-induced increase in pulmonary accumulation of leukocytes. NM did not inhibit the increase in the plasma concentration of tumor necrosis factor-alpha (TNF-alpha) observed after administration of LPS. NM did not inhibit the function of activated neutrophils in vitro. Plasma values of total serum hemolytic complement (CH50) were markedly decreased after the administration of LPS. NM inhibited the LPS-induced decrease in plasma CH50 values. Findings suggest that NM may reduce the pulmonary vascular injury as well as the coagulation abnormalities induced by LPS. The former effect may be independent of the anticoagulant effect but dependent on the inhibitory effect of the activation of the complement system in rats administered LPS.

CD95 (Fas/Apo-1) mediates apoptosis in cells of various types. Expression of CD95 and its function were investigated in myeloma cells and most plasma cell lines have been found to be CD95-positive. Anti-CD95 antibody induced apoptosis in these cell lines in a manner dependent on intensity of CD95 expression. Bel-2 expression levels in these cell lines were not correlated with sensitivity to CD95-induced apoptosis. Myeloma cells from approximately 70% of cases analyzed expressed CD95, suggesting a high frequency of CD95 expression of myeloma cells. The incidence of CD95 expression in samples from extramedullary lesions were higher than those of bone marrow myeloma cells. Anti-CD95 antibody did not induce apoptosis in some freshly isolated myeloma cells despite the expression of CD95 induced apoptosis in myeloma cell lines in a manner dependent on intensity of CD95 expression. In fresh samples, intensity of CD95 expression was not correlated with apoptosis evoked by anti-CD95 antibody. Interestingly, isolated myeloma cells with an high-level expression of CD95 showed spontaneous apoptosis during in vitro incubation. This was observed in samples from cases with high serum levels of lactate dehydrogenase (LDH), suggesting that the LDH was derived from cells undergoing spontaneous apoptosis in vivo. Taken together, CD95 is expressed in most myeloma cell lines and in many freshly isolated myeloma cells. However, some of the latter escape from CD95-dependent apoptosis despite the expression of CD95. Moreover, some myeloma cells from cases with aggressive disease may undergo apoptosis in a CD95-independent manner. In this review, the significance of CD95 and apoptosis of myeloma cells are discussed.

Adult T cell leukemia/lymphoma (ATL) is derived from CD4+ T cells and has a poor prognosis because of its resistance to chemotherapy. To evaluate the effectiveness of gene therapy for ATL, the effect of ganciclovir on ATL cell lines transfected with the thymidine kinase gene of herpes simplex virus type 1 (HSV-TK) was analyzed. To transfer the HSV-TK gene to ATL cells, a human immunodeficiency virus (HIV) vector that has specific infectivity to CD4+ cells was used. HSV-TK was inserted into the long terminal repeats of HIV-1 and driven by the SL3 promoter HXBSL3TK. HXBSL3TK was co-transfected with HXBCAT as a reporter into MT2 or HUT102 cells by DEAE-dextran. The cells were incubated with ganciclovir, and chloramphenicol acetyltransferase (CAT) activity was analyzed. The CAT activity of the MT2 cells and HUT102 cells transfected with HXBSL3TK decreased dose-dependently with ganciclovir. HXBSL3TK was also co-transfected into COS cells with an HIV-1 packaging vector that has gag, pol, and env driven by a cytomegalovirus promoter. The supernatant was transferred to MT2 cells or Raji cells and incubated with ganciclovir. Ninety percent of the MT2 cells transduced by HXBSL3TK and incubated with ganciclovir were killed, but Raji cells were not killed. In addition, HXBTK that expresses the HSV-TK gene and Tat gene driven by the LTR of HIV-1 was constructed. HXBTK had a higher expression of the HSV-TK gene and higher sensitivity to ganciclovir than did HXBSL3TK.

Objective: To investigate the role of granulocyte elastase in ischemia/reperfusion injury of liver, the effect of ONO-5046, a granulocyte elastase inhibitor, was examined in ischemia/reperfusion-induced liver injury in rats., Design: Prospective, randomized, controlled study., Setting: Research laboratory at a university medical center., Subjects: Male Wistar rats, weighing 220 to 280 g., Interventions: Animals receiving continuous intravenous infusion of ONO-5046 (50 mg/kg/hr) were subjected to hepatic ischemia/reperfusion. Hepatic damage was evaluated by effects on bile formation capacity, plasma clearance of indocyanine green, and serum aminotransferase concentrations after ischemia/reperfusion., Measurements and Main Results: Hepatic dysfunction, observed after 60 mins of ischemia/reperfusion, led to a reduction in bile flow and to a decrease in the plasma clearance of indocyanine green. These indicators of hepatic dysfunction were prevented, to a large extent, by administration of ONO-5046. Serum concentrations of aminotransferases increased after hepatic ischemia/reperfusion, peaking at 12 hrs of reperfusion. Increases in serum concentrations of aminotransferases were significantly inhibited by ONO-5046., Conclusion: Granulocyte elastase derived from activated leukocytes may play a critical role in hepatic dysfunction and the subsequent hepatic injury induced by ischemia/reperfusion.

Adult T-cell leukemia (ATL), an aggressive neoplasm of mature helper T cells, is etiologically linked with human T lymphotropic virus type I (HTLV-1). After infection, HTLV-I randomly integrates its provirus into chromosomal DNA. Since ATL is the clonal proliferation of HTLV-I-infected T lymphocytes, molecular methods facilitate the detection of clonal integration of HTLV-I provirus in ATL cells. Using Southern blot analyses and long polymerase chain reaction (PCR) we examined HTLV-I provirus in 72 cases of ATL, of various clinical subtypes. Southern blot analyses revealed that ATL cells in 18 cases had only one long terminal repeat (LTR). Long PCR with LTR primers showed bands shorter than for the complete virus (7.7 kb) or no bands in ATL cells with defective virus. Thus, defective virus was evident in 40 of 72 cases (56%). Two types of defective virus were identified: the first type (type 1) defective virus retained both LTRs and lacked internal sequences, which were mainly the 5' region of provirus, such as gag and pol. Type 1 defective virus was found in 43% of all defective viruses. The second form (type 2) of defective virus had only one LTR, and 5'-LTR was preferentially deleted. This type of defective virus was more frequently detected in cases of acute and lymphoma-type ATL (21/54 cases) than in the chronic type (1/18 cases). The high frequency of this defective virus in the aggressive form of ATL suggests that it may be caused by the genetic instability of HTLV-I provirus, and cells with this defective virus are selected because they escape from immune surveillance systems.

A 53-year-old female infected with human T lymphotropic virus type-I (HTLV-I) suffered from chronic renal failure, aplastic anemia, myelopathy, uveitis, Sjögren's syndrome and Weber-Christian disease. Although HTLV-I antibody was negative in cerebrospinal fluid, she was diagnosed as HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) based on clinical and histological findings. Though to date there is no direct evidence, other complications have also been reported to be HTLV-I related diseases. This case provided the unique opportunity to observe various HTLV-I related diseases.

Acute respiratory distress syndrome (ARDS) is a serious complication of sepsis. Thrombomodulin, an important endothelial anticoagulant, binds thrombin to generate activated protein C (APC). We have previously demonstrated that APC prevents endotoxin (ET)-induced pulmonary vascular injury by inhibiting activated leukocytes. We therefore examined whether recombinant human soluble thrombomodulin (rhs-TM) prevents activated leukocyte-induced pulmonary vascular injury in rats receiving ET. Intravenous administration of rhs-TM prevented ET-induced pulmonary accumulation of leukocytes and increase in pulmonary vascular permeability, as well as ET-induced histological changes, such as leukocyte infiltration and pulmonary interstitial edema. Dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, did not prevent these effects of ET. rhs-TM did not prevent ET-induced pulmonary accumulation of leukocytes and pulmonary vascular injury in rats pretreated with DEGR-Xa. These results suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes and that this effect may be mediated primarily by APC generation.

We present a 15-year-old woman with acute myelomonocytic leukemia without marrow eosinophilia, M4 in the FAB classification. She was admitted to our hospital with nausea and headaches. Upon admission, the leukocyte count was 284,000/microliters with 95% leukemic cells. The bone marrow aspirate was hypercellular with 74.8% blasts and 0.2% eosinophils. Leukemic cells were positive for myeloperoxidase and esterase staining. Initially, the karyotype of the bone marrow cells on admission was considered to be normal. However, the PEBP2 beta/MYH11 fusion transcript was detected in the bone marrow mononuclear cells by using the reverse transcriptase-polymerase chain reaction (RT-PCR). Reevaluation of karyotypes showed a t(16;16) (p13;q22) in the bone marrow cells. After achieving complete remission, she was treated with low-dose etoposide. Chromosome analysis showed a normal karyotype and no amplified chimeric transcripts were observed. This case indicates that the molecular analysis of PEBP2 beta and MYH11 genes is a useful tool to detect inv (16) and t(16;16) which were often difficult to find, and that leukemic cells from some cases of M4 without marrow eosinophilia have these chromosome abnormalities.

In paroxysmal nocturnal hemoglobinuria (PNH), little is known about the molecular events leading to the clinical manifestations except for the hemolysis. To unfold the complex pathophysiology, it is necessary to elucidate the nature of the PNH clone. PNH exhibits an acquired stem cell disorder, a clonal expansion of affected cells, concomitant depression of normal hematopoiesis in bone marrow (BM), and, although infrequently, the development of leukemia. The PNH clone is thus expected to exhibit some neoplastic features. We report here that CD34+ hematopoietic progenitor cells of PNH-BM yielded blood cells of three lineages with PNH phenotype alone when transplanted into sublethally irradiated severe combined immunedeficient mice. The hematopoiesis persisted for more than 10 months and did not always need human cytokines. In contrast, the hematopoiesis by control grafts obtained from healthy volunteers required an intense cytokine treatment. This in vivo model defines the preferential hematopoiesis of pluripotent PNH progenitor cells, indicating the intrinsic growth abnormality of PNH clone.

We evaluated the effects of antithrombin III (AT III) on the pulmonary vascular injury induced by injecting rats with lipopolysaccharide (LPS) to investigate the possible usefulness of AT III as a treatment for acute respiratory distress syndrome. The intravenous administration of AT III prevented the pulmonary accumulation of leukocytes (as evaluated by myeloperoxidase activity) and the increase in pulmonary vascular permeability to 125I-bovine serum albumin induced by LPS. The increase in pulmonary vascular permeability induced by LPS administration was unaffected by various anticoagulants but was inhibited by the leukocytopenia induced by nitrogen mustard or by the administration of a granulocyte elastase inhibitor, ONO-5046. AT III given alone, but not heparin plus AT III or Trp49-modified AT III, which lacks affinity for heparin, significantly increased the plasma concentration of 6-keto-prostaglandin F1alpha, suggesting that the interaction of AT III with heparin-like substances at the endothelial cell surface promotes the release of prostacyclin from endothelial cells in vivo. Trp49-modified AT III failed to prevent the LPS-induced accumulation of leukocytes and vascular injury. The pulmonary accumulation of leukocytes and vascular injury induced by LPS were not prevented by administering AT III to rats that were pretreated with indomethacin. The continuous intravenous infusion of prostacyclin prevented the LPS-induced pulmonary accumulation of leukocytes and vascular injury. Findings suggest that AT III depends on its ability to promote the release of prostacyclin, a potent inhibitor of leukocyte activation, from endothelial cells to prevent pulmonary vascular injury induced by LPS.

Adult respiratory distress syndrome and disseminated intravascular coagulation are important pathologic conditions affecting the outcome of patients with sepsis. To elucidate the possible therapeutic efficacy of SM-12502, a novel platelet activating factor antagonist, on acute lung injury and disseminated intravascular coagulation in sepsis, we investigated the effect of SM-12502 on an endotoxin (ET)-induced septic model in rats. SM-12502 prevented ET-induced increases in pulmonary vascular permeability and ET-induced histologic changes, such as leukocyte infiltration and pulmonary interstitial edema, 6 h following the administration of ET (5 mg/kg). SM-12502 also inhibited the decrease in fibrinogen and the increase in fibrin and fibrinogen degradation products observed following ET administration. SM-12502 prevented increases in the serum concentration of tumor necrosis factor (TNF) 90 min following ET administration in vivo, and significantly inhibited the production of TNF-alpha by ET-stimulated monocytes in vitro. These findings suggest that SM-12502 attenuates the actions of endotoxin by the inhibition of TNF production.

Objective: In order to determine whether gabexate mesilate, a synthetic protease inhibitor with anticoagulant properties, is useful for the treatment of adult respiratory distress syndrome, we examined its effect on endotoxin-induced pulmonary vascular injury in rats., Design: Prospective, randomized, controlled study., Setting: Research laboratory at a university medical center., Subjects: Male Wistar rats (180 to 220 g.), Interventions: Animals received intravenous infusions of endotoxin (5 mg/kg iv) or saline (control). Pulmonary vascular injury was assessed 6 hrs after administration of endotoxin in terms of the increase in vascular permeability. Rats received gabexate mesilate (10 mg/kg ip), heparin, antithrombin III, an inactive derivative of activated factor X (a selective inhibitor of thrombin generation), or N-[2-[4-(2,2-dimethyl-propionyloxy) phenylsulfonylamino] benzoyl] aminoacetic acid (ONO-5046) (a potent granulocyte elastase inhibitor) 30 mins before endotoxin administration. Leukocytopenia was induced by administration of methotrexate. The effects of the gabexate mesilate on the function of activated neutrophils and the production of tumor necrosis factor -alpha (TNF-alpha) by endotoxin-stimulated monocytes were examined in vitro using neutrophils and monocytes prepared from healthy human volunteers., Measurements and Main Results: Pulmonary vascular permeability was determined by measuring the vascular leakage of intravenously administered 125I-labeled bovine serum albumin. Intravenous administration of endotoxin significantly increased pulmonary vascular permeability. Gabexate mesilate significantly inhibited pulmonary vascular injury observed 6 hrs after the administration of endotoxin. Pulmonary vascular injury was not attenuated by the administration of heparin, heparin plus antithrombin III, or the inactive derivative of activated factor X, but pulmonary vascular injury was significantly attenuated in animals with methotrexate-induced leukocytopenia and in those animals treated with N-[2-[4-(2,2-dimethyl-propionyloxy) phenylsulfonylamino] benzoyl] aminoacetic acid. Gabexate mesilate in concentrations of 10(-4) to 10(-3) M inhibited the release of granulocyte elastase and leukocyte aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine and the opsonized zymosan-activated production of superoxide radical by neutrophils in vitro. Gabexate mesilate significantly inhibited the endotoxin-induced increase in the serum concentration of TNF-alpha in vivo and, at a concentration of 10(-8) M, the production of TNF-alpha by endotoxin-stimulated monocytes in vitro., Conclusion: Our findings suggest that gabexate mesilate attenuated endotoxin-induced pulmonary vascular injury mainly by inhibiting TNF-alpha production by monocytes, which may play a central role in sepsis-related lung injury.

Summary: An intensive combination chemotherapy regimen supported by granulocyte colony-stimulating factor (G-CSF) was evaluated in adult T-cell leukemia/lymphoma (ATLL) patients in a multiinstitutional, cooperative study. Vincristine 1 mg/m2 i.v. day 1, Adriamycin 40 mg/m2 i.v. day 1, cyclophosphamide 400 mg/m2 i.v. day 1, prednisolone 40 mg/m2 i.v. days 1 to 3 and 8 to 10, etoposide 35 mg/m2 i.v. days 1 to 8, vindesine 2 mg/m2 i.v. day 8, ranimustine 50 mg/m2 i.v. day 8, mitoxantrone 7 mg/m2 i.v. day 8, and G-CSF 50 mg/m2 s.c. days 9 to 21 were given for 2 to 4 courses every 3 weeks to 83 patients with ATLL. Complete remission (CR) and partial remission (PR) were achieved in 35.8 and 38.3 percent, respectively, of 81 evaluable patients. The median survival of all patients was 8.5 months, with a predicted 3-year survival of 13.5 percent by the Kaplan-Meier method. The median duration of response was 7.6 months (range 0.2-42.7), and 13 patients were alive. Their median survival time was 29.1 months (range 19.2-44.7). In 67.6 percent of courses, white blood cell (WBC) nadirs were < 1.0 x 10(9)/L. Days required for the recovery of WBC from the nadir to > 1.0 x 10(9)/L were <5 days in 71.4 percent of the treatment courses. The G-CSF supported an intensified chemotherapy regimen for ATLL and yielded better response rate and longer survival compared to previous reports in Japan. Because duration of remission is still short, further studies of postremission therapy or other strategies are warranted.

To investigate whether the P-selectin-mediated leukocyte adhesion to the endothelial cells is involved in pulmonary vascular injury after hemorrhagic shock, we examined the effect of an anti-P-selectin monoclonal antibody (MAb PB1.3) on the pulmonary accumulation of leukocytes and the subsequent pulmonary vascular injury observed after hemorrhagic shock in rats. Two hours after hemorrhagic shock, pulmonary accumulation of leukocytes, as evaluated by measuring myeloperoxidase activity, began to increase and peaked after 6 hours. Pulmonary vascular injury, as evaluated by the extravascular leakage of 125I-albumin, was significantly increased 6 hours after hemorrhagic shock. MAb PB1.3 significantly prevented both the pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury. MAb PNB1.6, an anti-P-selectin monoclonal antibody incapable of inhibiting P-selectin-mediated leukocyte adhesion, did not prevent either of these effects. These observations strongly suggest that the pulmonary sequestration of leukocytes and the subsequent pulmonary vascular injury after hemorrhagic shock are mediated by P-selectin.

We measured plasma levels of soluble fibrin (SF) in 98 patients suspected of having disseminated intravascular coagulation (DIC) using a newly developed enzyme-linked immunosorbent assay (ELISA) and investigated the correlations between SF determinations and measurements of other hemostatic molecular markers to determine the diagnostic usefulness of determinations of SF. Patients were classified into four groups according to their clinical and laboratory findings: overt DIC (n =33), subclinical DIC (n =23) hypercoagulability (n =22), and non-DIC (n =20). SF levels were significantly higher in patients with overt DIC compared with the other three groups and were significantly higher in the subclinical DIC and hypercoagulability groups compared with the non-DIC patients. SF levels increased significantly with each increase in the clinical stage. Although levels of thrombin-antithrombin III complex (TAT), prothrombin fragment 1 + 2 (PF 1+2), cross-linked fibrin degradation products (XDP), and plasmin-antiplasmin complex (PAP) were significantly increased in patients with overt DIC compared with non-DIC patients, the values of these hemostatic molecular markers did not consistently show an increase in association with advances in the disease stage. Plasma levels of SF in patients with overt DIC showed a positive correlation with levels of TAT, XDP,and FDP(E), but not with PF1+2 and PAP. Analysis of receiver-operating characteristic curves showed that the sensitivity and specificity of SF were similar to those of XDP for diagnosis of DIC. The sensitivity and specificity of SF for diagnosis of overt DIC were both above 90% when the cut-off value was set at 65 mu g/ml.plasma levels of SF were also increased in patients with extravascular fibrin formation without DIC. Our findings suggest that measurement of plasma levels of SF by this ELISA method is useful for the diagnosis of DIC and the evaluation of the patient's clinical status.

The synthetic glucocorticoid, dexamethasone, and glucagon cooperatively elevated the level of mRNA for the transcription factor CCAAT/enhancer binding protein beta (C/EBP beta) in primary-cultured rat hepatocytes. In response to dexamethasone and/or glucagon, C/EBP beta mRNA started to increase as early as 30 min, reached a maximum within 2 h, and then gradually decreased. The administration of cycloheximide, a protein synthesis inhibitor, led rather to an increase in C/EBP beta mRNA, which suggested that a labile negative protein factor(s) is involved in regulation of the C/EBP beta mRNA level. Cycloheximide further augmented the increases in C/EBP beta mRNA by dexamethasone and/or glucagon. Therefore, C/EBP beta mRNA accumulation in response to these hormones is apparently independent of ongoing protein synthesis. The elevation of the C/EBP beta mRNA level by these hormones was accounted for by increases in the rate of transcription of the C/EBP beta gene, as deduced on nuclear run-on analysis. Gel mobility shift analysis revealed that the DNA-binding activity of C/EBP beta was increased cooperatively by dexamethasone and glucagon. These results suggest that the C/EBP beta gene is primarily induced by glucocorticoids and/or glucagon and that the accumulated C/EBP beta protein is then involved in secondary activation of target genes in response to these hormones in the liver.

To examine the relationship between cell cycle progression and differentiation in megakaryocytes, variances in the cell cycle during PMA (phorbol 12-myristate-13-acetate)-induced differentiation were analysed in synchronized growing CMK cells. The cells stimulated with PMA in early S phase showed cell cycle arrest in G2 phase. In addition, the cells stimulated with PMA in early G1 phase showed cell cycle arrest in late G1. The expression of gpIIb/IIIa, megakaryocytic differentiation marker, was markedly induced in G2 phase when the cells were stimulated in early S phase, and was also induced in late G1 phase when cells were stimulated in early G1 phase. Therefore, the induction of gpIIb/IIIa expression seems to be associated with cell cycle blockage in both G2 and late G1. Expression of the transcription factor GATA-1 in cells stimulated in early S phase was much higher than that in controls at G2 phase. Furthermore, GATA-1 expression in cells stimulated in early G1 tended to be higher than that in controls at late G1. These periods corresponded to the time that the cell cycle arrest and gpIIb/IIIa expression were induced by PMA. These results suggest that the cell cycle in human megakaryocytic lineage cells may have two regulation points for differentiation.

Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.

Adult T cell leukemia (ATL), a neoplasm of mature helper T lymphocytes is etiologically associated with human T lymphotropic virus type-I (HTLV-I). ATL cells infiltrate various organs, the lung, skin, central nervous system, gastrointestinal tract, and bone, causing various clinical manifestations. Two unusual cases of ATL, in which lytic bone lesion was the primary site of ATL, are described. One patient had multiple lytic lesions in bones without any involvement of other organs, and the other patient had a bone lesion in the right radius, which disappeared after chemotherapy. In both cases, monoclonal integration of HTLV-I provirus was demonstrated in the genomic DNA from each bone lesion. Although their clinical courses and pathological findings were different, ATL in both patients began as a bone lesion, showing that primary lymphoma of bone can be manifested in ATL cases.

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

Genetic manipulation of somatic cells may be of therapeutic value in a variety of infectious diseases, particularly in human immunodeficiency virus (HIV) infection. Stable insertion of "resistance genes" into cells, susceptible to HIV, could reduce the viral burden in infected individuals and potentially retard the characteristic progressive immune dysfunction. Alternatively, ectopic expression of genes that encode viral antigens, might induce potent antiviral immune responses and form the basis for novel prophylactic and therapeutic vaccines. While laboratory studies have proved that the approach works in principle, preclinical and clinical studies will be necessary to evaluate the therapeutic benefits of such gene-based therapies. Currently, more than 400 patients have already been treated by this innovative therapeutic strategy in the US. In Japan, the Expert Committee on Gene therapy was set up in the council on Science and the Public Health and Welfare in 1991. Recently, gene therapy for ADA has been approved. It is thought that the first gene therapy against HIV infection in Japan is not far away.

We examined the relationship between the amino acid sequences of the V2 and V3 regions of the envelope protein and the biological properties of ten human immunodeficiency virus type 1 (HIV-1) primary isolates. The infectivity, cytopathic effect (CPE), and syncytium forming activity of these primary isolates were tested against three T cell lines (CEM, MT2, and MOLT4/CL.8 cells), CD8-depleted peripheral blood mononuclear cells (PBMC), and primary monocyte-derived macrophages (MDM) from seronegative donors. In addition to the viral groups which had the syncytium inducing/T-cell line tropic (SI/TT) phenotype or non-syncytium inducing/non-T cell line tropic (NSI/NT) phenotype (including the NSI/macrophage tropic (NSI/MT) phenotype), there was a group of viruses that infected one or two T cell lines and PBMC but could not mediate syncytium formation. We therefore classified this group of viruses as a non-syncytium inducing/partial T-cell line tropic (NSI/pTT) virus. To investigate the relationship between these viral phenotypes and the sequence variability of the V2 and V3 regions of the envelope, we cloned the viral gene segment and sequenced the individual isolates. The sequence data suggested that the SI/TT type changes in the V3 sequence alone mediate a partial T cell line tropism and mild cytopathic effect and that an isolate became more virulent (SI/TT phenotype) if there were additional changes in the V2 or other regions. On the other hand, sequence changes in the V2 region alone could not mediate phenotypic changes but some additional changes in the other variable regions (for example, V3) might be required for the phenotypic changes in combination with changes in V2. These findings also suggested that amino acid changes in both the V2 and V3 region are required for the development of virulent variants of HIV-1 that outgrow during advanced stages of the disease.

Human T-lymphotropic virus type I (HTLV-I) was the first retrovirus which was directly associated with adult T-cell leukemia (ATL). Infection with HTLV-I can also lead to various other diseases, e.g. HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and HTLV-I uveitis, possibly via induction of immunodeficiency or hyperreactivity against HTLV-I-infected cells. Epidemiological data have shown that patients who developed these diseases represent a small percentage of HTLV-I-infected individuals living in restricted geographical areas. The identification of HTLV-I-infected individuals using serological and DNA-diagnostic methods is important because knowledge of HTLV-I seropositivity may help to prevent the transmission between sexual partners, as well as transmission from mother to child and blood transfusion. It also assists in establishing a diagnosis of ATL, HAM/ TSP and other HTLV-I-associated diseases.

A case of CD5-positive diffuse large cell lymphoma in a patient with autoimmune hemolytic anemia (AIHA) is reported. The patient was diagnosed with AIHA in December 1988. Three and a half years later, the patient complained of fever and left sided flank pain. Abnormal lymphocytes appeared in the peripheral blood and were positive for HLA-DR, CD5, CD19, CD20, and surface immunoglobulin (mu, lambda). The pathological diagnosis of the cervical lymphnode was non-Hodgkin lymphoma; diffuse large cell type with a starry sky-like appearance. Although the 8q24 translocation was not detected by karyotypic analysis of the peripheral blood mononuclear cells (PBMNC), Southern blot analysis revealed that the c-myc rearrangements had occurred. This case showed two rearranged bands with Eco RI, Bam HI, or Bgl II digestion, and a germline band with Hin dIII digestion using a second exon fragment of the c-myc gene as a probe. Despite intensive chemotherapy, this patient died 6 months after being diagnosed with malignant lymphoma. We discuss the c-myc rearrangements in this aggressive CD5-positive diffuse large B cell lymphoma.

Adult T-cell leukemia (ATL) was first reported in Japan, where it has a high incidence in the southwestern region. The retrovirus, human T-lymphotropic virus type I (HTLV-I), is found to be the causative agent of ATL. In ATL-endemic areas, the rate of HTLV-I carriers is high. A definite diagnosis of ATL is based on the presence of HTLV-I proviral DNA in the tumor cell DNA. ATL cells originate from the CD4 subset of peripheral T cells. ATL shows diverse clinical features but can be divided into four subtypes: the acute, chronic, smoldering, and lymphoma types. Chemotherapy is not effective; the acute and lymphoma types have a poor prognosis. Familial occurrence of ATL is common. HTLV-I infection is caused by transmission of live infected lymphocytes from mother to child, or from man to woman, or by blood transfusion.

Adult respiratory distress syndrome (ARDS) is a serious complication of disseminated intravascular coagulation (DIC) or multiple organ failure. To determine whether recombinant soluble human thrombomodulin (rsTM) may be useful in treating ARDS due to sepsis, we investigated the effect of rsTM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intravenous administration of rsTM prevented the increase in pulmonary vascular permeability induced by LPS. Neither heparin plus antithrombin III (AT III) nor dansyl Glu Gly Arg chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury. The agents rsTM, heparin plus AT III, and DEGR-Xa all significantly inhibited the LPS-induced intravascular coagulation. Recombinant soluble TM pretreated with a monoclonal antibody (moAb) that inhibits protein C activation by rsTM did not prevent the LPS-induced vascular injury; in contrast, rsTM pretreated with a moAb that does not affect thrombin binding or protein C activation by rsTM prevented vascular injury. Administration of activated protein C (APC) also prevented vascular injury. LPS-induced pulmonary vascular injury was significantly reduced in rats with leukopenia induced by nitrogen mustard and by ONO-5046, a potent inhibitor of granulocyte elastase. Results suggest that rsTM prevents LPS-induced pulmonary vascular injury via protein C activation and that the APC-induced prevention of vascular injury is independent of its anticoagulant activity, but dependent on its ability to inhibit leukocyte activation.

We evaluated the effect of antithrombin III (AT III) on the plasma level of 6-keto-PGF1 alpha in rats to determine whether AT III may promote the release of prostacyclin (PGl2) from endothelial cells in vivo. The intravenous administration of AT III (250 U/kg) significantly increased the plasma levels of 6-keto-PGF1 alpha, with a peak seen 90 min post-administration. Neither Trp49-modified AT III, which lacks affinity for heparin but retains an inhibitory capacity for thrombin, nor heparin plus AT III, increased the plasma level of 6-keto-PGF1 alpha 90 min after administration. Indomethacin pretreatment inhibited the increase in plasma levels of 6-keto-PGF1 alpha produced by AT III. Observations suggest that AT III may promote the release of PGl2 from endothelial cells by interacting with heparin-like glycosaminoglycans in vivo, consistent with previous observations in cultured endothelial cells.

L-selectin is an adhesion molecule of the selectin family that mediates the initial step of leukocyte adhesion to vascular endothelium. Upon cellular activation, expression of the L-selectin gene is downregulated at both the protein and mRNA levels. To understand the mechanism of leukemic cell infiltration into organs, we studied the expression and regulation of L-selectin mRNA in fresh leukemic cells of adult T-cell leukemia (ATL) patients and investigated the response of the L-selectin promoter to human T-cell lymphotropic virus type 1 (HTLV-1) Tax, which is a viral transcriptional transactivator. Flow cytometry showed that L-selectin was expressed on fresh ATL cells along with other activation antigens. Northern blot analysis showed that ATL cells overexpressed that L-selectin mRNA and that the level was aberrantly upregulated after PMA stimulation. Studies using in situ hybridization showed expression of the L-selectin mRNA in the infiltrating leukemic cells in the liver of two ATL patients. Intravenous injection of a rat T-cell line that overexpresses L-selectin showed increased organ infiltration. The induction of Tax expression in JPX9 cells resulted in about a twofold increase in the mRNA expression levels compared with the basal level. Chloramphenicol acetyltransferase (CAT) assay after transient cotransfection showed about a fivefold transactivation of the L-selectin promoter by Tax. The serum level of the shed form of L-selectin was significantly increased in ATL patients (mean +/- SD, 4,215.4 +/- 4,111 ng/mL) compared with those of asymptomatic carriers and healthy blood donors (mean +/- SD, 1,148.0 +/- 269.0 ng/mL and 991.9 +/- 224 ng/mL, respectively). These results indicated that ATL cells constitutively overexpress the L-selectin gene that can be transactivated by HTLV-1 Tax. The overexpression of L-selectin, as well as of inflammatory cytokines, by ATL cells may provide a basis for ATL cells to attach the vascular endothelium, leading to transmigration and organ infitration.

Recently, two genes, AML1 and ETO have been isolated from the chromosomal breakpoint of t(8;21). In this study, we isolated and identified fusion transcripts from a leukemic cell line carrying t(8;21). We demonstrated by PCR analysis that these transcripts are consistently expressed in fresh leukemic cells with t(8;21). On the other hand, the wild type of ETO is expressed in several hematopoietic cells from different lineage, while the expression of AML1 was present in all hematopoietic cells investigated. These widespread expression suggests these molecules play an essential role in hematopoiesis.

Adult T-cell leukemia (ATL) was first reported in Japan, where it has a high incidence in the southwestern region. The retrovirus, human T-lymphotropic virus type I (HTLV-I), is the causative agent of ATL. In ATL-endemic areas, the rate of HTLV-I carriers is high. A definite diagnosis of ATL is based on the presence of HTLV-I proviral DNA in the tumor cell DNA. ATL cells originate from the CD4 subset of peripheral T cells. ATL shows diverse clinical features but can be divided into four subtypes:acute, chronic, smoldering, and lymphoma type. Chemotherapy is not effective; the acute and lymphoma types have a poor prognosis. Familial occurrence of ATL is common. HTLV-I infection is caused by transmission of live infected lymphocytes from mother to child, from man to woman, or by blood transfusion. Infection with HTLV-I can lead to other diseases, including HTLV-I-associated myelopathy/tropical spastic paraparesis and HTLV-I uveitis.

The proto-oncogene, c-kit, encodes a transmembrane tyrosine kinase receptor (KIT) and plays an important role in haemopoiesis. We have identified a 95 kD soluble form of KIT (S-KIT) in culture supernatant of human megakaryoblastic cell line, CMK. To study the physiological significance of S-KIT, we have established a sensitive sandwich ELISA system. Serum samples from healthy individuals contained detectable amounts of S-KIT. Next, we determined a total of 220 samples from 134 patients with haemopoietic disorders. A considerable number of patients with acute myeloid leukaemia (AML), especially those with more immature phenotypes (M0, M1 or M2) had elevated levels of serum S-KIT. Those levels decreased to the normal range after effective chemotherapy. In chronic myeloid leukaemia, patients with myeloid blastic crisis showed markedly elevated levels of serum S-KIT. In contrast, S-KIT levels decreased in cases with either acute or chronic lymphoid leukaemia. There was a tendency for patients with severe aplastic anaemia to show decreased levels, but it was not significant. In myelodysplastic syndrome, S-KIT levels appeared to vary by subsets, with higher concentration in more advanced forms of the disease. Although the functional role of S-KIT is not yet elucidated, these results suggest that the serum S-KIT levels may reflect the pathological states of various haematological disorders.