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Protein kinase C (PKC) has been shown to be involved in the mitogenic response and in oncogenic cell transformation in many experimental models. We analyzed the expression of PKC in both highly purified leukemic T cells freshly isolated from adult T-cell leukemia (ATL) patients and control T lymphocytes obtained from healthy volunteers. PKC activity was decreased in the ATL cells as compared with the control T cells. Cytosolic PKC activity in the ATL cells was remarkably decreased, whereas particulate membrane PKC activity was similar to the control level. The percentage of PKC activity in the particulate fraction was 34% in the ATL cells and 19% in the control cells. Regarding the altered subcellular localization of PKC activity, phorbol ester-induced translocation of cytosolic PKC was inhibited in some ATL cases. Similarly to the decrease in PKC activity, there was a decrease in the expression of the major PKC isozymes II(beta) and III(alpha) in ATL cells. These results suggest impaired regulation of PKC expression in ATL as well as in many experimental cancers.

We investigated the effects of interleukin 11 (IL-11) on murine megakaryopoiesis in serum-free cultures, using nonadherent, nonphagocytic, and T-cell-depleted bone marrow cells. IL-11 alone had no influence on megakaryocyte (Meg) colony formation in serum-free methylcellulose cultures, but it significantly enhanced the growth of Meg and granulocyte-macrophage-Meg colonies supported by optimal and suboptimal concentrations of interleukin 3 (IL-3). IL-11 also increased the size of IL-3-dependent Meg colonies as well as increasing the size and DNA content of constituent Meg. In liquid cultures, IL-11 alone did not increase the number of Meg, but it enhanced their size and acetylcholinesterase (AchE) levels. The addition of IL-11 to cultures containing suboptimal concentrations of IL-3 resulted in a synergistic increase of Meg AchE. These results suggest that IL-11, similarly to interleukin 6, has an effect on Meg and acts synergistically with IL-3 to augment murine megakaryopoiesis in vitro.

Adult T-cell leukemia (ATL)-derived factor (ADF) is a multifunctional protein homologous to thioredoxin (TRX) with co-cytokine and thiol-dependent reducing activities. ADF/thioredoxin production is enhanced in T cells transformed by HTLV-I. We have examined the effect of HIV-1 infection on ADF/TRX expression using specific antibody against ADF/TRX. Lymph nodes from 5 AIDS and 1 AIDS-related complex (ARC) patients were examined. As a control, 8 HIV noninfected lymph nodes, including 3 cases with hyperplasia, were also examined. Immunohistopathological studies using normal HIV noninfected lymph nodes showed that ADF/TRX high-producer (ADFh) cells were macrophages and cells with dendritic morphology in the paracortical area. Abundant ADFh cells were observed in HIV noninfected hyperplastic lymph nodes. The number of ADFh cells was low in hyperplastic lymph nodes from an ARC patient. All of the lymph nodes of 5 AIDS cases were atrophic and the number of ADFh cells were extremely low. To verify these histochemical studies, we examined the effect of in vitro HIV infection on ADF/TRX expression in HTLV-I (+) T-cell lines. Western blot analysis showed that a reduction of ADF/TRX in HIV-1-infected SKT-1B and MT-2 cells, and the reduction inversely correlated with p24 antigen level. On the basis of the above in vivo and in vitro findings, we imply that the levels of ADF/TRX were down-regulated by HIV-1 infection and that the down-regulation may play a role for pathophysiology of HIV-infected individuals.

Seroepidemiologic, clinical, and virologic studies were performed to determine whether human T-cell lymphotropic virus type I was closely associated with uveitis in two hospitals. One hospital was in an endemic area of the virus (Miyakonojo, Miyazaki) and the other hospital was in a less endemic area (Kurume). In the endemic area, the seroprevalence of the virus in patients with uveitis without defined causes (35.4%, 62 of 175 patients) was significantly higher than that in patients with nonuveitic ocular diseases (16.1%, 42 of 261 patients), or in patients with uveitis with defined causes (10.3%, eight of 78 patients). The seroprevalence in younger patients (20 to 49 years of age) with uveitis without defined causes in the area was 44.8% (30 of 67 patients), whereas it was only 9.3% (ten of 107 patients) in the other two groups. A similar observation was recorded even in the less endemic area (Kurume). Because the seroprevalence of the virus in the general population is known to be low in younger patients and to increase with age, these findings were interpreted to indicate that the association of human T-cell lymphotropic virus type I with uveitis was significant. Most patients, particularly those aged 20 through 49 years, had an intermediate uveitis characterized by a moderate inflammation in the vitreous body accompanied by an iritis and retinal vasculitis. The ocular symptoms in the patients differed from those of other types of uveitis common in Japan (Behçet's disease, Vogt-Koyanagi-Harada's disease, and toxoplasmosis, for example).(ABSTRACT TRUNCATED AT 250 WORDS)

We report a case of chronic myeloid leukemia (CML) in myelomonocytic transformation associated with bone marrow (BM) eosinophilia. At diagnosis, all BM cells showed a Ph chromosome. At the time of blastic phase, more than 50% of Ph+ cells had a pericentric inversion of chromosome 16, inv(16)(p13q22). This case confirms that blastic transformation of CML can involve any committed progenitor, and myelomonocytic leukemia with BM eosinophilia is specifically associated with rearrangement of chromosome 16 at band p13 and q22.

Although the possible occurrence of systemic fibrinogenolysis has been suggested in patients with metastasising prostatic cancer (MPC), direct evidence is lacking. We report on a patient with MPC whose laboratory data were consistent with hyperfibrinolysis: marked decrease of alpha 2-antiplasmin (AP) level (less than 50% of normal), increase of plasmin-alpha 2-antiplasmin complex, D-fragment of fibrin and fibrinogen degradation products [FDP(D)] and cross-linked fibrin degradation products (XDP). The patient neither showed laboratory nor clinical evidence for consumption coagulopathy except for a slight increase in thrombin-antithrombin III complex level. Immunoblotting of the patient's serum using an anti-fibrinogen antibody revealed the presence of a 250 kDa protein in addition to DD fragments. Following reduction of this protein by 2-mercaptoethanol after extraction from SDS-PAGE gel, gamma-chain of fibrinogen (47 kDa) was found by immunoblotting using a monoclonal antibody recognising a 86-302 residue of the gamma-remnant of fibrinogen. Moreover, the 250 kDa protein did not bind to Sepharose 4B to which a monoclonal antibody recognising the N-terminus of fragment D was conjugated. These findings indicated that this protein was not fragment DY, but rather fibrinogen fragment X. With the retraction of the prostatic tumour by an effective therapy, the patient's AP level increased gradually. When the plasma AP level rose to 60% of normal, the fragment X was no longer detectable. These findings suggested that systemic fibrinogenolysis occurred in the patient with MPC only when AP levels were markedly decreased.

Expression of multidrug resistance (mdr 1) gene, which encodes a transmembrane efflux pump referred to P-glycoprotein, leads to the decreased intracellular accumulation of various lipophilic drugs, such as vinca alkaloids, anthracyclines and epipodophyllotoxins. As these drugs are commonly used in chemotherapy for acute leukemia, it is of importance to determine whether mdr 1/P-glycoprotein expression is associated with clinical resistance. In several reports, some leukemia cells from untreated patients have expression of mdr 1/P-glycoprotein. We quantitatively detected low levels of mdr 1 expression in all cases of untreated acute leukemia and normal hematopoietic cells, using the reverse transcriptase-polymerase chain reaction. Carefully designed clinical trials including mdr 1 reversing agents may have significant consequences for the treatment of acute leukemia.

A chimeric mouse-human antibody (C beta 1) was constructed that recognized the principal neutralizing domain (PND) of human immunodeficiency virus type 1 (HIV-1) gp120. The constant (C) immunoglobulin regions (C gamma 1 and C kappa) of a mouse monoclonal antibody, 0.5 beta, were substituted for the human C gamma 1 and C kappa by recombining the DNA modules encoding variable or C regions. The DNA constructs were then transfected into X63 Ag8.653 myeloma cells. A clone with a high production of the chimeric antibody (C beta 1) was selected. This antibody was tested for its biological activity against HIV-1. It bound to the surface of HTLV-IIIB-infected cells and reacted with gp120/160 with equal affinity and specificity to that of the parental 0.5 beta murine monoclonal antibody in a Western blot assay. Neutralization and/or enhancement of HIV infection were evaluated with C beta 1 and 0.5 beta. Both C beta 1 and 0.5 beta neutralized cell-to-cell infection and cell-free virus infection by HTLV-IIIB. Antibody-dependence enhancement of HIV infection was not observed with either C beta 1 or 0.5 beta in the presence or absence of human complement. Antibody-dependent cell-mediated cytolysis (ADCC) and antibody-dependent complement-mediated cytolysis (ACC) were observed with C beta 1 but not with the parental 0.5 beta. These findings suggest that the neutralizing antibodies to PND may neutralize but not enhance HIV infection. Furthermore, the high levels of ACC and ADCC shown against HIV-infected cells by C beta 1 indicate that the clinical application of such monoclonal antibodies may be possible.

We investigated whether protein tyrosine phosphorylation was induced by erythropoietin (Epo) in two murine Epo-responsive cell lines (ELM-I-1, which proliferates autonomously and is induced to differentiate by Epo, and DA-1ER, which grows in a manner dependent on Epo or interleukin-3 (IL-3) without differentiation). In ELM-I-1, Epo induced the tyrosine phosphorylation of a protein of about 80-85 kDa (py80) which appeared in the Triton-X soluble fraction of the cell lysate in a time- and concentration-dependent manner. Maximal levels of phosphorylation were obtained within 5-10 min at Epo concentrations above 0.1 U/ml. IL-3 is known to promote the proliferation of this cell line, but it did not induce py80 phosphorylation. In DA-1ER, tyrosine phosphorylation of py80 was not induced by either Epo or IL-3. These findings suggest that there are multiple pathways of Epo signaling and that one of them could be via tyrosine kinase activation. Furthermore, it is possible that the tyrosine phosphorylation of py80 is involved in the pathway leading only to erythroid differentiation but not to cellular proliferation.

The antigenic profile of basal cell carcinoma (BCC) cells was analyzed by immunoperoxidase staining of 27 surgically removed BCC lesions with antiganglioside, antiadhesion molecule, and antihistocompatibility locus antigen (HLA) monoclonal antibodies (MoAb). The large majority of BCC lesions were stained by antiganglioside MoAb; among the latter the anti-GD3 ganglioside MoAb R24 displayed the broadest reactivity. The GD3 ganglioside expression by BCC cells, which was corroborated by thin layer chromatography immunostaining with MoAb R24, appears to be a proliferation dependent phenomenon. Among the adhesion molecules tested vitronectin receptor and CDw44 were found in up to 70% of the lesions tested, while intercellular adhesion molecule 1 (ICAM-1) was detected in only a low percentage of BCC cells in one lesion. ICAM-1 was not induced on BCC cells in five and three lesions removed 24 and 48 h, respectively, following the intralesional injection of gamma-interferon. The latter enhanced HLA Class I antigen expression and induced ICAM-1 expression by the surrounding keratinocytes; furthermore gamma-interferon induced HLA Class II antigen expression by a small percentage of BCC cells in three lesions. These results suggest that malignant transformation of keratinocytes is associated with a selective loss of susceptibility to induction by cytokines of ICAM-1 expression. Besides confirming the low HLA Class I and Class II antigen expression by BCC cells, the present investigation has shown a differential expression of distinct monomorphic determinants of HLA Class I antigens and a lower expression of HLA-A antigens than of HLA-B antigens by BCC cells. Furthermore, the present study has shown that HLA Class II antigens can be induced on BCC cells by cytokines.

A stable cell line, KHM-3S, was established from a patient with small cell lung cancer (SCLC), who had a high serum level of soluble interleukin 2 receptors (sIL2-R) and was seropositive for human T cell leukemia virus (HTLV)-1. KHM-3S cells were positive for IL2-R (Tac) and NKH-1, but negative for other lymphocytic markers such as OKT 11, OKT 4, OKT 8, T cell receptor (WT 31), B 1, and B 4. Moreover, the KHM-3S cells were negative for leukocyte common antigen and strongly positive for neuron-specific enolase (NSE). Secretion of sIL2-R and NSE by the KHM-3S line was detected by an enzyme-linked immunosorbent assay. Rearrangement of the T cell receptor gene and monoclonal HTLV-1 integration were found by Southern blot analysis of KHM-3S DNA. However, Northern blot analysis showed no T cell receptor mRNA. KHM-3S may be useful for studies on the role of HTLV-1 in carcinogenesis and IL2-R expression in SCLC.

The annual age- and sex-specific human T-cell leukemia virus type I carrier rate of blood donors in Kumamoto, Kyushu, Japan from 1986 to 1990 revealed that the carrier rates of all the age groups below 50 years declined linearly in both sexes (P less than 0.005). Furthermore, the annual declining rates relative to the carrier rates of 16-19-year-old and 20-29-year-old males were higher than those of all of the older males (P less than 0.02), and all female age groups below 50 years had higher relative declining rates than 50-64-year-old females (P less than 0.05). Although several factors, such as a notification program at obstetric clinics, methodological and technical improvement of the assays, wider knowledge of human T-cell leukemia virus type I infection in the latter years, and immigration of individuals from a nonendemic area, might cause an absolute decline of the carrier rate of the blood donors, these factors could not explain the acceleration of the relative declining rate among younger donors. Therefore, this acceleration represents the tendency of the general population.

Decay-accelerating factor (DAF) is a complement regulatory membrane protein that is often absent from the cell surface of blood cells in paroxysmal nocturnal hemoglobinuria (PNH). DAF has also recently been found in the body fluids of healthy individuals. However, its precise structure and biological significance are not yet clear. To clarify the clinical and pathological implications of free DAF, we measured plasma DAF levels in PNH patients, using a newly developed quantitative enzyme-linked immunosorbent assay (ELISA), with a measurable range between 0.2-12 ng, for soluble DAF. ELISA assays revealed significantly increased plasma DAF levels in PNH patients (258 +/- 150 ng/ml, mean +/- S.D., n = 9) as compared with healthy controls (80 +/- 41 ng/ml, n = 17) (p less than 0.01). Taken together with the finding that DAF is synthesized in and released extracellularly from affected PNH cells, plasma DAF levels would be useful for clinical diagnosis and for the quantitative evaluation of the clonal expansion of affected cells in PNH.

A patient with Münchhausen syndrome who had severe iron deficiency anemia (IDA) is reported. A 31 year-old female presented with irregular genital bleeding. However, gynecological examination disclosed no evidence of specific disorders causing bleeding. Tests for bleeding tendency and hemolysis were all negative. Although massive bleeding was absent, she had three episodes of a rapid fall in Hb level associated with marked fatigue and weakness, and subsequently rapidly developed serious IDA afterwards. Factitious bleeding was strongly suspected. Blood transfusion performed when the patient was not being watched failed to increase Hb, while transfusions given while the patient was watched were effective. She was not cooperative throughout the hospital course and was discharged on her own request. In outpatient clinic, the diagnosis was confirmed by the evidence that she diluted her own blood samples.

LD78 is a small secreted protein that has a sequence similar to a number of other polypeptides, including murine macrophage inflammatory protein 1 alpha (MIP-1 alpha), interleukin 8 (IL-8), Act-2, monocyte chemoattractant protein 1 (MCP-1), and others. These polypeptides are members of a novel cytokine superfamily that is involved in the inflammatory response, wound healing, hematopoiesis, and tumorigenesis. Specific receptors for purified clonal LD78 protein were measured using four cell lines (HL-60, U937, Jurkat, and MJ). 125I-labeled recombinant LD78 bound most efficiently to U937 cells. We therefore characterized the receptors as being on the surface of U937 cells. Binding reached an equilibrium after incubation for 60 min at 4 degrees C. Scatchard analysis showed that there were two classes of binding sites on U937 cells, high affinity sites (Kd = 5.3 x 10(-9) M) and low affinity sites (Kd = 9.3 x 10(-8) M), with the average number of binding sites per cell being approximately 30,000 and approximately 90,000, respectively. These receptors for LD78 were distinct from the receptors for gamma-IFN and for IL-8. SDS-PAGE analysis of chemically crosslinked 125I-labeled LD78 receptor complexes identified a single band of 52 kDa. The ability to detect specific LD78 receptors should prove valuable in efforts to molecularly clone these receptors and to dissect the biological actions of LD78.

In our previous study, we found that serum beta 2-microglobulin (beta 2M) levels were elevated in the active, but not in the inactive, phase of adult T-cell leukemia (ATL), suggesting a correlation between the beta 2M level and the clinical severity of this disease. In this study we examined the mechanisms underlying the elevation of serum beta 2M levels in ATL. First, the production of beta 2M by ATL cells was investigated in vitro. High levels of beta 2M were detected in the conditioned culture medium (CM) of ATL cells from seven out of nine patients. Second, we assessed the effects of the CM on the release of beta 2M by three human cell lines unrelated to ATL (NCTC 2544, Chang liver, and L 132; originating from the skin, the liver, and the fetal lung, respectively). Most of the CM definitely promoted beta 2M production by these cell lines. beta 2M production by the cell lines was markedly promoted by exogenous interferon-gamma (IFN-gamma), a well-known potent inducer of class I HLA antigen expression. We then investigated whether an antibody directed against IFN-gamma could attenuate the activity of three ATL CM. The anti-IFN-gamma antibody reduced the stimulatory activity of the CM to 28-65% of the original level, but did not affect basal beta 2M production by these cell lines. These data suggest that there are at least two mechanisms causing the elevation of serum beta 2M levels in ATL; direct production by tumor cells, and production by non-malignant cells that is mediated via humoral factors secreted by the ATL cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A cell line of plasma cells with high ammonia (NH3) production (KHM-4) was established from a patient with multiple myeloma complicated by hyperammonemia and abnormal serum concentrations of amino acids. Surface marker studies of KHM-4 cells showed that the cells were positive for cytoplasmic immunoglobulins (IgA kappa), HLA-DR, and T 10. Secretion of ammonia by the KHM-4 cells was detected by the addition of L-glutamine and L-arginine into the culture medium of amino acid-free RPMI 1640. In the presence of L-glutamine, KHM-4 cells secreted a greater amount of ammonia than the T cell line, CEM. However, production of ammonia by L-arginine was not observed in other cell lines. These observations provide evidence for the existence of a peculiar amino acid metabolism in the myeloma cells causing hyperammonemia and serum amino acid disturbance.

Seroepidemiological, clinical and virological studies were carried out in an HTLV-I endemic area to find out if HTLV-I caused an intraocular inflammatory disorder, uveitis. The seroprevalence in patients with uveitis without defined etiologies (62/175, 35.4%) was significantly higher than that in patients with non-uveitic ocular diseases (42/261, 16.1%) or in patients with uveitis with defined etiologies (8/78, 10.3%). Moreover, the seroprevalence in young adults (20-49 years) with uveitis without defined etiologies was 30/67 (44.8%), whereas it was only 10/107 (9.3%) in the other two groups. The uveitis in HTLV-I carriers was characterized clinically by a moderate inflammation of the vitreous body accompanied by a mild iritis and retinal vasculitis. The proviral DNA of HTLV-I was detected by polymerase chain reaction from the inflammatory cells in the anterior chamber in 9 out of 9 seropositive patients with the uveitis, but not in any of the tested patients with other types of uveitis. These data, thus, indicate that HTLV-I causes a specific type of intraocular inflammation, uveitis.

A neutrophil chemotactic factor (human interleukin 8, human granulocyte-macrophage colony-stimulating factor)-producing cell line, named KHM-5M, was established from a patient with an undifferentiated thyroid carcinoma, neutrophilia, and malignant pleurisy with many neutrophils and a few malignant cells. The cell line was transplanted into nude rats, and the infiltration of neutrophils was observed in and around the transplanted tumor tissue. Neutrophil chemotactic activity was predicted from the clinical features and pathological findings in this case. The extreme chemotactic activity of the neutrophils was demonstrated in conditioned medium from KHM-5M cells using the modified Boyden chamber technique. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least two neutrophil chemotactic activities in conditioned medium from the cell line were observed. The levels of these activities derived from KHM-5M cells were screened by measuring conditioned medium from the COS cells, which expressed a complementary DNA library from the KHM-5M cells. Chemotactic activities (human interleukin 8, human granulocyte-macrophage colony-stimulating factor) were identified by DNA cloning. These results show that the KHM-5M cells derived from an undifferentiated thyroid carcinoma produce multicytokines and suggest that those cytokines modified some pathological features in this case.

The p53 tumor suppressor gene was examined by direct sequencing of polymerase chain reaction-amplified DNA from fresh tumor cells of 10 patients with adult T-cell leukemia (ATL). Samples included nine patients with acute or lymphomatous ATL, and one patient in whom samples were examined in both his acute and chronic stages of ATL. Four missense mutations and one silent point mutation in the coding region of the p53 gene were found in cells from five patients with either acute or lymphomatous ATL. The missense mutations were homozygous and occurred in evolutionarily highly conserved regions of p53. One patient had no p53 mutation in his leukemic cells during chronic phase of ATL, but had a homozygous point mutation at codon 273 (Arg to His) when he progressed to acute ATL. In summary, we show that p53 is frequently mutated in the acute phase of ATL and one informative case suggests that p53 mutations may be associated with the transition from chronic to acute ATL.

In vitro immunomodulatory properties of gangliosides have been well characterized such as the ganglioside-induced modulation of CD4 on T lymphocytes and inhibition of lectin-induced proliferative response of lymphocytes. These findings have led to an interesting suggestion that gangliosides play a role as in vivo immune modulators, although this possibility is not clearly defined yet. We then first confirmed in vitro effects of gangliosides on murine immunocytes and examined in vivo effects of gangliosides on immune response in mice. Murine spleen cells that were treated with a ganglioside mixture (GS) purified from bovine brain exhibited a marked decrease in CD4 expression, while CD8 expression was slightly suppressed. Transplantation of GS-untreated control immunocytes that were isolated from syngeneic mice into the immune suppressed mice by X-ray irradiation restored in vivo immune responses, while GS-treated cells could not. Immune response was assayed by the evaluation of footpad swelling which was induced by immunization with sheep erythrocytes as antigens. Moreover, intramuscular administration of gangliosides into mice suppressed both immediate (Arthus)-type and delayed-type allergic reactions. These results suggest that gangliosides would be potential in vivo immune modulators.

Recombinant human erythropoietin (rHuEpo) was tested for its ability to stimulate rat megakaryopoiesis in vivo. Groups of Sprague-Dawley rats were injected with rHuEpo at a daily dose of 20, 80, or 200 U for 5 days. Significant thrombocytosis (a 30 to 40% increase over the control level) was found only in the rats that received 200 U/day, but some changes in the megakaryopoietic parameters were observed not only in the rats given 200 U/day, but also in those receiving 80 or 20 U/day. rHuEpo induced a dose-dependent elevation of megakaryocyte ploidy, with the maximum 45% increase in the mean ploidy over the control level seen in rats given 200 U/day. The size of the marrow megakaryocytes also increased dose-dependently. rHuEpo did not increase bone marrow megakaryocyte numbers, but it increased those in the spleen in a dose-dependent manner. A change of these parameters was seen as early as day 1 at 24 h after initiating the Epo injections at a time when significant thrombocytosis was already present. Moreover, a significant increase in the ratio of small acetylcholinesterase-positive bone marrow cells was also found, with the greatest response noted on day 1. Administration of a large dose of iron did not alter the thrombopoietic effect of rHuEpo. These results suggest that the in vivo administration of rHuEpo stimulates the maturation of mature as well as immature megakaryocytes already present in the bone marrow.

Using sheep erythrocytes and liposomes, an inhibitory effect of gangliosides has been shown on the activation of the alternative pathway of complement. However, in studies using human erythrocytes, we found that gangliosides had hemolytic activity that was possibly mediated through activation of the alternative pathway. Pretreatment of human erythrocytes obtained from healthy volunteers or paroxysmal nocturnal hemoglobinuria (PNH) patients with a ganglioside mixture purified from human erythrocytes enhanced their susceptibility to homologous human complement, and resulted in dose-dependent hemolysis. The enhancement was more marked in PNH erythrocytes than control cells. Protease treatment of the ganglioside mixture did not change its hemolytic activity, but sialidase treatment abolished the activity. Among the major erythrocyte gangliosides, II3NeuAc-LacCer (GM3) was the most potent hemolytic agent. Gangliosides purified from bovine brain were also active, while neither nonsialylated glycosphingolipids, the ceramide moiety, or sialic acid alone were active. Sialic acid residues in the ganglioside molecules were essential to this activity, but the amount of the residue or the source of the gangliosides seemed not to be important. Several treatments inhibiting the alternative but not classical complement pathway markedly reduced the ganglioside hemolytic activity. This novel bioactivity of gangliosides was thus suggested to be mediated partly by activation of the alternative pathway.

Dr Kira et al. (Lancet 1991, 338: 64-65) stress the high incidence of the co-infection with HTLV-I and HTLV-II in the HTLV-I associated myelopathy or tropical spastic paraparesis (HAM/TSP) detected by a polymerase chain reaction (PCR). They showed that 67% of HAM/TSP patients and 6% of healthy carriers had the co-infection. They suggested the co-infection might be important for the development of the myelopathy. Recently, we have analyzed the seropositivity of antibodies to HTLV-I and II in Adult T-cell Leukemia (ATL), HAM/TSP and carriers in Japan using SELECT-HTLV (IAF Biochem International Inc., Montreal, Canada). This system was a solid phase enzyme immunoassay utilizing synthetic peptides to differentiate between antibodies to type I and II. We examined 101 samples (31 ATL patients, 20 HAM/TSP and 50 carriers) which were all positive with the particle agglutination (PA, Fujirebio, Tokyo) and ELISA (Eisai, Tokyo). All samples were negative with HTLV-II and positive with HTLV-I. The results of our serological survey suggested that HTLV-II did not associate with the etiology of ATL and HAM/TSP in Japan.

Cell surface expression of the T-cell receptor (TCR)/CD3 complex on the cells from 11 acute type adult T-cell leukemia (ATL) and 4 lymphoma type ATL patients was examined by flow cytometry. Cells from 10 of 11 acute ATL patients were TCR alpha beta+ and CD3+, and their mean fluorescence intensities were low (TCR alpha beta, 25.3-84.6; CD3, 22.8-87.8). Cells from two of four lymphoma type ATL did not express this complex, and the other two were CD3+, TCR alpha beta-. In contrast, the mean fluorescence intensity of the TCR/CD3 complex in cells from a patient with T4 chronic lymphocytic leukemia was not low (TCR alpha beta, 129.9; CD3, 117.1). mRNA expressions of the TCR alpha, beta, and CD3 gamma, delta, epsilon, and zeta chains were examined by Northern blots. ATL cells from two acute and two lymphoma types expressed amounts of this complex equal to or greater than those expressed by T4 chronic lymphocytic leukemia. CD3 delta and TCR beta mRNA in ATL and T4 chronic lymphocytic leukemia cells were equally stable to actinomycin D treatment. The synthesis of CD3 zeta protein by ATL cells was detected by Western blotting assay. On the basis of these findings, we discuss the possible involvement of the TCR/CD3 complex in activation of ATL cells.

A principal neutralizing domain (PND) of the major envelope glycoprotein (gp120) of the HTLV-III BH10 strain of human immunodeficiency virus type 1 (HIV-1) has significant amino acid similarities to a reactive site of Kunitz-type basic proteinase inhibitors. We therefore thought that the PND may interact with cellular proteinase-like molecule(s) upon HIV-1 infection and measured the cellular proteolytic activities at the surface of intact Molt-4 clone 8 cells, which are highly susceptible to HIV-1 infection. The cells preferentially cleaved succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, a good substrate of chymotrypsin, and the activity was strongly inhibited by N-tosyl-L-phenylalanyl chloromethyl ketone (IC50 = 11.5 microM) and chymostatin (IC50 = 4.8 microM). A synthetic peptide of 24 residues (amino acids 308-331) that correspond to the PND also inhibited the cellular proteolytic activity in a dose-dependent manner (IC50 = 79.2 microM). The inhibition was still observed at low temperature (IC50 = 42.7 microM) and even after the peptide-treated cells were washed. We therefore think that the peptide interacts with proteinase-like molecule(s) located at the surface of the cells. The synthetic peptides from four other strains of HIV-1 corresponding to the PND similarly inhibited the proteolytic activity. These results may be helpful to clarify the novel mechanism(s) for HIV-1 infection.

Using the peptides from amino acids 100-130 of the HTLV-I gag protein, 175-199 of the HTLV-I env protein and the corresponding peptides of HTLV-II (amino acids 106 to 135 of the gag protein and 171 to 196 of the env protein), we tested for reactivity against antibodies by enzyme immunoassay in sera from HTLV-I and HTLV-II carriers. The peptides derived from the env proteins have high specificity for antibody binding. The peptide based on amino acids 175-199 of HTLV-I reacted with antibodies in sera from all HTLV-I carriers, and the peptide composed of amino acids 171-196 of HTLV-II reacted with antibodies in sera from all HTLV-II carriers. For the peptides derived from the gag proteins, we observed some cross-reactivity in sera from persons with anti-HTLV-I and anti-HTLV-II, due to antibody binding to the peptide corresponding to 12 amino acids from the C-terminal end of the gag protein. Separate enzyme immunoassays that used the four synthetic peptides as antigens clearly distinguished between serum with antibodies to HTLV-I or HTLV-II in various individuals and excluded false positive results using the particle agglutination assay that used a whole-virus lysate of HTLV-I as antigen.

We analysed the glycolipids of mouse thymocytes before and after Concanavalin A (Con A) or recombinant interleukin-2 (rIL-2) stimulation by TLC-immunostaining with carbohydrate-specific antiglycolipid antibodies. The thymocytes were cultured in serum-free medium in the presence of 500 ng ml-1 Con A, 10 U ml-1 rIL-2 or Con A plus rIL-2 for 6, 12, 24, 48, and 72 h, and were found to start proliferating 24 h after cultivation in the presence of Con A or Con A plus rIL-2, the maximum levels being reached at 72 h and 48 h, respectively, in a thymidine uptake experiment. The concentrations of II3Neu-Gg4Cer, Gg4Cer and IV3GalNAc alpha-Gb4Cer after 48 h Con A stimulation were found to be at almost the original levels. Conversely, II3Neu-Gg3Cer, which was not detected in the thymocytes at the start, began to appear after 48 h stimulation with Con A and Con A plus rIL-2, and IV3Neu-Gg5Cer in the cells 48 h after stimulation with Con A and Con A plus rIL-2 has increased to 41 and 44 times higher than in the original cells, respectively, as judged on TLC-immunostaining with monoclonal antibody YHD-06, which detects the GalNAc beta 1-4(NeuAc or NeuGc alpha 2-3)Gal beta-structure. These results indicate that the increased synthesis of both gangliosides, in other words, the activation of N-acetylgalactosaminyltransferase, is associated with the mitogen-induced proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effects of hypergravity on DNA synthesis and alkaline phosphatase (ALP) activity in cloned osteoblast-like cells, MC3T3-E1. Hypergravity (5 x g) stimulated DNA synthesis in these cells in a time-dependent manner and increased it approximately up to 150% of that of the control (1 x g). 12-O-Tetra-decanoylphorbol-13-acetate (TPA), a protein kinase C activator, and insulin-like growth factor I (IGF-I) enhanced DNA synthesis additively with hypergravity (5 x g). An increase in ALP activity induced by 10% fetal calf serum (FCS) was suppressed by hypergravity (2 x g, 5 x g). Five x g completely suppressed the increase in ALP activity. TPA and hypergravity (2 x g) suppressed the increase in ALP activity induced by FCS additively. Hypergravity (5 x g) showed no significant effect on cAMP nor cGMP production in these cells, but increased prostaglandin E2 (PGE2) production. Exogenous PGE2 stimulated DNA synthesis in these cells but had little effect on 10% FCS-induced ALP activity. These results suggest that hypergravity stimulates proliferation but suppresses differentiation of osteoblast-like cells through a pathway independent of the activation of protein kinase C and the production of cyclic nucleotides, and that hypergravity and IGF-I stimulate proliferation of these cells through an independent signal transduction pathway. Moreover, our data strongly suggest that PGE2 mediates the signalling of hypergravity on the proliferation of osteoblast-like cells.

It has been demonstrated that hyperprolactinemia is sometimes present even in patients with idiopathic diabetes insipidus (DI). In this study, we examined the responses of serum prolactin (PRL) to hypertonic saline infusion and TRH injection in 11 patients with idiopathic DI diagnosed by clinical examinations. Serum sodium in these patients (147.5 +/- 3.2 mEq/L) was significantly higher at baseline than in normal subjects (139.7 +/- 2.4 mEq/L). The plasma arginine vasopressin (AVP) level was significantly lower in DI (0.42 +/- 0.24 pg/ml) at baseline than in normal subjects (2.53 +/- 1.03 pg/ml). However, the serum PRL level in both groups did not differ significantly except in one patient with idiopathic DI (35.6 ng/ml). There was no significant correlation between the basal serum sodium and basal serum PRL in either group. After an infusion of hypertonic saline, the serum sodium level gradually increased to 155.6 +/- 3.4 mEq/L in DI and to 146.5 +/- 4.3 mEq/L in the normal subjects. However, this increase did not affect PRL secretion in either group. PRL response to TRH was essentially normal in all patients with idiopathic DI. These results indicate that the secretion of PRL is not generally affected by chronic mild hypernatremic hypovolemia in the patients with idiopathic DI.

ATL (adult T-cell leukemia) is the first human cancer known to be caused by a retrovirus. ATL cells show usually positive for CD2, CD3, CD4, CD25 and HLA-DR, but negative for CD8. They produce a variety of cytokines, including IL-1, IL-2, TNF, ADF and PTHrP. PTHrP is considered to be responsible for hypercalcemia which is frequently observed in ATL. Recently, we reported two unusual cases of HTLV-I associated malignancy; 1) a case of CD4 and 8 double negative tumor affecting mainly gastrointestinal tract and 2) a case mimicking small cell lung cancer. IL-2-toxin, a conjugate of IL-2 and diphtheria toxin, has been prepared as a recombinant product and evaluated for the suppressive effect to ATL cells. Clinical trail of IL-2-toxin is now anticipated.

We evaluated Retrovirus EIA kit (Roche Co.), in which virus components of three different viruses, HIV-1, HIV-2, and HTLV-I were used antigens. All the specimens from the patients with ATL, HAM, HTLV-I carrier, AIDS and AC, and from HIV-2 infected individuals were found to be positive. Four out of 40 specimens from hemophiliacs not infected with HIV-1 were found out to be positive. These specimens were confirmed to contain antibody against HTLV-I. Pseudo-negative or -positive reactions were not found in this study. Therefore, Retrovirus EIA kit should be evaluated more extensively, for the possible applications for screening of blood donors, and for selecting the sources of blood products as well as for clinical diagnoses.

When insulin and glucagon are administered to rats with severe liver injury, survival is enhanced with an attenuation of the liver injury compared to that of untreated controls. In rats with acute liver injury both hormones produce a rapid normalization of hepatic protein content following initiation of DNA synthesis. When rats receive both hormones after partial hepatectomy, the first burst of DNA synthesis reaches a maximum earlier than that seen in controls. Both hormones enhance the increment of hepatic putrescine essential for DNA synthesis through activation of ornithine decaroxylase and/or spermidine-N1-acetyltransferase. The enhancement of putrescine content by each hormone is additive. Putrescine supplementation promotes hepatic DNA synthesis after hepatectomy. Based on these data, we conclude that a combination of insulin and glucagon is effective in the therapy of acute hepatic failure in rats. The restoration of liver function as well as the stimulation of liver cell proliferation via putrescine production may contribute to this effect.

Erythropoietin (Epo) is a glycoprotein hormone that specifically regulates the proliferation and differentiation of erythroid progenitor cells. The effect of Epo on the phosphorylation of cellular proteins was investigated in a murine erythroleukemia cell line ELM-I-1 which differentiates to produce hemoglobin in response to Epo. The phosphorylation of a prominent 98 kDa phosphoprotein (pp98) (pI approximately 6.5) in the particulate fraction of ELM-I-1 cells decreased markedly upon exposure of the cells to recombinant human Epo. The effect was rapid and transient, occurring within 1 min after Epo exposure, and disappearing within 15-30 min. Interleukin 3, another hematopoietic factor which is known to act also on erythroid progenitors, did not induce this effect. Phosphoamino acid analysis revealed that pp98 is phosphorylated on serine residues. The results suggest that this rapid and transient alteration in protein phosphorylation may serve as critical trans-membrane signal for Epo.

To elucidate the role of leukocytes in intravascular coagulation in patients with septicemia, plasma levels of thrombin-antithrombin III complex (TAT), soluble fibrin monomer complex (SFMC) and fibrinogen (Fbg) were determined in 33 patients with septicemia. Twenty of 33 patients revealed marked leukopenia caused by suppression of hematopoiesis by the administration of chemotherapeutic agents for the treatment of hematological malignancies; the total leukocyte count of these patients was less than 1,000/microliters. Thirteen of 33 patients showed normal or increased leukocyte counts. Plasma levels of TAT and SFMC in septicemic patients without leukopenia were significantly higher than in patients with leukopenia. Although plasma TAT and SFMC levels correlated well with the number of leukocytes, a more significant positive correlation was found between the number of monocytes and the levels of TAT and SFMC. Plasma levels of Fbg were significantly lower in patients without leukopenia than in patients with leukopenia. No significant correlation was found between the number of leukocytes and the levels of Fbg. However, a significant negative correlation was found between the number of monocytes and the levels of Fbg. TAT levels did not correlate with the number of platelets. The fibrinolytic system was activated only in septicemic patients without leukopenia, which may be explained by secondary fibrinolysis following leukocyte-activated coagulation. These findings suggest that leukocytes, in particular monocytes, may play a critical role in the pathogenesis of intravascular coagulation in septicemia.

Because hepatocellular carcinoma treated by transcatheter arterial embolization often regains its size, routine follow-up is necessary. The usefulness of pulsed Doppler ultrasound for detection of this type of recurrence was compared with ultrasonography and computed tomography in 21 such hepatocellular carcinomas. Of 15 hepatocellular carcinomas diagnosed by angiography as showing recurrence, four were detected with ultrasonography and five were detected with computed tomography. Doppler signals were obtained in the peripheral portions corresponding to tumor vessels or stains on angiograms in 14 of these 15 hepatocellular carcinomas, but they were undetectable in six hepatocellular carcinomas with no recurrence. All signals disappeared after transcatheter arterial embolization. One false-negative hepatocellular carcinoma with pulsed Doppler ultrasound showed faint tumor stains on angiograms; these were also negative on ultrasonography and computed tomography. Pulsed Doppler ultrasound may be superior to ultrasonography and computed tomography as a routine procedure to detect the recurrence of hepatocellular carcinoma treated by transcatheter arterial embolization.