A novel clinical role for angiopoietin-1 in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumour associated with asbestos exposure that has only a limited response to conventional therapy; therefore, diagnosing MPM early is very important. We have previously reported that angiopoietin (Ang)-1 was correlated with bleomycin-induced pulmonary fibrosis. Here, we investigated the association of Ang-1 with the development of MPM cells, which originate from mesenchymal cells similar to lung fibroblasts, and demonstrated that Ang-1 stimulated the growth and migration of MPM cells in vitro. We also demonstrated that patients with MPM had significantly higher serum levels of Ang-1 in comparison to a population who had been exposed to asbestos but had not developed MPM. The patients with advanced-stage MPM showed higher levels of Ang-1 than the early-stage MPM patients and the Kaplan-Meier method revealed a significant correlation between serum Ang-1 levels and survival. We propose the possibility that Ang-1 plays an important role in MPM tumour growth and our data suggest that the serum concentration of Ang-1 could be useful as prognostic factor.

All-trans-retinoic acid inhibits tumour growth of malignant pleural mesothelioma in mice.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumour of mesothelial origin associated with asbestos exposure. Because MPM has limited response to conventional chemotherapy and radiotherapy, the prognosis is very poor. Several researchers have reported that cytokines such as interleukin (IL)-6 play an important role in the growth of MPM. Previously, it was reported that all-trans-retinoic acid (ATRA) inhibited the production and function of IL-6 and transforming growth factor (TGF)-beta1 in experiments using lung fibroblasts. We investigated whether ATRA had an inhibitory effect on the cell growth of MPM, the origin of which was mesenchymal cells similar to lung fibroblasts, using a subcutaneous xenograft mouse model. We estimated the tumour growth and performed quantitative measurements of IL-6, TGF-beta1 and platelet-derived growth factor (PDGF) receptor (PDGFR)-beta mRNA levels both of cultured MPM cells and cells grown in mice with or without the administration of ATRA. ATRA significantly inhibited MPM tumour growth. In vitro studies disclosed that the administration of ATRA reduced 1) mRNA levels of TGF-beta1, TGF-beta1 receptors and PDGFR-beta, and 2) TGF-beta1-dependent proliferation and PDGF-BB-dependent migration of MPM cells. These data may provide a rationale to explore the clinical use of ATRA for the treatment of MPM.

Expression of the T cell receptor Vbeta repertoire in a human T cell resistant to asbestos-induced apoptosis and peripheral blood T cells from patients with silica and asbestos-related diseases.

To explore the effects of asbestos and silica on the human immune system, an experimental model of low-dose and long-term exposure was established using a human HTLV-1-immortalized polyclonal T cell line, MT-2 (MT-2Org). MT-2 cells were continuously exposed to asbestos at a concentration (10 microg/ml) which does not induce complete cell death during short-term exposure. After acquiring resistance to CB-induced apoptosis (designated MT-2Rst), an immunological comparison was made between the MT-2Org and MT-2Rst lines in terms of T cell receptor-Vbeta (TcR-Vbeta) expression. MT-2Rst cells showed excess expression of various TcR-Vbeta, although TcR-Vbeta-overpresenting cells were characterized as undergoing apoptosis due to first contact with CB. Patients with asbestos-related diseases (ARD), such as asbestosis and malignant mesothelioma, were compared with silicosis (SIL) patients as a disease control and with healthy donors (HD). SIL and ARD not only differed in their causative materials, silica and asbestos as mineral silicates, but also in terms of complications; autoimmune disorders in SIL and tumors in ARD. ARD patients showed a restricted overpresentation of TcR-Vbeta without clonal expansion, whereas SIL patients revealed significant overpresentation of TcR-Vbeta 7.2. These experimental and clinical analyses indicate the superantigenic and dysregulation of autoimmunity-inducing effects of asbestos and silica, respectively.

Strong magnetic field effect on the dissolution process of tetragonal lysozyme crystals.

Either a homogeneous or inhomogeneous magnetic field has been known to dampen the protein crystal growth. To date the mechanism is not clear. However, it was generally proposed that the magnetic field may dampen the convection in the solution, resulting in a reduced crystal growth rate and possibly a good crystal quality, similar to the case of protein crystal growth in space. To understand the mechanism of the magnetic field effect on protein crystal growth, further explorations on the magnetic field effect on protein solution, on the processes of crystal growth and dissolution, and on different crystallization (solution) systems, should be valuable. In this paper we present our recent efforts to study magnetic field effects on the dissolution processes of tetragonal lysozyme crystals under a strong magnetic field. A layer of oriented tetragonal lysozyme crystals was prepared under a temperature gradient and magnetic field, after that the crystals were dissolved by increasing the temperature of the solution. The lysozyme molecules will diffuse upwards due to the steep concentration gradient at the lower side of the cell caused by the dissolution. The evolution of the concentration in the solution was measured in-situ using a Mach-Zehnder interferometer. The results confirmed that the dissolution process of the crystals was slowed by the magnetic field. Judging from the concentration evolution versus time at different positions in the solution, we concluded that the apparent diffusion coefficient of lysozyme molecules was decreased by the magnetic field. The results were discussed using a suspended crystal model in the initial dissolution stage.

Autoimmune hemolytic anemia as a first manifestation of primary effusion lymphoma.

A 55-year-old man developing transfusion-dependant anemia was diagnosed with autoimmune hemolytic anemia (AIHA). Although he received prednisolone (PSL) (daily 60 mg), his hemoglobin level continued to decrease. After 3 weeks of treatment, he presented with a distension of the abdomen. Cytological examination of ascitic fluid revealed large, immunoblastic lymphocytes with plasmacytoid features and abundant IgM chains on the cellular surface; this was diagnosed as primary effusion lymphoma (PEL). Administration of CHOP (cyclophosphamide, Adriamycin, vincristine, and PSL) chemotherapy elicited regression of ascites as well as recovery of hemoglobin level. We hypothesize that PEL cells generated antibodies against red blood cells, resulting in AIHA resistance to PSL.

Role of interleukin-12 in the regulation of CD4+ T cell apoptosis in a mouse model of asthma.

Allergic asthma, a chronic inflammatory disease of the airways, is characterized by the presence of T helper 2 cells and eosinophils in sputum, bronchoalveolar lavage, and mucosal biopsy specimens. Although the T helper 1-promoting cytokine, interleukin-12, is capable of inhibiting the T helper 2-driven asthma symptoms and bronchial responsiveness, the specific mechanisms underlying these interleukin-12 actions are unclear. The anti-allergic response to interleukin-12 is only partially dependent on interferon-gamma, which induces apoptosis by enhancing expression of Fas antigen. We therefore investigated in vivo whether the anti-allergic action of interleukin-12 is mediated through induction of apoptosis. C57BL/6 mice immunized to ovalbumin by intraperitoneal injection were challenged three times with an ovalbumin aerosol every second day for 7 days. Recombinant interleukin-12 was administered intravenously after the final challenge. After the last ovalbumin challenge, mice were examined for effects of interleukin-12 on inflammatory cell infiltration and apoptosis in the lung as detected by terminal deoxynucleotidyl transferase-mediated deoxyribonucleoside triphosphate nick end-labelling. Administration of interleukin-12 reduced ovalbumin-induced pulmonary eosinophilia (P < 0.01) and CD4+ T cell infiltration (P < 0.01). Moreover, treatment with interleukin-12 shortly after ovalbumin inhalation resulted in both increased interferon-gamma production (P < 0.01) and enhanced apoptosis of CD4+ T cells in allergic airway infiltrates (P < 0.05). These results suggest that the beneficial effects of interleukin-12 in asthma may include enhancement of apoptosis of CD4+ T cells in airways.

Successful treatment of advanced peripheral T-cell lymphoma with an angiocentric growth pattern complicated with hemophagocytic syndrome by high-dose chemotherapy and autologous peripheral blood stem cell transplantation.

Peripheral T-cell lymphomas (PTCL) account for about 10% of all lymphomas in Western countries, respond poorly to therapy, and have short survival with no sustained remission. Furthermore, the complication of hemophagocytic syndrome (HPS) sometimes makes the prognosis of this disease extremely worse. We report here a case of PTCL with an angiocentric growth pattern complicated with HPS successfully treated by high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Our case suggests this approach is an excellent candidate for the treatment of this disease.

An investigation of magnetic field effects on the dissolution of lysozyme crystal and related phenomena.

It is now widely known that a magnetic field, either homogeneous or inhomogeneous, depresses the growth process of protein crystals. In this report, the dissolution process of tetragonal lysozyme crystals is also confirmed to be depressed by a homogeneous magnetic field (inhomogeneity <1.5%). The dissolution process was monitored using a Mach-Zehnder interferometer. The results showed that the concentration change during the dissolution process was slowed in a magnetic field compared with that in the absence of a magnetic field. It was concluded that the diffusion coefficient of the lysozyme molecules in the solution was decreased by the magnetic field. The decrease in the diffusion coefficient may contribute to the slowed growth process. The changes in the spatial concentration distribution under a vertical temperature gradient before crystallization in the absence of a magnetic field was also studied. The concentration in the lower, colder part of the cell increased, while it decreased in the upper, hotter part, a similar phenomenon to that discovered by previous investigators in an isothermal supersaturated solution system. Aggregated domain formation is proposed to explain the concentration redistribution before crystal growth and a suspended crystal model is proposed to explain the decrease of diffusivity in a magnetic field.

HER2 peptide-specific CD8(+) T cells are proportionally detectable long after multiple DNA vaccinations.

We prepared a plasmid encoding 147 amino acid residues from the N terminus of c-erbB-2/HER2/neu (HER2), which included both a cytotoxic T lymphocyte (CTL) epitope (HER2p63) and a helper epitope (HER2p1), using the mammalian expression vector pCAGGS-New (pCAGGS147HER2). In a parallel analysis with a Tetramer assay and CTL assay, good specificity and sensitivity of a quantitative enzyme-linked immunospot (ELISPOT) assay to detect functional HER2p63-specific CD8(+) T cells were demonstrated after intramuscular immunization of pCAGGS147HER2. In an ELISPOT assay for HER2p63, spots of IFN gamma-producing cells were first detected 10 days after the first immunization, and additional immunizations increased the number of spots. HER2p63-specific CD8(+) T cells were detected over a period of more than 10 months after the last immunization. In hosts receiving more than three immunizations, surprisingly high numbers of specific CD8(+) T cells were persistently detectable. HER2 protein-specific antibodies of IgG class with dominance of IgG2a remain detectable 6 months after single or multiple immunizations. The antibodies however, were not reactive with cell surface HER2 antigens. Total suppression of tumor growth was observed when syngeneic HER2(+) tumor cells (2 x 10(6)) were injected subcutaneously 14 days after a single immunization with pCAGGS147HER2. Furthermore, the number of pulmonary metastases decreased significantly when DNA vaccination was initiated on the day of, or 3 days after, intravenous injection (1 x 10(6) cells).

Effects of post-inhalation treatment with interleukin-12 on airway hyper-reactivity, eosinophilia and interleukin-18 receptor expression in a mouse model of asthma.

BACKGROUND: Correcting Th1/Th2 imbalance with administration of IL-12 before and during antigen challenge holds therapeutic promise in asthma. However, the effects of IL-12 on the established asthmatic responses have not fully been examined. OBJECTIVE: We investigated whether IL-12 administered after antigen challenge could diminish airway hyper-reactivity (AHR) and eosinophilia in mice actively sensitized to ovalbumin. We also have investigated the ability of administered IL-12 to induce IL-18 receptor (IL-18R) expression that may lead possible synergic action of IL-12 with endogenous IL-18. METHODS: C57BL/6 mice immunized to ovalbumin (OVA) by intraperitoneal (i.p.) injection, were challenged three times with an aerosol of OVA every second day for 8 days. Recombinant IL-12 (500 ng) was intravenously administered on a single occasion 1 h after the final challenge of mice. Mice were analysed for effects of IL-12 on AHR, inflammatory cell infiltration and cytokine levels in lung tissue as well as serum immunoglobulin (Ig) E levels. Immunohistochemistry for IL-18R was performed using rat monoclonal antibody specific for murine IL-18Ralpha (IL-1 receptor related protein; IL-1Rrp). RESULTS: An intravenous IL-12 administration diminished AHR, pulmonary eosinophilia and T lymphocyte infiltration, serum IgE, IL-4 and IL-13 in lung tissue. Expression of IL-18R was induced in the mononuclear cells in the lung of mice exposed to OVA. IL-12 administration enhanced the IL-18R expression compared with the control. CONCLUSION: These data indicate that IL-12 can attenuate established antigen-induced AHR and inflammation. In this mechanism it would be interpreted as follows: IL-12 administration in OVA-challenged mice decreased IL-4 production and IgE production thereafter through direct effect on inhibiting the activation of established Th2 cells response and also combined effect with up-regulation of IL-18R expression by inflammatory cells in the lung.

Role of SEREX-defined immunogenic wild-type cellular molecules in the development of tumor-specific immunity.

Recognition of altered self-antigens in tumor cells by lymphocytes forms the basis for antitumor immune responses. The effector cells in most experimental tumor systems are CD8(+) T cells that recognize MHC class I binding peptides derived from molecules with altered expression in tumor cells. Although the need for CD4(+) helper T cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumor responses remain unclear. We examined whether broadly expressed wild-type molecules in murine tumor cells eliciting humoral immunity contributed to the generation of CD8(+) T cells and protective antitumor immune responses to unrelated tumor-specific antigens [mutated ERK2 (mERK2) and c-erbB2/HER/neu (HER2)]. The immunogenic wild-type molecules, presumably dependent on recognition by CD4(+) helper T cells, were defined by serological analysis of recombinant cDNA expression libraries (SEREX) using tumor-derived lambda phage libraries screened with IgG antibodies of hosts bearing transplanted 3-methylchoranthrene-induced tumors. Coimmunization of mice with plasmids encoding SEREX-defined murine wild-type molecules and mERK2 or HER2 led to a profound increase in CD8(+) T cells specific for mERK2 or HER2 peptides. This heightened response depended on CD4(+) T cells and copresentation of SEREX-defined molecules and CD8(+) T cell epitopes. In tumor protection assays, immunization with SEREX-defined wild-type molecules and mERK2 resulted in an inhibition of pulmonary metastasis, which was not achieved by immunization with mERK2 alone.

Heme oxygenase-1 (HO-1) protein induction in a mouse model of asthma.

BACKGROUND AND OBJECTIVE: Carbon monoxide (CO) is known to be present in measurable quantities in the exhalation of asthmatic patients. Corticosteroid treatment resulted in a decrease in exhaled CO levels in asthmatic patients, raising the possibility that an increase in exhaled CO concentration reflects inflammation of the asthmatic airway. Heme oxygenase-1 (HO-1) protein, also called HSP32, is the rate-limiting enzyme in the catabolism of heme to biliverdin, free iron and CO. However, it is unknown whether an expression of HO-1 within the lung tissue is related to allergic airway inflammation. We studied the expression of HO-1 in lung tissue and bronchoalveolar lavage cells in a mouse model of asthma. METHODS: Ovalbumin (OVA)-sensitized C57BL/6 mice were challenged with aerosolized OVA. HO-1 positive cells were identified by immunostaining in lung tissue and bronchoalveolar lavage fluid (BALF) after the challenge. RESULTS: HO-1 positive cell numbers increased in the subepithelium of the bronchi after OVA challenge. In cytospin preparations from BALF after OVA challenge, HO-1 was localized to alveolar macrophages. Inside the macrophages, HO-1 reactivity was expressed in the cytoplasm, and the perinuclear region in particular. CONCLUSION: The expression of HO-1 is increased within the lung tissue in allergic airway inflammation. Measurement of HO-1 activity may be clinically useful in the management of asthma.

[Effects of adjuvants on the function of antigen presenting cells in the production of IgE in mice].

In an examination of the effects of adjuvants on the production of IgE, amounts of mRNAs for cytokines in antigen presenting cells(APCs) were assayed by RT-PCR and expressions of surface molecules on the cells were analyzed by flowcytometry when primed with antigen plus adjuvant. When mice were primed with ovalbumin plus alum, the levels of total and specific IgE were higher than those of mice primed with ovalbumin plus CFA. The APCs from mice primed with ovalbumin plus alum expressed higher levels of IL-1 mRNA than those in APCs from mice primed with ovalbumin plus CFA. B7 molecules were more expressed on the surface of the APCs from mice primed with ovalbumin plus CFA. The results suggested that these modulations of the functions of APCs affected the induction of helper T cell subsets in mice primed with different adjuvant.

Anti-metastatic gene therapy utilizing subcutaneous inoculation of EC-SOD gene transduced autologous fibroblast suppressed lung metastasis of Meth-A cells and 3LL cells in mice.

We have previously reported that superoxide stimulates the motility of tumor cells and the administration of Cu-Zn superoxide dismutase (SOD) significantly suppresses metastasis. However, ideally, anti-metastatic therapy should be long-lasting, systemically effective and have low toxicity. The half-life of Cu-Zn SOD in plasma is so short that it cannot provide long-lasting effects. Therefore, in this study we have developed a gene therapy in a mouse model utilizing extracellular SOD (EC-SOD), which is the most prevalent SOD isoenzyme in extracellular fluids. We retrovirally transfected fibroblasts (syngeneic) with the EC-SOD gene and established EC-SOD-secreting fibroblasts. Inoculation of EC-SOD-secreting fibroblasts suppressed both artificial and spontaneous metastatic lung nodules in mouse metastasis models. These data indicate the feasibility of anti-metastatic gene therapy utilizing the EC-SOD gene.

[Pure red cell aplasia induced by clomipramine hydrochloride in a patient with SLE].

A 48-year-old woman, who had been suffering from systemic lupus erythematosus (SLE), developed normochromic normocytic anemia after receiving clomipramine hydrochloride. Her reticulocyte count was low, and a bone marrow aspirate revealed erythroid hypoplasia without involvement of other cell lines. Thus a diagnosis of pure red cell aplasia (PRCA) was made. The anemia gradually resolved following withdrawal of the drug. Although several drugs are known to cause PRCA, this is the first time that clomipramine hydrochloride has been reported to have such an effect. The underlying SLE in this case suggested the possible immunological pathogenesis of drug-induced PRCA.

Role of leukocytes in radicular pain secondary to herniated nucleus pulposus.

Some studies have assessed inflammatory cells such as macrophages, lymphocytes, and neutrophils in herniated lumbar disc tissues using histologic analysis. However, there is no consensus regarding the relationships between clinical symptoms, including radicular pain and the presence of inflammatory cells. It has been shown that autologous nucleus pulposus relocated on the lumbar nerve root in rats produces time dependent and reversible mechanical hyperalgesia, which is thought to be a pain related behavior in peripheral neuropathic pain models. The purpose of this study was to determine whether leukocytes play a role in the mechanical hyperalgesia induced by the nucleus pulposus and to characterize the role of leukocytes in radicular pain attributable to lumbar disc herniation. Nitrogen mustard was used to induce and evaluate leukocytopenia in rats. Sensitivity to mechanical noxious stimuli was measured quantitatively, and inflammatory cells in granulation tissue around the nerve root were examined histologically. The nucleus pulposus produced neither mechanical hyperalgesia nor abundant inflammatory cells in rats with nitrogen mustard induced leukocytopenia. Neuropathic pain produced by the nucleus pulposus, when placed on the nerve root, may be related to inflammatory cell infiltration induced by relocation of the nucleus pulposus, rather than the nucleus pulposus itself.

Resistance to Friend murine leukemia virus infection conferred by the Fv-4 gene is recessive but appears dominant from the effect of the immune system.

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). However, the resistance caused by Fv-4 is recessive in nude mice, which suggests that immunological effects play important roles in this resistance in vivo (K. Higo, Y. Kubo, Y. Iwatani, T. Ono, M. Maeda, H. Hiai, T. Masuda, K. Kuribayashi, F. Zhang, T. Lamin, A. Adachi, and A. Ishimoto, J. Virol. 71:750-754, 1997). To determine the immunological effect on the resistance in vivo, we infected immunologically immature newborn mice homozygous (Fv-4(r/r)) and heterozygous (Fv-4(r/-)) for Fv-4. Although the Fv-4(r/r) mice showed complete resistance to F-MuLV whether infected neonatally or as adolescents, the Fv-4(r/-) mice showed high sensitivity to viral proliferation and disease induction when infected as newborns but complete resistance when infected as adolescent mice. To confirm the immunological effect on the resistance in adolescent mice with the Fv-4(r/r) and Fv-4(r/-) genotypes, we examined the effect of an immunosuppressant drug, FK506, on the resistance. The mice with the Fv-4(r/r) genotype treated with FK506 still showed resistance, but the mice with the Fv-4(r/-) genotype became highly sensitive to F-MuLV infection. Flow cytometric analysis to detect the Fv-4 gene product showed that the Fv-4 gene product was expressed on the cells from newborn and adolescent mice. The Fv-4 gene product was also detected on the cells from the FK506-treated mice as well as on those from untreated mice. However, a quantitative difference in the gene product between the cells with the Fv-4(r/r) and Fv-4(r/-) genotypes was detected by indirect staining for flow cytometry. These results show that the resistance to F-MuLV infection conferred by the Fv-4 gene is originally recessive, but it looks dominant in adolescent mice mainly because of the effect of the immune system.

Survival-promoting activity of IL-7 on IL-2-dependent cytotoxic T lymphocyte clones: resultant induction of G1 arrest.

Interleukin-7 (IL-7) is essential for both T cell and B cell development. Recent studies have suggested that IL-7 also functions as a survival-promoting factor for resting and activated T cells. In this study we examined the effects of IL-7 on survival and cytotoxicity of tumor-specific CD8(+) cytotoxic T lymphocyte (CTL) clones established and maintained either with IL-2 alone or with a combination of IL-2 and IL-7. While the CTL clones cultured in IL-2 alone died around day 10, the CTL clones cultured in the presence of IL-2 and IL-7 survived for more than 4 weeks after seeding. The long-term survival of the latter was correlated with the presence of IL-7 in the medium. In addition, IL-7 alone prolonged survival of other IL-2-dependent CTL clones after the removal of IL-2. IL-7 maintained the CTLs in G1 arrest after a slight proliferation during the initial phase during which low-level but sustained DNA synthesis was observed. However, there was no direct correlation between DNA synthesis and enhancement of long-term survival by IL-7 as demonstrated by the inhibiting proliferation of the CTL clones with the protein kinase inhibitor genistein. During long-term survival in the presence of IL-7, the cytotoxic activities of the CTL clones decreased gradually to background levels although they were restored soon after the next passage. These results suggested that IL-7 had the ability to set machinery in motion against apoptosis in the IL-2-dependent CTL clones. Such an effect of IL-7 might play a role in vivo in the process leading activated T cells to the resting, that is, memory state.