Electron diffraction based analysis of phase fractions and texture in nanocrystalline thin films, part III: application examples.

In this series of articles, a method is presented that performs (semi)quantitative phase analysis for nanocrystalline transmission electron microscope samples from selected area electron diffraction (SAED) patterns. Volume fractions and degree of fiber texture are determined for the nanocrystalline components. The effect of the amorphous component is minimized by empirical background interpolation. First, the two-dimensional SAED pattern is converted into a one-dimensional distribution similar to X-ray diffraction. Volume fractions of the nanocrystalline components are determined by fitting the spectral components, calculated for the previously identified phases with a priori known structures. These Markers are calculated not only for kinematic conditions, but the Blackwell correction is also applied to take into account dynamic effects for medium thicknesses. Peak shapes and experimental parameters (camera length, etc.) are refined during the fitting iterations. Parameter space is explored with the help of the Downhill-SIMPLEX. The method is implemented in a computer program that runs under the Windows operating system. Part I presented the principles, while part II elaborated current implementation. The present part III demonstrates the usage and efficiency of the computer program by numerous examples. The suggested experimental protocol should be of benefit in experiments aimed at phase analysis using electron diffraction methods.

An open-label trial of rituximab therapy in pulmonary alveolar proteinosis.

Rituximab, a monoclonal antibody directed against the B-lymphocyte antigen CD20, has shown promise in several autoimmune disorders. Pulmonary alveolar proteinosis (PAP) is an autoimmune disorder characterised by autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF). An open-label, proof-of-concept phase II clinical trial was conducted in 10 PAP patients. The intervention consisted of two intravenous infusions of rituximab (1,000 mg) 15 days apart. Bronchoalveolar lavage (BAL) fluid and peripheral blood samples were collected. The primary outcome was improvement in arterial blood oxygenation. Both arterial oxygen tension and alveolar-arterial oxygen tension difference in room air improved in seven out of the nine patients completing the study. Lung function and high-resolution computed tomography scans, which were secondary outcomes, also improved. Peripheral blood CD19+ B-lymphocytes decreased from mean ± sem 15 ± 2% to <0.05% (n = 10) 15 days post-therapy. This decrease persisted for 3 months in all patients; at 6 months, CD19+ B-cells were detected in four out of seven patients (5 ± 2%). Total anti-GM-CSF immunoglobulin (Ig)G levels from baseline to 6 months were decreased in BAL fluids (n = 8) but unchanged in sera (n = 9). In this PAP cohort: 1) rituximab was well-tolerated and effectively ameliorated lung disease; and 2) reduction in anti-GM-CSF IgG levels in the lung correlated with disease changes, suggesting that disease pathogenesis is related to autoantibody levels in the target organ.

Immunological activity of regional lymph nodes in tumor-bearing mice.

Regional (popliteal) lymph node cells (RLNC) from A/Jax mice, inoculated in each hind foot with isogeneic Sarcoma 1 tumor cells, demonstrated cytotoxicity in vitro at Day 14 of tumor growth but lost this ability by Day 21. These noncytotoxic RLNC were capable of suppressing activity of other cytotoxic lymphoid cells, but after incubation for 24 hr in vitro their cytotoxicity was restored and their suppressive activity was abrogated. RLNC responsible for cytotoxicity were removed by treatment with anti-theta serum plus complement. The suppressive effect of RLNC was found to be mediated by a soluble "blocking" factor which was released into the culture medium after 24 hr incubation. The factor was not detected in culture media from RLNC pretreated with anti-theta serum plus complement prior to incubation. Absorption of RLNC culture supernatants with tumor-bearer spleen cells, but not with normal spleen cells, completely removed the blocking factor, while absorption by Sarcoma 1 cells significantly reduced blocking activity. The factor was trypsin sensitive, was retained on an Amicon XM100 filter, and did not demonstrate the presence of antibody to Sarcoma 1 in a radioimmunoassay. Although the exact nature of the factor has not been established, it appears to be a receptor antigen complex from T-cells of tumor-bearing animals.

Activation of human monocyte tumoricidal activity by C-reactive protein.

We examined the effect of purified human C-reactive protein (CRP) on induction of human peripheral blood monocyte (Mo)-mediated cytotoxicity (CTX) and oxidative metabolism. Exposure of Mo to acute phase serum levels of CRP in vitro resulted in dose-dependent expression of CTX against human tumor cell lines. Nonneoplastic human fibroblasts and glial cells were not affected by CRP-exposed Mo, and treatment of Mo monolayers with anti-Leu 11b (a natural killer marker) and complement did not abrogate or diminish CTX. Tumoricidal activity was observed after 20-44 h of Mo exposure to CRP, and after 48-72 h of coculture with radiolabeled target tumor cells. Mo exposed to CRP for 48 h also demonstrated elevated superoxide anion production when challenged with phorbol myristate acetate. Unlike CTX induced by lipopolysaccharide, CRP-induced CTX was completely inhibited by preincubation of CRP with phosphorylcholine, a CRP ligand, at a concentration of 5.5 molecules phosphorylcholine per molecule CRP. Further, when Mo medium (which contained 5% human AB serum) was preincubated with immobilized CRP, exposure of Mo to CRP in such medium did not result in CTX. In contrast, LPS-induced CTX was not affected. CRP-induced Mo CTX was observed, however, when Mo were exposed to CRP in medium preincubated with phosphorylcholine-treated immobilized CRP, suggesting that an active serum component which complexed with CRP was not removed. These findings indicate that one of the functions of the acute phase protein, CRP, may be to active Mo and that the process may require a CRP-binding serum component.

Inhibition of lung metastases in mice bearing a malignant fibrosarcoma by treatment with liposomes containing human C-reactive protein.

Recent studies suggest an immune modulator role for C-reactive protein (CRP). We have tested the effect of CRP in a tumor system designed for study of metastases. Fibrosarcoma T241 was implanted on one hind foot of syngeneic C57BL/6 mice. After 17 days, the tumor-bearing feet were amputated, and i.v. therapy of liposomes containing CRP or control reagents was started. Examination of the lungs on Day 35 showed that CRP:liposome-treated animals had significantly fewer and smaller metastases as compared with those in the control groups. Moreover, 38% of the animals in the former groups were completely free of metastases as compared with 0 to 2% of the controls. Significantly, enhanced survival was also noted in the CRP liposome-treated group. CRP may have biological response modifier" function of value in cancer therapy.

Induction of in vitro tumoricidal activity in alveolar macrophages and monocytes from patients with lung cancer.

Human alveolar macrophages (AMs) and blood monocytes were obtained from 65 smoking and nonsmoking normal volunteers and 29 patients with lung cancer. The oxidative metabolic response of these cells was measured by superoxide anion production after incubation with lipopolysaccharide. In addition, tumoricidal activity of AMs and monocytes was assessed against [3H]thymidine-labeled tumor target cells. Smoking was associated with depressed AM superoxide anion responses in normals but not in patients. In contrast, smoking appeared to slightly elevate monocyte superoxide anion activity. AMs and monocytes exposed to lipopolysaccharide or recombinant gamma-interferon showed tumoricidal activity in all groups. Mean cytotoxicity values of smoking patients versus smoking normals and exsmoking patients versus nonsmoking normals were not significantly different. Smoking, however, in both patients and normals was associated with significantly (P less than 0.005) depressed AM cytotoxicity levels (less than 40%) compared to nonsmoking volunteers and exsmoking patients. Activated AMs from cancer patients and normals were cytotoxic against three different tumorigenic cell lines but not against a nontumorigenic line. No correlation between monocyte and AM cytotoxic activity within single individuals was found. We conclude that AM and monocytes from smoking and exsmoking patients can be activated after exposure to immunomodulators; however, smoking may be slightly suppressive to cytotoxic responses. These studies provide a rationale for clinical trials of immunomodulators in patients with lung cancer.

Differential effect of recombinant granulocyte macrophage colony-stimulating factor on human monocytes and alveolar macrophages.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), a pluripotent cytokine, on tumoricidal activity of alveolar macrophages and monocytes from nonsmoking normal volunteers was compared using [3H]thymidine-labeled human tumor cells (SK-MEL-28, melanoma) as targets. A dose-response study (500-5000 units/ml) of recombinant GM-CSF indicated dramatic differences between cytotoxicity of alveolar macrophages and blood monocytes. Macrophages exhibited significant (P less than 0.01) tumoricidal activity at all GM-CSF doses tested. In contrast, monocytes showed no significant tumoricidal activity at 500 units/ml and significantly (P less than 0.01) less activity than alveolar macrophages at doses of 1000-5000 units/ml. Maximal activity in alveolar macrophages occurred 72-96 h after exposure to 1000-5000 units/ml GM-CSF. Tumoricidal activity may be related to the state of maturation, because monocytes matured in vitro for 7 days displayed enhanced tumoricidal activity after GM-CSF exposure. Tumor necrosis factor alpha and interleukin 1 beta were measured in supernatant fluids of 24-h GM-CSF-treated cells. No significant increase in either cytokine was detected after GM-CSF treatment of alveolar macrophages. Monocyte interleukin 1 beta secretion was not enhanced by GM-CSF; however, tumor necrosis factor alpha secretion was enhanced in some donors (three of five). Superoxide anion production of alveolar macrophages was not enhanced by GM-CSF. These data suggest that alveolar macrophage tumoricidal activity is induced by GM-CSF and is not dependent on oxidative metabolism or secreted forms of interleukin 1 beta or tumor necrosis factor alpha.

2-5A antisense telomerase RNA therapy for intracranial malignant gliomas.

Malignant gliomas are the most common intracranial tumors and are considered incurable. Therefore, exploration of novel therapeutic modalities is essential. Telomerase is a ribonucleoprotein enzyme that is detected in the vast majority of malignant gliomas but not in normal brain tissues. We, therefore, hypothesized that telomerase inhibition could be a very promising approach for the targeted therapy of malignant gliomas. Thus, 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked antisense against human telomerase RNA component (2-5A-anti-hTER) was investigated for its antitumor effect on an intracranial malignant glioma model. 2-5A is a mediator of one pathway of IFN actions by activating RNase L, resulting in RNA degradation. By linking 2-5A to antisense, RNase L degrades the targeted RNA specifically and effectively. Prior to the experiments using intracranial tumor models in nude mice, we modified the in vitro and in vivo treatment modality of 2-5A-anti-hTER using a cationic liposome to enhance the effect of 2-5A-anti-hTER. Here we demonstrate that 2-5A-anti-hTER complexed with a cationic liposome reduced the viability of five malignant glioma cell lines to 20-43% within 4 days but did not influence the viability of cultured astrocytes lacking telomerase. Furthermore, treatment of intracranial malignant gliomas in nude mice with 2-5A-anti-hTER was therapeutically effective compared with the control (P < 0.01). These findings clearly suggest the therapeutic potentiality of 2-5A-anti-hTER as a novel approach for the treatment of intracranial malignant gliomas.

Interleukin-1 beta-converting enzyme mediates cisplatin-induced apoptosis in malignant glioma cells.

Increasing the susceptibility of tumor cells to apoptotic cell death following chemotherapy is of importance to the outcome of cancer treatment. Although the tumor suppressor gene p53 is required for efficient induction of apoptosis by chemotherapeutic agents, it is not the only apoptosis mediator gene. The molecular mechanisms mediating apoptosis following chemotherapy via p53-dependent or p53-independent pathways remain unclear. We show here that cis-diamminedichloroplatinum (cisplatin) induces the expression of interleukin-1 beta-converting enzyme (ICE), a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, in murine and human malignant glioma cells during apoptosis regardless of their p53 status. Furthermore, overexpression of the murine ICE gene induces apoptosis in these tumor cells. The apoptosis induced by cisplatin treatment or murine ICE overexpression can be suppressed by the tetrapeptide ICE inhibitor Ac-YVAD-CMK or the apoptosis inhibitors bcl-2 or bcl-2-related bcl-XL gene. These findings suggest that ICE may mediate apoptosis induced by chemotherapy, and its induction could represent a novel approach for the effective treatment of malignant glioma.

Macrophage activation and generation of tumoricidal activity by liposome-associated human C-reactive protein.

The effect of human C-reactive protein incorporated into multilamellar vesicles (CRP-MLV) was studied in assays of macrophage function. Peritoneal exudate macrophages from C57BL/6 mice phagocytosed CRP-MLV in vitro more rapidly than multilamellar vesicles bearing comparable amounts of immunoglobulin G. Exposure of peritoneal exudate macrophages in vitro to CRP-MLV resulted in development of tumoricidal activity against syngeneic T241 fibrosarcoma and B-16 melanoma cells and against allogeneic Sarcoma 1 cells. Peritoneal exudate macrophages obtained from mice given CRP-MLV i.p. demonstrated antitumor activity against the syngeneic T241 fibrosarcoma in a Winn-type assay, and when challenged in vitro with phorbol myristate acetate, they showed elevated superoxide anion production. Administration of CRP-MLV i.p. did not enhance natural killer activity of spleen cells, however. In superoxide anion assays, CRP-MLV were approximately 10 to 100 times more effective than free C-reactive protein. Results indicate that C-reactive protein is capable of activating macrophages, thus supporting the concept of C-reactive protein as an immunomodulator.

Induction of cytokine messenger RNA and secretion in alveolar macrophages and blood monocytes from patients with lung cancer receiving granulocyte-macrophage colony-stimulating factor therapy.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes the proliferation and differentiation of hematopoietic progenitor cells. Although preliminary data are available from clinical trials, the effect of GM-CSF on gene expression of immunocompetent cells in treated patients has not been studied. We previously demonstrated that in vitro treatment with GM-CSF also enhances maturation-related anti-tumor activities in mononuclear phagocytes. The purpose of the present study was to examine the effects of in vivo recombinant GM-CSF therapy on alveolar macrophages and blood monocytes, to determine if these cells demonstrated differential expression of cytokine genes, cytokine production, and tumoricidal activity. Alveolar macrophages and blood monocytes were isolated from 13 patients receiving a range of GM-CSF doses (60-250 micrograms/m2/day) by continuous infusion over a 2-week period. Both monocytes and macrophages were isolated prior to therapy and at day 10 of the infusion. Monocytes, in addition, were isolated on day 3 of infusion. Results indicated that GM-CSF therapy enhanced expression of tumor necrosis factor, interleukin 1, and interleukin 6 mRNA in both monocytes and alveolar macrophages. Differential responses, however, were observed in cytokine secretion; monocytes demonstrated enhanced secretion of all three cytokines by day 3 of treatment, but alveolar macrophages showed only enhanced interleukin 6 secretion at day 10. Monocyte tumoricidal activity after in vitro lipopolysaccharide stimulation was also significantly elevated by day 3 of treatment, but at day 10 activity was not statistically different from pretreatment values in either monocytes or alveolar macrophages. These data indicate that GM-CSF exerts striking time-dependent modulatory effects on gene expression and functional activities of monocytes and alveolar macrophages in vivo, although the responses of the two cell types differ with respect to cytokine secretion.

Retroviral transfer of CPP32beta gene into malignant gliomas in vitro and in vivo.

Malignant gliomas are highly aggressive neoplasms that are very resistant to current therapeutic approaches, including irradiation, chemotherapy, and immunotherapy. To improve the prognosis, it is absolutely essential to explore novel modalities of treatment. Recently, we have demonstrated that interleukin 1beta-converting enzyme (ICE), a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, induces apoptotic cell death in malignant glioma cells. To date, ICE and ICE-like proteases (the ICE family), such as Ich-1L, CPP32beta, Mch2alpha, and Mch3alpha, have been shown to mediate apoptosis in some cells. The purpose of this study is to determine whether the ICE gene family functions as a useful tool for the treatment of malignant glioma cells through induction of apoptosis. The transient transfection assays showed that CPP32beta and Mch2alpha genes induced apoptotic cell death in malignant glioma cells more effectively than did the ICE, Ich-1L, and Mch3alpha genes. To improve the efficiency of gene transfer into malignant glioma cells, we constructed the retroviral vectors containing the ICE gene family. The retroviral transfer of CPP32beta or Mch2alpha gene effectively induced apoptosis in malignant glioma cells in vitro. Furthermore, treatment of tumors grown in mice with retrovirus containing CPP32beta significantly inhibited growth of the tumors through induction of apoptosis. The retroviral transfer of CPP32beta or Mch2alpha, therefore, may be a novel and promising approach for the treatment of malignant glioma, an invariably fatal tumor.

Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter.

Because the apoptotic pathway is often disrupted in tumor cells, its genetic restoration is a very attractive approach for the treatment of tumors. To treat malignant gliomas with this approach, it would be preferred to restrict induction of apoptosis to tumor cells by establishing a tumor-specific expression system. Telomerase is an attractive target because the vast majority of malignant gliomas have telomerase activity whereas normal brain cells do not. Activation of telomerase is tightly regulated at the transcriptional level of the telomerase catalytic subunit [human telomerase reverse transcriptase, (hTERT)]. Therefore, we hypothesized that using a hTERT promoter-driven vector system, an apoptosis-inducible gene may be preferentially restricted to telomerase- or hTERT-positive tumor cells. In this study, we constructed an expression vector consisting of the constitutively active caspase-6 (rev-caspase-6) under the hTERT promoter (hTERT/rev-caspase-6) and then investigated its antitumor effect on malignant glioma cells. The rationale for using the rev-caspase-6 gene is because it induces apoptosis independent of the initiator caspases. We demonstrated that the hTERT/rev-caspase-6 construct induced apoptosis in hTERT-positive malignant glioma cells, but not in hTERT-negative astrocytes, fibroblasts, and alternative lengthening of telomeres cells. In addition, the growth of s.c. tumors in nude mice was significantly suppressed by the treatment with hTERT/rev-caspase-6 construct. The present results strongly suggest that the telomerase-specific transfer of the rev-caspase-6 gene under the hTERT promoter is a novel targeting approach for the treatment of malignant gliomas.

Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis.

Malignant glioblastomas grow very rapidly and are generally resistant to either DNA-damaging drugs or gamma-irradiation. If tumor cells could be made more susceptible to cell death with treatments, this would clearly represent a significant improvement in the success of treatment. Recently, telomerase has become a focus of interest among oncologists as a target for treating cancer cells. Telomerase elongates telomeric DNA repeats (TTAGGG)n and is important in protecting and replicating DNA. The vast majority of tumor cells, indeed, express telomerase activity whereas normal somatic cells, except for a few cells, do not. Since telomerase is essential for protecting DNA, we may be able to make tumors more sensitive to treatments with DNA-damaging drugs by inhibiting telomerase activity. In this study, we used cis-diamminedichloroplatinum (cisplatin)-sensitive U87-MG cells and cisplatin-resistant U251-MG of human malignant glioblastoma cell lines. U87-MG cells did not express telomerase activity, whereas telomerase was highly detected in U251-MG cells. Interestingly, inhibition of telomerase with an antisense telomerase expression vector not only decreased telomerase activity but also increased susceptibility to cisplatin-induced apoptotic cell death in U251-MG cells. These findings suggest that treatment with antisense telomerase may represent a new chemosensitisation for tumors resistant to anticancer drugs.

The transforming activities of MDM2 in cultured neonatal rat astrocytes.

Although the molecular events regulating the pathogenesis of malignant astrocytomas remains unclear, the inactivation of tumor suppressor genes may be a key factor. The inactivation of p53 by mutation or deletion, however, is not the only obligatory step in astrocytoma genesis. The MDM2 protein has been shown to bind to and downmodulate p53 function, and to have oncogenic capacity. The MDM2 gene is also amplified and overexpressed in a subset of malignant astrocytomas without p53 mutation. Here we show that overexpression of MDM2 promoted the DNA synthesis of cultured neonatal rat astrocytes (RNB cells), abrogated the transcriptional activity of wild-type p53, conferred invasive activity, and subsequently induced the transformation from astrocytes to high-grade astrocytomas. Intriguingly, MDM2 enhanced the expression of angiogenic mitogens; basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) in RNB cells. These results indicate that MDM2 may play an important role in the progression of astrocytomas, by not only conferring invasive activity but also stimulating the expression of angiogenic growth factors.

Anti-sense oligonucleotide of p21(waf1/cip1) prevents interleukin 4-mediated elevation of p27(kip1) in low grade astrocytoma cells.

Elevation of the cyclin-dependent kinase (cdk) inhibitor, p27(kip1) is necessary for Interleukin (IL)-4-mediated growth arrest of human low grade astrocytoma (RTLGA) cells and occurs at 24 h of treatment. Pathways involved in IL4 alteration of p27(kip1) are unknown, however. Here we investigated whether other cdk inhibitors contributed to the actions of IL-4 on RTLGA cells. By 12 h of IL-4 treatment, both cdk4 and cdk2 kinase activities against the retinoblastoma protein (pRb) were reduced and nuclear entry of pRb was prohibited. Twelve-hour cdk complexes contained elevated p21(waf1/cip1) but not p27(kip1), p15(ink4B) or p16(ink4A). IL-4 increased p21(waf1/cip1) but not p27(kip1) mRNA levels, and stimulated luciferase activity of a p21(waf1/cip1) promoter-luciferase reporter. In p53-mutant WITG3 cells, IL-4 did not alter p21(waf1/cip1) mRNA and promoter-luciferase activity or p27(kipl) protein, suggesting a need for functional p53. STAT6 phosphorylation by IL-4, however, occurred in both p53-mutant WITG3 and p53-functional RTLGA cells. Pre-treatment of RTLGA with anti-sense but not missense p21(waf1/cip1) oligonucleotide prior to IL-4: (a) restored cdk activities; (b) reduced cdk4-associated p21(waf1/cip1) levels; (c) prevented p27(kipl) elevation; and (d) reversed growth arrest. These results are the first to suggest that p21(waf1/cip1) is essential for IL-4-mediated elevation of p27(kip) and growth arrest of astrocytoma cells.

WAF1/CIP1 increases the susceptibility of p53 non-functional malignant glioma cells to cisplatin-induced apoptosis.

Induction of apoptosis in tumor cells is an important determinant in the outcome of therapy. Molecular details of the apoptosis pathway, however, are still poorly defined. The recently discovered WAF1/CIP1 gene is a potent inhibitor of cyclin-dependent kinases and a mediator of tumor-suppressor p53-dependent apoptosis by DNA damage. In addition, WAF1/CIP1 expression is shown to be triggered through the p53-independent pathway. The relationship between WAF1/CIP1 and p53-independent apoptosis by DNA damage, however, remains unclear. In this study, we show that WAF1/CIP1 was induced in p53-dependent apoptosis of U87-MG glioma cells by cis-diamminedichloroplatinum (cisplatin), and overexpression of WAF1/CIP1 induced apoptosis in U87-MG cells without cisplatin treatment. In contrast, the p53-independent apoptosis of GB-1 glioma cells by cisplatin did not express WAF1/CIP1. Overexpression of WAF1/CIP1 inhibited DNA synthesis in GB-1 cells, but did not induce apoptosis. Interestingly, WAF1/CIP1 increased the susceptibility of GB-1 cells to cisplatin-induced apoptosis. These results suggest that overexpression of WAF1/CIP1 may have potential for the treatment of tumors with non-functional p53.

Human alveolar macrophage function: differences between smokers and nonsmokers.

Human alveolar macrophages and peripheral blood monocytes were obtained from smoking and nonsmoking normal volunteers. The macrophages and monocytes were incubated in vitro with bacterial lipopolysaccharide (LPS). The oxidative metabolic response of these cells was measured by superoxide anion production. Macrophages from smokers were suppressed in their superoxide anion response to LPS activation as compared to macrophages from nonsmokers. Monocytes from smokers and nonsmokers were not different. The cytotoxic properties of these macrophages and monocytes were assessed by an in vitro 3H-thymidine release assay against various allogeneic target cells. Macrophages and monocytes exposed to LPS were rendered tumoricidal. Macrophages from nonsmokers appeared to generate greater cytotoxic activity than macrophages from smokers. Macrophages from both smokers and nonsmokers were cytotoxic for three different tumorigenic cell lines but not for a nontumorigenic cell line. Monocytes from smokers and nonsmokers were not different in cytotoxic activity. We conclude that macrophages from both smokers and nonsmokers can be activated after exposure to LPS; however, macrophages from smokers may be slightly suppressed in their responses.

Activation of alveolar macrophage TNF and MCP-1 expression in vivo by a synthetic peptide of C-reactive protein.

Administration of multilamellar vesicles (MLV) encapsulating a synthetic peptide (RS-83277) derived from human C-reactive protein (CRP) augments anti-tumor activity of murine alveolar macrophages and reduces established pulmonary metastases of experimental tumors. To explore mechanisms involved in these phenomena, we investigated cytokine and integrin (CDllb) expression of bronchoalveolar lavage (BAL)-derived alveolar macrophages in control (blank MLV) and RS-83277-MLV-treated C57BI mice. Alveolar macrophage production of tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant bioactivity increased at 48 h after treatment with RS-83277-MLV but not control MLV. Chemoattractant activity was neutralized by antibody to monocyte chemoattractant protein-1 (MCP-1), but not irrelevant immunoglobulin G(IgG). Changes were reflected by augmented TNF-alpha and MCP-1 mRNA levels in pulmonary tissue and enhanced CD11b expression on mononuclear leukocytes derived from total lung tissue, but not on BAL-derived alveolar macrophages. Results suggest that RS-83277-MLV treatment is associated with activation of alveolar macrophage TNF-alpha and MCP-1 production and up-regulation of adhesion molecules on pulmonary mononuclear leukocytes but not on alveolar macrophages.

FADD gene therapy for malignant gliomas in vitro and in vivo.

Fas/APO-1 (CD95), a cell surface cytokine receptor, triggers apoptotic cell death by specific agonist antibody, suggesting that Fas/APO-1 may be a promising target for treatment of tumors. In this study, we show that treatment with anti-Fas antibody effectively induced apoptosis in malignant glioma cell lines with high expression of Fas/APO-1 (n = 3). Malignant glioma cells with low or undetectable expression of Fas/APO-1 (n = 6), however, were resistant to Fas/APO-1-dependent cytotoxicity. The purpose of this study, therefore, was to determine whether resistant tumors could be made susceptible to apoptosis. FADD/MORT1 constitutes a novel protein that associates specifically with the cytoplasmic death domain of Fas/APO-1 and induces apoptosis. We investigated whether overexpression of FADD would induce apoptosis in malignant glioma cells without activating Fas/APO-1. Results indicated that about 85% of malignant glioma cells, regardless of Fas/APO-1 expression levels, underwent apoptosis after transient transfection with FADD expression vector. To further improve gene transfer of FADD into malignant glioma cells, we constructed a retroviral vector containing the FADD gene. The retroviral transfer of FADD gene significantly enhanced the transduction efficiency and effectively inhibited both in vitro and in vivo survival of malignant glioma cells through induction of apoptosis. These findings suggest that the FADD gene is a novel and useful tool for the treatment of malignant gliomas.