Mebendazole therapy of parenteral trichinellosis.

Mebendazole was highly effective against the helminth parasite Trichinella spiralis in mice subjected to a 3-day course of treatment during the invasive and encystment phases of experimental trichinellosis. When treatment began either 2 or 4 weeks after the mice were inoculated with parasites, the number of larvae developing in the host musculature was greatly reduced by twice-daily oral administration of 3.125, 6.25, or 12.5 milligrams of mebendazole per kilogram of body weight.

Modulation of microRNA associated with ovarian cancer cells by genistein.

PURPOSE: Role of microRNAs in malignancies is well established due their regulatory role in cellular differentiation, proliferation and cell cycle control. Our purpose was to determine miRNA profiles of serially established ovarian cancer cell lines and the effect of genistein treatment. METHODS: Cell lines (UL-3A, UL-3B) were established from one patient during progression of disease. miRNA profiling was performed in untreated and genistein-treated cells. Estrogen receptors (ER) were studied with real-time polymerase chain reaction (RT-PCR) and Western immunoblotting. In vitro migration and invasion assays were utilized. RESULTS: While 108 miRNAs were expressed equally in both cell lines and their genistein-treated counterparts, an additional 53 miRNAs were differentially expressed. Genistein resulted in induction of ERalpha and ERbeta in ovarian cancer cells. A significant reduction in migration and invasion of UL-3A and UL-3B was demonstrated in genistein-treated cells. CONCLUSION: Common and unique miRNA profiles were demonstrated between the two cell lines, some of which were altered by genistein.

Inhibition of macrophage Ia antigen expression by shed plasma membrane vesicles from metastatic murine melanoma lines.

Shed plasma membrane-derived vesicles from metastatic variants of the murine B16 melanoma were examined for their ability to inhibit the induction of murine immune region-associated (Ia) antigen expression on macrophages, the initial step in the formation of an immune response. Membrane material that appears as a greater than 50 million-dalton fraction on column chromatography is found only in conditioned media from tumor cells and not in culture media from normal cells, such as murine 3T3 cells. Membrane vesicles from both metastatic variants B16-F1 (low lung colonizing) and B16-F10 (high lung colonizing) were taken up by macrophages; however, only membrane vesicles isolated from the B16-F10 cultures exhibited significant inhibitory activity for Ia induction. This inhibition appears to result from enhanced prostaglandin synthesis, since treatment with aspirin can reverse the membrane vesicle-induced inhibition. The inhibitory component(s) released into the media was demonstrated to be predominantly associated with membrane vesicles; however, the component(s) retained its activity after Triton X-100 treatment, indicating that the intact membrane vesicle was not necessary for the action of the inhibitory material. Treatments with heat (65 degrees C) and proteases (papain) indicated that the inhibitory component(s) is a heat-labile protein.

Suppression of wound healing in tumor bearing animals as a model for tumor-host interaction: mechanism of suppression.

Tumor wound healing was explored as a possible model for tumor-host interactions. Wound healing within tumors progressed normally through the hemorrhagic and inflammatory stages but failed at the mesenchymal ingrowth phase. Due to this failure of mesenchymal ingrowth, no significant collagen deposition could be detected within tumor wounds. Fluid collected from tumor wounds markedly altered fibroblast cytoskeletal structures and profoundly inhibited fibroblast proliferation and collagen synthesis. This suppression did not appear to be the direct consequence of tumor products, since tumor conditioned media enhanced fibroblast proliferation and had no effects on collagen synthesis and fibroblast cytoskeleton. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysed fibroblasts demonstrated that two polypeptides (Mr 280,000 and 240,000) were induced in or adherent to fibroblasts exposed to fluid from the tumor wound but not in fibroblasts exposed to fluid obtained from wounds in normal tissue or tumor conditioned media. These findings suggest that tumor wound healing is a model for mesenchymal inhibition within tumors but that the inhibitors are not tumor derived products.

Binding of specific peroxidase-labeled antibody to placental-type phosphatase on tumor-derived membrane fragments.

Monospecific antiserum to human placental alkaline phosphatase was purified by immunoabsorption and labeled with horseradish peroxidase. The binding of this labeled antibody to membrane fragments prepared from placental and tumor tissue was measured using agarose gel filtration to separate bound antibody. The antibody bound only to membrane fragments which contained placental phosphatase, and the amount bound varied in the order ascitic fluid membrane fragments greater than tumor extracts greater than placental extracts. Absorption of the antibody with crude placental membrane yielded a population of antibody which reacted with tumor tissue and pure placental enzyme, but only slightly with placental membranes. These results are interpreted to suggest that some antigenic sites are exposed in tumor tissue membranes which are not in placental membranes.

Identification of a human tumor-derived lipolysis-promoting factor.

Our previous studies have demonstrated the production and release of a tumor-derived factor that promoted lipolysis in normal adipocytes. We further demonstrated that this in vitro lipolysis was correlated with the in vivo loss of total carcass lipids induced by the presence of the same tumor. This study identified and isolated this "lipolysis-promoting" factor (LPF), released into the extracellular environment (conditioned media) by the human A375 melanoma cell line, which appears to be responsible for the previously demonstrated induction of in vitro and in vivo lipolytic activity. Unlike previously described non-tumor-derived molecules, such as tumor necrosis factor-alpha/cachectin, which have been implicated in cancer cachexia, the LPF induces alterations in lipid metabolism similar to those observed in cancer patients. The biochemical nature of human tumor-derived LPF appears to be a heat-stable molecule with an apparent molecular weight of approximately 6000. The lipolysis-promoting activity was trichloroacetic acid precipitable, but not precipitable with protamine sulfate or extractable with chloroform:methanol. Its activity appears to be resistant to enzymatic treatments with protease K, trypsin, Pronase, RNase, and DNase, as well as to periodate oxidation. Immunochemically, LPF appears to be distinct from tumor necrosis factor-alpha/cachectin. Furthermore, in contrast to the mechanism of action of tumor necrosis factor-alpha/cachectin, the mechanism of "lipolysis promotion" by LPF appears to be by the induction of cellular lipase activity.

Humoral immune responses to cathepsin D and glucose-regulated protein 78 in ovarian cancer patients.

Many cancer patients develop tumor-reactive immune responses against antigens that are either expressed on the surface of tumor cells or released from them into the peripheral circulation. In this study, tumor-reactive immunoglobulins, present in the sera of ovarian cancer patients, were used to identify commonly recognized tumor-associated antigens on ovarian tumor cells. Western immunoblot analysis of cellular proteins, obtained from UL-1 ovarian tumor cell line, demonstrated several commonly recognized immunoreactive proteins. Two of these proteins (Mr 32,000 and 71,000) were selected for further investigation. Cellular proteins isolated from normal human ovarian epithelia, in a similar fashion, failed to exhibit corresponding immunoreactivity to these proteins. As an additional control, sera from normal (nontumor-bearing) individuals failed to identify these proteins on Western immunoblots. Furthermore, the absorption of the ovarian cancer patients' sera with normal ovarian epithelial tissue did not remove the reactivity of these two proteins. The Mr 32,000 and 71,000 proteins were subsequently purified by reverse-phase high-performance liquid chromatography, separated by SDS-PAGE, transferred to the polyvinylidene difluoride membrane, and digested with trypsin. These resulting tryptic fragments were separated by microbore reverse-phase high-performance liquid chromatography, and selected fragments were sequenced by mass spectrometry. This sequence analysis identified the Mr 32,000 protein as cathepsin D and the Mr 71,000 as glucose-regulated protein 78 (member of the heat shock protein family). The identities of cathepsin D and glucose-regulated protein 78 were confirmed by Western blot analysis. Additionally, the presence of cathepsin D was demonstrated in association with immune complexes in vivo. Currently, the common antigenic epitopes of these proteins are being defined.

Aerosolized gamma-interferon and lipopolysaccharide enhances cytotoxicity of murine pulmonary alveolar macrophages.

In vivo stimulation of pulmonary alveolar macrophages (PAMs) may enhance tumor cell cytotoxicity. A model using aerosolized gamma-interferon (gamma-IFN) and lipopolysaccharide (LPS) was developed to induce enhanced PAM activation in vivo in C57BL/6 mice. Mice received four doses of aerosol (2 doses/day) consisting of gamma-IFN (10(4) microU/mouse) and LPS (100 micrograms/mouse). Other groups received either gamma-IFN alone, LPS alone, or saline (control). Cells were harvested by bronchoalveolar lavage. Macrophage cell count demonstrated an increase in macrophage recruitment in the gamma-IFN and LPS group. PAMs were evaluated for in vitro cytotoxicity against B16-F10 melanoma cells. Treatment groups demonstrated enhanced cytotoxicity over controls, and the combination (gamma-IFN plus LPS) was significantly better in cell killing than either treatment modality alone (p less than or equal to 0.02). Activated PAMs selectively killed tumor cells, but did not kill the 3T3 fibroblast cell line. Peritoneal macrophages from mice treated by inhalational gamma-IFN + LPS were enhanced (indicating a systemic effect), but not to the same extent as PAMs. These studies suggest that inhalation of gamma-IFN + LPS can selectively enhance in vivo cytotoxicity of murine PAMs. This may potentially be applicable to human tumor management.

Clonal heterogeneity of p53 mutations in ovarian cancer.

We analyzed clonal populations of ovarian cancer cells for heterogeneity in p53 mutations (exons 4-9) and chemosensitivity. UL-3A cells were developed from a patient with stage IIIC ovarian adenocarcinoma. Heterogeneity in p53 mutations was demonstrated, ranging from point mutations to deletions in exons 4, 6 and 7. UL-3A cells contained two point mutations, in codon 248 of exon 7 and in codon 76 of exon 4. Five groups of clones were identified according to the p53 mutations. UL-3A clones with low p53 levels were more sensitive to CDDP (LD50 <8.0 microg/ml). Heterogeneity of p53 mutations may provide growth advantage during disease progression or chemotherapy.

Taxol-induced bcl-2 phosphorylation in ovarian cancer cell monolayer and spheroids.

Expression of Bcl-2, Bax, p53 and induction of apoptosis were studied in cisplatin or Taxol treated monolayer and spheroid cultures of ovarian cancer cell lines (SKOV-3, UL-1, UL-3C). While cisplatin (15-75 microg/ml) induced apoptosis in monolayer and spheroid cultures, Taxol (100-800 nM) induced fragmentation in monolayers only. Cisplatin induced up to 5-fold DNA fragmentation in monolayers, while 3-fold (UL-3C, SKOV-3), and 1.5-fold (UL-1) in spheroids. Taxol treatment of monolayers resulted in the characteristic phosphorylation of Bcl-2, which was not demonstrated in spheroid cultures. Bax expression was reduced in spheroids following cisplatin or Taxol treatment, while p53 levels remained unchanged.

Effects of retinoic acid on lipolytic activity of tumor cells.

Tumor-producing substances that promote lipolysis in vitro may also account for fat mobilization in cachectic cancer patients. Cachexia might improve if this lipolytic action of cancer cells could be halted. This study examined the lipolytic activities of media from four tumor cell lines after treatment with retinoic acid (RA), a cell differentiation inducer. An in vitro adipocyte bioassay measured lipolysis. All four tumor cell lines were intrinsically lipolytic, with elevated baseline lipolytic activities relative to fibroblast-conditioned controls (128% to 287% of control, p less than 0.05). After a 2-week exposure to RA in culture medium followed by 3 days of continued growth in fresh medium, two of four cell lines (both rat prostatic adenocarcinomas) showed significantly reduced lipolytic activities (16% and 61% of corresponding untreated controls, p less than 0.05). These reductions in lipolytic activity after RA treatment were not generalized phenomena; nor were they simply caused by cell differentiation, as the other cell lines (human malignant melanoma and human ovarian teratocarcinoma) showed no reductions despite evidence of cell differentiation. No effect on lipolytic activity was seen after only a 24-hour exposure to RA. We conclude that RA can affect the lipolytic activity of certain tumor cells in vitro, perhaps by influencing tumor-producing lipolytic factor(s).

Altered cord serum lipid levels associated with small for gestational age infants.

OBJECTIVE: To determine whether small for gestational age (SGA) infants show changes in lipid metabolism that could distinguish growth-restricted subpopulations. METHODS: Sera from the arterial cord blood from 38 SGA infants were analyzed for apolipoprotein A-I level, total lipid content, and distribution of those lipids as triglycerides, diglycerides, free fatty acids, and phospholipids. Comparisons were made between appropriate for gestational age (AGA) controls (n = 25), SGA infants with a ponderal index below the tenth percentile (SGA I, n = 20), and SGA infants with a ponderal index above the tenth percentile (SGA II, n = 18). RESULTS: Total cord serum lipid content was markedly decreased in all SGA infants compared with AGA infants (2.8 times lower). Although SGA infants showed total lipid concentration decreases, SGA I and SGA II infants showed distinct characteristics. Infants in the SGA I group had higher triglyceride levels (1.8 times higher) and lower free fatty acid levels (1.4 times lower), compared with AGA infants (P < .001). The lipid subclass distribution in SGA II infants was not significantly different from that in AGA infants, with the exception of an increase in triglyceride concentrations (1.3 times higher). Although the 22-kD placenta-derived apolipoprotein A-I was similar in all groups, the level of fetal liver-derived 28-kD apolipoprotein A-I was 6.5 times lower in SGA I infants than in AGA or SGA II infants (P < .001). CONCLUSION: The SGA I infants appeared to have impaired utilization of circulating triglycerides, consistent with peripheral adipose depletion. Diminished fetus-derived apolipoprotein A-I levels with normal levels of placenta-derived apolipoprotein A-I levels might indicate a defect in the production or secretion of apolipoproteins associated with growth restriction.

Lymphokine-activated killer cell suppressor factor in malignant effusions.

We examined the possibility that tumor-released products inhibit lymphokine-activated killer cell activation. Lymphokine-activated killer cells from human peripheral blood lymphocytes were activated with recombinant interleukin 2 for 4 days in the presence of malignant effusions or conditioned media from cultured cell lines (10% vol/vol). Eight of 10 malignant effusions/media suppressed the induction of lymphokine-activated killer cell cytotoxicity, as measured in a 4-hour sodium chromate release assay. Seven of 10 effusions/media inhibited lymphokine-activated killer cell proliferation. Suppression was both dose and time dependent. A representative suppressive effusion was fractionated by agarose gel chromatography, treated with detergents disruptive of ionic bonds and lipids, and refractionated using polyacrylamide gel chromatography. Seven suppressive fractions ranging in molecular weight from 1 x 10(5) to 3 x 10(5) d were isolated. It is speculated that this suppressor factor may represent a large multimeric structure with ionic-bonded individual suppressive components.

Alterations in humoral immune responses associated with recurrent pregnancy loss.

OBJECTIVE: To investigate the reactivity of maternal antibodies with endometrium-derived antigens and to correlate their association with recurrent pregnancy loss (RPL). DESIGN: Prevalence study. SETTING: Academic research center. PATIENT(S): Nulliparous women (n = 10), women with RPL (n = 15), pregnant women (n = 8), and multiparous women with a normal obstetric history (n = 20). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Reactive antibodies were analyzed by Western immunoblot techniques and quantitated by densitometry. RESULT(S): Antibodies from women with RPL and multiparous women recognized antigens ranging from 10-120 kd on normal endometrium and endometrial tumors. Antibodies from most women with RPL (10/15) and from multiparous women (15/20) recognized 65-kd and 80-kd proteins in normal endometrium. Antibodies from women with RPL recognized 21-kd and 28-kd antigens (12/15 and 13/15, respectively) in endometrial tumors at a significantly greater rate (than did antibodies from multiparous women (5/20 and 8/20, respectively). Women with RPL had significantly lower levels of asymmetric IgG compared with controls. CONCLUSION(S): Recurrent pregnancy loss may be linked with the failure to elicit asymmetric IgG and a unique immunologic recognition of endometrial antigens.

Expression and shedding of CD44 variant isoforms in patients with gynecologic malignancies.

OBJECTIVES: The presence of CD44 isoforms was evaluated in ascitic fluid and serum samples of patients with gynecologic malignancies. Previously, the shedding of tumor-associated cell surface antigens has been demonstrated in the blood and malignant effusions of gynecologic cancer patients. Thus, the shedding of CD44 was also studied in ascitic fluids and sera of these patients, to address variant isoform expression as a biomarker of gynecologic cancer. METHODS: The expression of CD44 isoforms by ovarian tumor cells was examined by flow cytometry using variant-specific monoclonal antibodies. The release of these isoforms into the peripheral circulation and ascites was assayed by Western immunoblot analysis. RESULTS: Flow cytometric analysis of ovarian tumor cell lines revealed a strong expression of CD44 with significant levels of v4/5 and v6 isoforms. The presence of circulating CD44 isoforms was detectable in the sera of six of eight cancer patients, as well as in 12 of 16 ascitic fluids. Of the CD44-positive specimens, all six positive sera expressed detectable levels of variant CD44. The CD44v6 was present in all of the positive sera samples tested. In the ascites, the "shed" CD44 appeared to be associated predominantly with shed particles (vesicles) of plasma membranes (membrane fragments). Of ten CD44-positive ascites samples, all expressed significant levels of variant CD44. CONCLUSIONS: In addition to mediating metastasis, the differential expression and shedding of CD44 isoforms into the circulation may represent important determinants in the escape of tumors from immune surveillance, and their detection may be a diagnostic or prognostic marker.

Inhibition of endometrial cancer cell lines by mifepristone (RU 486).

OBJECTIVE: To determine the role of mifepristone (RU 486) in the growth of endometrial cancer cell lines, and the mechanism associated with this regulation. METHODS: Three endometrial cancer cell lines (Hec-1A, KLE, and RL95-2) were used in this study. Growth inhibition was demonstrated by sulforhodamine B cytotoxicity assay. Mode of inhibition by RU 486 was studied by induction of DNA fragmentation. The effect of RU 486 on steady-state accumulation of the progesterone and glucocorticoid receptors (PRs and GRs, respectively) and apoptosis-associated gene products was studied by Western blotting. RESULTS: We demonstrated a dose-dependent inhibition of growth in all of the three endometrial cancer cell lines. Following treatment with 5.0 micrograms/mL of RU 486, there was 39.3%, 66.3%, and 75.5% inhibition of KLE, Hec-1A, and RL95-2 cells, respectively. Decreased expression of GR in RL95-2 (0.1-10 micrograms/mL) and in KLE cells (10 micrograms/mL) was observed. A marked decrease of PR was seen with RL95-2 cells at 10 micrograms/mL, there was no change in the KLE cells, and a dose-dependent decrease was seen with Hec-1A cells. Various levels of apoptosis were demonstrated by DNA fragmentation in all three cell lines. Of the genes associated with apoptosis, dose-dependent reduction of bax expression was demonstrated in KLE cells, while induction of WAF-1 was seen in Hec-1A and RL95-2 cells, and reduction of bcl-2 was demonstrated in RL95-2 cells. CONCLUSION: Clinically achievable doses of RU 486 inhibit endometrial cancer cell lines. The mechanism of inhibition involves apoptosis, and regulation of bax, bcl-2, and WAF-1 is demonstrated. Therapeutic application of these findings remains to be determined.

Induction of immune responses to ovarian tumor antigens by multiparity.

OBJECTIVE: Because epidemiologic data indicate a reduction in ovarian cancer risk with increased parity, the occurrence of maternal immunization against ovarian tumor-associated antigens during pregnancy was investigated. METHODS: Sera were obtained from nulligravid and multiparous women and from men. Cellular proteins were isolated from four ovarian tumor cell lines as well as from normal ovaries. These proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of cellular proteins reactive with each individual's serum was assessed by Western immunoblot. Tumor-reactive antibodies from two multiparous women were used to prepare immunoaffinity columns for the isolation of reactive proteins from ovarian tumor cells. These immunoaffinity-purified antigens were transferred electrophoretically to nitrocellulose membranes, stained with Ponceau S, and identified by amino acid sequencing. RESULTS: Western immunoblot analysis of the cellular proteins from four established ovarian tumor cell lines using sera from multiparous women as the primary antibody indicated that these samples recognized multiple bands on ovarian tumors, ranging from 30 to 150 kD. Two commonly recognized proteins were isolated and subjected to microsequencing, which identified the 56-kD band protein as elongation factor-1 alpha and the 38-kD protein as nucleophosmin/B23 protein. Both of these proteins play integral roles in cell growth. CONCLUSION: These findings suggest that certain antigens expressed by the fetus immunize women during pregnancy. This immune response may protect these women from the subsequent development of cancer.

Tumor-reactive immunoglobulins in ovarian cancer: diagnostic and therapeutic significance? (review).

Inhibition of the immune system has been observed in association with most stages of ovarian cancer; however, the mechanisms involved in the induction and maintenance of this chronic immune unresponsiveness associated with cancer progression are poorly understood. This immunosuppressed state is primarily defined as the failure to eradicate the tumor. This immunosuppressed state is generally associated with decreased numbers and reactivity of lymphoid cells in women with ovarian cancer. The degree of immune dysfunction in ovarian cancer patients has been demonstrated to correlate with patient survival. While ovarian cancer patients generally fail to exhibit effective immunosurveillance, as manifested by continued tumor growth and progression, the presence of tumor-reactive immunoglobulins can be demonstrated in these women, indicating the continued presence of immune recognition. We have not only demonstrated the presence of tumor-reactive antibodies in ovarian cancer patients, but have also shown that the levels of these antibodies increase as the disease progresses. The antigens recognized by the patients' humoral response have been identified as either membrane-associated or intra-cellular. In general, the localization of these antigens tend to be linked to the patient's prognosis. The presence of a humoral response against intracellular proteins are correlated with poor prognosis, while autoantibodies reactive with surface components appear to have a better prognosis. In addition to general antigen recognition, these reactive antibodies have been utilized to define specific epitopes on tumor-associated proteins. Certain specific antigenic epitopes exhibit common recognition among patients with the same tumor type. The specific recognition of certain epitopes can provide early evidence of aberrant protein expression and this aberrant expression of certain proteins, such as procathepsin D, appear to be linked to the tumor's acquisition of specific malignant characteristics, including metastasis formation and chemoresistance. Despite the existence of circulating tumor-reactive immunoglobulins, their presence correlates, in general, with poor prognosis and poor host survival. Since tumor-reactive immunoglobulins are elicited and can be detected early in the development of tumors and their enhanced synthesis is induced prior to the clinical manifestation of recurrence, the assessment of the tumor-reactive immune response against specific antigenic epitopes should represent an early significant diagnostic and prognostic marker in ovarian cancer.

Evaluation of cell proliferation and cell death based assays in chemosensitivity testing.

BACKGROUND: Drug sensitivity testing (DST) is used to predict the clinical response to chemotherapy with limited success. Our objective was to evaluate assays that measure cell proliferation or apoptosis in determining sensitivity of ovarian cancer cells to cisplatin and paclitaxel. MATERIALS AND METHODS: Four ovarian cancer lines were used. LD10-LD90 doses were determined by viability assays. Assays measuring cell proliferation, sulforhodamine-B (SRB), tritiated thymidine; and cell death, diphenylamine or DNA histone ELISA were compared. RESULTS: SRB assay was consistent and sensitive. Histone ELISA correlated with the viability assay at high doses. The [3H] thymidine test was not sensitive and resulted in false positive responses. While less sensitive, detection of apoptosis by diphenylamine assay shows a similar trend to histone ELISA. CONCLUSIONS: We demonstrate significant differences between the various assays. Most of these assays are better predictors of resistance. Further studies are needed to determine the best correlation between in vitro testing and responses in vivo.

Microsomal activation to mutagens of antischistosomal methyl thioxanthenones and initial tests on a possibly non-mutagenic analogue.

Five methylthioxanthenone and methylbenzothiopyranoindazole analogues, including lucanthone (Miracil D), are non-mutagenic for Salmonella typhimurium but are activated to mutagens by a rat liver microsome preparation. Hydroxymethyl analogues, including hycathone (Etrenol), are mutagenic in the absence of microsomes. It seems reasonable to assume that the hydroxymethyl derivatives are the more proximal mutagens and that Salmonella is unable to carry out the hydroxylation necessary for mutagen activation. During the pase 24 years, several million patients with schistosomiasis have been treated with lucanthone, and in recent years about 700 000 persons with hycanthone. The possible long-term deleterious effects of these agents for man even now remain to be determined. Our studies indicate that particular modifications in the structure of thioxanthenones drastically alter their mutagenicity. One apparently non-mutagenic thioxanthenone has been found. A number of the less mutagenic compounds also exhibit decreased acute toxicity in the mouse while retaining appreciable antischistosomal activity, suggesting that genetic and schistosomicidal activities may be dissociated from each other.