Effects of Malignant Effusions on the Mitotic Index of L Strain Mouse Cells Grown in Tissue Culture

By employing a clone of L strain mouse fibroblasts (L_E) which does not exhibit cell clumping and lysis (cytolytic antibody reaction), it was possible to screen for the presence of growth-regulating factors in human sera and effusions, exclusive of an antigen-antibody reaction. Under conditions of the test a mitotic index greater than 20% indicated the presence of a growth-promoting factor.A total of 11 pleural effusions was tested. Four of the eight malignant effusions possessed a growth-promoting factor, while none of the three non-malignant effusions or the one sample of human umbilical cord serum possessed such a factor. Overnight storage of the unfiltered effusions at 5° C. resulted in complete loss of the growthpromoting activity.     

The Effect of Nouaual on Dipodascus aggregatus

The growth of a strain of Dipodascus aggrrgatus Francke-Grosmann was strongly promoted by the aliphatic aldehyde nonanal. The highest effect was found with 80–160 μmol of nonanal per 1. The growth-promoting activity of nonanal is principally the result of an ability to shorten the lag phase. Neither the maximum value for growth nor the growth rate seem to be increased. The growth-promoting activity of nonanal could be observed only if the cells used for inoculation were taken from a culture in the phase of accelerated growth. The highest growth-promoting activity was observed when the nonanal was added before inoculation, a large effect was still observed when it was added 24 hours after inoculation, but there was no effect when it was added 33 hours after inoculation. The growth-promoting activity of nonanal remained unchanged when a mixture of 15 vitamins and growth factors was given to the medium. Nonanol and nonanoic acid stimulated growth, although to a lower degree than nonanal. There was a gradual increase in the growth-promoting effect of nonanal as the pH of the medium was increased between 3.0 and 8.0, showing that this effect is most pronounced at the higher pH values.     

Weak telomerase activity in malignant cells in metastatic serous effusions: correlation to short survival time

OBJECTIVE: The purpose of this study was to evaluate whether the intensity of telomerase activity measured on the cellular level in effusions correlates to survival time in cases of metastatic spread to the serous cavities. STUDY DESIGN: Telomere repeat amplification protocol (TRAP) in situ was applied to 46 effusions containing metastatic cancer cells. RESULTS: Weak telomerase activity in tumor cells was seen in 7 cases, and moderate or strong activity in 39 cases. The intensity of the enzyme activity correlated significantly to survival (Kaplan-Meier survival analysis), the median survival time being 18 days for patients with weakly positive tumors and 100 days for patients with moderately or strongly positive tumors (Kruskal-Wallis test, p = 0.021). CONCLUSION: The strong relationship between telomerase activity in tumor cells in effusions and survival time has never been described before. Determination of telomerase activity on the cellular level provides a way to identify a subgroup of patients with a high probability of short survival.     

Suppressor cells for natural killer activity in carcinomatous pleural effusions of cancer patients

Summary Carcinomatous pleural effusions of 25 of 32 patients with lung cancer, which had markedly low or no natural killer (NK) activity against K562 cells in a 4 h chromium release assay, contained cells capable of suppressing the lytic function of blood NK cells from normal donors and cancer patients. Suppressor cells were found to be Sephadex G-10- and serum coated plastic dish-adherent monocyte/macrophages in 21 of 25 patients and nylon wool-nonadherent lymphocytes in the other four cases. Nonmalignant pleural effusions did not contain any type of suppressor cells. Twenty-four-hour preincubation of suppressor cells with effector cells was required for mediation of the suppressor function. Neither culture supernatants of effusion cells and NK cells nor effusion supernatants suppressed NK activity. The presence of indomethacin during the preincubation and cytotoxicity assay did not abrogate suppressor function. Suppressor cells did not reduce the number of lymphocyte/K562 conjugates. Contaminating tumor cells were not responsible for the suppression of cytotoxic activity. NK cells precultured with suppressor cells were not able to show cytotoxic function even after removal of the suppressor cells. When effusion mononuclear cells were passed through a Sephadex G-10 column and then preincubated for 24 h, these cells showed a significant increase in NK activity. The results suggest that carcinomatous pleural effusions contain at least two classes of suppressor cells for NK activity, monocyte/macrophages, and nylon wool-nonadherent lymphocytes, which could be one of the causes of impaired NK activity in carcinomatous pleural effusions.     

Natural killer cells in carcinomatous pleural effusions

Summary Effector cells in carcinomatous pleural effusions of patients with primary or secondary lung cancer were examined for natural killer (NK) activity against K562 cells in a 4-h chromium release assay, and for mitogenic responses and lymphocyte subpopulation constitutions. NK activity of lymphocyte-rich mononuclear cells isolated from carcinomatous pleural effusions by centrifugation on a discontinuous gradient of Ficoll-Hypaque was markedly low in seven of 40 patients studied, and absent in the other 33 cases. NK activity of peripheral blood mononuclear cells from the patients was lower than that of cells from normal donors, but always higher than that of effusion cells from the same patients. NK cells in the peripheral blood and in pleural effusions had some characteristics in common, in that they lacked a capacity to bind sheep erythrocytes, were nonadherent to Sephadex G-10 beads and nylon wool, and belonged to large granular lymphocytes. On the other hand, nonmalignant effusions of patients with congestive heart failure had significant NK activity. The effector cells in the effusions included a higher frequency of T cells than those in the peripheral blood of the same patients. Proliferative responses to phytohemagglutinin and concanavalin A of effusion cells were comparable to those of normal blood cells and were higher than those of blood cells from the same patients. The reason for low NK activity and high mitogenic response in carcinomatous pleural effusions is as yet undefined.     

Effect of Nocardia rubra cell wall skeleton on augmentation of cytotoxicity function in human pleural macrophages

Summary The ability of Nocardia rubra cell wall skeleton (N-CWS) to augment macrophage cytotoxicity function was examined using human pleural macrophages prepared from 32 malignant pleural effusions and 53 pleural washings. The cytostatic activity of pleural macrophages for human lung cancer cells (PC-9) was augmented following incubation of pleural mononuclear cells with 10 g/ml N-CWS for 24 h. Macrophage activity was increased by direct interaction of macrophages with N-CWS or by incubation of macrophages with supernatant culture fluids from pleural lymphocytes with N-CWS. The cytotoxic potential of the pleural macrophages obtained from patients treated with 500 g of N-CWS intrapleurally was also increased. The heat and acid stability studies revealed that the culture fluids from pleural lymphocytes treated with N-CWS contained macrophage activation factor in addition to interferon-. These results suggest that direct and indirect macrophage activation is part of the mechanism in which N-CWS has a clinical effect on malignant pleural effusions.     

MN/CA9: a potential gene marker for detection of malignant cells in effusions

Many cancers cause malignant effusions. The presence of malignant cells in effusions has implications in diagnosis, tumour staging and prognosis. The detection of malignant cells currently presents a challenge for cytopathologists. New adjunctive methods are needed. Although the effusions provide excellent materials for molecular assay, the available molecular markers are extremely limited, which hinders its clinical application. MN/CA9 has proved to be a valuable marker in many cancers such as lung, breast, colon, kidney, etc. The present study was to evaluate MN/CA9 as a new molecular marker for the detection of cancer cells in pleural effusions. Seventy-one pleural effusions including 59 malignant effusions from patients with cancer, and 12 patients with benign diseases as a control, were subjected to RT-PCR for detection of MN/CA9 gene expression. MN/CA9 gene expression was detected in 53/59 (89.8%) pleural effusions from cancer patients (15/16 for breast cancers, 10/11 for lung cancers, 4/4 for ovary cancers, 2/3 for colon–rectal cancers, 5/6 for cancers of unknown site, 7/8 for mesothelioma and 10/11 for other cancers). Furthermore, MN/CA9 was positive in 13/18 (72.2%) of cytologically negative effusions of cancer patients. MN/CA9 was detected in only 1/12 (8.3%) effusions from the control patients (p<0.01). The sensitivity and specificity of MN/CA9 gene expression were, respectively, 89.8% and 91.7%. Our preliminary results suggest that MN/CA9 could be a potential marker for the detection of malignant cells in effusions. A large-scale study is needed to confirm these results.     

Effects of lithium on thrombopoiesis in patients with low platelet cell counts following chemotherapy or radiotherapy

Therapy for neoplasma is limited by hematological side effects of tumor-destructive therapy and, in part, makes expensive supportive care necessary to overcome and treat leukopenia and thrombocytopenia and their consequences. Thrombocytopenia is a major clinical problem caused by chemotherapy and radiotherapy. An effective and very cost-effective option for treating moderate neutropenia is the administration of lithium carbonate. Lithium induces the release of colony-stimulating factors (CSF) and therefore stimulates proliferation of neutrophil granulocytes. Other cytokines, such as interleukin-1 (IL-1), IL-6, and tumor-necrosis factor-α (TNF-α), are also stimulated. Apart from granulocyte-macrophage-CSF (GM-CSF), there have as yet been no reports of lithium salts inducing early activating factors for the megakaryocytic lineage, such as IL-3, IL-11, stem cell factor and flt-3 ligand, or maturation factors, such as thrombopoietin (TPO). A statistically significant increase in the mean number of platelets for patients with cell counts below 150,000/μL on the commencement of treatment with lithium carbonate could be observed. Patient tolerability of lithium carbonate therapy is very good. Patients with persistent leukopenia and thrombocytopenia following chemotherapy or radiotherapy can be treated with this trace element very cost-effectively. Unfortunately this treatment has not gained acceptance in clinical oncology in the face of extremely cost-intensive treatment with recombinant GM-CSF, IL-11 or, potentially, thrombopoietin.     

Determination of serum and plasma erythropoietin in mice with phenylhydrazine-induced anemia

The simple, specific and sensitive erythropoietin bioassay in serum and plasma from phenylhydrazine treated mice is described, based on H3-thymidine incorporation into divided hemopoietic cells. Spleen cells taken from mice on the third day following the second of 2 daily injections of phenylhydrazine were cultured in 24 hours in the presence of test material. Following incubation for 2 hours with H3-thymidine solution radioactivity was measured.     

Demonstration of competence and progression activities for human fibroblasts

Human embryonic lung fibroblasts (HEL) completed 4.5 population doublings in 6 days when maintained in DMEM supplemented with 10% human whole blood serum (WBS), plasma-derived serum (PDS) or defibrinogenated plasma containing 10 mM CaCl₂. Plasma in the absence of additional calcium promoted less growth. Sera and plasma chromatographed through carboxymethyl Sephadex (CMS) supported only one population doubling. Increased growth resulting in three doublings was observed in CMS-treated WBS or PDS supplemented with commercially prepared platelet-derived growth factor (PDGF). The magnitude of this PDGF response was dependent on serum concentration. A significant increase in the proportion of cells incorporating [³H]thymidine was observed in confluent cultures exposed to PDGF prior to incubation in WBS-CMS or PDS-CMS indicating competence and progression activities for human fibroblasts. In contrast, cells maintained in the presence of plasma-CMS failed to grow in response to PDGF. Factors bound to CMS columns restored growth-promoting activity to PDGF-supplemented WBS-CMS, PDS-CMS and plasma-CMS. However, growth-promoting CMS-bound components from plasma were lost during dialysis through membranes excluding materials above 12000 MW.     

Intrapleural administration of OK432 in cancer patients: Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes

Summary Ten patients with carcinomatous pleural effusions were treated with single intrapleural (i.pl.) injections of the streptococcal preparation OK432 on day 0 and the effects of i.pl. OK432 on the lysis of fresh or cryopreserved autologous tumor cells isolated from the pleural effusions were observed on day 7. In eight patients tumor cells in the effusions had decreased or disappeared by day 7. The other two patients, however, had no clinical evidence of therapeutic benefit from i.pl. OK432. Effusion tumor cells were relatively resistant to lysis by autologous lymphocytes when tested in a 4-h ⁵¹Cr-release assay. Positive reactions were recorded for blood and effusion lymphocytes in two of ten untreated patients. Injection of OK432 i.pl. resulted in an induction or augmentation of cytotoxicity against autologous tumor cells and K562 in the effusions of seven of ten subjects by day 7. In contrast, autologous tumor killing activity of blood lymphocytes was not always modified by i.pl. OK432. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation enriched autologous tumor killing activity, with no reactivity in LGL-depleted, small T lymphocytes. Significant lysis of autologous tumor cells was observed with effusion LGL from seven of ten untreated patients. Seven days after i.pl. OK432 injection, effusion LGL expressed enhanced cytotoxicity against autologous effusion tumor cells, whereas T cells were still not cytotoxic to autologous tumor cells on day 7. The frequency of LGL among effusion lymphocytes was not altered by i. pl. OK432. Adherent effusion cells were not involved in lysis of autologous effusion tumor cells in either untreated or OK432-treated patients. In vitro treatment of blood and effusion lymphocytes with OK432 induced an enhancement of autologous tumor-killing activity in patients who subsequently responded to i.pl. OK432 treatment. OK432 augmented in vitro autologous tumor killing activity of LGL, whereas T cells failed to lyse autologous tumor cells even after in vitro activation with OK432. These results indicate that i.pl. administration of OK432 to cancer patients will result in an augmentation of autologous tumor killing activity of LGL in the pleural effusions, and that this could be responsible for the antitumor activity of i.pl. OK432 therapy.     

The stimulation by epidermal growth factor (Urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones

Epidermal growth factor (EGF) added in a single dose (between 10^–16 and 1.7 ± 10^–9M) to neonatal rat hepatocytes in primary culture with subsequent incubation for 12 and 24 hours in Eagle's MEM fortified with 10% (v/v) FBS stimulated their entry into S and M phases, as shown by (³H)thymidine labeling and autoradiography and by a 1-hour exposure to colchicine (0.1 mM). Growth stimulation by EGF was detectable after 4 hours, peaking between 12 and 16 hours, and thereafter declining in intensity. Rat hepatocytes exposed for 72 hours (between the fourth and the seventh day in vitro) to no serum or to 10% fresh FBS possessed similar growth rates and absolute numbers in the cultures. A 24-hour exposure to 20 to 50% FBS stimulated hepatocytic DNA synthesis and mitotic activity and resulted (except for the 50% FBS treatment) in increased hepatocytes' numbers, which were relatively greater than the concurrent increases in connective tissue cell numbers. In serum-devoid medium EGF (10^–11M) enhanced hepatocytic mitotic, but not DNA-synthetic activity. To be fully effective EGF required a 10% FBS addition to the medium, then eliciting within 24 hours a marked increase in hepatocytes' number with respect to cultures incubated with 10% serum only. When associated with 20 to 30% FBS, EGF stimulated parenchymal cell growth at rates slightly higher, but not significantly different, than those elicited by the same serum concentrations alone. However, when used in conjunction with 10 to 30% FBS, EGF preferentially increased the number of hepatocytes rather than that of non-parenchymal cells. Moreover, comparative proliferation kinetic studies showed that in the presence of 10% FBS, an equimolar (10^–14M) mixture of EGF, insulin, and glucagon promoted an early and marked increase in the DNA-synthetic and mitotic activities of hepatocytes, which peaked after 8 hours. Within a 24-hour time lag this growth stimulation was as effective in increasing the final hepatocytes' number as was a 1000-fold higher EGF concentration, and was twice as active as either an equimolar (10^–14M) mixture of the two pancreatic hormones or EGF by itself at 10^–14M. These results show that the growth-promoting effect of EGF on primary neonatal rat hepatocytes is modulated by serum factor(s) and can be additively amplified by the simultaneous administration of subphysiological doses of glucagon and insulin.     

MN/CA9: a potential gene marker for detection of malignant cells in effusions.

Many cancers cause malignant effusions. The presence of malignant cells in effusions has implications in diagnosis, tumour staging and prognosis. The detection of malignant cells currently presents a challenge for cytopathologists. New adjunctive methods are needed. Although the effusions provide excellent materials for molecular assay, the available molecular markers are extremely limited, which hinders its clinical application. MN/CA9 has proved to be a valuable marker in many cancers such as lung, breast, colon, kidney, etc. The present study was to evaluate MN/CA9 as a new molecular marker for the detection of cancer cells in pleural effusions. Seventy-one pleural effusions including 59 malignant effusions from patients with cancer, and 12 patients with benign diseases as a control, were subjected to RT-PCR for detection of MN/CA9 gene expression. MN/CA9 gene expression was detected in 53/59 (89.8%) pleural effusions from cancer patients (15/16 for breast cancers, 10/11 for lung cancers, 4/4 for ovary cancers, 2/3 for colon-rectal cancers, 5/6 for cancers of unknown site, 7/8 for mesothelioma and 10/11 for other cancers). Furthermore, MN/CA9 was positive in 13/18 (72.2%) of cytologically negative effusions of cancer patients. MN/CA9 was detected in only 1/12 (8.3%) effusions from the control patients (p < 0.01). The sensitivity and specificity of MN/CA9 gene expression were, respectively, 89.8% and 91.7%. Our preliminary results suggest that MN/CA9 could be a potential marker for the detection of malignant cells in effusions. A large-scale study is needed to confirm these results.     

Effects of the lysosomal fraction of polymorphonuclear leukocytes on proliferation of cultured vascular cells

The effects of the lysosomal fraction isolated from polymorphonuclear leukocytes (PLF) on the growth of cultivated aortic medial smooth muscle cells (SMCs) and arterial endothelial cells (ECs) were studied by assaying DNA synthesis and counting the numbers of cells. PLF proved to promote the growth of cultivated SMCs and ECs. There was a positive correlation between an increase in DNA synthesis and the dose of PLF. The growth-promoting effect was observed in sparsely cultivated SMCs and ECs, in densely cultivated SMCs, but not in confluently cultivated ECs. The difference in response between SMCs and ECs seems to depend on their biological characteristics. Because a small amount of PLF showed potent growth-promoting activity in the presence of 10% fetal bovine serum which possesses a high protease blocking activity, the mechanism of this promoting activity is suggested to be independent of the proteases contained in PLF.     

Cytotoxicity Evaluation of Mycotoxins by an MTT-Bioassay

A colorimetric tetrazolium (MTT) cleavage test was modified and established as a bioassay for the cytotoxicity of mycotoxins. Using the human erythroleukemia cell line K562 and porcine white blood cells (lymphocytes and granulocytes) we evaluated the influence of deoxynivalenol, ochratoxin A, and zearalenone on cellular MTT cleavage activity. The yellow MTT is reduced by mitochondrial enzymes of metabolically active cells into a dark blue formazan product, the optical density (OD) of which can be measured by an ELISA reader. After an exposure time of 24hours, concentrations of deoxynivalenol and ochratoxin A as low as 0.4 \gmg/mL were found to inhibit significantly the cleavage activity in K562 cells. Cytotoxicity in lymphocytes and granulocytes was observed at concentrations of 0.8 up to 0.4 \gmg/mL for deoxynivalenol and 3.1 and 0.8 \gmg/mL for ochratoxin A, respectively. Zearalenone concentrations of 25.0 to 12.5 \gm/mL inhibited the mitochondrial cleavage activity of lymphocytes and of K562 cells significantly, whereas in granulocytes none of the concentrations tested was proved to be toxic. Morphological findings on the ultrastractural level showed that toxin incubation (28 hours) resulted in massive cell damage. Similar alterations were observed in about 15% of control cells. This indicates, that the massive cytotoxic effect of the mycotoxin deoxynivalenol is more likely to be an unspecific than a specific one. The modified MTT cleavage assay was found to be a quick (28 hours) and efficient colorimetric test for examining the cytotoxicity of three mycotoxins. The simplicity and speed of the procedure, which allows the simultaneous testing of various parameters and the possibility of objective data analysis could establish this test as an additional bioassay for the evaluation of cytotoxicity of mycotoxins.     

Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis

When bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37°C, washed extensively with phosphate-buffered saline (PBS) and incubated in bFGF-free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin-like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long-term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCI and incubated in bFGF-free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF-free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long-term (24-48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF-containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4°C, incubated in bFGF-free medium for 24 hours at 37°C, and assayed for ³H-thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4°C was carried out in the presence of heparin. Thus, long-term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrixmay act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF.     

Autoantibodies against p53 are not increased in human ascites and pleural effusions

 Mutated human p53 may give rise to the formation of autoantibodies and may be a marker for a worse prognosis. We speculated that ascites or pleural effusions may enhance the formation of such autoantibodies in cancer patients and, therefore, we measured the presence of autoantibodies in the ascites or pleural effusion of 40 patients with advanced malignancies. As controls, p53 autoantibodies were measured in 15 patients with effusions who did not have a malignancy. Using a specific enzyme-linked immunosorbent assay, p53 autoantibodies could only be detected in the effusions of 5/40 patients (12.5%) with known malignancies. The formation of autoantibodies did not correlate with the presence or absence of tumor cells in the effusion. The effusions of the patients without tumor were all negative for p53 autoantibodies. Our study shows that malignant or reactive effusions do not stimulate the local or systemic production of autoantibodies against p53. Received: 14 November 1995 / Accepted: 8 February 1996     

A bioassay for thyroid stimulating immunoglobulins of patients with Graves' disease using porcine thyroid monolayer cells

A bioassay for thyroid stimulating immunoglobulins (TSI) of patients with Graves' disease was developed by porcine thyroid monolayer cells. Thyroid cells were prepared by dispersion using collagenase and trypsin. Aliquots of the cell suspension (2 X 10(6) cells/1.5 ml/dish) in Ham's F-12 medium (pH 7.2) containing 10% calf serum and 1.5 mM Hepes were seeded and cultured in air at 36 C. On day 6 of culture, cells were incubated with test samples (IgG or bTSH) in 1 ml of serum-free, 0.5 mM IMX-included fresh medium for an additional time, and cAMP in the cells was measured by radioimmunoassay. Intracellular cAMP was increased within 5 minutes after the addition of bTSH and the maximal increase was observed after 30 min. Responses of cAMP were in a dose-related manner up to 10 mU/ml of bTSH. With the addition of IgG from untreated Graves' patients, dose-related increases in cAMP were also observed up to 10 mg/ml IgG and the maximal response was seen at 2 hours incubation. Thyroid stimulating activity in IgG's from normal subjects and patients with Graves' disease was tested with a dose of 10 mg/ml and 2 hours incubation and the activity was expressed as a percent of the control (incubated in the same experiment without IgG). One hundred forty one of 145 untreated patients showed higher activity (228 +/- 51.8%, mean +/- SD; 127-393%, range) than normal subjects (103 +/- 13.3%, mean +/- SD, n = 24; 80-129%, range). Sequential changes in TSI activity in 27 patients after initiating thionamide drugs were studied for 24 months. Initially all 27 patients showed positive TSI and 6 months later 15 remained positive. At 6 months after that, 10 of 23, 4 of 16, and 2 of 6 followed patients showed positive TSI. These results indicate that this bioassay is clinically useful for detecting TSI.     

Aeromonas spp.-mediated cell-contact cytotoxicity is associated with the presence of type III secretion system

In the study we examined the production of cytotonic and cytotoxic toxins and the presence of a type III secretion system (TTSS) in 64 Aeromonas spp. strains isolated from fecal specimens of patients with gastroenteritis. We observed that contact of the bacteria with host epithelial cells is a prerequisite for their cytotoxicity at 3 h incubation. Cell-contact cytotoxic activity of the strains was strongly associated with the presence of the TTSS. Culture supernatants of the strains induced low cytotoxicity effects at the same time of incubation. Cell-free supernatants of 61 (95%) isolates expressed cytotoxic activity which caused the destruction of HEp-2 cells at 24 h. Moreover, 44% strains were cytotonic towards CHO cells and 46% of strains invaded epithelial cells.     

Neuroblastoma Growth Factors Derived from Neurofibroma (NF1): Participation of Uridine in a Neuroblastoma Growth

Abstract: Human glioma cell extracts were found to elicit a marked growth-promoting activity on human neuroblastoma cells. This activity was also detected in the extracts of neurofibroma type 1 (NF1; von Recklinghausen neurofibromatosis) comprising aberrant Schwann cell growth. The purified substance from the NF1 extracts by HPLC on ODS columns was identical to a pyrimidine nucleoside, uridine, the chemical structure of which was identified by gas chromatography-mass spectrometry. The authentic uridine showed a strong growth-promoting activity on human neuroblastoma cells. Other purine or pyrimidine nucleotides, their derivatives, and ribose sources for their syntheses were employed to test the activity; a purine nucleoside, adenosine, showed a stronger activity than uridine. The current study raises the possibility that human neuroblastoma cells may be affected by dysfunctions of the de novo pathway of both purine and pyrimidine nucleotide biosyntheses.