Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

Role of neutrophils in gastric damage induced by platelet activating factor

Summary Platelet-activating factor (PAF) has recently been shown to be a potent ulcerogenic agent in the stomach and intestinal mucosa. Its exact mechanism of action is not yet known although histological studies suggest that vasocongestion is an important feature of PAF-induced damage. We have therefore studied the activity of various agents with different modes of action toward PAF-induced gastrointestinal lesions in the rat (PAF 2 g/kg i.v. ; macroscopic lesions of tissues scored 20 min later; arbitrary scale from 0 to 4). Drugs were administered either i. m., s. c. (5 min) or orally (30 min) before PAF injection. PAF-induced gastric lesions were strongly inhibited by the natural PAF-antagonist BN 52021 as well as by atropine sulphate and cimetidine which implicates cholinergic stimulation in the ulcerogenic activity of PAF. The somatostatin analog BIM 23014 was also very potent against PAF, perhaps by reducing the parasympathetic stimulation in the gastric wall as described for somatostatin. Allopurinol, which is a free radical scavenger also almost totally inhibited PAF-induced gastric damage, suggesting that neutrophils are involved in the mucosal lesions. The considerable inhibition of the gastric effects of PAF found in neutrophil-depleted animal supports this hypothesis. Theophylline and disodium cromoglycate, mast cell stabilizing drugs which were also active in our model, could act by protecting mast cell degranulation induced by free radicals released from activated neutrophils. A multifunctional process seems to determine the mucosal gastric damage induced by PAF, but parasympathetic stimulation and neutrophil activation play a major role in this pathology.Send offprint requests to A. Etienne at the above address     

Polymorphisms of tumor necrosis factor receptor 2 are not associated with insulin-dependent diabetes mellitus or Graves' disease

Insulin-dependent diabetes mellitus (IDDM) and Graves' disease (GD) are autoimmune endocrinopathies and associated with distinct HLA-DR and -DQ alleles as well as several tumor necrosis factor a (TNF-α) and β (TNF-β) alleles. TNF-α and TNF-β interact with TNF receptor (TNF-R), of which two subtypes have been described: TNF-R1 and TNF-R2. We investigated TNF-R2 alleles in 90 patients with IDDM, 101 with GD and 70 healthy controls. Genomic DNA was amplified with specific flanking primers for the untranslated 3 region of TNF-R2. SSCP analysis revealed two alleles by different fragment patterns: TNF-R2*1 and TNF-R2*2. Patients with IDDM or Graves' disease and controls did not differ significantly: TNF-R2*1/*1:IDDM(8%)/GD(2%)/KO(4%); TNF-R2*2/*2:IDDM(34%)/GD(48%)/KO(42%), heterozygosity TNF-R2*1/*2:IDDM(58%)/GD(50%)/KO(54%) (IDDM vs KO: P =0.46, χ²=1.57; GD vs KO: P =0.59, χ²=1.05). In conclusion, the studied polymorphism of TNF-R2 was associated with neither IDDM nor GD in a German population.     

Immunocytochemical Evidence of Amyloid-enhancing Factor (AEF) in Polymorphonuclear Leukocytes

Amyloid enhancing factor (AEF) was extracted from spleens of mice that had received amyloidogenic stimulation. Sephacryl S 300 gel filtration of the crude AEF yielded five fractions, among which strong AEF activity was present in the first peak (Fl), and confirmed by an amyloid induction experiment. An anti AEF antiserum was obtained from a rabbit by immunization with Fl. This antibody reacted strongly with splenic polymorphonuclear leukocytes (PML) from mice given amyloidogenic stimulation, and weakly with those from normal untreated mice. Isoelectric focusing (IEF) analysis of both Fl and sera from mice given amyloidogenic stimulation was performed. A single band was observed on IEF analysis of Fl, whereas many bands were seen on IEF analysis of the sera. After the substances in the gel had been transferred to nitrocellulose membranes by capillary blotting, the membranes were made to react with the anti-AEF antiserum. The results suggested that AEF is a high molecular-weight substance derived from PML and increases in the serum at the time of, or shortly prior to, amyloid deposition in the spleen. Acta Pathol Jpn 39: 349∼355, 1989.     

A Study of Early Pregnancy Factor Activity in Preimplantation

PROBLEM and METHOD: Early pregnancy factor (EPF), an Immunosuppressive substance, which appears in pregnant women's sera 48 h after fertilization, is a kind of pregnancy-specific protein. To determine whether the EPF activity could be a super early indicator of pregnancy, we used rosette inhibition assay to detect EPF activity in the sera, collected from 70 women 2–7 days after ovulation intending to conceive monitored by ultrasonography. Simultaneously we selected 40 non-pregnant sera and 12 early-pregnant sera as negative control and positive control, respectively. RESULTS: The results of this study demonstrated that EPF activity is detected in 35 women's sera out of 70 women within 2–7 days after ovulation, and 28 women out of the 35 were pregnant, which was known by follow-up, and 7 were not pregnant, possibly due to either false positive results or embryo loss because of preimplantation failure, thus causing no pregnancy. The other 35 out of 70 had no EPF activity and 34 of them were not pregnant, which was known by follow-up, but one case became pregnant, which was false negative result. Our study showed that diagnosis of the super early pregnancy could be made by detecting EPF activity in maternal serum within the time of preimplantation. The accuracy of pregnancy diagnosis by this method is 88.6%, with a false negative rate of 3.4% and a false positive rate of 17.1%. The β-HCG level was measured from the above 70 women's sera in order to contrast EPF activity. All of the sera collected 2–6 days following ovulation indicated that there were lower β-HCG values in very early pregnancy (≥a5 mIU/ml). On the seventh day after ovulation, EPF activity was detected in 11 out of 15 sera with only 2 of them with a b-HCG level that reached or slightly surpassed that of the early pregnancy diagnosis (5 mIU/ml and 5.4 mIU/ml, respectively). This demonstrated that β-HCG is not the earliest signal of pregnancy; otherwise the EPF activity is one that appears 2–6 days earlier than β-HCG appears. We measured the progesterone level of the 48 sera from the 70 collected above within 2–7 days postovulation and found most of them reached the level of progesterone in the luteal phase (7.5–98.3 nmol/L). This indicated that ovulation had taken place in these women, which was in accordance with observations by ultrasonography. CONCLUSIONS: Our study showed that diagnosis (of 88.6%) of super early pregnancy could be made with an accuracy of 88.6% by detecting EPF activity in maternal serum within 2-days after ovulation. This offers a basis for pregnancy diagnosis for the women who attempt to terminate their pregnancy safely or who conceive unexpectedly, and it contributes to family-planning.     

Protective actions of human recombinant basic fibroblast growth factor on MPTP-lesioned nigrostriatal dopamine neurons after intraventricular infusion

Basic fibroblast growth factor (bFGF, FGF-2) is a trophic factor for neurons and astrocytes and has recently been demonstrated in the vast majority of dopamine (DA) neurons of the ventral midbrain of the rat. Potential neuroprotective actions of FGF-2 in the l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) model have also been reported. The actions of the FGF-2 have now been further analyzed in a combined morphological and behavioural analysis in the MPTP model of the adult black mouse, using a continuous human recombinant FGF-2 (hrFGF-2) intraventricular (i.v.t.) administration in a heparin-containing (10 IU heparin/ml) mock cerebrospinal fluid (CSF) solution. Tyrosine hydroxylase (TH) immunocytochemistry in combination with computer assisted microdensitometry demonstrated a counteraction of the MPTP-induced disappearance of neostriatal TH-immunoreactive (ir) nerve terminals following the FGF-2 treatment. Unbiased estimates of the total number of nigral TH ir neurons, using stereological methods involving the optical disector (Olympus), showed that the MPTP-induced reduction in the number of nigral TH ir nerve cell bodies counterstained with cresyl violet (CV; by 56%) was partially counteracted by the FGF-2 treatment (by 26%). The behavioral analysis demonstrated an almost full recovery of the MPTP-induced reduction of the locomotor activity after FGF-2 treatment. This action was maintained also 1 week after cessation of treatment. The hrFGF-2 produced an astroglial reaction as determined in the lateral neostriatum and in the substantia nigra (SN) far from the site of the infusion, indicating that the growth factor may have reached these regions by diffusion to activate the astroglia. Immunocytochemistry revealed FGF-2 immunoreactivity (IR) in the nuclei of the astroglia cell population in the dorsomedial striatum and the microdensitometric and morphometric evaluation demonstrated an increase in the number, but not in the intensity, of these profiles on the cannulated side, suggesting the possibility that hrFGF-2 stimulates FGF-2 synthesis in astroglial cells with low endogenous FGF-2 IR. These results indicate that hrFGF-2, directly and/or indirectly via astroglia, upon i.v.t. infusion exerts trophic effects on the nigrostriatal DA system and may increase survival of nigrostriatal DA nerve cells exposed to the MPTP neurotoxin.     

Tumor necrosis factor-mediated interactions between inflammatory response and tumor vascular bed

Summary: Solid tumor therapy with chemotherapeutics greatly depends on the efficiency with which drugs are delivered to tumor cells. The typical characteristics of the tumor physiology promote but also appose accumulation of blood-borne agents. The leaky tumor vasculature allows easy passage of drugs. However, the disorganized vasculature causes heterogeneous blood flow, and together with the often-elevated interstitial fluid pressure, this state results in poor intratumoral drug levels and failure of treatment. Manipulation of the tumor vasculature could overcome these barriers and promote drug delivery. Targeting the vasculature has several advantages. The endothelial lining is readily accessible and the first to be encountered after systemic injection. Second, endothelial cells tend to be more stable than tumor cells and thus less likely to develop resistance to therapy. Third, targeting the tumor vasculature can have dual effects: (i) manipulation of the vasculature can enhance concomitant chemotherapy, and (ii) subsequent destruction of the vasculature can help to kill the tumor. In particular, tumor necrosis factor α is studied. Its action on solid tumors, both directly through tumor cell killing and destruction of the tumor vasculature and indirectly through manipulation of the tumor physiology, is complex. Understanding the mechanism of TNF and agents with comparable action on solid tumors is an important focus to further develop combination immunotherapy strategies.     

Guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction

Summary:  Members of the cytohesin protein family, a group of guanine nucleotide exchange factors for adenosine diphosphate ribosylation factor (ARF) guanosine triphosphatases, have recently emerged as important regulators of signal transduction in vertebrate and invertebrate biology. These proteins share a modular domain structure, comprising carboxy-terminal membrane recruitment elements, a Sec7 homology effector domain, and an amino-terminal coiled-coil domain that serve as a platform for their integration into larger signaling complexes. Although these proteins have a highly similar overall build, their individual biological functions appear to be at least partly specific. Cytohesin-1 had been identified as a regulator of β2 integrin inside-out regulation in immune cells and was subsequently shown to be involved in mitogen-associated protein kinase signaling in tumor cell proliferation as well as in T-helper cell activation and differentiation. Cytohesin-3, which had been discovered to be strongly associated with T-cell anergy, was very recently described as an essential component of insulin signal transduction in Drosophila and in human and murine liver cells. Future work will aim to dissect the mechanistic details of the modes of action of the cytohesins as well as to define the precise roles of these versatile proteins in vertebrates at the genetic level.     

Role of the stromal microenvironment in regulation of bone-marrow hematopoiesis after administration of dipyridamole

I. P. Pavlov First Leningrad Medical Institute. Central Roentgeno-Radiological Research Institute, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 7, pp. 98–100, July, 1990.