Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

The effect of substance P on the regional lymph node antibody response to antigenic stimulation in capsaicin-pretreated rats

The undecapeptide substance P (SP) contained in primary afferent nerves is thought to mediate that part of the neurogenic inflammatory response consisting of vasodilation and plasma extravasation. This response is diminished in rats pretreated as neonates with the neurotoxin capsaicin. It is not known whether primary afferent nerves influence cellular responses of the immune response to antigenic stimulation. Using 6- to 12-wk-old Sprague-Dawley rats pretreated as neonates with capsaicin, we examined the regional lymph node response to a s.c. antigenic stimulus of sheep red blood cells. The number of cells secreting antigen-specific antibody in these animals was reduced by more than 80% using direct and indirect plaque assay methods. The reduced antibody response in capsaicin-pretreated animals was reversed by a s.c. infusion of SP given over a 4-hr period at the injection site immediately after antigen stimulation. This response had a threshold at approximately 1.0 X 10(-5) M SP. SP1-7 (1.0 X 10(-5) M) was without effect but an infusion of SP5-11 (1.0 X 10(-5) M) reversed the effects of capsaicin treatment indicating a carboxyl-terminal effect of SP. The results suggest that the reduced response of capsaicin-treated animals to an antigenic stimulus is due to an effect of capsaicin on the SP-containing primary afferent nerves rather than a toxic effect of capsaicin on the immune system.     

The effect of the binding of the antigenic determinant of tetrodotoxin-sensitive protein on the phytohemagglutinin-induced proliferative response of the mononuclear cells in mouse spleen]

Mammalian brain-derived "tetrodotoxin-sensitive protein" has been shown to share an epitope with as yet unidentified structure of the human and rodent lymphocyte surface. Previously obtained observations that a monoclonal antibody to this epitope induces a proliferative response of murine splenic mononuclear cells are confirmed. However, this antibody fails to modify the phytohemagglutinin-induced response. Moreover, lectin with submitogenic concentration inhibited the antibody-induced response provided that monocytes were present in the culture. The antibody-induced proliferation appeared to be less monocyte-dependent than the lectin-induced one. Taken together, these findings argue against hypothesis that a lymphocyte structure with epitope of the "tetrodotoxin-sensitive protein" is associated with either T cell receptor for antigen or interleukin-2 receptor.     

Induction of an immune response in athymic nude mice to thymus-dependent antigens by Pseudomonas aeruginosa exotoxin A

Exposure of spleen cells from athymic nude mice to Pseudomonas aeruginosa exotoxin A induces these cells to respond to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC). The response induced by toxin is dose dependent, antigen specific, and not due to polyclonal B-cell activation. Enhancement of the anti-SRBC response can be observed when toxin addition precedes antigen stimulation by 24-48 hr, which decreases when toxin administration follows antigen stimulation. A significant response is also observed when toxin and antigen are added simultaneously. A significant anti-SRBC response can be observed out to Day 10 postantigen and toxin stimulation after attaining a peak response at Day 5. Cultures exposed to toxin in the presence or absence of antigen exhibited a higher cell number and relative number of B cells as compared to control cultures. Exposure of T-cell depleted B cells from euthymic +/nu mice to toxin plus antigen does not result in an anti-SRBC response indicating that exotoxin A alone is not sufficient to induce B-cell responsiveness to T-dependent antigens and that other cells and/or factors are involved in the toxin-induced responsiveness of nude mice to T-dependent antigens.     

Epithelial modulation of tracheal smooth muscle response to antigenic stimulation

The role of the epithelium has been studied in the contractile responses of rat trachea. The different modulations observed are discussed in respect to vagal components of the epithelial layer. Responses of rat trachea to immunologic stimulation are shown to be dependent on the presence of the epithelium, which prolongs the relaxation stage without affecting the contractions. This prolongation is abolished by neonatal capsaicin pretreatment, whereas substance P induces a significantly greater relaxation of serotonin-precontracted intact than deepithelialized trachea. Serotonin concentration-response curves are shifted to the right in intact preparations, which is partly reversed by neonatal capsaicin pretreatment, but a hyporeactivity of the tissue exists. A relaxing factor released by the epithelium is hypothesized, possibly dependent on substance P-ergic innervation. Muscarinic cholinergic innervation slightly modulates the contractions but not the relaxations in antigen-induced responses, independently on the presence of the epithelial layer. 4-Aminopyridine induces epithelium-dependent potentiations of contractions to antigen and to serotonin, which involves acetylcholine at one step of the reaction cascade. Epithelial-dependent contracting and relaxing factors are thus suggested in rat trachea.     

The regulatory effect of macrophages on the antigen-induced lymph node cell proliferative response

Employing an antigen-induced T cell-dependent lymph node cell (LNC) proliferative assay in the mouse we observed differences in the capacity of unstimulated and thioglycollate-activated peritoneal exudate cells (PECs) to present antigen. Antigen-primed LNCs can be induced to proliferate by a brief (2 hr) antigen exposure and the addition of various numbers of thioglycollate PECs (thio-PECs) modifies the proliferative response depending on the ratio of $\text{PEC}\text{LNC}$ in cultures. With ratios of 3–12% both DNA and protein synthesis were enhanced, but at ratios greater than 12% suppression was significant. Treatment of thio-PECs with mitomycin C, irradiation (3000 rad), anti-Thy 1.2, or anti-Ia plus complement did not alter suppression, suggesting the possibility that the Ia negative macrophages present in the PECs were involved in the suppression. An enhancing effect on the proliferative response was noted following the addition of small numbers of thio-PECs. This was comparable to that seen with an equivalent concentration of supernatant from thio-PECs suggesting that soluble factors play an important role. Two enhancing fractions (separated on a G-75 column) which were themselves mitogenic were identified which eluted with approximate molecular weights of 15,000 and 60,000.     

Magnetic field inhibits isolated lymphocytes' proliferative response to mitogen stimulation

We aimed to find out how the exposure of isolated lymphocytes to a pulsed magnetic field (MF) affected their in vitro proliferative response to mitogenic stimulation. Cells were exposed to MF of various intensities (0.3, 0.6, and 1.2 T) at a constant frequency of 30 Hz, for a period of 60, 180, and 330 s. Then, the proliferative response of splenocytes was induced by optimal concentrations of concanavalin A (Con A; mitogenic toward T cells), bacterial lipopolysaccharide (LPS; mitogenic toward B cells), or pokeweed mitogen (PWM; mitogenic toward both populations). We found that the exposure of lymphocytes to the MF profoundly inhibited their proliferative response to mitogens. The suppressive action of the MF on B and T cell proliferation was intensified when a cooperative response of those two lymphocyte populations was simultaneously induced by PWM. The inhibitory effect of MF depended on the exposure time and MF intensity. Prolonged exposure and/or a stronger intensity of the MF weakened its inhibitory influence on the response of lymphocyte to mitogenic stimulation. The data show that an exposure to MF may influence the activity of lymphocytes in their response to mitogenic stimuli.     

Effect of antigenic stimulation on acid deoxyribonuclease activity in lymphocytes: I. Antibody-induced response

The effect of antigen-induced stimulation on acid deoxyribonuclease (DNase) activity in BALB/c mouse lymphoid cells was determined. Increase in acid DNase activity was found in intact spleen cell populations of mice from the second or fourth day after immunization with pneumococcal polysaccharide type III and from the fourth day after immunization with SRBC. DNase determinations performed with spleen cell fractions prepared from SRBC-immunized mice, showed that the rise in the enzyme activity was confined to the fraction containing the antibody-forming cells. The DNase activity was also increased in spleen cell cultures, stimulated with SRBC in vitro. Rise in the activity of this enzyme was also observed in peritoneal cell populations taken from SRBC-immunized mice. This change was maximal on the second day after immunization, when no appreciable increase in DNase activity of spleen cells was yet detected. The results obtained suggest, that acid DNase is an enzyme involved in the proliferative/maturation response to antigenic stimulation. It is a consequence of antigenic stimulation rather than being involved in the process of afferent stimulation.     

The effect of alpha 1 antitrypsin on the proliferative response of human peripheral blood lymphocytes

In evaluating immune aberrations in patients with alpha 1 antitrypsin (alpha 1 AT) deficiency, we have previously shown that they exhibit enhanced lymphocyte responsiveness to PHA that is serum mediated. In this study, we demonstrate suppression of the PHA response by using purified alpha 1 AT, and also similar but less marked suppression of the Con A response. alpha 1 AT, however, has no effect whatsoever on PWM-induced proliferation. This effect is demonstrable provided alpha 1 AT is added within 4 hr of mitogen activation and is mediated by its action on adherent cells rather than on proliferating lymphocytes. Adherent cells still exhibit this effect if pulsed with alpha 1 AT, then thoroughly washed before their activation. This suggests that it may be inhibiting a membrane serine esterase already activated before the addition of PHA. Thus alpha 1 AT may modulate the activation of T cells through its effect on monocytes, leading to abnormalities in immunoregulation, and hence a predisposition to the development of a variety of immunologic disorders in alpha 1 AT-deficient subjects.     

The effect of sulfinpyrazone on the response to sympathetic nervous system stimulation

The purpose of this study was to determine if sulfinpyrazone has a direct action on sympathetic nerve endings to prevent release of the transmitter. Pre-synaptic and post-synaptic events as well as direct sympathetic nervous system stimulation were tested in 15 α-chloralose anesthetized cats before and 1 hour after sulfinpyrazone (100 mg·kg⁻¹, i.v.). Heart rate response to cardiac accelerator nerve stimulation or to increasing doses of isoproterenol was not significantly depressed by sulfinpyrazone. In addition, no alteration in the reflex activation of the sympathetic nervous system in response to histamine was observed following sulfinpyrazone. Both norepinephrine and epinephrine levels were similar to those levels reported previously by Smith and Robinson (7) for untreated cats. We conclude sulfinpyrazone has no direct depressing effect on the sympathetic nerve endings and that this mechanism cannot explain the reported beneficial effect of sulfinpyrazone on coronary occlusion induced arrhythmias.