Rat monoclonal antibodies. VII. Enhancement of ascites production and yield of monoclonal antibodies in rats following pretreatment with pristane and Freund's adjuvant.

The effect of intraperitoneal injections of pristane, incomplete Freund's adjuvant (IFA) and a v/v mixture of pristane and IFA (called PIFA) on ascites production and the yield of monoclonal antibodies has been studied in Louvain rats. The best results were obtained following injection of 2 ml PIFA at the moment of i.p. transfer of hybridoma or immunocytoma cells. Ascites production was increased by as much as 4.7 times and monoclonal antibody production by more than six times compared with untreated control rats.

Immunodetection of small cell lung cancer metastases in bone marrow using three monoclonal antibodies.

Detection of bone marrow metastases by indirect immunofluorescence methods was investigated using three monoclonal antibodies (MoAbs) raised against small cell lung cancer (SCLC). These antibodies, designated anti-LCA1, -LCA2 and -LCA3, recognize three different antigens on the surface of SCLC cells. Eighty-four bone marrow samples from 74 different patients were studied. Whereas tumor cells were found in 32 (38%) by MoAb staining, only 10 (12%) were positively identified using conventional morphological methods. Nine out of the morphologically positive specimens showed reactivity with at least two monoclonal antibodies. Among the 32 samples proven positive by immunofluorescence, an important antigenic variability was noted. Anti-LCA1 recognized tumor cells in 62%, anti-LCA2 and anti-LCA3 in 53%. Due to the recognition of bone marrow involvement by fluorescence methods in 26% of the 34 patients classified as limited disease, a new subgroup of limited disease patients was defined whose prognosis remains undetermined. Our results confirm the utility of immunodetection in the diagnosis of SCLC bone marrow metastases and emphasize the advantage of using a panel of MoAbs with different antigenic specificities. Further study is needed to determine the prognostic significance of bone marrow involvement established by immunodetection.

Detection of small cell lung cancer bone marrow metastases by immunofluorescence.

It is well known that small cell lung cancer (SCLC) has a high propensity to metastasize to the bone marrow and that such involvement has a prognostic significance. A more accurate detection of these bone marrow metastases is thus mandatory. In this study, we analysed the results of the detection of these metastases using an indirect immunofluorescence test. For this purpose, 3 anti-SCLC rat monoclonal antibodies (MoAbs) specific for 3 different antigens (LCA1, LCA2, LCA3) have been utilized to examine 59 bone marrow samples from patients at time of diagnosis and 20 samples from chemotherapy treated patients. Eight patients had bone marrow clearly involved by morphological analysis. They all had fluorescent cells recognized at immunodetection with at least 2 MoAbs positive for 7 out of the 8 samples. Fifteen samples, negative by morphological analysis, were proven positive by immunofluorescence. In 12 cases, involvement was detected only by 1 MoAb (6 anti-LCA1, 2 anti-LCA2, 4 anti-LCA3). A correlation was found between the number of samples proven positive by morphological analysis and the number of positive MoAbs for these samples (p less than 0.005). Among the bone marrow samples provided by the 32 limited disease patients, LCA positive cells were detected in 9 (28%) compared to 14 out of the 27 (52%) samples from extensive disease patients (p less than 0.05). We concluded that the indirect immunofluorescence with a panel of MoAbs increases the rate of detection of bone marrow SCLC metastases.

Neonatal IgE injections do not modify Nippostrongylus brasiliensis-induced and mercuric chloride-induced IgE increase in the rat.

The effect of neonatal IgE injections on total IgE responses was studied in two rat models. After Nippostrongylus brasiliensis infection, no differences in serum IgE level and surface or cytoplasmic IgE expression were observed between IgE-treated and control LOU/C rats. Likewise, after HgCl2 injection, IgE-treated Brown-Norway rats and controls showed no difference in serum IgE level or in the development of autoimmune glomerulonephritis and proteinuria. It has been concluded that IgE-class-restricted tolerance, induced by neonatal injections of IgE, cannot be observed in rats after strong IgE stimulation.

Fc epsilon receptors on rat B-lymphocytes: specificity and binding kinetics.

The binding kinetics of radiolabelled rat IgE to Fc epsilon receptors (R) of rat B-cells have already been studied in IgE-stimulated animals. The receptors expressed after Nippostrongylus brasiliensis infection or 2 injections of 5 mg IgE/100 g body wt were class-specific: IgE binding was not hindered by rat IgM, IgD, IgA, IgG1, IgG2a, IgG2b and IgG2c in immunocompetitive-binding assays. The rat B-cell Fc epsilon R were not species-specific, since mouse IgE competes with rat IgE for binding to these receptors. The apparent number of Fc epsilon R on rat mesenteric lymph node B-cells varied with temp and was 1.1-2.4 X 10(5) at 4 degrees C and 5.9-7.7 X 10(5) at 37 degrees C. The experimental Ka was not influenced by temp and had an average value of 1.38 X 10(8) M-1. At 4 degrees C the IgE binding to B-cell Fc epsilon R had an association rate of 4.9 X 10(3) M-1 sec-1 and a dissociation rate of 4.65 X 10(-5) sec-1. After a very strong stimulation produced by injecting 5 mg IgE/100 g body wt every 24 hr for 5 days, the equilibrium binding curve became diphasic, indicating a probable heterogeneity of the B-cell Fc epsilon R.

Nippostrongylus brasiliensis infection can induce IgE production in rnu/rnu rats.

Although rnu/rnu homozygous athymic rats are generally considered to be entirely deficient in mature T cells and unable to produce any T-cell-dependent immune response, we were able to induce slight IgE production in some Nude rats after Nippostrongylus brasiliensis infection. This report stresses the importance of taking the greatest care in the use of congenitally athymic rnu/rnu rats, as strong stimulation can, in some individuals, induce reactions which are generally considered to be T-cell-dependent. Hypotheses explaining this phenomenon are suggested.

In vivo kinetics and nature of rat IgE-bearing lymphocytes after IgE stimulation.

Surface IgE-bearing (sIgE+) cells were studied in BN and rnu/rnu athymic rats after Nippostrongylus brasiliensis infection or i.p. injection of unpurified or purified IgE from plasmacytoma ascitic fluid. The number of sIgE+ cells increased markedly after a serum IgE increase without change in the proportion of sIgM+ and sIgD+ cells. A high percentage of the sIgE+ cells bore cytophilic IgE. Receptors for IgE were induced with a 2.56-micrograms IgE injection/100 g body weight and reached a maximum with 1.6 mg IgE/100 g body weight. They appeared less than 4 hr after a single injection of purified IgE. The number of IgE receptor-bearing cells reached a maximum plateau at 24 hr to day 3 after injection and declined thereafter, to reach the control level on day 9 or 11 after injection. Nearly all the sIgE+ cells of BN rats also bore sIgD, but the number of triple sIgE-sIgM-sIgD+ cells varied in a wide range. Maximum 4.5% of the sIgE+ cells of euthymic rats were T cells. More than 98% of the sIgE+ cells of nude rats were triple sIgM-sIgD-sIgE+ cells, and the majority were cytophilic IgE+. For the most part, the sIgM-sIgD-sIgE+ cells are probably not cells that can differentiate, as generally accepted, in IgE-producing cells. New interpretations of the role of these triple sIgM-sIgD-sIgE+ cells in IgE immune responses are necessary.

[Ontogenesis and differentiation of IgE B lymphocytes in the rat]. / Ontogenèse et différenciation des lymphocytes B à IgE chez le rat.

In fetal liver, the stem cells of the B lineage transform into pre-B cells with intracytoplasmic mu-chains and thereafter into mature B cells with IgMc and IgMm. The other immunoglobulin classes appear later. The IgEm B cells appear on the day of birth. They bear IgMm and IgDm. Anti-IgM or anti-IgD suppression, performed from birth, prevents IgE synthesis. IgM and IgD seem to be necessary to IgE lymphocyte differentiation. Neonatal anti-IgE suppression seems to have no influence on IgE lymphocytes, but this result has to be confirmed. IgE immune responses are T-dependent. Neonatally thymectomized animals or rnu/rnu rats are unable to produce IgE immune responses. Axenic rats possess a great number of IgEm-IgAm-bearing cells in Peyer's patches; this is not observed in conventional animals. Nevertheless, the major problems are due to cells with receptors for IgE which must be differentiated from cells with intrinsic IgE. Work is under way to solve these problems.