Microenvironmental factors regulate Ly-6 A/E expression on PyV-transformed BALB/c 3T3 cells

Ouabain-induced changes of the free cytoplasmic Na+ concentration ([Na+]i) were monitored in aggregates of cells prepared from beta-cell-rich pancreatic mouse islets and the results were compared with the total islet content of sodium. The steady-state [Na+]i was lower in 20 mM glucose (11 mM) than in 3 mM glucose (14 mM). In the presence of 3 mM glucose the addition of 1 mM ouabain resulted in a rise in [Na+]i with an initial rate of 1.5 mM/min. However, the increase of total sodium corresponded to 2.8 mM/min, suggesting that rapid binding and/or sequestration of Na+ are prominent features for pancreatic beta-cells. Elevation of the glucose concentration to 20 mM increased the rate of ouabain-dependent rise of [Na+]i. The effect of glucose was mimicked by 1 mM tolbutamide or 100 microM carbachol and was counteracted by 100 nM of the alpha 2-adrenergic agonist clonidine. Glucose also accelerated the lowering of [Na+]i after withdrawal of ouabain. In promoting not only the entry but also the extrusion of Na+, glucose actually enhances the turnover of the ion in pancreatic beta-cells.     

Growth suppression of transformed BALB/c 3T3 cells by transforming growth factor beta 1 occurs only in the presence of their normal counterparts

Transforming growth factor beta 1 (TGF-beta 1) enhances the yield of transformed foci of BALB/c 3T3 cells, but the continuous presence of TGF-beta 1 after foci formation inhibits the growth of transformed foci. The focus-forming ability of Ha-ras-, v-src- and PyMT-transformed cells growing on a monolayer of non-transformed cells was completely suppressed by TGF-beta 1, whereas growth of the transformed cells was little inhibited by TGF-beta 1 in the absence of their normal counterparts. The inhibition by TGF-beta 1 of focus formation by transformed BALB/c 3T3 cells on a normal cell monolayer remained when TGF-beta 1 was removed from the culture medium after 2 weeks. However, the transformed cells were not killed, since they grew in culture conditions under which only transformed cells are able to grow (soft agar). These results suggest that TGF-beta 1 suppresses growth of transformed cells in the presence of normal cells. Furthermore, when non-transformed cells were treated with TGF-beta 1 before co-culture with Ha-ras-transformed cells, formation of transformed foci was inhibited. When normal and transformed cells were cultured in the same dish but separated physically, focus formation was still inhibited. On the other hand, TGF-beta 1 enhanced the growth and changed the morphology of non-transformed cells only in the presence of transformed counterparts. The growth inhibitory effect of TGF-beta 1 on transformed cells and its growth stimulatory effect on non-transformed cells in co-culture conditions suggest the induction of reciprocal paracrine growth regulatory factors. As TGF-beta 1 inhibits the growth of transformed BALB/c 3T3 cells only in the presence of their normal counterparts, a paracrine negative growth control mechanism appears to be operating.     

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.     

Effects of an acute exposure to lindane (gamma-hexachlorocyclohexane) on brain and liver carbohydrate metabolism of rainbow trout

The ZR-75-1 ER positive breast cancer cell line, xenografted in female nude mice, has been used to determine the effect of tamoxifen on cell proliferation (as measured by mitosis) and cell death (as evidenced by apoptosis and necrosis). After 2 days treatment, there was a significant rise in apoptosis (p < 0.05), whereas a fall in mitosis was not apparent until 7 days (p < 0.05). Furthermore there was an increase in the apoptotic:mitotic ratio on day 7 (p < 0.05). These changes antedated tumour regression, which did not reach not significance until day 14. Tamoxifen did not increase necrosis (which significantly decreased in treated tumours once they had regressed (p < 0.01). In contrast tamoxifen treatment of xenografted MDA-MB-231 ER-negative breast cancer cells produced no significant effects on growth, apoptosis, or mitosis. This study presents clear evidence for tamoxifen inducing apoptosis in ZR-75-1 xenografts (but not MDA-MB-231 tumours). Since changes in apoptosis and mitosis antedate tumour regression, their assessment may provide the potential by which to predict tumour response to tamoxifen therapy.     

Lack of transforming activity of fumonisin B1 in BALB/3T3 A31-1-1 mouse embryo cells

The capacity of fumonisin B1 (FB1) to induce morphological transformation of cultured mammalian cells was assessed using BALB/3T3 A31-1-1 mouse embryo cells. FB1 with 90% purity was prepared from Fusarium proliferatum grown on whole corn. Cell growth was not inhibited by 48 hr of exposure at concentrations up to 1000 micrograms/ml. Moderate inhibition was induced by 6 days of exposure. In transformation assays with a 48-hr exposure, increases in transformed foci were observed at some concentrations; however, the responses were not reproducible. Prolonged exposure for up to 4 wk at 10, 100 and 500 micrograms/ml failed to induce increases in transformed foci. Analysis of combined results showed that only the increase induced by a 48-hr exposure at 500 micrograms/ml was significant. A trend test indicated the lack of a dose response for concentrations of 10-1000 micrograms/ml. FB1 seems to lack in vitro transforming activity.     

Initiating activity of quinones in the two-stage transformation of BALB/3T3 cells

2-tert-Butyl-1,4-benzoquinone (BQ), a metabolite of the rodent carcinogen 3-tert-butyl-4-hydroxyanisole (3-BHA), has been shown previously to have initiating activity for cell transformation. In this paper, we examined the initiating activity of quinones in a two-stage transformation assay using BALB/3T3 cells. Cells were treated first with a quinone and then with the tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA). The quinones tested were 1,4-benzoquinone (pQ), phenyl-1,4-benzoquinone (PhQ), menadione and 2,5-di-tert-butyl-1,4-benzoquinone (DBQ) in addition to BQ. pQ is a metabolite of benzene and phenacetin, and PhQ is a metabolite of o-phenylphenol (OPP) and sodium o-phenylphenate (OPP-Na). All of the tested quinones induced transformation in the presence of TPA but not in its absence. The extent of transformation caused by quinones followed by TPA was weak but statistically significant. Thus these quinones were shown to act as initiators in the transformation of BALB/3T3 cells. This result suggests that BQ, pQ and PhQ may be involved in carcinogenesis by 3-BHA, benzene, phenacetin, OPP and OPP-Na in vivo. Menadione has been reported to cause cytotoxic effects and mutations through active oxygen generation from semiquinone radicals. DBQ has two bulky substitutes which interfere with covalent bonds with DNA. Menadione and DBQ exhibited initiating activity in the present study. This result suggests that active oxygens generated from semiquinone radicals may play a role in the initiation of cell transformation.     

Neoplastic transformation by quartz in the BALB/3T3/A31-1-1 cell line and the effects of associated minerals

Quartz, the most common form of crystalline silica, was tested quantitatively for neoplastic transformation in the mouse embryo cell line, BALB/3T3/A31-1-1. Five quartz dust samples of respirable size [Min-U-Sil 5 (MQZ); hydrofluoric-acid-etched MQZ (HFMQZ); Chinese standard quartz (CSQZ); DQ12; and F600] all induced significant levels of neoplastic transformation, showing dose-dependent increases in the frequency of morphologically transformed foci at lower tested doses and a plateau level of response at higher doses. The plateau levels reached by the five tested samples did not differ substantially (maximum transformation frequencies per 10(5) cells ranging from 53.2 for MQZ to 28.3 for HFMQZ). F600 had minimal cytotoxicity but transforming activity comparable to the other samples. Cells from all tested transformed foci, when injected s.c. in nude mice, grew as sarcomas. Cytogenetic analysis showed that all tested silica-transformed cell lines had acquired one to five additional marker chromosomes, of types not seen in untreated control lines, indicative of induced chromosomal translocations and amplification. Increased expression of one or more of five genes (p53, myc, H-ras, K-ras, and abl) was observed in several quartz-transformed cell lines. No transforming activity was found for hematite and anatase (both nontoxic), and for rutile (more toxic than MQZ). Combined exposure (1:1 w/w per unit culture area) of each of these dusts with MQZ showed that hematite and anatase inhibited MQZ toxicity as well as transformation, whereas rutile markedly enhanced MQZ toxicity but not MQZ-induced transformation.     

Metabolic pathways of WR-2721 (ethyol, amifostine) in the BALB/c mouse

This study investigated the metabolism of the radio- and chemoprotector compound, WR-2721 [amifostine; s-2-(3- aminopropylamino)ethylphosphorothioate], in the Balb/c mouse. The latter was selected for these studies because considerable radiation protection data have been published for this mouse strain using the WR-2721 dose, route of administration, and optimal time for protection following intraperitoneal injection used herein. It is known that protection requires conversion of the parent drug to its free thiol metabolite, WR-1065, in cultured cells. Because it is possible that metabolites of WR-1065 could be involved in protection and because thiols are metabolically very reactive molecules, we investigated the metabolism of WR-2721 using electrochemical detection-HPLC methods. The following are the major findings in this study: 1) WR-2721 drug was rapidly cleared from the bloodstream. Blood concentration of the parent drug decreased 10-fold 30 min after administration from the maximal observed value at 5 min 2) WR-1065 rapidly appeared in the perchloric acid (PCA)-soluble fraction of normal solid tissues. The highest WR-1065 concentrations in liver and kidney were 965 and 2195 mumol/kg, respectively, 10 min after parent drug administration, whereas for heart and small intestine the highest values were 739 and 410 mumol/kg at 30 min. 3) WR-1065 accumulated in the PCA-soluble fraction of two experimental tumors at a lower rate than for the other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of BALB/c donor CD8+ lymphocytes in graft-versus-host disease in (BALB/c x A/J)F1 (CAF1) mice

To investigate the role of donor T lymphocyte subsets in the development of chronic graft-vs-host disease (GVHD) induced in (BALB/c x A/J)F1 (CAF1) mice by injecting BALB/c lymphoid cells, we analyzed the effect that CD8+ cell removal from donor inoculum has on the manifestation of the disease. Compared with age- and sex-matched CAF1 mice injected with whole lymphocyte inoculum, CAF1 mice injected with CD8(+)-depleted inoculum exhibited: 1) a higher incidence and exacerbation of nephritis by immunocomplexes; 2) higher (five- to sevenfold) spontaneous IL-4 production; 3) higher frequency titer and precocity of anti-dsDNA, anti-histone, and IgM and IgG rheumatoid factors; 4) a dramatic change in the frequency and titer of anti-U1 small nuclear ribonucleoprotein Abs; and 5) a markedly decreased engraftment (10- to 15-fold) on BALB/c donor lymphocytes. In contrast, rheumatoid arthritis-like disease, a later clinical manifestation of the GVHD in CAF1 + BALB/c model, is not present in the CD8(+)-depleted model (CAF1 + CD8-BALB/c). Considered together, these data suggest that CD8+ donor T lymphocytes play an important role in the degree of chimerism, modulation of the response to autoantigens, and clinical aspects developed in the GVHD model presented here.     

Multiple pathways lead to activation of the survival mechanism in quiescent BALB/c-3T3 cells

We investigated the effects of human anti-sporozoite antibodies on the sporogonic development of Plasmodium falciparum in Anopheles stephensi. Equal volumes of washed human erythrocytes and human sera from 1) volunteers with protective immunity induced by immunization with irradiated P. falciparum sporozoites, 2) the same volunteers before immunization, or 3) Kenyans exposed to natural sporozoite transmission, were fed to cohorts of P. falciparum-infected A. stephensi on either day 5, 8, or 11 after infection. A fourth group of infected mosquitoes from the same cohort were not refed. In two experiments, the effects of anti-sporozoite antibodies were evaluated by determining the infection rates and parasite densities for oocysts and salivary gland sporozoites. There was no evidence that anti-sporozoite antibodies had any effect on the development or intensity of P. falciparum infection in A. stephensi. However, accelerated oocyst maturation was associated with mosquitoes taking a second blood meal, independent of serum source. Salivary gland sporozoites from mosquitoes that fed on immune human sera contained bound human IgG, which was detectable by indirect immunofluorescence assay. The infectivity and transmission potential of human IgG-coated sporozoites is unknown.     

Cross-reactivity of the NPa and NPb idiotypic responses of BALB/c and C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl (NP)

A comparative antigenic analysis was carried out to determine whether cross-reactivity exists between the major idiotypic responses to (4-hydroxy-3-nitrophenyl)acetyl (NP) in BALB/c and C57BL/6 mice. Extensive cross-reactivity exists between the NPa (BALB/c) and NPb (C57BL/6) allotype-linked idiotypic responses to NP. The cross-reactive determinants of the NPb idiotype are confined to one particular group of NPb-positive monoclonal antibodies. The extent of cross-reactivity between this group of C57BL/6 antibodies and idiotype-positive monoclonal antibodies of BALB/c is so great that they cannot be readily distinguished as NPb- or NPa-positive antibodies with polyclonal anti-idiotypic reagents. That this cross-reactivity is not unique to monoclonal antibodies was confirmed by the demonstration of these cross-reactive determinants in the immunesera of individual BALB/c and C57BL/6 mice. Additionally, evidence was obtained from these experiments and from earlier ones from this laboratory which suggests that the BALB/c idiotypic response to NP-protein conjugate is more homogeneous than the C57BL/6 idiotypic responses.     

T cell receptor (TCR) engagement in apoptosis-defective, but interleukin 2 (IL-2)-producing, T cells results in impaired ZAP70/CD3-zeta association

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-beta results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-beta also resulted in impaired association of CD3-zeta, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-zeta were detected, although the overall level of tyrosine-phosphorylated CD3-zeta was low. More strikingly, inducible association of ZAP70 to CD3-zeta was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-zeta is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.     

Cytotoxic and anti-proliferative effects of high-energy pulsed ultrasound (HEPUS) on human squamous cell carcinoma cells as compared to connective tissue fibroblasts

The cytotoxic and anti-proliferative effects of high-energy pulsed ultrasound (HEPUS) on human squamous cell carcinoma cells cloned from the hypopharynx (FaDu) and benign connective tissue cells (fibroblasts) were investigated in vitro. Sonication was carried out using an experimental piezoelectric, self-focusing burst-signal transducer. To increase the induction of cavitation, the transducer used was specifically designed to produce multiple oscillations with a high negative pressure amplitude. In both cell lines tested, the application of 100, 800 and 2000 pulses resulted in a high reduction of vital cells. After 2000 pulses, 4.0 +/- 1.1% of the fibroblasts but only 2.0 +/- 0.4% of the FaDu cells survived HEPUS exposure. A postexposure inhibiting effect of HEPUS for 10 days on the proliferation of surviving cells was noted for the FaDu cells exposed to 2000 pulses, but not as much for the fibroblasts. These findings support the hypothesis that human squamous cell carcinoma cells of the hypopharynx might be more sensitive to HEPUS than fibroblasts and that total tumor cell ablation might be possible in vitro given a sufficient number of HEPUS pulses.     

Determination of adhesive properties of variant metastatic melanoma cells to BALB/3T3 cells and their virus-transformed derivatives by a monolayer attachment assay

The role of phospholipids in the activity of UDP-D-galactose: D-xylose galactosyltransferase (galactosyltransferase I) from embryonic chick cartilage was investigated. Phospholipase C treatment of particulate galactosyltransferase I caused inactivation of this enzyme to the extent of 60 to 70% as well as hydrolysis of 75 to 80% of the membrane phospholipids. Addition of phospholipid restored activity to nearly control levels. The order of effectiveness of various phopholipids in reactivating phospholipase C-treated galactosyltransferase I was as follows: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylcholine. The effect of phospholipase A on galactosyltransferase I activity was also examined and was found to be concentration-dependent. At concentrations less than 10 mug/mg of pellet protein, phospholipase A slightly activated galactosyltransferase I. whereas at higher concentrations it inhibited the activity in a manner similar to phospholipase C. Galactosyltransferase I was activated moderately and also solubilized by treatment with Nonidet P-40 in the presence of 0.5 M KCl. Following solubilization and purification by gel filtration and affinity chromatography, galactosyltransferase I could be inactivated by detergent removal by dialysis and subsequently reactivated by addition of detergent. Neither phospholipase C treatment nor exogenous phospholipid had any significant effect on three of the other chondroitin sulfate glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase, UDP-D-glucuronic acid:3-O-beta-D-galactosyl-D-galactose glucuronosyltransferase, and UDP-N-acetyl-D-galactosamine:(BlcUA-GalNAc-4-sulfate)j N-acetylgalactosaminyltransferase). On lipid analysis by thin layer chromatography, phosphatidylcholine and phosphatidylethanolamine were found to be the major phospholipids of particulate and solubilized glycosyltransferase preparations from embryonic chick cartilage, while lysopholipids of particulate and solubilized glycosyltransferase preparations from embryonic chick cartilage, while lysophosphatidylcholine and lysophosphatidylethanolamine were barely detectable components. The concentration of these specific phospholipids was diminished greatly following phospholipase C treatment.     

Cytotoxic mechanisms of new antitumor nucleoside analogues, 3'-ethynylcytidine (ECyd) and 3'-ethynyluridine (EUrd)

The cytotoxic mechanisms of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil (EUrd) were studied with mouse mammary tumor FM3A cells and human fibrosarcoma HT1080 cells. ECyd and EUrd are converted to ECyd 5'-triphosphate (ECTP) in the cells. ECTP has also outstanding stability in the cells; the half life of ECTP in FM3A cells was more than 3 days. The metabolisms and mechanisms of these analogues may play a key role in a potent antitumor activities against slow-growing solid tumors.     

Comparison of propylene oxide and epichlorohydrin effects in two transformation tests (C3H/10T1/2 and SHE cells)

The neoplastic cell transformation induced by propylene oxide (PO) and epichlorohydrin (ECH) was studied in two in vitro assays, mouse embryo fibroblasts (C3H/10T1/2) and Syrian hamster embryo (SHE) cells. In C3H/10T1/2 cells treated with PO (2.5-10 mM), the transformation frequencies were enhanced about 2-4 times in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with the transformation frequencies in the absence of TPA. In SHE cells, an even higher increase (about 6-9 times) was reached at concentrations of 2.5-20 mM. The presence of TPA strongly influenced the ability of ECH to induce the morphological transformation at low-moderate concentrations (0.25-1 mM). At the highest concentrations applied, 1 mM in C3H/10T1/2 cells and 0.5 mM in SHE cells, 41- and 4-fold increases, respectively, were observed. In C3H/10T1/2 cells, the rad-equivalence (rad/mMh) of PO and ECH in the presence of TPA was calculated to be 36 +/- 8 and 296 +/- 65 (mean +/- S.E.), respectively.     

Dose-dependent promoting effects of catechol on glandular stomach carcinogenesis in BALB/c mice initiated with N-methyl-N-nitrosourea

The effects of catechol administration in the diet on stomach carcinogenesis in mice after initiation with N-methyl-N-nitrosourea (MNU) in the drinking water were investigated in a development trial for a new experimental protocol. Male 6-week-old BALB/c mice were given MNU in the drinking water intermittently for a total of three one-week periods, with one-week intervals, at the concentration of 120 ppm (groups 1 and 2). Groups 3 and 4 served as non initiated controls. From week 7, groups 1 and 3 were divided into three subgroups and the mice were fed on diet containing 0.05% (groups 1a and 3a), 0.2% (groups 1b and 3b), 08% (groups 1c and 3c) or 0% (groups 2 and 4) catechol for 29 weeks. At week 20, appreciably enhanced development of pepsinogen 1-altered pyloric glands was noted in all catechol-treated groups, in a partially dose-dependent manner (12.8 +/- 12.5, 13.8 +/- 11.7, and 24.0 +/- 12.7/100 pyloric glands respectively, for groups 1, 2 and 3). The incidences of adenomas (groups 1, 2 and 3) were also increased. At week 35, dose-dependent induction of adenocarcinomas in groups 1 (3/19), 2 (3/19) and 3 (14/20) was evident. In addition, the depth of invasion of the adenocarcinomas was enhanced by catechol in a dose-dependent manner, though the histological type was not influenced. Thus, the administration of catechol in the diet strongly enhanced the preneoplastic and neoplastic lesions in mouse glandular stomach induced by MNU in the drinking water, in a dose-dependent manner.     

Transfer of thyroiditis, with syngeneic spleen cells sensitized with the human thyrotropin receptor, to naive BALB/c and NOD mice

In previous studies we have induced TSH binding-inhibiting Igs and thyroiditis in BALB/c mice and thyroiditis alone in NOD mice immunized with the extracellular domain of the human TSH receptor produced as a maltose-binding protein fusion in bacteria (MBP-ECD). In this study, our aim was to determine whether thyroiditis can be transferred to syngeneic naive recipients with in vivo and in vitro primed spleen cells. Groups of 6-week-old female BALB/c and NOD mice were immunized ip with MBP-ECD in an adjuvant of alum plus attenuated Bordetella pertussis toxin, on days 0 (100 micrograms), 14, 28, and 35 (50 micrograms). These mice (in vivo primed) and nontreated age- and sex-matched controls were killed on day 43, and their spleens and thyroids were removed, the latter to verify the induction of thyroiditis in the antigen-treated mice. Splenocytes were disrupted mechanically and cultured at 3 x 10(6)/ml in RPMI supplemented with 20 micrograms/ml MBP-ECD for 48-64 h. After this in vitro priming, some of the splenocytes received no further treatment, but a portion was fractionated into a CD4+-enriched population. Groups of 6-week-old female BALB/c and NOD mice were immunized into the tail vein with 100-200 microliters PBS containing approximately 10(5)-10(7) unfractionated T cells (both in vivo primed and not) and CD4+-enriched (in vivo primed) splenocyte populations. The animals were killed 16 days later, and their thyroids were examined histologically and by immunohistochemistry. In addition, levels of antibody to the MBP-ECD priming antigen were assessed by enzyme-linked immunosorbent assay in the antigen- and spleen-treated mice. In the donor animals, in vivo priming resulted in an extensive lymphocytic infiltration of the thyroids in both BALB/c and NOD mice and follicular destruction in the latter. There was no evidence of thyroiditis in all 9 BALB/c mice and all 4 NOD mice who received unfractionated T cells from mice that had not been primed in vivo. In contrast, transfer of MBP-ECD in vivo primed unfractionated T cells resulted in thyroiditis in 9 of 13 BALB/c mice and 5 of 6 NOD mice; similarly, the equivalent CD4+-enriched population produced extensive thyroiditis in 2 of 3 BALB/c mice and all three NOD mice. The most striking difference between the antigen- and spleen-treated mice was in the quantity of the infiltrate, which was much greater in the latter and extended throughout the thyroid glands of these animals. In common with mice treated directly with the MBP-ECD antigen, the infiltrates of both BALB/c and NOD recipient mice contained large numbers of activated T cells expressing the receptor for interleukin-2, and macrophages and dendritic cells were plentiful, particularly in the BALB/c mice, in which B cells and interleukin-10-positive T cells were also present. The most abundant infiltrates, containing numerous CD8+ T cells and follicular destruction, were observed in NOD mice receiving primed unfractionated T cells or CD4+-enriched T cells. In contrast to the donors, none of the recipient animals had circulating antibodies to the MBP-ECD antigen. In conclusion, we have shown that it is possible to transfer thyroiditis with spleen cells from mice primed in vivo with a human TSH receptor preparation. Furthermore, the thyroiditogenic activity appears to reside in the CD4+ population.     

Suppression of the benzylpenicilloyl- (BPO) specific IgE formation with isologous anti-idiotypic antibodies in BALB/c mice

In vivo effects of actively produced or passively administered isologous anti-idiotypic antisera (aId) on the benzylpenicilloyl- (BPO) specific IgE and IgG formation in BALB/c mice have been studied. Isologous anti-BPO aId were raised in BALB/c mice by immunization with purified anti-BPO antibodies isolated from ascites induced with BPO-bovine gamma-globulin in the same mouse strain. Mice producing isologous anti-BPO aId exhibited long-term suppression of BPO-specific IgE and IgG antibody responses induced by BPO-ovalbumin (BPO-OVA) in aluminum hydroxide. Simultaneously, they produced increased amounts of anti-BPO aId after each challenge with the BPO-OVA antigens. Passive administration of isologous anti-BPO aId into syngeneic mice previously sensitized with BPO-OVA caused depression of BPO-specific IgE antibody levels for 2 to 3 weeks. When anti-BPO IgE had again reached its previous level, passively administered aId had decreased to the level of untreated mice. Passive administration of anti-BPO aId also depressed the primary anti-BPO IgE formation for 2 to 3 weeks. In all these experiments the IgE antibody formation against the carrier proteins used for BPO-antigens was not affected. These results show that IgE and IgG antibodies share major idiotypic determinants and that IgE production is accessible to regulation by aId.     

Effects of pyrethroid insecticides on gap junctional intercellular communications in Balb/c3T3 cells by dye-transfer assay

The effects of fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin, p-chlorophenylisovaleric acid (CPIA, major metabolite of fenvalerate) and DDT, a liver tumor promoter, on gap junctional intercellular communication (GJIC) were examined in Balb/c3T3 cells by dye-transfer assay. Separate groups of Balb/c3T3 cells were exposed to the chemicals for 1 day. On the following day, GJIC was measured by counting the number of dye-transferring cells per injection of Lucifer Yellow under a fluorescent microscope. Fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin and DDT inhibited GJIC at noncytotoxic concentrations, while CPIA did not inhibit GJIC even at a cytotoxic concentration. It is concluded that the examined pyrethyroid insecticides, but not a metabolite, have inhibitory effects on GJIC in Balb/c3T3 cells.