The bovine papillomavirus type 1 (BPV1) replication protein E1 modulates transcriptional activation by interacting with BPV1 E2.

The study of bovine papillomavirus type 1 (BPV1) DNA replication has shown that E1 and E2 are the only viral proteins required for this process. Both E1 and E2 interact with the viral origin of replication (ori). The BPV1 E2 protein is also a well-characterized transcriptional regulator. We show in this report that E1 can modulate transcription by interactions with E2. At low concentrations, E1 enhanced the E2-mediated transactivation of heterologous promoters containing the BPV1 ori by promoting cooperative binding of both E1 and E2 to the DNA. In contrast, in the presence of excess E1, transactivation by E2 is repressed. This last process, however, does not require cooperative DNA binding of the two proteins. These results imply that the balance between these two distinct types of interaction is crucial both for control of replication and for early viral transcription.

Mouse T-lymphocyte activation by Urtica dioica agglutinin. I.--Delineation of two lymphocyte subsets.

Urtica dioica agglutinin (UDA) is a mouse T-lymphocyte-specific mitogen endowed with proliferative characteristics different from ConA, the prototypic T-lymphocyte mitogen. In particular, UDA induces 2-3-fold-reduced thymidine incorporation as compared to ConA. In an attempt to define the basis of this reduced proliferation, we analysed whether UDA binds to a unique subset of T lymphocytes or whether it activates only a T-cell subset. Cytofluorimetric analysis showed that this lectin binds uniformly to all T lymphocytes and does not, on this criterion, distinguish a particular T-cell subset. We next analysed whether UDA provokes the activation of all T lymphocytes. This was carried out by measuring the increase in cell size and the induction of the p55 chain of the IL2 receptor. The analysis showed that, throughout the kinetics of cell activation, only one subset of T lymphocytes increased in size and expressed the p55 chain of the IL2 receptor, suggesting that UDA activates only a subpopulation of T cells. This conclusion was strengthened by the analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA of UDA-activated cells. Two populations were easily identifiable: a BrdU-negative subset consisting of all the small p55-negative lymphocytes, and a BrdU-labelled subset including all the large p55-positive cells. BrdU was incorporated in both CD4+ and CD8+ cells, indicating that UDA did not distinguish helper from cytotoxic T lymphocytes. In addition to the p55 chain of the IL2R, all cycling cells expressed the Pgp-1 activation marker. The T lymphocytes, which bound UDA but did not proliferate, remained fully susceptible to subsequent stimulation by ConA. In conclusion, the capacity to proliferate upon UDA binding differentiates a UDA-sensitive from a UDA-refractory subset among splenic mouse T lymphocytes.

Mouse T-lymphocyte activation by Urtica dioica agglutinin. II.--Original pattern of cell activation and cytokine production induced by UDA.

Urtica dioica agglutinin (UDA) is a T-lymphocyte-specific polyclonal activator that differs from ConA, the classical mouse T-cell mitogen, by inducing a late and limited proliferation of a distinct T-cell subset recruited among both CD4+ and CD8+ lymphocytes. We investigated the possibility that the particular kinetics may originate from UDA-specific activation processes in which the known early mandatory signals were completed only after an extended delay. We report that the time of contact required between lectin and the cell membrane to acquire the capacity to proceed into cell cycle was much longer (36-40 h) for UDA than for ConA (8-10 h). Addition of phorbol ester, which artificially induces PKC translocation, or ionomycin, which provokes Ca2+ mobilization, did not accelerate the proliferative kinetics, suggesting that these early mandatory signals are not the limiting factors in the delayed proliferation. The induction of c-myc was retarded in the UDA group, and there was a good correlation between the kinetics of c-myc induction and the kinetics of cell proliferation. The comparison of the level of transcription of the genes encoding different cytokines revealed additional differences between the two mitogens: the whole wave of cytokine gene expression was delayed with UDA. In particular, IL2, IL3 and IFN gamma gene expression was retarded compared to the ConA-induced single wave. An even later transcriptional wave took place at around 72 h for IL4 and IL5. Finally, this particular kinetics corresponded to an unusually high level of IL3 and IFN gamma and a low level of IL4 and IL5 gene transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional heterogeneity of virgin and memory B murine lymphocytes revealed by the utilization of cyclosporin A: an overview.

The effects of cyclosporin A on the generation and revelation of B memory cells by thymus-independent (TI) antigens was investigated. A class 1 (TNP-LPS) and a class 2 (TNP-Ficoll) TI antigens were used for priming an elicitation. Evidence is presented that cyclosporin A does not interfere with the generation of hapten-specific (TNP) B memory cells by TNP-LPS or DNP-Ficoll. Cyclosporin A does not affect the revelation of B memory cells by TNP-LPS, but inhibits their revelation by TNP-Ficoll. These findings are discussed in terms of two distinct B cell lineages leading to antibody-forming cells and memory cells precursors, and in terms of heterogeneity of B memory cells.

Improvement of an ELISA bioassay for the routine titration of murine interferon-gamma.

We improved an ELISA bioassay for murine IFN-gamma (MuIFN-gamma) based on measurement of Ia antigen on P388D1, a mouse macrophagic tumour line. Cells were cultured in microtitre plates in medium containing dilutions of IFN-gamma source. They were then washed and stained with a rat anti-mouse I amAb followed by mouse anti-rat peroxidase-labelled antibody. After incubation with substrate, the OD was read directly from microtitre plates. Standard curves obtained with reference NIH MuIFN-gamma showed that this assay allowed for the definition of unit values (giving 50% of the maximal effect) comparable to NIH international units (IU). It detected as low as 0.2 IU/ml of MuIFN-gamma and, in contrast to antiviral assays, was insensitive to IFN-alpha/beta. We used a concanavalin A-conditioned supernatant, which is a mixed source of lymphokines, to assess the specificity of our assay. Indeed, Ia expression induced by ConA-conditioned supernatant was fully inhibited by preincubation with anti-MuIFN-gamma antibodies. Using a stable indicator cell line, the present cell surface assay is easier to perform than other ELISA using bone-marrow-derived macrophages, and does not require cell fixation; its high sensitivity and specificity are comparable to that of immunoradiometric assays. It is thus valuable for routine MuIFN-gamma quantitations in culture supernatant and biological fluids.

Brain neocortex modulation of mitogen-induced interleukin 2, but not interleukin 1, production.

Interleukin 2 (IL-2) production by splenic T cells stimulated by Concanavalin A was studied in mice after unilateral or bilateral brain neocortex ablation. The brain cortex was shown to modulate IL-2 production in an asymmetrical way. IL-2 levels were higher in animals with a right cortical lesion (group R) and lower in mice with a symmetrical lesion (group L) as compared to controls, differences between groups R and L being significant. Such variations of IL-2 production that were observed after unilateral lesions were abolished with bilateral cortical ablations. These results extend the immunoregulatory roles of the brain neocortex to IL-2 production by splenic T cells and may provide molecular support for neuro-immunological networks.

Urtica dioica agglutinin, a new mitogen for murine T lymphocytes: unaltered interleukin-1 production but late interleukin 2-mediated proliferation.

Urtica dioica agglutinin, a small-molecular-weight lectin purified from stinging nettle rhizomes, induces murine cell proliferation. U. dioica agglutinin is a specific T-cell mitogen for both thymocytes and spleen T lymphocytes; its mitogenic properties are strictly dependent on the presence of accessory cells. The kinetics of proliferation are markedly different from those of the classical T-cell mitogen concanavalin A, with a 2 to 3-day delay for both splenic and thymic populations and a rate of DNA synthesis twofold lower than that observed with concanavalin A. The late T-lymphocyte proliferation induced by U. dioica agglutinin correlates well with (i) the observed late interleukin-2 production and interleukin-2 receptor expression, and (ii) the long-lasting cyclosporin A-sensitive early activation period. In contrast, the production of interleukin-1 is not different, both in terms of concentration and kinetics, from that observed with concanavalin A.

A sensitive, IL-2-independent, assay for IL-1.

The thymocyte costimulator (LAF) assay, the standard biological test used for IL-1 titration, has a low sensitivity and lacks specificity since it can be potentiated by the IL-2 which is frequently present in IL-1-containing biological fluids. We describe here a new IL-1 titration method which takes advantage of the capacity of a thymoma line, EL4-6.1, to differentiate and express IL-2 receptors upon stimulation by IL-1 in the presence of a suboptimal dose of phorbol diester. Membrane IL-2R measurement on this indicator cell line permits the detection of 1-2 X 10(-4) ng/ml IL-1, compared to 5 X 10(-2) ng/ml in the LAF assay. In addition, rIL-2 up to 250 U/ml has no effect on IL-1 measurement by this assay, which also exhibits a 100-fold lower sensitivity to inhibitory effects of prostaglandin, compared to the LAF assay. Finally, tumor necrosis factor alpha only exerts a weak costimulation effect at very high doses. A flow cytometry technique and an ELISA are described for IL-2 receptor detection. Due to its high sensitivity and specificity, this novel assay should now permit reliable IL-1 titration in biological fluids such as IL-2-rich lymphocyte culture supernatants.

Murine primary in vivo response to TNP-Ficoll: multigenic control involving H-2.

Since the primary PFC response to TNP-Ficoll, a thymus-independent type 2 antigen, displays an important variability in vivo among diverse inbred mouse strains, we used, in the present report, H-2 congenic strains possessing different genetic backgrounds to show that the amplitude of this response is governed by MHC genes, with one regulating locus situated in or near the centromeric part of the I-A subregion. In addition, this H-2 control was largely modulated by gene(s) located outside MHC and IgH haplotypes, as evidenced by the response of recombinant inbred strains (BXH) between the high responder C3H/HeJ and the low responder C57BL/6J. Our results are discussed in terms of humoral regulations and the requirement for self-recognition in cellular interactions which lead to activation of B lymphocytes in the in vivo primary response towards TI-2 antigens.

Immune memory expression to Tnp-Ficoll in CB.20 mice: evidence for a multigenic control.

Trinitrophenyl (Tnp)-Ficoll, a class 2 thymus-independent (TI) antigen, generates in most mouse strains Tnp-specific B-memory cells which can be detected in situ 1 week after priming by a heterologous stimulation with Tnp-lipopolysaccharide (LPS) but not by a homologous Tnp-Ficoll challenge. We have investigated the secondary responses raised in CB.20 congenic mice by a homologous challenge in situ occurring at various time intervals after priming. We report that a memory-type response is obtained, culminating when the challenge is performed at 4 weeks; this finding assesses definitely the ability of TI-2 antigens to produce immunological memory under standard conditions. However, the same immunization procedure elicits no memory-type response in the majority of other mouse strains, suggesting a possible genetic control of the expression of memory to class 2 TI antigens. The utilization of F1 hybrids between C57BL/6 and BALB/c and of appropriate congenic strains shows indeed that this memory expression is under multigenic control: Igh-V or closely linked genes are clearly involved but a complementation with other gene(s), located outside the H-2 complex, is required for a memory-type response to Tnp-Ficoll. We have also analyzed the secondary heterologous response to Tnp-LPS in CB.20 mice at different times after Tnp-Ficoll priming. The difference in the kinetic profile of the heterologous (TI-2----TI-1) versus homologous (TI-2----TI-2) secondary responses is discussed in terms of B-memory-cell ontogeny and humoral regulation.

Study on B-memory generation by Tnp-Ficoll: induction but not expression is observed among various inbred mouse strains.

The primary and secondary responses to Tnp-Ficoll, a class 2 thymus-independent antigen, were assessed in various inbred strains of mice. The eventual implication of H-2 or IgH linked genes was searched for. Contrasting with our previous reports using Tnp-LPS, a class 1 thymus-independent antigen, no homologous memory-type response to Tnp-Ficoll and consequently no genetic control was observed. However Tnp-specific B-memory lymphocytes were induced in most strains since a heterologous challenge with Tnp-LPS evoked a typical memory type response characterized by an increased number of antibody-secreting cells and/or significant amount of anti-Tnp antibodies of the IgG isotype. The lack of memory revelation by Tnp-Ficoll is discussed in terms of a possible humoral or cellular regulation and of B-memory cell generation and maturation.