A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.

Helper/inducer T cell clones specific for protein antigens and class II MHC determinants consist of two nonoverlapping subsets. One (called Th1) secretes IL 2 and IFN-gamma and the other (Th2) produces BSF1 upon stimulation with antigen or polyclonal activators. By using hapten-binding normal B cells and the B lymphoma line WEHI-279 as assays for B cell helper (maturation) factors, we have shown that Th2 clone supernatants (SN) induce differentiation to antibody secretion, whereas Th1 SN do not. The failure of Th1 SN to activate B cells is due to inhibitory effects of IFN-gamma, because it can be reversed by a neutralizing monoclonal antibody specific for IFN-gamma. Thus, in the presence of this antibody, even Th1 SN stimulate B cell maturation maximally. Conversely, recombinant IFN-gamma inhibits proliferation and differentiation of B cells induced by active Th2 SN. These results demonstrate that IFN-gamma is a potent inhibitor of B lymphocyte activation and can be distinguished from growth and maturation-inducing helper factors that are produced by both subsets of helper T cells.

The findings presented in this study provide evidence that BSF1 receptors and mIg transmit signals via dissimilar transduction mechanisms that result in a common biologic response, hyper-Ia expression. Specifically, BSF1-containing supernatant does not induce PtdInsP2 hydrolysis as determined by measurement of PtdOH and InsP3. Additionally, BSF1 does not stimulate Ca2+ mobilization, PKC translocation from cytosol to membrane, or membrane depolarization. All of these metabolic events appear to play a central role in hyper-Ia expression mediated by mIg and are initiated after treatment of resting B cells with anti-Ig antibodies. In vitro phosphorylation studies with partially purified plasma membranes from resting B cells revealed that BSF1 interaction with membrane receptors stimulates a membrane-associated protein kinase that phosphorylates an endogenous protein of 44 KDa. Anti-Ig does not stimulate phosphorylation of the 44 KDa protein, suggesting that it does not activate the membrane-associated protein kinase. This observation provides the first evidence of a signal transduction mechanism associated with BSF1-receptor ligation. It indicates that although BSF1 does not modulate events associated with PKC activation, it may function via activation of a membrane-associated protein kinase. This provides a focal point for further studies directed at elucidating signal transduction resulting from BSF1-receptor interaction.

The presence of receptors specific for murine B cell stimulatory factor 1 (BSF1) was demonstrated by utilizing an internally radiolabeled recombinant BSF1. Radiolabeled BSF1 was efficiently produced in Xenopus laevis oocytes injected with a cloned mRNA for BSF1 and 35S-methionine. The labeled BSF1 specifically bound to splenic B cells. A Scatchard analysis indicated the existence of one class of receptor sites. BSF1 receptors were found to be distributed on a wide range of hematopoietic lineage cells, including B cells, T cells, macrophages, and mast cells. B cells from CBA/N mice with the xid gene defect had a similar level of BSF1 binding capacity compared with BALB/c strain B cells, and responded well to insoluble anti-Ig and BSF1 in proliferation assays, indicating that CBA/N B cells express functional BSF1 receptors at normal levels. Pre-B cell lines showed low levels of BSF1 binding, suggesting that cells in the B cell lineage acquire BSF1 responsiveness early in development.

Three factors with distinct function are involved in the regulation of the B-cell response into antibody-producing cells: (1) a factor for the activation of resting B cells (BSF1/IL-4), (2) a factor for the growth of activated B cells (BCGFII/IL-5), and (3) a factor for the final maturation of B cells into antibody-producing cells (BSF2/IL-6). The cDNAs for these three molecules have been cloned, and studies with recombinant molecules demonstrated that their individual functions were not confined to the B-lineage cells; they were found to have a wide variety of biological functions. A typical example of a pleiotropic function of such interleukins is BSF2/IL-6. BSF2/IL-6 is not only a B-cell-differentiation factor, but also is a potent growth factor for myeloma cells. Moreover, BSF2/IL-6 acts as hepatocyte-stimulating factor to induce acute-phase proteins and multicolony-stimulating factor to activate hematopoietic stem cells. It appears that BSF2/IL-6 plays an essential role in the host defense mechanisms against infections, inflammation, and injury. Receptors for BSFs have not yet been molecularly cloned because of the low density of receptor molecules. Recently, the cloning of the cDNA for BSF2/IL-6 receptor has been achieved and its molecular structure and the signals transduced through it are described herein.

The rat GABAA receptor delta subunit gene has been isolated and its promoter mapped to facilitate studies of neuron-specific gene transcription. The gene is transcribed from a single promoter with several clustered transcriptional initiation sites. The major start site maps to a prototype initiator sequence located in the center of a TATA-less CpG-rich island. Analysis of the 5'-flanking region of the GABAA receptor delta subunit gene revealed a novel tandemly repeated purine sequence element. Furthermore, gel retardation assays identified several tissue-specific and ubiquitous proteins that bind to this sequence. One of these factors, brain-specific factor (BSF) 1, was only observed in extracts from brain and was distinct from BETA and PAL, two DNA-binding proteins with similar purine-rich recognition sequences. The distribution of BSF1 in brain closely matched the expression pattern previously determined for the delta subunit mRNA, and its binding activity appeared simultaneously with GABAA receptor delta subunit mRNA during differentiation of cerebellar granule cells in vitro. Our data therefore suggest that BSF1 is a novel brain-specific and neuronal cell type-enriched factor possibly involved in differential transcription of the GABAA receptor delta subunit gene.

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by "veto cells" contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)

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This experiment aimed to evaluate substitution of soybean meal (SBM) by black soldier fly (BSF) larvae meal in a napier grass diet as performed by an in vitro rumen fermentation system.

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Samples of napier grass, SBM, and BSF larvae age 1 week (BSF1) and 2 weeks (BSF2) were arranged according to the following dietary treatments (dry matter [DM] basis): T1, 100% napier grass; T2, 60% napier grass + 40% SBM; T3, 60% napier grass + 40% BSF1; T4, 60% napier grass + 40% BSF2; T5, 60% napier grass + 20% SBM + 20% BSF1; and T6, 60% napier grass + 20% SBM + 20% BSF2. The samples were determined for their chemical composition and were incubated in vitro using buffered rumen fluid for 48 h at 39°C. In vitro incubation was carried out in three runs and represented by two incubation bottles per run.

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Supplementation of BSF, both BSF1 and BSF2, increased ether extract, neutral- and acid-detergent insoluble crude protein contents of T3-T6 diets. The T3 or T4 diet resulted in lower ruminal ammonia concentration, in vitro DM digestibility, and in vitro organic matter (OM) digestibility as compared to those in T2 (p<0.05). Diet supplemented with BSF produced lower methane emission in comparison to that of supplemented with SBM (p<0.05). Diet containing BSF2 produced lower methane and methane per digestible OM than that containing BSF1 (p<0.05).

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Substitution of SBM by BSF in ruminant diet results in a lower nutritional value in vitro but with an advantage of lowering ruminal methane emission.

We investigated the effect of a lymphokine termed 'suppressive B-cell factor' (SBF), which is produced by FcR gamma (Fc receptor for IgG)-stimulated B cells or hybridoma TS4.44, and is known to suppress B-cell responses in vivo and in vitro by inhibiting their proliferation. Small B cells, fractionated by Percoll density gradient, from athymic nude mice (BALB/c) secreted SBF after binding EA (sheep erythrocytes sensitized with IgG mouse anti-sheep erythrocyte antibody), and the proliferation of small but not large B cells was preferentially suppressed by SBF in response to LPS in vitro. Proliferation of purified B cells from BALB/c nu/nu mice, induced by a synergistic interaction between F(ab')2 fragment of goat anti-mouse IgM antibody and B-cell stimulating factor (BSF1), was almost completely abrogated by the co-existence of SBF during the 72-hr culture period. However, the co-culture with SBF for the last 24 or 48 hr, as well as of B cells pretreated with SBF for 1 hr at 37 degrees, partially inhibited the growth response. These findings suggest that SBF operates on resting B cells and holds them in a resting state. This notion would be further supported by the fact that SBF inhibited G0-G1 transition. Taken together, we conclude that SBF acts on the early step of B-cell activation, thereby inhibiting B-cell growth. Arrest of resting B cells in the G0 phase and failure of an increase in functional receptors for BSF1 seem to be responsible for the suppression of B-cell responses.

We studied the effect of immune complexes (IC) on the responses of polyclonally activated murine B cells. For this, normal resting B cells were stimulated with the F(ab')2 fraction of goat anti-mouse mu-chain antibody and B-cell stimulating factor 1 (BSF1) after preculturing them with IC. Next, the relative membrane potential changes and the subsequent proliferative response were analysed. IC, particularly in antibody excess, inhibited both membrane depolarization and while those in antigen excess did poorly. Neither antigen nor antibody alone was effective. Inhibition was mediated via binding of IC to FcR gamma on B cells in a dose-dependent manner. Kinetic experiments showed that at least 6 hr was necessary for inducing the suppression of B-cell responses after binding of IC. We conclude that IC bound to FcR gamma on B cells regulates B-cell responses by acting on the initial step of activation.

A rat monoclonal antibody (NIM-R3) was found to be reactive with activated mouse B cells but neither activated T cells nor small resting lymphocytes, T or B. The antibody stains antibody-forming cell precursors within 48 hr of primary or secondary immunization in vivo, but not at longer times (greater than 14 days) immunization. When added to cultures of spleen cells responding in vitro to dinitrophenylated bovine, IgG, the number of antibody-forming cells was increased. NIM-R3 also maintained the proliferation of purified B blasts in the absence of lipopolysaccharide, but did not activate small resting B cells in the presence of anti-Ig. NIM-R3 could replace B-cell growth factor (BCGF II) in cultures of the murine B-cell lymphoma BCL1 inducing proliferation but not differentiation. Finally, competition was demonstrated for binding sites on BCL1 cells between BCGF II and NIM-R3, but not between NIM-R3 and T-cell replacing factor (B15-TRF). We suggest that NIM-R3 may represent a novel specificity and may be directed against the cell surface receptor for BCGF II, and in turn this receptor may be independent of the B15-TRF and BSF1 receptors.