Effects of starvation,chilling, and injury on endocrine gland function in Galleria mellonella

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain-centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands. The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects. None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.     

The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity.

Signals delivered to B cells via CD40 can synergize with those provided by other B cell surface receptors to induce B cell proliferation and antibody class switching as well as modulate cytokine production and cell adhesion. Recently, it has been shown that the ligand for CD40 is a cell surface protein of approximately 39 kDa expressed by activated T cells, gp39. Here we report on the isolation and characterization of a cDNA clone encoding human gp39, a type II membrane protein with homology to TNF, and the construction and characterization of a soluble recombinant form of gp39. COS cell transfectants expressing gp39 synergized with either anti-CD20 mAb or PMA to drive strong B cell proliferation and alone were able to drive B cells to proliferate weakly. In all cases the B cell proliferation induced by gp39-expressing COS cells was reduced to background levels by the addition of soluble CD40. Unlike gp39-expressing COS cells, recombinant soluble gp39 was not mitogenic alone and required co-stimulation to drive B cell proliferation. These results suggest that B cells require a second signal besides gp39-CD40 to drive proliferation and that soluble gp39 alone in a non-membrane bound form is able to provide co-stimulatory signals to B cells.     

Triangularis sterni: a primary muscle of breathing in the dog

The isolated action, pattern of neural activation, and mechanical contribution to eupnea of the triangularis sterni (transversus thoracis) muscle were studied in supine anesthetized dogs. Linear displacement transducers were used to measure the axial displacements of the ribs and sternum. Tetanic stimulation of the triangularis sterni in the apneic animal caused a marked caudal displacement of the ribs, a moderate cranial displacement of the sternum, and a decrease in lung volume. During quiet breathing, there was invariably a rhythmic activation of the muscle in phase with expiration that was independent of the presence or absence of activity in the abdominal and internal interosseous intercostal muscles. This phasic expiratory activity in the triangularis sterni was of large amplitude and caused the ribs to be more caudal and the sternum to be more cranial during the spontaneous expiratory pause than during relaxation. Additional studies on awake animals showed that rhythmic activation of the triangularis sterni occurs in all body positions and is not caused by anesthesia. These findings indicate that expiration in the dog is not a passive process and that the end-expiratory volume of the rib cage is not determined by an equilibrium of static forces alone. Rather, it is actively determined and maintained below its relaxation volume by contraction of the triangularis sterni throughout expiration. The use of this muscle is likely to facilitate inspiration by increasing the length of the parasternal intercostals and taking on a portion of their work.     

Activation of antigen-enriched B cells. II. Role of linked recognition in B cell proliferation to thymus-dependent antigens

The responsiveness to T-dependent (TD) and T-independent (TI) TNP-antigens of murine splenic B cells previously enriched for antigen-binding cells (ABC) was examined. TNP-TI antigens induced B cell proliferation. TNP-TD antigens did not induce a proliferative response regardless of the physical form or nature of the TNP-TD antigen (e.g., soluble vs particulate, low or high haptenation of carrier, TNP on various insoluble matrices, etc.). TNP-TD antigens were effective in enhancing the response of the TNP-ABC to all concentrations of lipopolysaccharide (LPS) tested, indicating that binding of antigen to surface immunoglobulin alters the LPS responsiveness of the cell. Irradiated, keyhole limpet hemocyanin- (KLH) primed T cells induced a threefold to fourfold greater B cell proliferative response with TNP-KLH than with fluoresceinated KLH (FLU-KLH) or FLU-KLH together with TNP-human serum albumin (TNP-HSA). Therefore, linked recognition appears essential for optimal T cell-mediated B cell proliferation, whereas the induction of B cell proliferation via nonlinked, carrier-activated T cells is a minor component of the response.     

Abdominal muscle use during breathing in unanesthetized dogs

The pattern of abdominal muscle use during breathing in unanesthetized dogs is unknown. Therefore, we have recorded the electromyograms of the rectus abdominis, external oblique, and transversus abdominis in eight conscious animals breathing quietly in the sitting, standing, and prone postures. During quiet breathing in the sitting posture, all animals invariably had a large amount of phasic expiratory activity in the transversus abdominis. In contrast, only four animals showed some expiratory activity in the external oblique, and only one animal had expiratory activity in the rectus abdominis. A similar pattern was observed when the animals were standing or lying prone, although the amount of expiratory activity was less in this posture. Bilateral cervical vagotomy in four animals did not affect the degree of transversus abdominis expiratory activation or the influence of posture. We conclude that in conscious dogs 1) the abdominal muscles play an important role during breathing and make spontaneous quiet expiration a very active process, 2) the transversus abdominis is the primary respiratory muscle of the abdomen, and 3) unlike in anesthetized animals, extrapulmonary receptors play a major role in promoting abdominal expiratory contraction.     

Membrane Ig-cytoskeletal interactions. III. Receptor cross-linking results in the formation of extensive filamentous arrays of vimentin

The proposed function of intermediate filaments is to provide a cell type-specific structural framework that maintains cell shape and organelle distribution and mediates signal transduction through its connections with the plasma membrane and the nucleus. Vimentin is the intermediate filament protein expressed in B lymphocytes. Immunocytochemical analysis of the high salt-stable cytoskeletons from B cells stimulated with anti-Ig revealed an increased accumulation of vimentin in the cytoskeleton compared to nontreated controls. This increased accumulation of vimentin in the cytoskeleton was manifested by the organization of vimentin into extensive filamentous arrays (EFA) as viewed in the fluorescent microscope. In contrast to the effects of anti-Ig, activation of B cells with LPS did not induce the organization of vimentin into EFA. This suggested that signals unique to anti-Ig directed EFA formation. Immunocytochemical results were verified by biochemical analysis showing that vimentin was more abundant in isolated cytoskeletons from anti-Ig activated B cells, than cytoskeletons isolated from LPS-activated B cells. These observations established a relationship between increased content of vimentin in the cytoskeleton and the formation of EFA. By testing a wide variety of activating agents, we were able to correlate increased vimentin expression in the cytoskeleton to activating agents that cross-link membrane Ig. It appeared that treatment of B cells with LPS prohibited the induction of EFA by anti-Ig because cotreatment with both anti-Ig and LPS resulted in decreased vimentin accumulation in the cytoskeleton to a level less than that in resting cells. The significance of these results with regard to B cell biology is discussed.     

Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines.

Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN-gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL-5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells.     

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.     

Co-localization of ecdysteroid receptors and c-fos-like protein in the brain of Manduca sexta larvae

Summary The presence of c-fos, a marker for cell activation, was investigated in cerebral neurons actively expressing ecdysteroid receptors during larval-pupal development in the tobacco hornworm, Manduca sexta. Colocalization was accomplished by ecdysteroid autoradiography using the tritiated high affinity 20-hydroxyecdysone agonist ponasterone A and immunocytochemistry with an antibody to a peptide sequence which is highly conserved in both human and murine c-fos. Immunoreactivity to a c-fos-like protein(s) was present in nuclei of many neurons of all the developmental stages examined. However, with the exception of the optic lobe, cells expressing nuclear ecdysteroid receptors were more immunoreactive than non-ecdysteroid-binding neurons. These data suggest that ecdysteroid-induced gene activation and translation may involve c-fos expression. Offprint requests to: H.-J. Bidmon