Pathways and regulation of B-cell responsiveness and tolerance.
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Animals↗
Biomedical subjects
D W Scott
Publications and source records attributed to D W Scott.
Failure of terfenadine as an antipruritic agent in atopic dogs: results of a double-blinded, placebo-controlled study.
Terfenadine (5 mg/kg body weight, q12h) and placebo (0.5 grain/dog q12h) were both administered orally as individual agents to 18 dogs with atopy in a double-blinded study. No dog improved. Hyperactivity, polyphagia, lethargy, anorexia, increased pruritus, or ocular discharge were seen in three dogs treated with terfenadine. Under the conditions of the study, terfenadine was not a useful antipruritic agent for the atopic dog.
Administration, Oral↗
Distribution and characterization of tumor necrosis factor-alpha-like immunoreactivity in the murine central nervous system.
Tumor necrosis factor-alpha (TNF alpha) is a protein released from macrophages during infection and inflammation. Recent studies suggest that it has several effects within the central nervous system, including generation of fever, enhancement of slow wave sleep, and stimulation of pituitary hormone secretion. We have proposed that TNF alpha may be synthesized by neurons in the CNS and used as a neuromodulator in the pathways involved in the central control of these activities. To test this hypothesis, we have used an antiserum raised against recombinant murine (rm) TNF alpha with an indirect immunoperoxidase technique to stain the murine CNS immunohistochemically. Western blot analysis of mouse brain homogenates revealed one band with electrophoretic mobility identical to that of rmTNF alpha. We identified TNF alpha-like immunoreactive (ir) neurons in the hypothalamus, in the bed nucleus of the stria terminalis, in the caudal raphe nuclei, and along the ventral pontine and medullary surface. TNF alpha ir innervation was widespread within the CNS, particularly in areas involved in autonomic and endocrine regulation, including the hypothalamus, amygdala, bed nucleus of the stria terminalis, parabrachial nucleus, dorsal vagal complex, nucleus ambiguus, and thoracic sympathetic preganglionic cell column. Our data suggest that TNF alpha may serve as a neuromodulator in central pathways involved in the regulation of the autonomic, endocrine and behavioral components of the acute-phase response to inflammation and infection.
Animals↗
Clinical efficacy of milbemycin oxime in the treatment of generalized demodicosis in adult dogs.
Thirty adult dogs with chronic generalized demodicosis were treated with oral administration of milbemycin oxime (0.52 to 3.8 mg/kg of body weight, q 24 h). Results of skin scrapings were used to determine whether administration of milbemycin should be continued or discontinued. Dogs that were free of clinical signs of demodicosis 12 months after administration of milbemycin was discontinued were considered cured. Sixteen dogs were cured, 5 dogs were never cleared of mites, and 9 dogs were cleared of mites, but relapsed after administration of milbemycin was discontinued. Adverse effects of milbemycin were rare and transient.
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Antisense oligodeoxynucleotides to the blk tyrosine kinase prevent anti-mu-chain-mediated growth inhibition and apoptosis in a B-cell lymphoma.
Crosslinking of membrane immunoglobulin (mIg) receptors by anti-Ig causes growth inhibition and subsequent cell death due to apoptosis in a murine B-cell lymphoma model. The earliest signal transduction via mIg has recently been shown to be dependent on the activation of one or more protein tyrosine kinases (PTKs). In this study, we utilized the CH31 lymphoma, which is extremely sensitive to growth inhibition by anti-Ig, to examine the role of PTKs in cell cycle arrest. This cell line expresses multiple PTKs, whose activities are stimulated by crosslinking mIg. To determine whether PTK activity is essential for the inhibition of cell growth, we exposed CH31 cells to antisense oligodeoxynucleotides for the blk PTK prior to the growth inhibition assay. We found that exposure of CH31 cells to blk antisense effectively prevented anti-mu-chain-mediated growth inhibition and subsequent apoptosis. Corresponding blk sense or antisense oligonucleotides for other PTKs had no protective effect against anti-mu. Moreover, antisense blk oligonucleotides had no effect on transforming growth factor beta-mediated growth arrest and apoptosis. Further experiments showed significantly reduced endogenous p55blk in blk antisense-treated cells. In addition, anti-mu stimulation of antisense-treated cells failed to induce any detectable increase in kinase activity of p55blk, a result suggesting the uncoupling of blk proteins from normal signal pathways that are essential for growth inhibition. These results implicate a role of blk kinase in anti-mu-mediated pathway to cell cycle arrest.
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B cell tolerance induction by cross-linking of membrane IgM, but not IgD, and synergy by cross-linking of both isotypes.
Previous studies in our laboratory demonstrated that overnight exposure of adult splenic B cells to anti-Ig resulted in an unresponsive state characterized by decreased antibody synthesis but normal mitogen-driven proliferation (i.e., energy). Because both anti-F(ab')2 and anti-mu were equally effective at inducing tolerance, it was important to determine whether cross-linking of IgD together with or separately from IgM influenced the induction of unresponsiveness. Although anti-mu induced significant unresponsiveness, treatment of adult splenic B cells with anti-delta alone generally failed to reduce the subsequent response to either LPS or fluoresceinated Brucella abortus. Interestingly, anti-delta synergized with suboptimal concentrations of anti-mu to induce tolerance. Synergy could be observed in this system when anti-delta was added either simultaneously with or before (but not after) anti-mu; moreover, anti-delta was effective in a pretreatment (wash-out) protocol. To investigate the role of protein tyrosine kinase (PTK) activity in tolerance induction, splenic B cells were treated with tyrphostin before treatment with either anti-mu or anti-delta. We found that pretreatment with tyrphostin for 2 h before the addition of anti-mu prevented the induction of unresponsiveness with this antibody, whereas this PTK inhibitor facilitated tolerance when used with anti-delta treatment only. We propose that cross-linking of surface IgM directly or indirectly invokes a tyrphostin-sensitive, PTK-dependent pathway leading to the early events in tolerance induction, which can be augmented under limiting conditions by anti-IgD. Because cross-linking of either receptor initiates several common pathways, simultaneous cross-linking can lead to synergy and a dominance of the IgM signal. In contrast, IgD alone may fail to elicit tolerance because this isotype may also be associated with different PTK that cause positive signaling.
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Inhibition of protein tyrosine kinase activity by herbimycin A prevents anti-mu but not LPS-mediated cell cycle progression and differentiation of splenic B lymphocytes.
Protein tyrosine kinases (PTKs) have been implicated in signal transduction in a variety of cell types. B lymphocytes express the genes encoding for eight members of the src family of nonreceptor PTKs. Four of these PTKs (p55blk, p53/56lyn, p59fyn, and p56lck) are activated by the ligation of mIg receptors. The functional roles of these PTKs in membrane-bound immunoglobulins (mIg) receptor-mediated activation of resting B lymphocytes were examined using the PTK inhibitor, herbimycin A. Here we show that mIg receptor-mediated B-cell proliferation and differentiation were inhibited by treatment with herbimycin A, while inhibitor-treated B cells retained LPS (mitogen) responsiveness for proliferation and antibody formation. Further studies demonstrated that herbimycin A blocked the G0 to G1 transition during B-cell activation. When the effects of herbimycin A were directly examined by a kinase activity assay, the enzymatic activity of each PTK was inhibited to varying degrees. The inhibition of PTK activity was also reflected by reduced tyrosine phosphorylation of intracellular substrates, including phospholipase C-gamma. These results implicate PTK-dependent signaling pathways in the mIg receptor-mediated functional activation of B lymphocytes.
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Analysis of B cell tolerance in vitro.
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Animals↗
Expression of protein tyrosine kinases in the Ig complex of anti-mu-sensitive and anti-mu-resistant B-cell lymphomas: role of the p55blk kinase in signaling growth arrest and apoptosis.
The src family of non-receptor protein tyrosine kinases (PTKs), including the blk, fyn, lyn and lck kinases, is expressed in B-lineage cells, may associate with the immunoglobulin receptor complex and, therefore, play a role in signal transduction via membrane IgM. To establish which of these PTKs is involved in growth inhibition of B-cell lymphomas by anti-mu, we examined the expression pattern and state of activation of these kinases in nine B-cell lymphomas. Tyrosine-phosphorylated p55blk was constitutively expressed in all growth-inhibitable lymphomas; furthermore, anti-mu caused a relative increase of tyrosine phosphorylation in p55blk and a 2- to 3-fold increase in its kinase activity in these cells within minutes. In contrast, p55blk was not present in three of five anti-mu-resistant lymphomas and there was no detectable increase of blk activity in one of the resistant cell lines tested. Thus, we proposed that activatable blk kinase in the IgM complex is essential for the growth inhibitory effect of anti-mu. To test this hypothesis, CH31 lymphoma cells were treated with antisense oligos for the blk kinase and found to be resistant to anti-mu-mediated growth inhibition and subsequent apoptosis. These studies implicate the blk kinase as an integral part of the growth inhibitory pathway leading to arrest and apoptosis. Transfectants of blk gene constructs are being generated to further test this hypothesis.
Animals↗
Medical management of allergic pruritus in the cat, with emphasis on feline atopy.
Pruritus associated with allergic skin diseases is an important reason for consultation in feline medicine. The pathophysiology of pruritus is poorly understood in the cat, but histamine and eicosanoids appear to be important mediators. The medical management of allergic pruritus in cats, especially those with atopy, using glucocorticoids, antihistamines, omega-3/omega-6 fatty acid-containing products, and combinations thereof is discussed.
Animals↗
Nonsteroidal anti-inflammatory agents in the management of canine allergic pruritus.
Pruritus associated with allergic skin diseases is an important reason for consultation in canine medicine. The pathophysiology of pruritus is poorly understood in the dog, but histamine, leukotrienes, and prostaglandins appear to be important mediators. The medical management of allergic pruritus in dogs with nonsteroidal anti-inflammatory agents is discussed. Antihistamines, tricyclic antidepressants, and omega-3/omega-6 fatty acid-containing products are often useful, either as single agents, in combinations, or for reducing glucocorticoid requirements.
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Synergy between TGF-beta and anti-IgM in growth inhibition of CD5+ B-cell lymphomas.
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Animals↗
Dermatologic disorders of Chinese Shar Peis: 58 cases (1981-1989).
A retrospective study on the skin diseases of Chinese Shar Peis was conducted over a 9-year period. Skin disease was found in 58 (49.2%) of the 118 dogs studied. Folliculitis was the most common clinical finding (43 of 58 dogs). In 6 dogs, there was no apparent reason for the folliculitis; however, it was secondary to allergic dermatitis, demodicosis, IgA deficiency, or hypothyroidism in the other dogs. Approximately 20% of the dogs had more than one of these disorders. Serum IgA concentration was measured in 7 dogs and was low in all 7.
Animals↗
Evaluation of an allergy screening test for use in atopic dogs.
An in-office allergy screening kit was evaluated for its repeatability, sensitivity, and specificity in 21 atopic dogs. Results of this testing were compared with results of a full regionalized allergy test offered by the same manufacturer. Results of the screening kit were repeatable and had high specificity but poor sensitivity. The kit as presently marketed is of questionable value as a diagnostic aid for use in dogs with atopy.
Animals↗
Cross-linking of surface IgM or IgD causes differential biological effects in spite of overlap in tyrosine (de)phosphorylation profile.
Although displaying similar amounts of surface IgM and IgD, ECH 408-1 cells only succumb to apoptosis after cross-linking of IgM (not IgD), suggesting that different signaling pathways couple to both receptors. Immunoprecipitation studies revealed the presence of several proteins selectively associated with IgM and IgD, thus ruling out that the lack of inhibitory signaling mediated by IgD might be due to membrane expression in the absence of associated proteins belonging to the B cell receptor complex. 32P metabolic labeling and immunoprecipitation studies demonstrated that IgM and IgD are associated with phosphoproteins of 32-33 kDa in an isotype-specific fashion. Kinetic analyses of tyrosine kinase activity showed that cross-linking of surface IgM or IgD resulted in the rapid (1-3 min) phosphorylation of several protein substrates on tyrosine residues, followed by a dephosphorylation step. Isotype-specific changes of the phosphorylation status specifically affected molecules in the 32-33 kDa range, i.e. IgM (not IgD) cross-linking affected a approximately 32-kDa protein, whereas IgD (not IgM) cross-linking induced phosphorylation of a protein exhibiting a slightly lower mobility (33 kDa). These results suggest that isotype-specific immunoglobulin-associated molecules could be involved in the second messenger cascade leading to different biological effects upon IgM and IgD cross-linking.
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Effect of priming with a thymus-independent antigen on susceptibility to B-cell tolerance.
The effects of priming on the susceptibility of B-cell subsets to tolerance induction have been tested in a model system in which anti-immunoglobulin (anti-Ig) has been employed as a surrogate for tolerogen. T-cell-depleted B cells were primed in vitro with fluorescein or trinitrophenylated Ficoll (a thymus-independent (TI) antigen) and then exposed overnight to anti-Ig to attempt to induce B-cell anergy. Primed cells were relatively resistant to this tolerance protocol and resistance was hapten specific. The dose response and kinetics suggested that this process was not due to receptor blockade or modulation, but was an active process. Moreover, this priming for resistance to tolerance was reproduced in vivo upon intraperitoneal treatment with haptenated Ficoll. Such in vivo priming for tolerance resistance was long-lasting and did not occur with a thymus-dependent priming protocol with fluoresceinated hemocyanin. These results are discussed in terms of TI priming to drive B cells into cycle and express novel functional and phenotypic properties.
Animals↗
Prostaglandin E2 induces apoptosis in immature normal and malignant B lymphocytes.
The purpose of this research was to determine whether prostaglandin E2 (PGE2), a major product of macrophages which can kill certain murine B cell lymphomas, induces death by a necrotic mechanism or by an alternate pathway called apoptosis. CH31 is a phenotypically "immature" B cell lymphoma which resembles immature neonatal B cells in its susceptibility to killing by reagents which cross-link surface immunoglobulin (sIg). In the present study we first show that PGE2, but not the closely related prostanoid, PGF2 alpha, kills CH31 lymphoma cells. In contrast, CH12, a phenotypically "mature" lymphoma which is not negatively affected by sIg cross-linking, is not induced to die after exposure to PGE2. Agarose gel electrophoresis demonstrated that the DNA of PGE2-treated CH31, but not CH12 cells, is cleaved into characteristic 200 base pair oligonucleosomal fragments indicative of an apoptotic mechanism of death. However, a necrotic form of death, indicated by random DNA cleavage which produces a smear following electrophoresis, could be induced by treatment of CH12 or CH31 with anti-class II MHC antibodies and complement. The apoptotic mechanism of CH31 cell killing by PGE2 was confirmed using scanning electron microscopy which demonstrated the unique membrane blebbing and bubbling pathognomonic of this form of death. Finally, using a recently devised flow cytometric method to study apoptosis in heterogeneous cell populations, we compared the ability of anti-IgM, PGE2, or PGF2 alpha to induce apoptosis in B lymphocytes from neonatal or adult mice. Anti-IgM, and to a lesser extent PGE2, but not PGF2 alpha, induces apoptosis in a fraction of neonatal B cells. None of these treatments induced cell death in B lymphocytes from mature mice. Overall, these observations suggest that PGE-secreting cells such as macrophages, which inhabit the B cell microenvironments of lymphoid organs, may eliminate a subset of immature B lymphocytes and may be important in controlling the spread of PGE-sensitive malignant B lymphoma cells.
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Characterization of "modified-self"-induced specific antibody hyporesponsiveness to herpes simplex virus.
Pretreatment of mice iv with syngeneic spleen cells modified with soluble HSV envelope antigens induced an anti-HSV antibody hyporesponsiveness following challenge with infectious virus. The epitope density on the HSV-modified spleen cells was quantitated using a photon-counting spectrofluorimeter so that observed immunological results could be correlated with the HSV antigen dose on the splenocytes. The degree of anti-HSV antibody hyporesponsiveness was found to be related to the epitope density on the HSV-modified spleen cells, but not the number of modified cells used in the pretreatment over the 16-fold range tested. Anti-HSV antibody hyporesponsiveness was induced if 7, but not 3, days had elapsed between pretreatment and challenge. This antibody hyporesponsiveness could be adoptively transferred with T cells. Only mice that had induced an anti-HSV antibody hyporesponsiveness following pretreatment with HSV-modified splenocytes were able to survive an LD50 challenge with infectious virus.
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