Induction Of Desensitization Research Articles

Allergen-specific B cell responses in oral immunotherapy-induced desensitization, remission, and natural outgrowth in cow's milk allergy.

Antigen-specific memory B cells play a key role in the induction of desensitization and remission to food allergens in oral immunotherapy and in the development of natural tolerance (NT). Here, we characterized milk allergen Bos d 9-specific B cells in oral allergen-specific immunotherapy (OIT) and in children spontaneously outgrowing cow's milk allergy (CMA) due to NT. Samples from children with CMA who received oral OIT (before, during, and after), children who naturally outgrew CMA (NT), and healthy individuals were received from Stanford biobank. Bos d 9-specific B cells were isolated by flow cytometry and RNA-sequencing was performed. Protein profile of Bos d 9-specific B cells was analyzed by proximity extension assay. Increased frequencies of circulating milk allergen Bos d 9-specific B cells were observed after OIT and NT. Milk-desensitized subjects showed the partial acquisition of phenotypic features of remission, suggesting that desensitization is an earlier stage of remission. Within these most significantly expressed genes, IL10RA and TGFB3 were highly expressed in desensitized OIT patients. In both the remission and desensitized groups, B cell activation-, Breg cells-, BCR-signaling-, and differentiation-related genes were upregulated. In NT, pathways associated with innate immunity characteristics, development of marginal zone B cells, and a more established suppressor function of B cells prevail that may play a role in long-term tolerance. The analyses of immunoglobulin heavy chain genes in specific B cells demonstrated that IgG2 in desensitization, IgG1, IgA1, IgA2, IgG4, and IgD in remission, and IgD in NT were predominating. Secreted proteins from allergen-specific B cells revealed higher levels of regulatory cytokines, IL-10, and TGF-β after OIT and NT. Allergen-specific B cells are essential elements in regulating food allergy towards remission in OIT-received and naturally resolved individuals.

Oral immunotherapy combined with omalizumab for high\u2013risk cow\u2019s milk allergy: a randomized controlled trial

We evaluated the efficacy and safety of oral immunotherapy (OIT) combined with 24 weeks of omalizumab (OMB) at inducing desensitization in children with cow’s milk allergy (CM) compared with an untreated group. The present study was a prospective randomized controlled trial. Sixteen patients (age, 6–14 years) with high IgE levels to CM were enrolled in the present study. Patients were randomized 1:1 to receive OMB-OIT group or untreated group. The primary outcome was the induction of desensitization at 8 weeks after OMB was discontinued in OMB-OIT treated group and at 32 weeks after study entry. None of the 6 children in the untreated group developed desensitization to CM while all of the 10 children in the OIT-OMB treated group achieved desensitization (P < 0.001). A significantly decreased wheal diameter in response to a skin prick test using CM was found in the OMB-OIT treated group (P < 0.05). These data suggest that OIT combined with OMB using microwave heated CM may help to induce desensitization for children with high-risk CM allergy. This prospective randomized controlled trial was intended for 50 participants but was prematurely discontinued due to overwhelming superiority of OMB combined with microwave heated OIT over CM avoidance.

Oral immunotherapy in clinical practice

Food avoidance is still considered the gold-standard treatment for food allergy (FA). However, recently it has been consolidating the theory that, where possible, the food intake might facilitate the induction of desensitization and tolerance, resulting more effective and safer -if we consider the possibility of accidental ingestion- than a strict avoidance diet [1]. Furthermore, specific oral immunotherapy (OIT) is the unique active treatment for IgE-mediated FAs [1].

Immunotherapy for food allergies: a myth or a reality?

Food allergy is a worldwide issue, with an estimated prevalence of 2-10%. An effective treatment is not available for people affected and the only management is the avoidance of the allergen. Oral immunotherapy and sublingual immunotherapy have been tested by several authors, in particular for milk, egg and peanuts allergy, with significant results in term of desensitization induction. The achievement of tolerance is by the contrary doubtful, with different results obtained. In this review, we reviewed protocols of oral and sublingual immunotherapy for food allergy published in literature, mainly against milk, egg and peanut. At present, immunotherapy does not represent the gold standard in the treatment of food allergy, even if it can desensitize patients.

Pharmacological characterization of desensitization in scratching behavior induced by intrathecal administration of hemokinin-1 in the rat

Desensitization is induced by the repeated administration of high doses of substance P (SP) or hemokinin-1 (HK-1). However, little information is available about the mechanisms involved in the induction of desensitization by these peptides. Thus, to characterize this desensitization, we examined the dose-dependent effect of these peptides, the effect of pretreatment with neurokinin 1(NK1) receptor antagonists, and the effect of pretreatment with inhibitors of protein kinases such as protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin kinase II (CaMKII) and mitogen-activated protein kinase kinase (MEK). The number of scratchings induced by 10(-3)M SP or HK-1 decreased following pretreatment with 10(-11)-10(-3)M SP or HK-1 with a marked reduction at 10(-3) and 10(-6)M SP or HK-1. The effect of NK1 receptor antagonists on desensitization induced by pretreatment with 10(-6)M SP was marked, whereas there was little effect of pretreatment with these antagonists on 10(-6)M HK-1-induced desensitization. Additionally, 10(-6)M SP- and HK-1-induced desensitization was attenuated by pretreatment with PKA, PKC and MEK inhibitors, except a CaMKII inhibitor that inhibited SP-induced desensitization. These results indicate that the receptor and kinases involved in HK-1-induced desensitization are partially different from those of SP.

Transient Resensitization Interrupting the Development of Carbachol-induced Desensitization in Smooth Muscle of Guinea-pig Taenia Caeci: Ca&lt;sup&gt;2+&lt;/sup&gt;-dependent Termination of Resensitization

It is important to clarify developmental mechanisms of desensitization because of their great significance in regulation of cellular responsiveness. We have found that carbachol-induced desensitization to carbachol develops in three successive phases in the presence of extracellular Ca(2+) in the smooth muscle of guinea pig taenia caeci: fast desensitization within 15 s, transient resensitization reaching a peak at 1 min and the subsequent re-development of desensitization to terminate resensitization for up to 30 min. In contrast, in the absence of extracellular Ca(2+), desensitization develops without resensitization. To further clarify the roles of Ca(2+) in the formation of the transient resensitization phase, we examined the developmental process of carbachol-induced desensitization in the absence of extracellular Ca(2+), following the induction of desensitization by a 15-s treatment with carbachol in the presence of extracellular Ca(2+). Desensitization to carbachol occurred due to pretreatment with 10(-4) M carbachol for 15 s in normal physiological solution, and continued pretreatment with carbachol in Ca(2+)-free solution containing 0.2 mM EGTA induced resensitization followed by the obscure progress of re-desensitization for up to 30 min resulting in a long-lasting phase of resensitization. These results suggest that resensitization is promptly terminated by the Ca(2+)-dependent development of subsequent desensitization for further regulation of cellular responsiveness via G(q) protein-coupled Ca(2+)-mobilizing receptors against sustained stimuli.

Phosphorylation and Activation of a cAMP-specific Phosphodiesterase by the cAMP-dependent Protein Kinase: INVOLVEMENT OF SERINE 54 IN THE ENZYME ACTIVATION

A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M.(1994) J. Biol. Chem. 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation. The PKA-dependent incorporation of phosphate in the partially purified, recombinant rat PDE4D3 followed a time course similar to that of activation. Half-maximal activation of the enzyme was obtained with 0.6 microM ATP and 30 nM of the catalytic subunit of PKA. Phosphorylation altered the Vmax of the PDE without affecting the Km for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram. Cyanogen bromide cleavage of the 32P-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell-free system. Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of 32P was reduced to the largest extent in mutants with both Ser13 --> Ala and Ser54 --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3. While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser54 completely suppressed the kinase activation. Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser54 --> Ala substitution could not be activated by the endogenous PKA in the intact cell. These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser54 in this domain plays a crucial role in activation.

Characterization of acute homologous desensitization of μ‐opioid receptor‐induced currents in locus coeruleus neurones

1. Acute homologous desensitization of mu-opioid receptor-induced currents was pharmacologically characterized in locus coeruleus (LC) neurones by use of intracellular and whole cell recording in superfused brain slices. 2. Following desensitization of opioid receptors by perfusion with a high concentration of [Met5] enkephalin (ME) for 5 min, there was a reduction in the maximum response and a rightward shift of the concentration-response curves for ME, [D-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO) and normorphine. 3. By simultaneously fitting the operational model to the paired pre- and post-desensitization concentration-response data for each agonist, estimates of the level of desensitization were obtained. The values obtained for the three agonists (between 88% and 96%) were similar and did not vary according to the efficacy of the agonist used. 4. Use of whole cell patch recording techniques caused a slow rundown in the amplitude of ME currents (approx. 40% reduction over 60 min) but did not greatly affect the expression of acute desensitization of opioid currents. 5. When included in the patch recording solution, the phosphatase inhibitors, microcystin (50 nM-4 microM) and okadaic acid (1 microM) had no effect on the induction of desensitization or the normal ability of opioid or alpha 2-adrenoceptors to produce currents. Microcystin decreased the rate of recovery of the ME (300 nM) currents following desensitization; however, okadaic acid had little effect on the rate of recovery from desensitization. 6. Strong calcium buffering with BAPTA (10-20 mM) had no effect on desensitization or the recovery from desensitization. 6. Strong calcium buffering with BAPTA (10-20 mM) had no effect on desensitization or the recovery from desensitization.7 These results suggest that acute homologous desensitization of micro-opioid receptors in LC neurones entails a rapid loss of responsiveness that involves a majority of the receptor population. The mechanism by which desensitization is reversed may involve a non-calcium-dependent protein phosphatase but the processess that cause desensitization remain unclear.

Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation

We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.

Molecular mechanisms in down-regulation of tumor necrosis factor expression.

Excessive production of tumor necrosis factor (TNF) after stimulation by lipopolysaccharide (LPS) may result in fever, intravascular coagulation, and lethal shock. An efficient way of preventing the excessive TNF production is desensitization of monocytes/macrophages to LPS. We have analyzed the molecular mechanisms involved in the induction of desensitization and the mechanisms operative in the desensitized, LPS-refractory cells by employing the human monocytic cell line Mono-Mac-6. Similar to human blood monocytes, treatment of Mono-Mac-6 cells with LPS (1 microgram/ml) results in a rapid and transient expression of TNF. When Mono-Mac-6 cells are precultured in medium containing low levels of LPS, they become refractory to subsequent LPS stimulation and show no or little secretion of TNF protein. Desensitization can be blocked by the inhibition of cyclooxygenase and protein kinase C; both prostaglandin E2 (together with a second signal) and phorbol 12-myristate 13-acetate can mimic desensitization. By employing prostaglandin E2 and low concentrations of phorbol 12-myristate 13-acetate, a synergism in the induction of desensitization can be demonstrated. Hence, our studies show that two distinct pathways are involved in the induction of hyporesponsiveness. In both LPS-responsive and LPS-desensitized Mono-Mac-6 cells, LPS was able to induce the transcription factor NF-kappa B in the nucleus. Still, the prevalence of TNF-specific mRNA was dramatically reduced in the desensitized cells. These data indicate that LPS-desensitized Mono-Mac-6 cells are able to activate initial steps of signal transduction up to the level of the NF-kappa B transcription factor. The absence of TNF transcripts, however, indicates that additional nuclear factors may be missing or that silencers may be active such that transcription of the TNF gene is prevented.

Agonist-induced desensitization of a P2Y-purinergic receptor-regulated phospholipase C

A guanine nucleotide-dependent P2Y-purinergic receptor-regulated phospholipase C activity of turkey erythrocyte membranes has been characterized in detail previously (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890). The occurrence of agonist-induced desensitization of this receptor-regulated phospholipase C is now described. Preincubation of turkey erythrocytes with the P2Y-purinergic receptor agonist ADP beta S resulted in a marked loss of capacity of ADP beta S plus GTP to stimulate phospholipase C in membranes derived from these cells. The half-time of occurrence of desensitization was 0.5-2.0 min, and within 10 min responsiveness had reached a new quasi-steady state level representing 40-55% of control. Transfer of agonist-preincubated erythrocytes to agonist-free medium resulted in recovery of agonist plus GTP responsiveness of the membrane phospholipase C activity to control levels with a half-time of 10-20 min. The change in ADP beta S plus GTP responsiveness occurred as a loss of maximal effect with little or no change in the apparent affinity of agonist for stimulation of inositol phosphate production. Induction of desensitization occurred with an agonist-specificity that followed that expected of a P2Y-purinergic receptor. Neither the rate of activation nor the final phospholipase C activity attained in the presence of GTP gamma S alone was altered in membranes from cells preincubated with ADP beta S for 15 min. AlF-4-stimulated inositol phosphate production was also not modified in membranes from agonist-preincubated erythrocytes. In contrast, the capacity of ADP beta S to increase the rate of activation of phospholipase C by GTP gamma S was markedly reduced in membranes from agonist-preincubated cells. The amount of 3H-radioactivity in phosphoinositides, as well as the ratio of labeling among the phosphoinositides, was not altered by incubation of erythrocytes with a P2Y-purinergic receptor agonist. Taken together these data suggest that P2Y-purinergic receptor agonist-induced desensitization occurs as a consequence of a modification at the level of the receptor or at the level of receptor-guanine nucleotide regulatory protein (G-protein) coupling with no change occurring in the capacity of the G-protein to activate phospholipase C.

Induction of desensitization by phorbol ester to β-adrenergic agonist stimulation in adenylate cyclase system of rat reticulocytes

Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in the desensitization of adenylate cyclase to the β-adrenergic agonist stimulation depending on the dose and period of the TPA treatment. Treatment of the reticulocytes with TPA caused approximately 40% reduction in the stimulation by β-adrenergic agonists of adenylate cyclase activity, whereas the treatment had little effect on the basal activity and the activation by fluoride and guanine nucleotide of the enzyme system. No change in the number of β-adrenergic receptors was observed after the TPA treatment. Treatment with 1-oleoyl-2-acetyl-glycerol (OAG), an activator of protein kinase C, also caused the desensitization of reticulocyte adenylate cyclase to isoproterenol. On the other hand, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C, prevented the desensitization induced by TPA. These results suggest the involvement of protein kinase C in a process of desensitization of adenylate cyclase system to β-adrenergic agonists in rat reticulocytes.

Regulation of insulin and insulin-like growth factor (IGF) responsiveness by IGFs in rat hepatoma cells.

We have previously reported that insulin induces a complete and reversible desensitization to the induction of tyrosine aminotransferase by insulin and insulin-like growth factors (IGFs) in HTC rat hepatoma cells. This loss of responsiveness cannot be accounted for by down-regulation of cell surface receptors, but occurs at a postbinding step in hormone action. Here we present evidence that IGF-I and IGF-II also induce desensitization to the actions of both IGFs and insulin and that this effect is mediated by IGF receptors. First, the concentration dependence for this effect is similar to that for the IGF-I and IGF-II induction of tyrosine aminotransferase, which has been shown to be mediated by IGF receptors. Second, antibody to the insulin receptor, which blocks insulin, but not IGF-II, binding to HTC cells, causes a rightward shift in the concentration dependence for insulin induction of desensitization, but does not significantly change the concentration dependence for IGF-II. These results indicate that IGF-II-induced desensitization to IGF-II is not mediated by the insulin receptor, but presumably by an IGF receptor. Although the IGFs do cause a moderate decrease in the binding of IGFs and insulin (to approximately 50-75% of the control value), this cannot account for the virtually complete desensitization to their actions. We conclude that IGFs, like insulin, induce a nearly complete loss of responsiveness to insulin and IGFs, that this effect is mediated via IGF receptors, and that desensitization occurs at a step distal to hormone binding which may be common to the actions of insulin and the IGFs.

Inhibition of protein synthesis on homologous and heterologous desensitization in the rat adipocyte

Desensitization of lipolysis was induced in isolated rat adipocytes by incubation with isoproterenol 10 −5M or ACTH 250 mU/ml for two and three hours, respectively. Those cells desensitized with isoproterenol were restimulated with either isoproterenol 10 −7M or ACTH 6 mU/ml and those cells desensitized with ACTH were restimulated with isoproterenol 10 −7M. Lipolysis was quantitated by the release of cyclic AMP and glycerol. No effect on either homologous or heterologous desensitization was observed when either cycloheximide 2 μg/ml or puromycin 10 −4M was included in the incubation media during the induction of desensitization. These findings support the conclusion that protein synthesis plays no role in the desensitization of lipolysis in the isolated rat adipocyte.

Refractoriness of the cyclic AMP response to catecholamines in rat parotid slices in vitro.

Parotid slices respond to norepinephrine with a rapid but transient accumulation of cyclic AMP. Continued exposure of the slices to norepinephrine results in a loss of responsiveness to subsequent application of norepinephrine (refractoriness or desensitization). Refractoriness could not be overcome by exposing the slices to a supra-maximum concentration of norepinephrine. The suppressed response was clearly seen at 15 min after the first exposure to norepinephrine, but about 60 min were required for reaching complete suppression. The refractoriness also depended on the concentration of agonists, and their order of potency correlated well with their agonistic activity. The induction of desensitization was beta- agonist specific, and was blocked by beta-adrenergic blocking agents such as atenolol and alprenolol. The recovery from desensitization was observed by removing norepinephrine from the medium. The recovery was not evident until 15 min after washing the slices, but was nearly complete after 1 hr. Although parallel changes were observed between the initial increase in cyclic AMP level and the degree of desensitization by norepinephrine, the induction of desensitization does not seem to be mediated by cyclic AMP. The mechanism underlying this phenomenon does not appear to involve the activation of phosphodiesterase, the formation of an inhibitory substance in the medium or an increase in the rate of excretion of cyclic AMP. Changes in the ATP level by norepinephrine did not always correlate with the degree of refractoriness. No significant changes in the amount of [3H]-dihydroalprenolol binding were found in the membrane fraction prepared from the slices incubated with norepinephrine. These results suggest that the induction of desensitization may be mediated by modulating the coupling process between the receptor and adenylate cyclase.