GRPR/Extracellular Signal-Regulated Kinase and NPRA/Extracellular Signal-Regulated Kinase Signaling Pathways Play a Critical Role in Spinal Transmission of Chronic Itch.

Intractable or recurrent chronic itch greatly reduces the patients' QOL and impairs their daily activities. In this study, we investigated whether there are certain key signaling molecules downstream of the recently identified peptides mediating itch in the spinal cord. RNA sequencing analysis of mouse spinal cord in chronic itch models induced by squaric acid dibutylester and imiquimod showed that extracellular signal-regulated kinase (ERK) 1/2 cascade is the most significantly upregulated gene cluster in both models. In four different mouse models of chronic itch, sustained ERK phosphorylation was detected mainly in spinal neurons, and MAPK/ERK kinase inhibitors significantly inhibited chronic itch in these models. Phosphorylated ERK was observed in the interneurons expressing the receptors of different neuropeptides for itch, including gastrin-releasing peptide receptor, natriuretic peptide receptor A, neuromedin B receptor, and sst2A. Blocking gastrin-releasing peptide receptor and natriuretic peptide receptor A by genetic approaches or toxins in mice significantly attenuated or ablated spinal phosphorylated ERK. When human embryonic kidney 293T cells transfected with these receptors were exposed to their respective agonists, ERK was the most significantly activated intracellular signaling molecule. Together, our work showed that phosphorylated ERK is a unique marker for itch signal transmission in the spinal cord and an attractive target for the treatment of chronic itch.

Spinal GRPR and NPRA Contribute to Chronic Itch in a Murine Model of Allergic Contact Dermatitis.

Recurrent and intractable chronic itch is a worldwide problem, but mechanisms, especially the neural mechanisms, underlying chronic itch still remain unclear. In this study, we investigated the peripheral and spinal mechanisms responsible for prolonged itch in a mouse model of allergic contact dermatitis induced by squaric acid dibutylester. We found that repeated exposure of mice to squaric acid dibutylester evoked persistent spontaneous scratching and significantly aberrant cutaneous and systemic immune responses lasting for weeks. Squaric acid dibutylester-induced itch requires both nonhistaminergic and histaminergic pathways, which are likely relayed by GRPR and NPRA in the spinal cord, respectively. Employing genetic, pharmacologic, RNAscope assay, and cell-specific ablation methods, we dissected a neural circuit for prolonged itch formed as Grpr+ neurons act downstream of Npr1+ neurons in the spinal cord. Taken together, our data suggested that targeting GRPR and NPRA may provide effective treatments for allergic contact dermatitis-associated chronic pruritus.

Conjugate Vaccines from Bacterial Antigens by Squaric Acid Chemistry: A Closer Look.

By using O-SP-core (O-SPcNH2 ) polysaccharide, isolated from Vibrio cholera O1 lipopolysaccharide (LPS) and related synthetic substances, a detailed study of factors that affect conjugation of bacterial polysaccharides to protein carriers through squaric acid chemistry to form conjugate vaccines has been carried out. Several previously unrecognized processes that take place during the squarate labeling of the O-SPcNH2 and subsequent conjugation of the formed squarate (O-SPcNH-SqOMe) have been identified. The efficiency of conjugation at pH 8.5, 9.0, and 9.5 to bovine serum albumin (BSA) and to the recombinant tetanus toxin fragment C (rTT-Hc) has been determined. The study led to a protocol for more efficient labeling of O-SPcNH2 antigen with the methyl squarate group, to yield a higher-quality, more potent squarate conjugation reagent. Its use resulted in about twofold increases in conjugation efficiency (from 23-26 % on BSA to 51 % on BSA and 55 % on rTT-Hc). The spent conjugation reagent could be recovered and regenerated by treatment with MeI in the absence of additional base. The immunological properties of the experimental vaccine made from the regenerated conjugation reagent were comparable with those of the immunogen made from the parent O-SPcNH-SqOMe.

Determination of in vivo dose response and allergen-specific T cells in subjects contact-sensitized to squaric acid dibutyl ester.

BACKGROUND: Squaric acid dibutyl ester (SADBE) is a known contact sensitizer, but dose-response data are not defined. OBJECTIVE: To determine the relationship between sensitization dose and contact hypersensitivity (CHS) response to SADBE in human volunteers. The study also aimed to investigate whether SADBE-reactive blood T cells could be detected using ex vivo mature dendritic cells (DCs) as antigen-presenting cells. METHOD: Forty healthy volunteers were sensitized to either 12.5, 25, 50, or 250 microg of SADBE in a 48 microL volume. This was followed by elicitation 2 weeks later with five doses (0, 0.2, 2, 20, and 200 microg in 20 microL). An additional 10 subjects received the elicitation doses without prior sensitization. Blood samples obtained after sensitization were purified into T cells and mature DCs. RESULTS: A direct relationship between sensitization dose and in vivo CHS response was observed. The SADBE dose that effectively sensitized 50% of the population (ED50) was 22 microg/cm2. Significant SADBE-specific T-cell proliferation in vitro was not observed 2 weeks after sensitization but became evident after elicitation. CONCLUSION: This study establishes the in vivo dose-response characteristics of immune reactivity to SADBE and antigen-specific T-cell reactivity.

The importance of myeloid-derived suppressor cells in the regulation of autoimmune effector cells by a chronic contact eczema.

Induction of a chronic eczema is a most efficient therapy for alopecia areata (AA). We had noted a reduction in regulatory T cells during AA induction and wondered whether regulatory T cells may become recruited or expanded during repeated skin sensitization or whether additional regulatory cells account for hair regrowth. AA could not be cured by the transfer of CD4(+)CD25(high) lymph node cells from mice repeatedly treated with a contact sensitizer. This obviously is a consequence of a dominance of freshly activated cells as compared with regulatory CD4(+)CD25(+) T cells. Instead, a population of Gr-1(+)CD11b(+) cells was significantly increased in skin and spleen of AA mice repeatedly treated with a contact sensitizer. Gr-1(+)CD11b(+) spleen cells mostly expressed CD31. Expression of several proinflammatory cytokines as well as of the IFN-gamma receptor and the TNF receptor I were increased. Particularly in the skin, Gr-1(+) cells expressed several chemokines and CCR8 at high levels. Gr-1(+)CD11b(+) cells most potently suppressed AA effector cell proliferation in vitro and promoted partial hair regrowth in vivo. When cocultured with CD4(+) or CD8(+) cells from AA mice, the Gr-1(+)CD11b(+) cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein expression in AA T cells, apoptosis induction remained unaltered. Instead, zeta-chain expression was strongly down-regulated, which was accompanied by a decrease in ZAP70 and ERK1/2 phosphorylation. Thus, a chronic eczema supports the expansion and activation of myeloid suppressor cells that, via zeta-chain down-regulation, contribute to autoreactive T cell silencing in vitro and in vivo.

Squaric monoamide monoester as a new class of reactive immunization hapten for catalytic antibodies.

A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclonal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance paraoxon hydrolysis over background. Kinetic analysis of these antibodies was performed and interestingly both undergo a single turnover event due to covalent modification within the antibody combining site. Because antibodies 2D4 and 3C8 result in covalent attachment and thus inactivation of paraoxon, they could be useful probes for investigating paraoxon intoxication.

Contact immunotherapy with squaric acid dibutylester for the treatment of recalcitrant warts.

BACKGROUND: Contact immunotherapy has been shown to be effective for warts. Two previous studies on the use of squaric acid dibutylester (SADBE) for warts have reported widely divergent cure rates (10% and 60%). OBJECTIVE: Our purpose was to determine the efficacy of SADBE in the treatment of recalcitrant warts. METHODS: We treated 29 patients with SADBE for warts that were resistant to other therapies. The patient population had warts for a mean duration of 2.1 years. Patients were sensitized with 1% or 2% SADBE in acetone under occlusion, then treated with 0.5% to 5% SADBE applied to their warts every 2 to 4 weeks in the office. RESULTS: Clearing of all warts was seen in 20 of 29 patients (69%), improvement in 3 patients (10%), and no change in 6 patients (21%). For the cured patients, mean duration of treatment was 4.2 months (range, 1 to 12 months) and mean number of treatments was 5.7 (range, 2 to 15). Adverse effects included acute contact dermatitis with 6 patients experiencing blisters and one experiencing hypopigmentation. CONCLUSION: SADBE treatment is worth considering in patients with recalcitrant warts, especially in those who tolerate painful procedures poorly.

Percutaneous penetration of squaric acid and its esters in hairless mouse and human skin in vitro.

Squaric acid diethylester and squaric acid dibutylester have been used in contact sensitization therapy of alopecia areata. This study investigated the application of these esters or squaric acid alone to hairless mouse and human skin in vitro to determine squaric acid flux from the various preparations. Measurable amounts of squaric acid were delivered through skin by squaric acid itself, but flux was lower than for that delivered by the two esters. These results support the proposal by Noster that the esters combine with a protein to form an antigen while squaric acid can not and that this explains why the esters are active in contact sensitization and the acid is not. We suggest that the results of previous studies showing that the diethyl ester of squaric acid was a less effective sensitizer than the dibutyl ester may have been due to decomposition of the ethyl ester to squaric acid.

Decreased in vitro lymphocyte stimulation and reduced sensitivity to IL-2 in patients with alopecia areata.

The response to the T-cell growth factor interleukin-2 (IL-2) and to phytohemagglutinin (PHA) and concanavalin A (Con-A) were investigated in 63 patients with alopecia areata (AA) and in control subjects. The proliferative response to mitogens and to IL-2 determined by measuring [3H]-thymidine incorporation 72 h after stimulation is generally decreased in AA patients. The response to mitogens and to IL-2 was related to the response to the topical sensitizer SADBE (squaric acid dibutylester) and patients with no allergic reaction to this substance showed a marked reduction in lymphocyte stimulation, especially with IL-2. HLA typing of 34 of the 63 AA patients was performed in order to investigate the immunogenetic basis of hyporesponsiveness to topical sensitization. The relationship between reduced in vitro response to mitogens and particularly to IL-2, and in vivo response to sensitization to SADBE and the presence of HLA-DR5 are discussed.