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New Approaches to the Treatment of Pemphigus

      In pemphigus vulgaris, treatment with systemic glucocorticosteroids is life saving; it may, however, cause severe side effects, including death. A patient with pemphigus vulgaris and myasthenia gravis was treated for approximately five years with the cholinomimetic Mestinon (pyridostigmine bromide), Imuran (azathioprine), and a topical corticosteroid gel before the need to introduce systemic glucocorticosteroids. Because activation of keratinocyte acetylcholine receptors also has been shown to abolish pemphigus IgG–induced acantholysis in cultured keratinocyte monolayers, a clinical trial of Mestinon was initiated in patients with active pemphigus vulgaris, pemphigus foliaceus, and paraneoplastic autoimmune multiorgan syndrome (also known as paraneoplastic pemphigus). First results indicate that nonsteroidal treatment of pemphigus is possible. Mestinon may be used to slow down progression of the disease and to treat mild cases with chronic lesions on limited areas. Stimulation of the keratinocyte– acetylcholine axis may lead to a therapeutic effect through any of the following mechanisms: (1) stimulating keratinocyte cell-to-cell attachment; (2) accelerating re-epithelialization; and (3) competing with the disease-causing pemphigus antibodies, preventing them from attachment to keratinocytes. Glucocorticosteroids and various types of steroid-sparing drugs used to treat pemphigus exhibit cholinergic side effects, including effects on expression and function of keratinocyte adhesion molecules, that are very similar to those produced by the cholinomimetic drugs. Further elucidation of the mechanisms underlying therapeutic efficacy of antiacantholytics may shed light on the immunopharmacological mechanisms of pemphigus antibody–induced acantholysis.

      Keywords

      Abbreviations:

      ACh
      acetylcholine
      AChE
      acetylcholinesterase
      AChR
      acetylcholine receptor
      ChAT
      choline acetyltransferase
      GS
      glucocorticosteroid hormone
      mAChR
      muscarinic acetylcholine receptor
      MG
      myasthenia gravis
      nAChR
      nicotinic acetylcholine receptor
      PAMS
      paraneoplastic autoimmune multiorgan syndrome
      PV
      pemphigus vulgaris
      The major objective of pemphigus research is development of a safer treatment regimen. Pemphigus patients need drugs that can replace the gluco-corticosteroid hormones (GS) that many of them must take for life. GS therapy is life saving, but patients suffer from severe side effects and complications. Alternative therapies that foster keratinocyte adhesion and/or specifically antagonize the effects of pemphigus antibodies are desperately needed. In the past, I developed treatment modalities (
      • Grando S.A.
      Combined immunosuppressive therapy of autoimmune bullous dermatoses (In Russian).
      ;
      • Grando S.A.
      • Glukhenky B.T.
      • Drannik G.N.
      • Kostromin A.P.
      • Romanenko A.B.
      The effect of experimental hemocarbofiltration upon activity of mononuclear cells from normal and autoimmune patients.
      ;
      • Grando S.A.
      • Drannik G.N.
      • Glukhenky B.T.
      • Kostromin A.P.
      • Romanenko A.B.
      • Chayun O.A.
      • Chernyavsky A.I.
      Clinical and laboratory evaluation of hemocarboadsorption in autoimmune bullous dermatoses.
      ;
      • Grando S.A.
      Decompensation in proteinase-inhibitor system and application of proteinase inhibitors in pemphigus and pemphigoid.
      ) that, in addition to GS, included: (1) ex-vivo filtration of patients' blood through carbon adsorbent, followed by plasmapheresis and administration of cytostatic drugs, to eliminate disease-causing pemphigus autoantibodies and suppress the synthesis of new ones; (2) antiproteases, antikinins, and antileukotrienes (quercetin, aprotinin, and ε-aminocaproic acid) to inactivate mediators of inflammation present in pemphigus blister fluid (
      • Grando S.A.
      Glukhen'kii BT, Romanenko AB: Role of endogenous proteinases and their inhibitors in the pathogenesis of pemphigus vulgaris (In Russian).
      ;
      • Grando S.A.
      • Glukhenky B.T.
      • Drannik G.N.
      • Epshtein E.V.
      • Kostromin A.P.
      • Korostash T.A.
      Mediators of inflammation in blister fluids from patients with pemphigus vulgaris and bullous pemphigoid.
      ;
      • Grando S.A.
      • Glukhenky B.T.
      • Drannik G.N.
      • Kostromin A.P.
      • Chernyavsky A.I.
      Cytotoxic proteases in blister fluid of pemphigus and pemphigoid patients.
      ); and (3) doxycycline to suppress the cell-mediated autoimmunity component of pemphigus' immunopathogenesis (
      • Grando S.A.
      • Glukhenky B.T.
      • Drannik G.N.
      • Kostromin A.P.
      • Boiko Y.
      • Senyuk O.F.
      Autoreactive cytotoxic T lymphocytes in pemphigus and pemphigoid.
      ). Unfortunately, none of these approaches allowed complete replacement of GS in pemphigus patients. Recent research results, however, suggest that novel antiacantholytic therapies may be developed by mimicking the antiacantholytic effects of GS with nonsteroidal drugs acting at the acetylcholine receptors (AChR) expressed by keratinocytes (
      • Grando S.A.
      • Pittelkow M.R.
      • Shultz L.D.
      • Dmochowski M.
      • Nguyen V.T.
      Pemphigus: An unfolding story.
      ). Keratinocyte adhesion is controlled, in part, by acetylcholine (ACh), a cytokine-like chemical (i.e., a cytotransmitter) that is locally produced by keratinocytes (reviewed in
      • Grando S.A.
      Biological functions of keratinocyte cholinergic receptors.
      ). ACh and its congeners (i.e., cholinomimetic drugs) can reverse pemphigus antibody–induced acantholysis both in vitro (
      • Grando S.A.
      • Dahl M.V.
      Activation of keratinocyte muscarinic acetylcholine receptors reverses pemphigus acantholysis.
      ) and in vivo.
      Nguyen VT, Grando SA: Novel animal model for testing antiacantholytic treatments of pemphigus. J Invest Dermatol 117:543, abstr. #919, 2001
      Taking into consideration that GS can prevent but not reverse pemphigus IgG-induced acantholysis (
      • Swanson D.L.
      • Dahl M.V.
      Methylprednisolone inhibits pemphigus acantholysis in skin cultures.
      ), these observations suggest that cholinomimetic drugs might be a novel and more efficient treatment for pemphigus. A case of pemphigus vulgaris (PV) that improved by cigarette smoking (
      • Mehta J.N.
      • Martin A.G.
      A case of pemphigus vulgaris improved by cigarette smoking.
      ), studies showing negative correlation between smoking and pemphigus (
      • Brenner S.
      • Tur E.
      • Shapiro J.
      • et al.
      Pemphigus vulgaris: Environmental factors. Occupational, behavioral, medical, and qualitative food frequency questionnaire.
      ), and successful use of nicotinamide as a steroid-sparing agent in pemphigus (
      • Chaffins M.L.
      • Collison D.
      • Fivenson D.P.
      Treatment of pemphigus and linear IgA dermatosis with nicotinamide and tetracycline: a review of 13 cases.
      ) hint that this expectation is realistic for the following reasons: (1) cigarette smoke contains cholinomimetic nicotine; and (2) nicotinamide exhibits cholinomimetic effects (
      • Romanenko A.V.
      The action of nicotinamide on neuromuscular transmission.
      ) due to both stimulation of ACh release (
      • Koeppen A.
      • Klein J.
      • Erb C.
      • Loeffelholz K.
      Acetylcholine release and choline availability in rat hippocampus: Effects of exogenous choline and nicotinamide.
      ) and inhibition of acetylcholinesterase (AChE) (
      • Stoytcheva M.
      • Zlatev R.
      Bioelectrocatalytical studies of the effect of some pharmaceuticals on the acetylcholinesterase activity.
      ). The rationale behind the use of cholinergic drugs also stems from data demonstrating involvement of keratinocyte AChR in pemphigus pathophysiology (reviewed in
      • Grando S.A.
      Autoimmunity to keratinocyte acetylcholine receptors in pemphigus.
      ). On these premises, I hypothesized that cholinomimetics should abort or alleviate pemphigus. This therapeutic approach should be successful even if keratinocyte AChR are not targeted by disease-causing pemphigus antibodies in a particular patient, because ACh and its muscarinic and nicotinic congeners can accelerate the rate of keratinocyte migration, thus fostering re-epithelialization of pemphigus erosions (
      • Grando S.A.
      Biological functions of keratinocyte cholinergic receptors.
      ). As a test of this hypothesis, an open clinical trial of a nontoxic AChE inhibitor, Mestinon (pyridostigmine bromide), was initiated approximately two years ago in the dermatology clinic of the University of California at Davis. In the clinical trial, Mestinon was chosen as a systemic cholinomimetic drug because an index patient with both PV and myasthenia gravis (MG) had successfully treated her pemphigus for almost five years, without using systemic GS, by manipulating her daily dose of Mestinon from 180 to 360 mg, taking Imuran (azathioprine) and applying a glucocorticoid gel to fresh lesions.
      1Nguyen VT, Grando SA: Novel animal model for testing antiacantholytic treatments of pemphigus. J Invest Dermatol 117:543, abstr. #919, 2001

      Results

      The results of the clinical trial reported herein were obtained in eight patients with active pemphigus without MG, including all three major clinical forms of autoimmune pemphigus. The diagnosis of pemphigus was based on the results of comprehensive clinical and histological examinations, together with immunological studies, following standard protocols (
      • Beutner E.H.
      • Chorzelski T.P.
      • Jablonska S.
      Immunofluorescence tests. Clinical significance of sera and skin in bullous diseases.
      ). This study had been approved by the University of alifornia, Davis, Human Subjects Review Committee.

      Treatment of PV without systemic GS in the index patient

      The index patient was a 39-year-old Ashkenazi Jewish female with both PV and MG. At the age of 18, she developed arthralgias and was given the diagnosis of lupus erythematosus on the basis of positive results of ANA, nDNA and VDRL antibody tests and LE cell preparations. She was treated with NSAIDs until the symptoms resolved by the age of 20 and the above tests became normal. After that, she had two pregnancies that resulted in miscarriages during the first trimester due to large subchorionic hemorrhages associated with borderline titers of anticardiolipin antibodies. A third pregnancy was complicated by another large subchorionic hemorrhage in the first trimester, but the pregnancy continued and resulted in a normal vaginal delivery by induction. At the age of 30, she developed symp-toms of nasal speech at five months into the next pregnancy. Three weeks prior to delivery of the second child, she developed ptosis of the left eyelid. Three days after delivery, her speech became much more slurred and she developed difficulty in deep breathing. The finding of an anti-AChR antibody at the titer of 25 and the results of a Tensilon test confirmed the clinical diag-nosis of MG. A chest CT scan revealed a hyperplastic thymus, but was negative for thymoma. She was started on 60 mg Mestinon three times a day. Initial improvement was followed by an increase of muscular weakness, difficulty swallowing, and worsening of slurred speech. At the age of 31, she had a thymectomy, which was preceded by five sessions of plasmapheresis. The postoperative course was relatively uncomplicated. Since then, she has been controlling her myasthenia symptoms with daily doses of Mestinon ranging between 120 and 210 mg.
      At the age of 33, this patient developed the initial symptoms of PV. She first noticed occasional bleeding and inflammation of her gums that did not disturb her much. Approximately one year later, multiple white and ulcerated areas on gingival crest and crevice, which bled readily, were found during an annual dental checkup. Superficial mucosa could be wiped away, indicating the presence of a positive direct Nikolskiy sign (
      • Grando S.A.
      • Grando A.A.
      • Glukhenky B.T.
      • Doguzov V.
      • Nguyen V.T.
      • Holubar K.
      History and clinical significance of mechanical symptoms in blistering dermatoses: A reappraisal.
      ). A crusted erosion of approximately 1×1 cm that bled easily upon mechanical stimulation was also found behind the right ear. She was referred to an oral surgeon, who made an incisional biopsy of the right posterior mandibular alveolar mucosa. The histopathological study revealed suprabasilar acantholysis consistent with PV. This clinical and histological diagnosis was confirmed by the results of immunological studies performed at the Beutner Laboratories (Buffalo, New York) during exacerbation of her skin disease. Intercellular antibodies were found in the perilesional skin by direct immunofluorescence, and low-titer intercellular antibodies were demonstrated by indirect immunofluorescence.
      During the first four years, the course of PV in this patient was rather mild. One to two new lesions on oral mucosa and/or skin would develop monthly and heal quickly, either spontaneously or upon the use of topical GS. These symptoms developed in the background of a maintenance dose of Mestinon. At the age of 37, the patient developed a moderately severe exacerbation characterized by the appearance of approximately 50 small, 1–2-cm-diameter, bullous lesions on skin and oral mucosa soon after these preceding events: (1) five sessions of intravenous gammaglobulin injections over a period of one week in an attempt to improve myasthenia symptoms; (2) slight tapering of Mestinon because improvement of myasthenia had been achieved; and (3) severe emotional stress. Systemic GS were not initiated because of the threat of a myasthenic crisis. Instead, the patient was treated with plasmapheresis. For several years after that, in addition to the maintenance dose of Mestinon, she took Imuran at a daily dose of 150 mg and used 0.05% clobetasol propionate gel (Temovate) on new pemphigus lesions that occasionally appeared on her skin and oral mucosa. Her pemphigus antibody titer remained at 1/320, as determined by indirect immunofluorescence using monkey esophagus as a substrate. She infrequently developed side effects from the Mestinon, such as skin flushes, sweating, and diarrhea. Otherwise, she had a good quality of life. After approximately five years of treatment, the lesions on her skin and oral mucosa began to develop more frequently, which required initiation of prednisone therapy to control her PV. Currently, she takes 20 mg prednisone per day.

      First results of clinical trial of mestinon in pemphigus patients

      The intriguing aspect of the management of the index patient was that conventional GS therapy was not instituted for the first five years of her disease. Although it is possible to maintain pemphigus patients in remission using immunosuppressive drugs without GS (
      • Lever W.F.
      • Schaumburg-Lever G.
      Immunosuppressants and prednisone in pemphigus vulgaris: Therapeutic results obtained in 63 patients between 1961 and 1975.
      ;
      • Lever W.F.
      • Schaumburg-Lever G.
      Treatment of pemphigus vulgaris. Results obtained in 84 patients between 1961 and 1982.
      ;
      • Stemm C.
      • Thivolet J.
      Weaning of systemic steroid treatment in pemphigus. A twelve-year retrospective study on 270 French patients.
      ), initial treatment of PV relies on systemic GS in a high dose (
      • Holubar K.
      • Fellner M.J.
      Pemphigus and related diseases.
      ;
      • Muller S.
      • Stanley J.R.
      Pemphigus: Pemphigus vulgaris and pemphigus foliaceus.
      ;
      • Carson P.J.
      • Hameed A.
      • Ahmed A.R.
      Influence of treatment on the clinical course of pemphigus vulgaris.
      ). To the best of my knowledge, neither Imuran alone nor Imuran in combination with plasmapheresis and/or Temovate gel has ever been reported to allow complete avoidance of systemic GS at the initial stage of pemphigus treatment. Therefore, I considered Mestinon as a therapeutic agent that ameliorated the natural course of disease in this patient, and I initiated an open clinical trial. An overall goal was to evaluate the efficacy of Mestinon in terminating the spread of pemphigus erosion and in fostering re-epithelialization of already existing lesions. Both new patients with pemphigus who had not received GS and established patients with disease exacerbation on the background of immunosuppressive therapy were enrolled. Patients with generalized, life-threatening forms of disease whose well being might be jeopardized by any delay in initiating systemic GS therapy were excluded from the study, as were children less then than 16 years of age, pregnant women, and nursing mothers.
      During approximately two years of the clinical trial, eight patients with active pemphigus used Mestinon for at least four weeks (Table 1). They took Mestinon tablets at a total daily dose of 360 mg. Three PV patients (patients 1, 6, and 8) and a patient with paraneoplastic autoimmune multiorgan syndrome [(PAMS; also known as paraneoplastic pemphigus (
      • Nguyen V.T.
      • Ndoye A.
      • Bassler K.D.
      • et al.
      Classification, clinical manifestations, and immunopathological mechanisms of the epithelial variant of paraneoplastic autoimmune multiorgan syndrome: A reappraisal of paraneoplastic pemphigus.
      )] showed a very good response (the patients are hereafter referred to as the responders). The other three PV patients and one pemphigus foliaceus patient showed no significant improvement. Among the responders, two PV patients (patients 1 and 6) and the PAMS patient were able to control their disease using Mestinon alone. One responder demonstrated a direct strong interrelationship between the use of Mestinon and the ability to control his PV (patient 1; Figure 1). After achieving stable control, he discontinued Mestinon. Approximately two weeks later, he reported redness and itching/burning sensations at the sites of the fully healed pre-existing lesions, which were followed by microvesiculation and advert lesional weeping. At this point, Mestinon was restarted. Within several days, the progression of the lesions aborted and the erosions began to dry. It took about three weeks for the lesions to completely heal, after which the patient slowly tapered his Mestinon daily dose to zero. He has remained free from lesions for almost 18 months without any need of medication for pemphigus. This patient provides a “proof of concept” case of the efficacy of Mestinon in pemphigus because he showed (1) rapid improvement of his disease at the time the drug was taken, although this might be attributed to a coincidental spontaneous improvement of the disease; (2) rapid exacerbation of PV after the drug had been abruptly discontinued; and (3) rapid reversal of the flare-up of his PV after the drug had been restarted.
      Table ISummary of the Results of Open Trial of Mestinon in Pemphigus Patients
      Abbreviations: GS, glucocorticosteroid hormones; IF, direct and/or indirect immunofluorescence; IS, immunosuppressors; ND, not done; PAMS, paraneoplastic autoimmune multiorgan syndrome; PF, pemphigus foliaceus; PV, pemphigus vulgaris.
      PatientAge/SexDiagnosisIF testsNikolskiy signPrior systemic therapySystemic treatment during the trialOutcome of the trialSystemic treatment after the trial
      145/MPVpositivepositiveGS, ISnonepermanent remissiontaper Mestinon over 3 months
      264/FPVpositivepositiveGS, ISGSno improvementGS, IS
      338/MPVpositivepositiveGS, ISGS (lower dose)no improvementGS, IS
      433/MPFNDpositiveGSGS (lower dose)no improvementGS, IS
      565/MPAMSpositivenegativeGS, ISnonetemporary remissionGS (lower dose); Mestinon
      653/FPVpositivepositivenonenoneimprovementMestinon
      751/FPVpositivepositiveGS, ISGS, ISno improvementGS, IS
      882/MPVpositiveNDGS, ISGS (lower dose)improvementGS (lower dose); Mestinon
      a Abbreviations: GS, glucocorticosteroid hormones; IF, direct and/or indirect immunofluorescence; IS, immunosuppressors; ND, not done; PAMS, paraneoplastic autoimmune multiorgan syndrome; PF, pemphigus foliaceus; PV, pemphigus vulgaris.
      Figure thumbnail gr1
      Figure 1Treatment of Pemphigus Vulgaris with Mestinon. (A) Before treatment. (B) Three months on Mestinon. (C) Three weeks after discontinuing Mestinon. (D) One month after restarting Mestinon.

      Discussion

      Possible mechanisms of the therapeutic efficacy of Mestinon in pemphigus

      The first results of the clinical trial of Mestinon are encouraging, indicating that this cholinomimetic can be used to slow down progression of disease in patients with acute pemphigus and to treat mild cases with limited areas of nonhealing erosions. No controls needed to be allocated to standard therapy because the success of the new treatment could be judged against the well-known standard prognosis. The course of pemphigus is well characterized, and it is well known that practically all pemphigus patients relapse if systemic GS are withdrawn during the disease's active phase (Herbst and Bystryn, 2000). In a classic example, Dr. Morton established the efficacy of anesthesia by demonstrating that one anesthetized patient felt none of the excruciating pain that had invariably accompanied surgery. Therefore, the “proof of concept” result with Mestinon in the PV patient shown in Figure 1 indicates that an approach to the treatment of pemphigus that employs cholinomimetics is practical. The obtained results do raise a question, however: How can Mestinon suppress acantholysis in pemphigus? The answer may provide a new lead in solving the pemphigus enigma.
      It is well known that Mestinon increases tissue levels of endogenous ACh because of the reversible inhibition of AChE that hydrolyzes ACh (
      • Taylor P.
      Anticholinesterase agents.
      ). Keratinocytes actively metabolize ACh, employing the synthesizing enzyme choline acetyltransferase (ChAT) and the degrading enzyme AChE, and they use ACh as an autocrine and paracrine hormone or cytotransmitter (reviewed in
      • Grando S.A.
      Biological functions of keratinocyte cholinergic receptors.
      ). Additionally, Mestinon can act directly on keratinocyte AChR because it has been shown to interact directly with cholinergic receptors as a weak agonist capable of inducing desensitization, both alone and when combined with ACh (
      • Akaike A.
      • Ikeda S.R.
      • Brookes N.
      • Pascuzzo G.J.
      • Rickett D.L.
      • Albuquerque E.X.
      The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor-ionic channel complex. II. Patch clamp studies.
      ). Human epidermal keratinocytes express both the ACh-gated ion channels—that is, the neuronal-type nicotinic AChR (nAChR), which can comprise α3, α5, α7, α9, α10, β2, and β4 subunits (
      • Grando S.A.
      • Horton R.M.
      • Pereira E.F.R.
      • Diethelm-Okita B.M.
      • George P.M.
      • Albuquerque E.X.
      • Conti-Fine B.M.
      A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.
      ;
      • Grando S.A.
      • Horton R.M.
      • Mauro T.M.
      • Kist D.A.
      • Lee T.X.
      • Dahl M.V.
      Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation.
      ;
      • Nguyen V.T.
      • Hall L.L.
      • Gallacher G.
      • et al.
      Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.
      ;
      • Nguyen V.T.
      • Ndoye A.
      • Grando S.A.
      Novel human α9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity.
      ;
      • Sgard F.
      • Charpantier E.
      • Bertrand S.
      • et al.
      A novel human nicotinic receptor subunit, α10, that confers functionality to the α9-subunit.
      )—and G protein–coupled muscarinic AChR (mAChR) of the M1, M3, M4, and M5 subtypes (
      • Ndoye A.
      • Buchli R.
      • Greenberg B.
      • et al.
      Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis.
      ). Oral keratinocytes also express the M2 mAChR subtype, but lack M1 (
      • Arredondo J.
      • Hall L.L.
      • Ndoye A.
      • Chernyavsky A.I.
      • Jolkovsky D.L.
      • Grando S.A.
      Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.
      ). Mestinon can interact with the ACh-ionic channel complex, blocking it in open conformation, via at least three distinct, although possibly interacting, mechanisms: (1) a weak agonist action; (2) the formation of desensitized receptor-complex intermediates; and (3) the alteration of the conductance properties of active channels (
      • Albuquerque E.X.
      • Akaike A.
      • Shaw K.P.
      • Rickett D.L.
      The interaction of anticholinesterase agents with the acetylcholine receptor-ionic channel complex.
      ;
      • Pascuzzo G.J.
      • Akaike A.
      • Maleque M.A.
      • Shaw K.P.
      • Aronstam R.S.
      • Rickett D.L.
      • Albuquerque E.X.
      The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor-ionic channel complex. I. Agonist, desensitizing, and binding properties.
      ).
      As will be detailed below, stimulation of the keratinocyte ACh axis with Mestinon might lead to a therapeutic effect in pemphigus through any one or a combination of the following mechanisms: (1) stimulating keratinocyte cell-to-cell attachment; (2) promoting faster re-epithelialization; and (3) competing with the disease-causing pemphigus antibodies, preventing them from attachment to keratinocyte AChR.

      ACh as a cytotransmitter regulating keratinocyte adhesion

      Mestinon might intercede at the intracellular signaling pathway that mediates the acantholytic effects of pemphigus antibodies. The binding of pemphigus IgG to keratinocytes leads to acantholysis through activation of a biochemical cascade that involves activation of phospholipase C, production of inositol 1,4,5-trisphosphate, Ca2+ influx and rapid transient increase of intracellular Ca2+, changes in the intracellular cAMP/cGMP ratios, and activation and translocation of protein kinase C from the cytosol to the particulate/cytoskeleton fractions (
      • Grando S.A.
      • Glukhenky B.T.
      • Romanenko A.B.
      • Demidov S.V.
      Pemphigus antibody-induced intercellular separation of cultivated murine epidermocytes is accompanied by change in ratio of intraepidermocytic cAMP/cGMP.
      ;
      • Esaki C.
      • Seishima M.
      • Yamada T.
      • Osada K.
      • Kitajima Y.
      Pharmacologic evidence for involvement of phospholipase C in pemphigus IgG-induced inositol 1,4,5-trisphosphate generation, intracellular calcium increase, and plasminogen activator secretion in DJM-1 cells, a squamous cell carcinoma line.
      ;
      • Lyubimov H.
      • Goldshmit D.
      • Michel B.
      • Oron Y.
      • Milner Y.
      Pemphigus: Identifying the autoantigen and its possible induction of epidermal acantholysis via Ca2+ signalling.
      ;
      • Seishima M.
      • Esaki C.
      • Osada K.
      • Mori S.
      • Hashimoto T.
      • Kitajima Y.
      Pemphigus IgG, but not bullous pemphigoid IgG, causes a transient increase in intracellular calcium and inositol 1,4,5-triphosphate in DJM-1 cells, a squamous cell carcinoma line.
      ;
      • Osada K.
      • Seishima M.
      • Kitajima Y.
      Pemphigus IgG activates and translocates protein kinase C from the cytosol to the particulate/cytoskeleton fractions in human keratinocytes.
      ). The other messenger systems used by ACh are the same as those used by pemphigus IgG (
      • Grando S.A.
      • Glukhenky B.T.
      • Romanenko A.B.
      • Demidov S.V.
      Pemphigus antibody-induced intercellular separation of cultivated murine epidermocytes is accompanied by change in ratio of intraepidermocytic cAMP/cGMP.
      ;
      • Esaki C.
      • Seishima M.
      • Yamada T.
      • Osada K.
      • Kitajima Y.
      Pharmacologic evidence for involvement of phospholipase C in pemphigus IgG-induced inositol 1,4,5-trisphosphate generation, intracellular calcium increase, and plasminogen activator secretion in DJM-1 cells, a squamous cell carcinoma line.
      ;
      • Lyubimov H.
      • Goldshmit D.
      • Michel B.
      • Oron Y.
      • Milner Y.
      Pemphigus: Identifying the autoantigen and its possible induction of epidermal acantholysis via Ca2+ signalling.
      ;
      • Seishima M.
      • Esaki C.
      • Osada K.
      • Mori S.
      • Hashimoto T.
      • Kitajima Y.
      Pemphigus IgG, but not bullous pemphigoid IgG, causes a transient increase in intracellular calcium and inositol 1,4,5-triphosphate in DJM-1 cells, a squamous cell carcinoma line.
      ;
      • Osada K.
      • Seishima M.
      • Kitajima Y.
      Pemphigus IgG activates and translocates protein kinase C from the cytosol to the particulate/cytoskeleton fractions in human keratinocytes.
      ). In effect, activation of keratinocyte AChR restores normal morphology of pemphigus IgG–treated acantholytic keratinocytes in cultures (
      • Grando S.A.
      • Dahl M.V.
      Activation of keratinocyte muscarinic acetylcholine receptors reverses pemphigus acantholysis.
      ). Therefore, a signal sent by ACh through activating keratinocyte AChR can override the signal evoked by pemphigus antibody binding to keratinocytes.
      It has been convincingly demonstrated that nicotinic and muscarinic drugs exhibit dramatic effects on cell-to-cell and cell-to-substrate cohesion of human epidermal and oral keratinocytes (
      • Grando S.A.
      • Dahl M.V.
      Activation of keratinocyte muscarinic acetylcholine receptors reverses pemphigus acantholysis.
      ;
      • Grando S.A.
      • Crosby A.M.
      • Zelickson B.D.
      • Dahl M.V.
      Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: Requirement of endogenous acetylcholine for outgrowth initiation.
      ;
      • Grando S.A.
      • Horton R.M.
      • Pereira E.F.R.
      • Diethelm-Okita B.M.
      • George P.M.
      • Albuquerque E.X.
      • Conti-Fine B.M.
      A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.
      ;
      • Nguyen V.T.
      • Hall L.L.
      • Gallacher G.
      • et al.
      Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.
      ;
      • Nguyen V.T.
      • Ndoye A.
      • Grando S.A.
      Novel human α9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity.
      ). Blocking AChR with either muscarinic or nicotinic antagonists—atropine and mecamylamine, respectively—in both cases results in acantholysis in keratinocyte monolayers (
      • Grando S.A.
      • Dahl M.V.
      Activation of keratinocyte muscarinic acetylcholine receptors reverses pemphigus acantholysis.
      ;
      • Grando S.A.
      • Horton R.M.
      • Pereira E.F.R.
      • Diethelm-Okita B.M.
      • George P.M.
      • Albuquerque E.X.
      • Conti-Fine B.M.
      A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.
      ). Notably, systemic use of atropine has exacerbated skin and oral blistering in one of our patients with PV. Results obtained in our pilot studies strongly suggest that cholinergic control of keratinocyte adhesion is exerted through receptor-mediated modifications of both expression and phosphorylation of adhesion molecules.
      Grando SA, Arredondo J, Chernyavsky A, Kitajima Y, Nguyen VT: Mechanisms of pharmacologic regulation of keratinocyte adhesion by cholinergic drugs. J Invest Dermatol 119:225, abstr. #107, 2002
      For instance, we demonstrated that the acantholytic effect of atropine is associated with increased phosphorylation of cadherins in DJM-1 cell monolayers. Phosphorylation of classical and desmosomal cadherins is known to be involved in regulation of cell-to-cell adhesion (
      • Parrish E.P.
      • Marston J.E.
      • Mattey D.L.
      • Measures H.R.
      • Venning R.
      • Garrod D.R.
      Size heterogeneity, phosphorylation and transmembrane organisation of desmosomal glycoproteins 2 and 3 (desmocollins) in MDCK cells.
      ;
      • Stappenbeck T.S.
      • Lamb J.A.
      • Corcoran C.M.
      • Green K.J.
      Phosphorylation of the desmoplakin COOH terminus negatively regulates its interaction with keratin intermediate filament networks.
      ;
      • Pasdar M.
      • Li Z.
      • Chan H.
      Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells.
      ;
      • Kowalczyk A.P.
      • Bornslaeger E.A.
      • Norvell S.M.
      • Palka H.L.
      • Green K.J.
      Desmosomes. Intercellular adhesive junctions specialized for attachment of intermediate filaments.
      ), and pemphigus IgG–induced acantholysis involves phosphorylation of desmoglein 3 and its dissociation from plakoglobin (
      • Aoyama Y.
      • Owada M.K.
      • Kitajima Y.
      A pathogenic autoantibody, pemphigus vulgaris-IgG, induces phosphorylation of desmoglein 3, and its dissociation from plakoglobin in cultured keratinocytes.
      ). Interestingly, ligation of α9 AChR has been reported to induce phosphorylation of cell membrane proteins with molecular weights of 120 and 220 kDa (
      • Szonyi M.
      • Csermely P.
      • Sziklai I.
      Acetylcholine-induced phosphorylation in isolated outer hair cells.
      ). These may represent adhesion molecules, such as the 120-kDa E-cadherin (
      • Mareel M.M.
      • Behrens J.
      • Birchmeier W.
      • et al.
      Down-regulation of E-cadherin expression in Madin Darby canine kidney (MDCK) cells inside tumors of nude mice.
      ) and the 220-kDa desmoplakin 2 (
      • Joly P.
      • Thomine E.
      • Gilbert D.
      • et al.
      Overlapping distribution of autoantibody specificities in paraneoplastic pemphigus and pemphigus vulgaris.
      ). Thus, Mestinon might stimulate keratinocyte adhesion by activating classical and desmosomal cadherins, including effects on the phosphorylation status of an adhesion molecule.

      ACh as a cytotransmitter regulating re-epithelialization

      Re-epithelialization of pemphigus erosions is a self-sustained process that can be regulated by endogenously secreted mediators such as ACh. ACh can facilitate keratinocyte outgrowth in culture. Both muscarinic and nicotinic agonists produce a stimulatory effect on keratinocyte spreading and migration, whereas inhibiting ACh synthesis and blocking AChR abrogate lateral migration of human keratinocytes (
      • Grando S.A.
      • Crosby A.M.
      • Zelickson B.D.
      • Dahl M.V.
      Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: Requirement of endogenous acetylcholine for outgrowth initiation.
      ;
      • Grando S.A.
      • Horton R.M.
      • Pereira E.F.R.
      • Diethelm-Okita B.M.
      • George P.M.
      • Albuquerque E.X.
      • Conti-Fine B.M.
      A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.
      ). In neurons, too, ACh regulates the direction of nerve growth cone extension, and blocking ACh signaling inhibits nerve outgrowth (
      • Zheng J.Q.
      • Felder M.
      • Poo M.M.
      Turning of nerve growth cones induced by neurotransmitters.
      ). To characterize cholinergic control of the metamorphosis of keratinocytes during wound healing, we developed an in vitro model of skin re-epithelialization that allows accurate evaluation of drug effects on lateral migration of keratinocytes (
      • Grando S.A.
      • Crosby A.M.
      • Zelickson B.D.
      • Dahl M.V.
      Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: Requirement of endogenous acetylcholine for outgrowth initiation.
      ). The cells exposed to carbachol, a muscarinic and nicotinic agonist and reversible AChE inhibitor, moved significantly farther compared to nonexposed keratinocytes.
      Lee TX, Horton RM, Grando SA: Cholinergic drugs stimulate chemokinesis of human epidermal keratinocytes. J Invest Dermatol 106:841, abstr. #215, 1996
      The response to carbachol was dose dependent and was seen starting from the nanomolar concentrations of the drug. Both the nicotinic antagonist mecamylamine and the muscarinic antagonist atropine abrogated the carbachol-induced keratinocyte migration. Recent results obtained in in vivo skin-wounding experiments in AChR knockout mice indicate that M4 mAChR plays a central role in mediating cholinergic control of keratinocyte migration by regulating integrin expression.
      Chernyavsky A, Arredondo J, Nguyen VT, Ndoye A, Zia S, Wess J, Grando SA: Molecular mechanisms of stimulatory effect of M4 muscarinic acetylcholine receptor on keratinocyte migration. J Invest Dermatol 119:225, abtsr. #108, 2002
      Therefore, the role of Mestinon as a stimulator of a basic regulatory pathway of keratinocyte migration that might help re-epithelialize pemphigus erosions merits further consideration.

      Immunopathological similarities of PV and MG

      Although experimental PV in neonatal mice can be induced with autoantibodies to the adhesion molecules desmoglein 1 and desmoglein 3 (
      • Amagai M.
      • Karpati S.
      • Prussick R.
      • Klaus-Kovtun V.
      • Stanley J.R.
      Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.
      ;
      • Arteaga L.A.
      • Prisayanh P.S.
      • Warren S.J.
      • Liu Z.
      • Diaz L.A.
      • Lin M.S.
      A subset of pemphigus foliaceus patients exhibits pathogenic autoantibodies against both desmoglein-1 and desmoglein-3.
      ), results obtained in my laboratory show that pemphigus symptoms can be induced in neonatal mice lacking desmoglein 3 with passive transfer of the PV IgG that lack desmoglein 1 antibody (
      • Nguyen V.T.
      • Lee T.X.
      • Ndoye A.
      • et al.
      The pathophysiological significance of non-desmoglein targets of pemphigus autoimmunity. Pemphigus vulgaris and foliaceus patients develop antibodies against keratinocyte cholinergic receptors.
      ). The pool of disease-causing pemphigus antibodies includes the autoantibodies to keratinocyte AChR, which are found in approximately 85% of patients (
      • Nguyen V.T.
      • Lee T.X.
      • Ndoye A.
      • et al.
      The pathophysiological significance of non-desmoglein targets of pemphigus autoimmunity. Pemphigus vulgaris and foliaceus patients develop antibodies against keratinocyte cholinergic receptors.
      ). The antigenic specificities of pemphigus antibodies include the novel human α AChR (
      • Nguyen V.T.
      • Ndoye A.
      • Grando S.A.
      Novel human α9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity.
      ), with mixed, nicotinic and muscarinic pharmacology (
      • Elgoyhen A.B.
      • Johnson D.S.
      • Boulter J.
      • Vetter D.E.
      • Heinemann S.
      α9: An acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.
      ), and pemphaxin, a human annexin that binds ACh (
      • Nguyen V.T.
      • Ndoye A.
      • Grando S.A.
      Pemphigus vulgaris antibody identifies pemphaxin: A novel keratinocyte annexin-like molecule binding acetylcholine.
      ). Indeed, pemphigus antibodies have been shown to compete directly with a cholinergic radioligand, [3H]atropine, for binding to keratinocytes (
      • Grando S.A.
      • Dahl M.V.
      Activation of keratinocyte muscarinic acetylcholine receptors reverses pemphigus acantholysis.
      ), indicating that binding of anti-AChR IgG to keratinocytes can produce an immunopharmacological effect.
      We reported a pemphigus patient with MG who developed an autoantibody binding to keratinocyte α3 nAChR.
      Grando SA, George PM, Dahl MV, Conti-Tronconi BM: Antibody against keratinocyte nicotinic acetylcholine receptor in patient with coexistent pemphigus foliaceus, myasthenia gravis and thymoma. J Invest Dermatol 102:609, abstr. #511, 1994
      Myasthenia and pemphigus may therefore share a common immunopathological pathway. MG is caused by autoantibodies against the nAChR expressed at the neuromuscular junction to mediate neuromuscular transmission (reviewed in
      • Conti-Tronconi B.M.
      • McLane K.E.
      • Raftery M.A.
      • Grando S.A.
      • Protti M.P.
      The nicotinic acetylcholine receptor structure and autoimmune pathology.
      ). These antibodies are heterogeneous and can be detected in approximately 85% of patients (
      • Tzartos S.J.
      • Seybold M.E.
      • Lindstrom J.M.
      Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies.
      ). It is not uncommon to find in some myasthenic patients clinical manifestations of pemphigus (reviewed in
      • Kaplan R.P.
      • Callen J.P.
      Pemphigus associated diseases and induced pemphigus.
      ). In such patients, autoantibodies can be directed against both desmosomal and neuromuscular antigens (
      • McKee P.H.
      • McClelland M.
      • Sandford J.C.
      Co-existence of pemphigus, anti-skeletal muscle antibody and a retroperitoneal paraganglioma.
      ;
      • Beutner E.H.
      • Pelton S.
      • Hashimoto T.
      • et al.
      A nonfatal case and 2 fatal cases of paraneoplastic pemphigus: Can a complement indirect immunofluorescent test help to identify fatal ‘group A’ paraneoplastic pemphigus cases?.
      ). Likewise, AChR accumulate at both desmosomal and neuromuscular junctions (
      • Engel A.G.
      • Lindstrom J.M.
      • Lambert E.H.
      • Lennon V.A.
      Ultrastructural localization of the acetylcholine receptor in myasthenia gravis and in its experimental autoimmune model.
      ;
      • Grando S.A.
      • Zelickson B.D.
      • Kist D.A.
      • et al.
      Keratinocyte muscarinic acetylcholine receptors. Immunolocalization and partial characterization.
      ). Recently, Dr. Beutner's group proposed that in pemphigus associated with malignancies, autoimmunity may serve primarily as a defense mechanism against such systemic complications, although some forms of it, such as the autoantibodies to AChR, can cause the death of patients with PAMS (
      • Beutner E.H.
      • Pelton S.
      • Hashimoto T.
      • et al.
      A nonfatal case and 2 fatal cases of paraneoplastic pemphigus: Can a complement indirect immunofluorescent test help to identify fatal ‘group A’ paraneoplastic pemphigus cases?.
      ). Approximately 70% of MG patients have thymitis, and approximately 10% develop thymoma (reviewed in
      • Marx A.
      • Osborn M.
      • Tzartos S.
      • et al.
      A striational muscle antigen and myasthenia gravis-associated thymomas share an acetylcholine-receptor epitope.
      ). Like myasthenia, pemphigus may be associated with a tumor of the thymus, and pemphigus, thymoma, and myasthenia may coexist in the same patient (reviewed in
      • Younus J.
      • Ahmed A.R.
      The relationship of pemphigus to neoplasia.
      ;
      • Sherer Y.
      • Bar-Dayan Y.
      • Shoenfeld Y.
      Thymoma, thymic hyperplasia, thymectomy and autoimmune diseases. Review.
      ). This raises the possibility that two seemingly disparate clinical conditions such as PV and MG may have similar immunopathological mechanisms. In addition to mAChR (
      • Maslinski W.
      • Grabczewska E.
      • Bartfai T.
      • Ryzewski J.
      Muscarinic antagonist binding to intact rat thymocytes.
      ;
      • Rinner I.
      • Porta S.
      • Schauenstein K.
      Characterization of 3H-N-methylscopolamine binding to intact rat thymocytes.
      ), the thymus expresses both the “muscle”α1 and the “neuronal” or “epithelial”α3, α5, α7, and β4 types of nAChR subunits (
      • Wheatley L.M.
      • Urso D.
      • Tumas K.
      • Maltzman J.
      • Loh E.
      • Levinson A.I.
      Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.
      ;
      • Navaneetham D.
      • Penn A.
      • Howard Jr, J.
      • Conti-Fine B.M.
      Expression of the α7 subunit of the nicotinic acetylcholine receptor in normal and myasthenic human thymuses.
      ;
      • Mihovilovic M.
      • Butterworth-Robinette J.
      Thymic epithelial cell line expresses transcripts encoding α3, α5 and β4 subunits of acetylcholine receptors, responds to cholinergic agents and expresses choline acetyl transferase. An in vitro system to investigate thymic cholinergic mechanisms.
      ). Therefore, these two autoimmune diseases might develop as a result of autoimmune responses triggered by autosensitization against the AChR expressed by the thymus. In myasthenia, such sensitization would focus on the AChR expressed by myocytes; in pemphigus, on the AChR expressed by keratinocytes. On the basis of this model, only those patients with thymoma/thymitis would be expected to develop a second or third disease whose antibodies are directed toward the epitope of thymic AChR shared by the AChR expressed in the muscle or skin. Future studies are needed to determine whether the keratinocyte self antigens targeted by autoantibodies in pemphigus patients with MG are the same as those targeted in patients with pemphigus without thymoma/thymitis and/or myasthenia.

      Cholinergic side effects of drugs used to treat pemphigus

      The enigma of pemphigus stems from the fact that the doses of GS required to stop blistering, as well as to sustain remission in many patients, are usually much higher compared to those ordinarily used to control other autoimmune diseases (
      • Myles A.B.
      • Daly J.R.
      ). In pemphigus, GS may work by (1) inhibiting antibody synthesis; (2) suppressing inflammation, especially eosinophilic spongiosis; and (3) stopping acantholysis via direct pharmacologic effect on keratinocyte, given that the addition of GS to skin organ cultures treated with pemphigus antibodies prevents pemphigus IgG–induced acantholysis (
      • Swanson D.L.
      • Dahl M.V.
      Methylprednisolone inhibits pemphigus acantholysis in skin cultures.
      ;
      • Jeffes E.
      • Kaplan R.P.
      • Ahmed A.R.
      Acantholysis produced in vitro with pemphigus serum. Hydrocortisone inhibits acantholysis, while dapsone and 6-mercaptopurine do not inhibit acantholysis.
      ). The last of the three mechanisms just listed, reported independently by two different groups, deserves particular attention because the use of very large doses of methylprednisolone (“pulse therapy”) suppresses pemphigus in patients within 48 hours (
      • Werth V.P.
      Treatment of pemphigus vulgaris with brief, high-dose intravenous glucocorticoids.
      ), and it is believed that the therapeutic effect is mediated by a direct pharmacologic effect of GS on keratinocytes (
      • Hashimoto K.
      • Singer K.
      • Lazarus G.S.
      The effect of corticosteroids, dapsone and gold upon plasminogen activator synthesis and secretion by human epidermal cells cultured with pemphigus antibody.
      ). We recently reported that PV IgG and methylprednisolone exhibit reciprocal effects on the transcription, translation, and phosphorylation of keratinocyte adhesion molecules.
      Nguyen VT, Arredondo J, Chernyavsky A, Pittelkow MR, Kitajima Y, Grando SA: Pemphigus vulgaris IgG (PVIgG) and a corticosteroid exhibit reciprocal effects on keratinocyte adhesion molecules. J Invest Dermatol 119:227, abstr. #116, 2002
      Methylprednisolone upregulated transcription of the genes encoding desmoglein 3, desmocollins, plakophilin, E-cadherin, p-cadherin, α-catenin, several protein phosphatases, protease inhibitors, and lipocortins, and also suppressed PVIgG-induced phosphorylation of adhesion molecules. Therefore, GS may block PVIgG-induced acantholysis via a complex of intracellular genomic and nongenomic events, some of which are also involved in mediating signaling from keratinocyte AChR. Further elucidation of the mechanisms underlying the therapeutic activity of GS and other drugs that have been or are successfully used to treat pemphigus may shed light on the pharmacologic mechanisms mediating pemphigus IgG–induced acantholysis.
      Nonsteroidal treatments of pemphigus reported to date include the following drugs (in chronological order): quinine and strychnine (reviewed in
      • Kartamyshev A.I.
      Pemphigus vulgaris. Chronic pemphigus.
      ), organic arsenic compounds (
      • Oppenheim M.
      Pemphigus chronicus serpiginosus.
      ), suramin (also known as germanin or nephuride) (
      • Veiel F.
      Die Behandlung des Pemphigus mit Germanin.
      ), vitamin D (
      • Ludy J.B.
      • DeValin C.M.
      Viosterol in the treatment of pemphigus.
      ), methotrexate (
      • Lever W.F.
      • Goldberg H.S.
      Treatment of pemphigus vulgaris with methotrexate.
      ), azathioprine (
      • Wolff K.
      • Schreiner E.
      Immunosuppressive therapy of pemphigus vulgaris. Preliminary results of azathioprine (Imuran) treatment (in German).
      ), cyclophosophamide (
      • Krain L.S.
      • Landau J.W.
      • Newcomer V.D.
      Cyclophosphamide in the treatment of pemphigus vulgaris and bullous pemphigoid.
      ), gold (
      • Penneys N.S.
      • Eaglstein W.H.
      • Indgin S.
      • Frost P.
      Gold sodium thiomalate treatment of pemphigus.
      ), dapsone (
      • Haim S.
      • Friedman-Birnbaum R.
      Dapsone in the treatment of pemphigus vulgaris.
      ), heparin (
      • Mashkilleyson N.A.
      Heparin action in pemphigus vulgaris. Clinical and immunologic studies.
      ), cyclosporine (
      • Balda B.R.
      • Rosenzweig D.
      Cyclosporin A in the treatment of pemphigus foliaceus and pemphigus erythematosus (In German).
      ), quercetin and doxycyline (
      • Grando S.A.
      Combined immunosuppressive therapy of autoimmune bullous dermatoses (In Russian).
      ), aprotinin and ε-aminocaproic acid (
      • Grando S.A.
      Decompensation in proteinase-inhibitor system and application of proteinase inhibitors in pemphigus and pemphigoid.
      ), nicotinamide and tetracycline (
      • Chaffins M.L.
      • Collison D.
      • Fivenson D.P.
      Treatment of pemphigus and linear IgA dermatosis with nicotinamide and tetracycline: a review of 13 cases.
      ), minocycline (
      • Sawai T.
      • Kitazawa K.
      • Danno K.
      • Sugie N.
      • Machizuki T.
      • Sugiura H.
      • Uehara M.
      Pemphigus vegetans with oesophageal involvement. Successful treatment with minocycline and nicotinamide.
      ), p-aminomethylbenzoic acid (
      • Dobrev H.
      • Popova L.
      • Vlashev D.
      Proteinase inhibitors and pemphigus vulgaris. An in vitro and in vivo study.
      ), mycophenolate mofetil (
      • Enk A.H.
      • Knop J.
      Treatment of pemphigus vulgaris with mycophenolate mofetil.
      ), and tranilast (
      • Miyamoto H.
      • Takahashi I.
      Successful treatment of pemphigus vulgaris with prednisolone and tranilast.
      ). Surprisingly, a single mechanism of action common for GS and many of the above listed nonsteroid drugs is cholinergic activity (Table 2). Is this a mere coincidence, or does the pharmacologic modulation of keratinocyte ACh axis provide a common denominator of antiacantholytic action of these drugs?
      Table IICholinergic “Side-effects” of Drugs Used to Treat Pemphigus
      GSVia their genomic effects, GS upregulate the cholinergic enzymes ChAT and AChE (
      • Kaufman H.
      • Vadasz C.
      • Lajtha A.
      Effects of estradiol and dexamethasone on choline acetyltransferase activity in various rat brain regions.
      ) [although this effect may vary depending on the cell type (
      • Tria M.A.
      • Vantini G.
      • Fiori M.G.
      • Rossi A.
      Choline acetyltransferase activity in murine thymus.
      ;
      • Hortnagl H.
      • Berger M.L.
      • Havelec L.
      • Hornykiewicz O.
      Role of glucocorticoids in the cholinergic degeneration in rat hippocampus induced by ethylcholine aziridinium AF64A.
      )] and increase expression of both nAChRs and mAChRs (
      • Ben-Baruch G.
      • Egozi Y.
      • Kloog Y.
      • Mashiach S.
      • Sokolovsky M.
      Altered ontogenesis of muscarinic cholinergic receptor in mouse brain: Effect of L-thyroxine and betamethasone.
      ;
      • Marquardt D.L.
      • Motulsky H.J.
      • Wasserman S.I.
      Rat lung cholinergic receptor. Characterization and regulation by corticosteroids.
      ;
      • Braun S.
      • Askanas V.
      • Engel W.K.
      • Ibrahim E.N.
      Long-term treatment with glucocorticoids increases synthesis and stability of junctional acetylcholine receptors on innervated cultured human muscle.
      ), which is proposed as a possible explanation for some of their therapeutic effects (
      • Vilquin J.T.
      • Braun S.
      • Labouret P.
      • Zuber G.
      • Tranchant C.
      • Poindron P.
      • Warter J.M.
      Specific effect of corticoids on acetylcholine receptor expression in rat skeletal muscle cell cultures.
      ). The non-genomic effects of GS are mediated by virtue of their ability to attach in a non-competitive manner to a site of ACh-gated ion channels on the outer cell membrane and alter ACh-induced inward currents (
      • Inoue M.
      • Kuriyama H.
      Glucocorticoids inhibit acetylcholine-induced current in chromaffin cells.
      ;
      • Bouzat C.B.
      • Barrantes F.J.
      Acute exposure of nicotinic acetylcholine receptors to the synthetic glucocorticoid dexamethasone alters single-channel gating properties.
      ;
      • Ke L.
      • Lukas R.J.
      Effects of steroid exposure on ligand binding and functional activities of diverse nicotinic acetylcholine receptor subtypes.
      ;
      • Nurowska E.
      • Ruzzier F.
      Corticosterone modifies the murine muscle acetylcholine receptor channel kinetics.
      ).
      CyclophosphamideBehaves like a classic nicotinic cholinergic ligand, because it specifically binds to the ligand-binding sites of both the muscle- and the neuronal-types nAChRs (
      • Minker E.
      • Blazso G.
      The effect of alkylating agents on the synaptic transmission in the frog's isolated sympathetic ganglion.
      ), but not mAChR (
      • Peroutka S.J.
      Chemotherapeutic agents do not interact with neurotransmitter receptors.
      ), and also reversibly inhibits AChE activity in a dose-dependent manner (
      • al-Jafari A.A.
      • Duhaiman A.S.
      • Kamal M.A.
      Inhibition of human acetylcholinesterase by cyclophosphamide.
      ).
      CyclosporinAugments synthesis of ACh (
      • Esquifino A.I.
      • Selgas L.
      • Maggiore V.D.
      • Castrillon P.O.
      • Cardinali D.P.
      Diurnal changes in cyclosporine effect on ornithine decarboxylase and noradrenergic and cholinergic activities in submaxillary lymph nodes.
      ), and also interferes with protein kinase C-mediated signal transduction from mAChR (
      • Hoecker M.
      • Waschulewski I.H.
      • Kern H.F.
      • Domagk K.A.
      • Schwarzhoff R.
      • Foelsch U.R.
      • Schmidt W.E.
      Cyclosporin A inhibits protein-kinase-C-mediated amylase release from isolated rat pancreatic acini.
      ).
      GoldAuranofin and other gold-containing compounds inhibit ACh-mediated effects on non-neuronal cells (
      • Ohlstein E.H.
      • Horohonich S.
      Selective inhibition of endothelium-dependent relaxation by gold-containing compounds.
      ;
      • Fontaine J.
      • Fang Z.Y.
      • Berkenboom G.
      • Famaey J.P.
      Effects of auranofin on endothelium dependent contractions in isolated rat aorta.
      ).
      NicotinamideIncreases tissue levels of choline [a metabolic precursor of ACh and pharmacologic agonist of AChRs [
      • Sterz R.
      • Peper K.
      • Simon J.
      • Ebert J.P.
      • Edge M.
      • Pagala M.
      • Bradley R.J.
      Agonist and blocking effects of choline at the neuromuscular junction.
      ;
      • Ulus I.H.
      • Millington W.R.
      • Buyukuysal R.L.
      • Kiran
      Choline as an agonist: Determination of its agonistic potency on cholinergic receptors.
      ]], leading to increased ACh release (
      • Koeppen A.
      • Klein J.
      • Schmidt B.H.
      • Van Der Staay F-J.
      • Loeffelholz K.
      Effects of nicotinamide on central cholinergic transmission and on spatial learning in rats.
      ;
      • Koeppen A.
      • Klein J.
      • Erb C.
      • Loeffelholz K.
      Acetylcholine release and choline availability in rat hippocampus: Effects of exogenous choline and nicotinamide.
      ), and also regulates mAChR-coupled K+ channel (
      • Higashida H.
      • Robbins J.
      • Egorova A.
      • Noda M.
      • Taketo M.
      • Ishizaka N.
      • Takasawa S.
      • Okamoto H.
      • Brown D.A.
      Nicotinamide-adenine dinucleotide regulates muscarinic receptor-coupled K+ (M) channels in rodent NG108-15 cells.
      ;
      • Higashida H.
      • Egorova A.
      • Hoshi N.
      Noda: Streptozotocin, an inducer of NAD+ decrease, attenuates M-potassium current inhibition by ATP, bradykinin, angiotensin II, endothelin 1 and acetylcholine in NG108-15 cells.
      ). Nicotinic acid (syn: niacin) acts as a competive inhibitor of AChE (
      • Stoytcheva M.
      • Zlatev R.
      Bioelectrocatalytical studies of the effect of some pharmaceuticals on the acetylcholinesterase activity.
      ), whereas its ester exhibits an ACh-like effect on smooth muscle contraction (
      • Winkelman R.K.
      • Sams Jr, W.M.
      • Bohr D.F.
      Effect of nicotinate ester, acetylcholine, and other vasodilating agents on cutaneous and mesenteric vascular smooth muscle.
      ).
      TetracyclinesTetracycline, chlortetracycline, minocycline and doxycycline cause a concentration-dependent inhibition of ACh release (
      • Anadon A.
      • Martinez-Larranaga M.R.
      An inhibitory action of tetracyclines on guinea-pig myenteric plexus.
      ).
      TranilastInhibits cholinergic neurotransmissions of guinea pig bronchial muscle in vitro (
      • Kamikawa Y.
      Inhibitory effect of anti-allergic drugs on cholinergic and non-cholinergic neurotransmissions of guinea pig bronchial muscle in vitro.
      ).
      AprotininInhibits activity of AChE (
      • Chasapakis G.
      • Augustaki O.
      • Kekis N.
      • Philippou P.
      • Moraitis H.
      • Floras A.
      • Makkous A.
      The influence of the kallikrein-trypsin inactivator trasylol on the serum cholinesterase.
      )—the enzyme that hydrolyses not only ACh but also various peptides, just like a professional trypsin-like endopeptidase (
      • Small D.H.
      • Ismael Z.
      • Chubb I.W.
      Acetylcholinesterase exhibits trypsin-like and metalloexopeptidase-like activity in cleaving a model peptide.
      ).
      QuercetinInhibits ACh release (
      • Lutterodt G.D.
      Inhibition of gastrointestinal release of acetylcholine by quercetin as a possible mode of action of Psidium guajava leaf extracts in the treatment of acute diarrhoeal disease.
      ).
      HeparinEnhances agonist binding to an inhibitory-type of mAChRs, due to disruption of the mAChR-G protein interactions (
      • Wang S.Z.
      • Edmundson R.
      • Zhu S.Z.
      • El-Fakahany E.E.
      Selective enhancement of antagonist ligand binding at muscarinic M-2 receptors by heparin due to receptor uncoupling.
      ), interferes with intracellular signalling from the stimulatory-type mAChR, due to inhibition of inositol 1,4,5-trisphosphate (
      • Olianas M.C.
      • Onali P.
      Impairment of muscarinic stimulation of adenylyl cyclase by heparin in rat olfactory bulb.
      ), and also inhibits AChR aggregation (
      • Hopf C.
      • Hoch W.
      Heparin inhibits acetylcholine receptor aggregation at two distinct steps in the agrin-induced pathway.
      ) and solubilizes AChE from the cell membrane (
      • Talesa V.
      • Principato G.B.
      • Giovannini E.
      • Di Giovanni M.V.
      • Rosi G.
      Dimeric forms of cholinesterase in Sipunculus-nudus.
      ).
      SuraminCompetitive agonist of nAChRs (
      • Henning R.H.
      • Nelemans A.
      • Scaf A.H.
      • Van Eekeren J.
      • Agoston S.
      • Den Hertog A.
      Suramin reverses non-depolarizing neuromuscular blockade in rat diaphragm.
      ).
      QuinineCauses a closed-channel block of nAChR (
      • Ssieb J.P.
      • Milone M.
      • Engel A.G.
      Effects of the quinoline derivatives quinine, quinidine, and chloroquine on neuromuscular transmission.
      ), inhibits mAChR-induced K+ currents (
      • Chen S.
      • Inoue R.
      • Ito Y.
      Pharmacological characterization of muscarinic receptor-activated cation channels in guinea-pig ileum.
      ), and also acts as both non-competitive inhibitor of AChE (
      • Stoytcheva M.
      • Zlatev R.
      Bioelectrocatalytical studies of the effect of some pharmaceuticals on the acetylcholinesterase activity.
      ) and a high affinity competitive inhibitor of choline transport (
      • Porter R.K.
      • Scott J.M.
      • Brand M.D.
      Choline transport into rat liver mitochondria. Characterization and kinetics of a specific transporter.
      ).
      StrychnineSpecific pharmacologic ligand of the novel α AChR that was first found in rat (
      • Elgoyhen A.B.
      • Johnson D.S.
      • Boulter J.
      • Vetter D.E.
      • Heinemann S.
      α9: An acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.
      ), and then cloned by us from human keratinocytes (
      • Nguyen V.T.
      • Ndoye A.
      • Grando S.A.
      Novel human α9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity.
      ).
      Arsenic compoundsInhibit mAChRs (
      • Fonseca M.I.
      • Lunt G.G.
      • Aguilar J.S.
      Inhibition of muscarinic cholinergic receptors by disulfide reducing agents and arsenicals. Differential effect on locust and rat.
      ) and both cholinergic enzymes, ChAT and AChE (
      • Kobayashi H.
      • Yuyama A.
      • Ishihara M.
      • Matsusaka N.
      Effects of arsenic on cholinergic parameters in brain in vitro.
      ;
      • Sheabar F.Z.
      • Yannai S.
      In vitro effects of cadmium and arsenite on glutathione peroxidase, aspartate and alanine aminotransferases, cholinesterase and glucose-6-phosphate dehydrogenase activities in blood.
      ).

      The post-corticosteroid era in the treatment of pemphigus: it is possible

      The results of the clinical trial of Mestinon suggest that nonsteroidal treatment of PV patients can be achieved by pharmacologically stimulating keratinocyte cell-to-cell adhesion through the keratinocyte ACh axis. Future studies in this direction will create an opportunity for pemphigus patients to obtain safer treatment of their disabling condition. A successful GS-free treatment regimen should be able to efficiently block the intracellular signaling elicited by pemphigus antibody binding to keratinocytes. Both an immediate and a more distant solution to this problem should be sought. An immediate solution will be to identify a pharmacological substitute for, or an adjunct to, GS that can efficiently control acantholysis. A more distant solution will be to prevent acantholysis by inhibiting synthesis of disease-causing pemphigus antibodies. In addition to their use as a tolerogen for T cell tolerization in the future, the sequences of the pathogenic epitopes of self antigens can be used for ex-vivo selective immunoadsorption of disease-causing pemphigus antibodies from patients' blood. Thus, successful development of nonsteroidal treatment of pemphigus will animate patient management, as shown in Figure 2.
      Figure thumbnail gr2
      Figure 2Hypothetical plan of treatment of pemphigus in the post-corticosteroid era.
      This work was supported by a research grant from the Robert Leet & Clara Guthrie Patterson Trust and by the International Pemphigus Research Fund.

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