The Association between Anti-Acetylcholine Receptor Antibodies and Pemphigus Vulgaris Clinical Status

doi:10.21203/rs.3.rs-7711793/v1

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The Association between Anti-Acetylcholine Receptor Antibodies and Pemphigus Vulgaris Clinical Status

https://doi.org/10.21203/rs.3.rs-7711793/v1

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Over the past three decades, research has examined the role of acetylcholine (ACh) in keratinocyte adhesion and acantholysis, with a focus on the pathogenesis of autoimmune blistering dermatoses. Flaccid blisters and non healing erosions of the oral mucosa and occasionally the skin are caused by autoantibody mediated suprabasilarintraepidermal splitting in pemphigus vulgaris (PV). In total, 15 patients with PV and 15 control patients (6 men, 9 women) who were planned to receive Rituximab, dexamethasone and cyclophosphamide pulse therapy were enrolled. Disease activity was assessed by using the Pemphigus Disease Area Index (PDAI) and Using ELISA, acetylcholine receptors (AchR) antibodies activity. Abs was determined at baseline and three months treatment of rituximab, dexamethasone and cyclophosphamide pulse therapy. At baseline, 83.50 ± 5.3 ng/dL and PDAI score 32.93 ± 2.7 for acetylcholine receptors (AchR) Abs, acetylcholine receptors (AchR) Abs values were dramatically decreased for the three month treatment of rituximab, dexamethasone and cyclophosphamide pulse therapy 15.92 ± 2.3 ng/dL(95% confidence interval)(p < 0.001) and PDAI score 15.25 ± 1.4. There was a significant positive correlation between disease activity and AchR values at baseline (P = 0.04), the reduction in antibody titers from baseline to the end of three month treatment was statistically significant (p = 0.04). The present study suggests that three month treatment of rituximab, dexamethasone and cyclophosphamide pulse therapy combination steroid and oral cyclophosphamide are similar in their ability to influence disease progression and plasma levels of reduction in AchR Abs with clinical improvement in this study may suggest a role for AchR Abs it could be an epiphenomenon.

antimuscarinic acetylcholine receptor antibody

pemphigus vulgaris

dexamethasone-cyclophosphamide/rituximab pulse therapy and disease activity

Pemphigus, a group of autoimmune blistering disorders affects the skin and mucousmembranes. It is characterized by the formation of antibodies against autoantigens on the cell surface of keratinocyte. Desmogleins (Dsg), a component of Desmosomes belonging to cadherin family, help in the cell-to-cell attachment along with other adhesion molecules such as desmocollins, plakoglobins, and desmoplakins. Dsg1 and Dsg3 are considered as the most important autoantigens of pemphigus and their titers often parallel the disease activity [1].

One of the main neurotransmitters of the central nervous system, acetylcholine (ACh) has historically been thought to function on muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs). However, more recently, it has been demonstrated that ACh is produced and that its receptors are expressed in a wide range of cells in various organisms, confirming the theory that ACh is an ontogenetically ancient, universal cytotransmitter that has only sporadically become specialized in the nervous system [2]. It is now well established that ACh metabolism plays a vital role in both neuronal and non neuronal cells, and its importance in keratinocyte function has been elucidated over the last three decades [3]. The highest concentration of free ACh is found in human skin when compared to other organs, such as the lungs, oral mucosa, intestine, and gallbladder. ACh is not only synthesized in human keratinocytes but also secreted as a cytotransmitter and important for keratinocyte vital functions [4]. For example, when human foreskin keratinocytes are treated with ACh receptor (AChR) antagonists, they undergo premature death [3]. In typical Pemphigus vulgaris, basal Keratinocytes are believed to be selectively targeted because they express a bulk of Dsg3, whereas in atypical Pemphigus vulgaris(non Dsg Pemphigus), the predominant pathogenic target on basal KCs remains unknown. Some researchers believe that in atypical Pemphigus vulgaris, anti-M3 acetyl choline receptors could be the predominant pathogenic target [5].

One study had revealed that the serum levels of anti-M3 AchR Abs reduced with therapy and could be used for monitoring disease activity [1]. While another study showed that anti-M3 AchR Abs were not higher in patients with pemphigus when compared to that in healthy people and that although anti-nAChR Abs were high in patients with Pemphigus, their levels did not decrease with treatment [6].

As per the previous report rituximab was introduced for the pemphigus treatment in 2001 [7]. In the last years, it has been considered the first line option to treat pemphigus in developed countries [8].Nevertheless, in Brazil, Rituximab has been prescribed as an exceptional option, for those cases of unresponsiveness to commoner drugs [9–10], which encouraged us to look for a diverse treatment in order to decrease corticosteroid side effects when chronically prescribed [11–12].

A persistent blistering condition affecting the skin and/or mucosa is called pemphigus vulgaris (PV).Pemphigus vulgaris (PV) is a common term for a persistent blistering illness of the skin and/or mucosa. The anti-Dsg1 and − 3 profile can predict the phenotype of pemphigus, according to the widely accepted desmoglein (Dsg) compensation theory. Nevertheless, the Dsg compensation hypothesis falls short in explaining the nature of PV. The importance of non Dsg antigens in PV pathogenesis has been reinforced by the identification of patients with PV in the active phase that did not have detectable anti-Dsg antibodies (Abs) [13–14].

The present study Pemphigus vulgaris, being an autoimmune blistering disease is characterized by autoantibodies against desmogleins (Dsg) and in recent years, new immunological targets in pemphigus vulgaris have been described including cholinergic receptors. Very few studies have been done involving anti AchR antibody titers with disease activity in pemphigus the treatment of rituximab dexamethasone and cyclophosphamide pulse therapyhas become a safe option to treat pemphigus: it avoids the long-term side effects of oral daily treatment with corticosteroids and/or immunosuppressant drugs.

Participants

The study was conducted on a total of 30 patients with pemphigus with active skin or mucosal lesions and 15 control samples at the time of presentation who attend the outpatient dermatology department of HIMS Hassan, Karnataka, India. Written informed consent was obtained from all study subjects before participation in the study. Patients were recruited as per the eligibility criteria. Patients with other autoimmune vesiculobullous disorders and patients with other autoimmune disease involving anti- AchR antibodies were excluded. 15 control subjects were recruited to assess the levels of anti-M3 AchR in the healthy population.

Inclusion criteria

All confirmed cases of pemphigus vulgaris with active mucosal and/or skin lesions at the time of presentation were included

Exclusion criteria

Patients with other autoimmune vesiculobullous disorders and Patients with other autoimmune disease involving anti-AchR antibodies like myasthenia gravis, thymoma were excluded

Clinical assessment of disease severity

A detailed medical history was taken and clinical examination was done. Disease activity was assessed by using Pemphigus Disease Area Index (PDAI) for skin and mucosa. Patients were followed up at 2 time points: baseline, and three months after therapy. PDAI is calculated individually for cutaneous and mucosal lesions, the total score is calculated by totalling the values. Patients were treated with either Rituximab or Dexamethasone Cyclophosphamide pulse therapy. Inj Rituximab 1g was given in 400mL Normal saline infusion. DCP therapy was given in pulses every month. Inj Dexamethasone 100mg was given in 5% Dextrose infusion on Day1 and Day3. On second day, Inj Cyclophosphamide 500mg was added to the same infusion. Patients were asked to take Cyclophosphamide 50mg tablets between the pulses.

Sample collection

5 mL of blood was collected from the patients at baseline and on follow-up. After collecting blood samples were allowed to clot at room temperature for 10 to 20 minutes. Then was centrifuged at 1500 rpm for 15 minutes for 4 ⁰C and supernatants of plasma were collected and stored at -80 ⁰C before processing the enzyme-linked immunosorbent assay (ELISA).

Anti-M3 AChR (Anti-M3 Acetylcholine Receptor Antibody)

The Anti-M3 AChR (Anti-M3 Acetylcholine Receptor Antibody) was estimated from plasma samples. Estimation was done using the ELISA method as per the procedure given by the manufacturer (Fine test ELISA kit code; EH14692, batch no. FN241231 L). The plasma was incubated in microtiter plates coated with monoclonal capture antigen for 90 minutes. The corresponding biotinylated detection antigen was added after this. Incubation was continued for a further period of 60minutes. After this, substrate was added. 2N sulphuric acid was added to stop the reaction. Finally, optical density (O.D) reading was taken (450nm). A standard curve was generated using the standards provided by the manufacturer. The levels of Anti-M3 AChR (Anti-M3 Acetylcholine Receptor Antibody) were expressed in ng/dL read from the standard curve. The analytical sensitivity is < 1 ng/dL and the Interassay CV < 10% and Intraassay CV < 10%.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics tool, version 29.02.00. The data were entered in MS excel 2020 and were analysed descriptive statistics are reported as means and standard deviations, Ab levels were compared between before treatment and after treatment disease groups (PV and three month PV) and Control group. And with Chi Square Test and Fisher’s Exact test if the cell values were small. Comparison was done with paired t test for repeated measures. Significance was accepted at 𝑝< 0.01. This calculation was based on the prevalence of pemphigus is around 0.09% to 1.8% which is lesser than the rest of the world. Considering 1.0% prevalence with 5% margin of error minimum sample needed to conduct this study is 15 cases. By using the following formula:

Sample Size =	Zα²	P (1-P)
	d²
Zα² = Std normal variate 1.96
P = Expected proportion from population
d = Absolute error

Patient characteristics

In total, 30 patients (15 control and 15 cases) with PV (12 men, 18 women) were enrolled in the study. The mean age of patients and the mean disease duration were 44.73 ± 05.85 years (range 23–65 years), respectively (Table 1).

Table 1
Demographic and clinical features of pemphigus cases.
Sl No	Control	All patients	Treatment patients
n	15	15	15
Male (%)	40%
Female (%)	60%
Avg Age (SD)	44.73 ± 2.34
Height	158.8
Weight	61.66
BMI	24.23
Stress	No

Disease severity

The average PDAI score at baseline for all patients was 32.93 ± 2.7. After three month treatment, the average PDAI score for all patients was 15.25 ± 1.4, documenting significant clinical improvement during treatment of drug (p < 0.0001) (Fig. 2).Drug therapy with Rituximab, dexamethasone and cyclophosphamide pulse therapy has improved the lesions as shown by the falling PDAI Score at the end of three months of treatment. The data is represented in the following (Fig. 2).

Antibody levels

The change in anti-M-AChR antibody titers from baseline to the end of three month treatment pulse therapy was analyzed in 15 patients. A statistically significant reduction in anti-M-AChR antibody titers was observed at the end of treatment compared to baseline (p = 0.04), as shown in (Fig. 2) at baseline, 83.50 ± 5.3 ng/dL to 15.92 ± 2.3 ng/dL (95% confidence interval) (P < 0.001). There was a significant positive correlation between disease activities.

Pemphigus vulgaris is caused by autoantibodies against epidermal antigens – most important among these antigens being desmosomal cadherins (Dsg1 and Dsg3); however many other antigens, including the epithelial acetylcholine receptors have recently been incriminated. Few experts have opined that Dsg autoantibodies are not necessary for the immune-pathogenesis of Pemphigus vulgaris [15].

The pathogenesis of pemphigus vulgraris has been a topic of interest recently and various studies have focused on autoantibodies involved in blister formation .The Dsg compensation hypothesis does not completely explain the clinical presentation of Pemphigus Vulgaris.The ‘multiple hittheory’ explains the pathogenesis of Pemphigus vulgaris in a better way, with Acetyl choline receptors being one of the most important non- Dsg antigens [16] The present study shows that there is a statistically significant (p < 0.0001) reduction in PDAI score after three months treatment compared to baseline score, significant clinical improvement has also been reported in other studies. Previous reported remission in 41 (82%) patients in their study of 50 patients of auto immune bullous disease treated with DCP therapy [17]. Previous study shows that achieved remission in all the 103 pemphigus patients treated with DCP therapy [18]. In this study, 15 patients with pemphigus vulgaris were treated with either Rituximab or Dexamethasone Cyclophosphamide pulse therapy. A significant decrease in disease activity from baseline to three month treatment, as evidenced by a decrease in PDAI score as well as a decrease in acetylcholine receptors antibody titres was seen in PV patients and was seen (Fig. 1 and Table 1).

As per the [1], reported that the relationship between disease severity and anti-M3 AchR Abs. According to [19] there is a weak association between baseline anti-M-AChR antibody titers and disease activity, as evaluated by the PDAI. Several factors may explain this weak correlation, including variability in anti M-AChR antibody levels between patients, the possibility that this antibody is not the sole or primary pathogenic driver of disease, and the limitations of single-point measurement, which may not capture peak immunologic activity. Furthermore, PDAI reflects clinical symptoms such as blisters, erosions, and new erythema, which can be affected not only by antibody-mediated pathology but also by non-antibody processes, the patient's healing capacity, and secondary infections.

Overall, our research indicates that Non Dsg antigens like Acetyl choline receptors do play a role in the pathogenesis of Pemhigus Vulgaris and also do correlate with the severity of the disease. Anti-M3 AchR Abs could bw used as a diagnostic and even prognostic marker for Pemphigus vulgaris.

This study looks into the role of plasma anti-M-AChR antibody levels in pemphigus. We conclude that, whereas these titers may not reflect disease activity at the time of diagnosis, they can be used as prognostic indicators to track changes in disease activity after treatment. However, larger investigations with more diverse populations and comparisons with anti-Dsgtiters are required to corroborate our findings and investigate the pathogenic and diagnostic potential of anti-M-AChR antibodies in pemphigus.

Limitations

This study has significant limitations, including a small sample size, recruiting from a single geographic region, and the lack of comparative anti-Dsg antibody titer data. Furthermore, 15% participants were lost to follow-up at the conclusion of three months of treatment due to the patient's follow-up, which may raise the chance of a type II error.

Authorship contribution:

Suresh M R: Research study, editing manuscript, and conceptualization and grant PI.

Keerthana H L: Co-Investigator in the grant and sample handling treatment and follow-up the patients, concept and design

Vittal B G: Editing manuscript, analysis, or interpretation of data

Yallappa B Saunshi: Manuscript draft, acquisition, analysis, or interpretation of data.

Madhan S: Data acquisition and analysis.

Funding

The first author also acknowledges the DHR/ICMR-Multi Disciplinary Research Unite, HIMS Hassan for supporting the project.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declaration of competing interest

All co-authors and the organizations where the research was carried out have given theirapproval for the manuscript to be submitted. I certify that this work is original and that none of the authors have any conflicting interests. It has not been published anywhere else and is not being considered for publication anywhere else right now.

Ethical statement:

The study was approved by the Institutional Ethical Committee (IEC/HIMS/RR-580/28-06-2024). All the participants gave their informed consent before participating in the study.

Acknowledgement

The authors gratefully acknowledge The Director, Hassan Institute of Medical Sciences, Hassan, India for providing necessary laboratory facilities. The authors sincerely thanks to MRU, Hassan Institute of Medical Sciences, Hassan for funding our study (Grant ID: DERM-01) and providing research facility to conduct the study.

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No competing interests reported.

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You are reading this latest preprint version

The Association between Anti-Acetylcholine Receptor Antibodies and Pemphigus Vulgaris Clinical Status

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Participants

Inclusion criteria

Exclusion criteria

Clinical assessment of disease severity

Sample collection

Anti-M3 AChR (Anti-M3 Acetylcholine Receptor Antibody)

Statistical analysis

Results

Patient characteristics

Disease severity

Antibody levels

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1