CERTIFICATE BY THE GUIDE

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COMPARATIVE ANALYSIS OF BIOCHIP MOSAIC BASED INDIRECT IMMUNOFLUORESCENCE WITH DIRECT IMMUNOFLUORESCENCE IN DIAGNOSIS OF

AUTOIMMUNE BULLOUS SKIN DISEASES

Dissertation Submitted to

THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY

In the fulfilment of the regulations for the award of the degree

M.D.

DERMATOLOGY, VENEREOLOGY AND LEPROLOGY

DEPARTMENT OF DERMATOLOGY, VENEROLOGY AND LEPROLOGY

PSG INSTITUTE OF MEDICAL SCIENCES AND RESEARCH THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI,

TAMILNADU

MAY 2020

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COMPARATIVE ANALYSIS OF BIOCHIP MOSAIC BASED INDIRECT IMMUNOFLUORESCENCE WITH DIRECT IMMUNOFLUORESCENCE IN DIAGNOSIS OF

AUTOIMMUNE BULLOUS SKIN DISEASES In the fulfilment of the regulations for the award of the degree

M.D.

DERMATOLOGY, VENEREOLOGY AND LEPROLOGY

GUIDE

Dr. REENA RAI, MD

DEPARTMENT OF DERMATOLOGY, VENEREOLOGY AND LEPROLOGY

PSG INSTITUTE OF MEDICAL SCIENCES AND RESEARCH THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI,

TAMILNADU

MAY 2020

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CERTIFICATE

This is to certify that the thesis entitled “COMPARATIVE

ANALYSIS OF BIOCHIP MOSAIC BASED INDIRECT IMMUNOFLUORESCENCE WITH DIRECT

IMMUNOFLUORESCENCE IN DIAGNOSIS OF AUTOIMMUNE

BULLOUS SKIN DISEASES” is a bonafide work of Dr. ARUNPRASATH P. done under the direct guidance and

supervision of Dr. REENA RAI, MD, in the department of Dermatology, Venereology and Leprology, PSG Institute of Medical Sciences and Research, Coimbatore in fulfillment of the regulations of Dr.MGR Medical University for the award of M.D. degree in Dermatology, Venereology and Leprology.

Dr. REENA RAI Dr. RAMALINGAM

Professor DEAN Dept. of DVL

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DECLARATION

I hereby declare that this dissertation entitled “COMPARATIVE ANALYSIS OF BIOCHIP MOSAIC BASED INDIRECT

IMMUNOFLUORESCENCE WITH DIRECT

BULLOUS SKIN DISEASES” was prepared by me under the direct guidance and supervision of Dr. REENA RAI, MD, PSG Institute

of Medical Sciences and Research, Coimbatore.

The dissertation is submitted to the Tamilnadu Dr.MGR Medical University in fulfillment of the University regulation for the award of M.D. degree in Dermatology, Venereology and Leprology. This dissertation has not been submitted for the award of any other Degree or Diploma.

DR. ARUNPRASATH P.

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CERTIFICATE BY THE GUIDE

This is to certify that the thesis entitled “COMPARATIVE ANALYSIS OF BIOCHIP MOSAIC BASED INDIRECT

IMMUNOFLUORESCENCE WITH DIRECT

BULLOUS SKIN DISEASES” is a bonafide work of Dr. ARUNPRASATH P. done under my direct guidance and

supervision in the department of Dermatology, Venereology and Leprology, PSG Institute of Medical Sciences and Research, Coimbatore in the fulfillment of the regulations of Dr.MGR Medical University for the award of MD degree in Dermatology, Venereology and Leprology.

Dr. REENA RAI Professor

Dept. of DVL

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PLAGIARISM TOOL

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CERTIFICATE – II

This is to certify that this dissertation work titled

“COMPARATIVE ANALYSIS OF BIOCHIP MOSAIC BASED INDIRECT IMMUNOFLUORESCENCE WITH DIRECT IMMUNOFLUORESCENCE IN DIAGNOSIS OF AUTOIMMUNE

BULLOUS SKIN DISEASES” of the candidate Dr. ARUNPRASATH P. with registration Number 201830351

for the award of In the branch of MD degree in Dermatology, Venereology and Leprology. I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion pages and results show 1 percentage of plagiarism in the dissertation.

Guide & Supervisor Sign with Seal,

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ACKNOWLEDGEMENT

I would like to express my sincere thanks to all the people who extended their support in successful completion of my dissertation.

I am very much thankful to my guide Dr Reena Rai for her guidance and support throughout the process of dissertation.

I thank my co guide Dr V. Chaitra for her support and her expertise to overcome the difficulties at various stages of dissertation.

I owe my thanks to my teachers Prof Dr. C. Shanmuga Sekar, Prof Dr. K. Mahadevan, Dr S. Priya, Dr Settu Mittal for their support and guidance.

I thank my senior and fellow colleagues for their constant support and encouragement.

I thank my junior colleagues for helping me complete my dissertation by taking care of my other responsibilities in the department.

Above all, I thank our patients for their support and co-operation for being part of our study.

I would also take this opportunity to thank my parents, my In-laws, my wife Dr P. Megavardhini and my daughter A. Srinika for their love and support.

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TABLE OF CONTENTS

SL.NO. CONTENT PAGE NO.

1 INTRODUCTION 1

2 AIM AND OBJECTIVES 2-3

3 REVIEW OF LITERATURE 4-58

4 MATERIALS AND METHODS 59-62

5 RESULTS 63-77

6 DISCUSSION 78-85

7 CONCLUSION 86

8 BIBLIOGRAPHY

9 ANNEXURES

Clinical Photographs Consent Forms Proforma Master chart

List of Abbreviations

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LIST OF TABLES

SL.NO. CONTENT PAGE NO.

1 ADHERENS JUNCTION AND DESMOSOMES 7

2 PEMPHIGUS DISEASES AND TARGET

ANTIGENS

10

3 PEMPHIGOID DISEASES AND TARGET

ANTIGENS

32 4 DISTRIBUTION OF STUDY PARTICIPANTS

BY GENDER

BY CLINICAL DIAGNOSIS

BY DIF

BY BIO-CHIP DIAGNOSIS

67 8 COMPARISON OF DIF WITH BIOCHIP

DIAGNOSIS

68 9 DISTRIBUTION OF PEMPHIGUS GROUP BY

PRIMATE OESOPHAGUS

DESMOGLEIN 1

DESMOGLEIN3

71 12 DISTRIBUTION OF PEMPHIGOID GROUP BY

PRIMATE OESOPHAGUS

SALT SPLIT SKIN

BULLOUS PEMPHIGOID 180

BULLOUS PEMPHIGOID 230

75 16 SENSITIVITY OF BIOCHIP TECHNIQUE IN

DIAGNOSIS OF PEMPHIGUS VULGARIS

76 17 SENSITIVITY OF BIOCHIP TECHNIQUE IN

DIAGNOSIS OF BULLOUS PEMPHIGOID

77

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LIST OF FIGURES

SL.NO. CONTENT PAGE NO.

1 ADHERENS JUNCTION AND DESMOSOMES 7

2 DISTRIBUTION OF STUDY PARTICIPANTS BY GENDER

BY CLINICAL DIAGNOSIS

BY DIF

BY BIOCHIP DIAGNOSIS

67 6 COMPARISON OF DIF WITH BIOCHIP

DIAGNOSIS

PRIMATE OESOPHAGUS

DESMOGLEIN 1

DESMOGLEIN3

71 10 DISTRIBUTION OF PEMPHIGOID GROUP

BY PRIMATE OESOPHAGUS

BY SALT SPLIT SKIN

BY BULLOUS PEMPHIGOID 180

BY BULLOUS PEMPHIGOID 230

75

14 SENSITIVITY OF BIOCHIP TECHNIQUE IN IN DIAGNOSIS OF PEMPHIGUS VULGARIS.

76 15 SENSITIVITY OF BIOCHIP TECHNIQUE IN IN

DIAGNOSIS OF BULLOUS PEMPHIGOID

77

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INTRODUCTION

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INTRODUCTION

Autoimmune bullous skin diseases (AIBD) include diverse group of skin diseases characterized by autoantibodies against desmosomal proteins in case of pemphigus group of diseases and components of basement membrane zone (BMZ) in pemphigoid diseases. Diagnosis of AIBD is based on the combination of characteristic clinical features, histopathological findings, direct immunofluorescence (DIF), indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) against target antigens. BIOCHIP mosaic, a novel diagnostic technique employs detection of target antigens and characteristic staining pattern, in a single miniature incubation field.¹

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AIM AND OBJECTIVE

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AIM AND OBJECTIVE

AIM

To compare the BIOCHIP mosaic based IIF with DIF in the diagnosis of autoimmune bullous skin diseases.

OBJECTIVE

To determine whether biochip method can be used as a first line tool in the diagnosis of autoimmune bullous skin diseases.

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NEED FOR THE STUDY

DIF study of the perilesional skin remains the gold standard in diagnosis of AIBD, however is an invasive and time consuming technique. A non-invasive technique with faster results will ease the diagnosis and also helps in institution of specific therapy at the earliest.

BIOCHIP mosaic is a novel diagnostic technique which unlike in DIF does need any invasive procedures and provides identification of the target antigens and characteristic staining pattern in single miniature incubation field.¹

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REVIEW OF

LITERATURE

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REVIEW OF LITERATURE

PEMPHIGUS GROUP OF DISEASES

The word ‘pemphigus’ has its origin from the Greek word

‘pemphix’ which means blister or bubble and it describes a group of blistering skin diseases. These diseases are characterized by autoantibodies against the components of desmosome which serves as a cementing substance between the keratinocytes, resulting in loss of cohesion between the keratinocytes. This process of loss of cell-cell adhesion is called acantholysis.² The common types of pemphigus encompass pemphigus vulgaris [PV] and pemphigus foliaceus [PF]. Less common types include Herpetiform pemphigus, paraneoplastic pemphigus, drug induced pemphigus and IgA pemphigus.²

PV is characterized by mucosal erosions and cutaneous blisters and erosions involving the deeper portion of the epidermis above the basal layer. Pemphigus vegetans is a variant of PV. PF have only cutaneous lesions sparing the mucosa where there is involvement of upper one third of epidermis involving the granular layer. Pemphigus erythematosus is a localised form and fogo selvagem is an endemic form of PF.

Paraneoplastic pemphigus has an association with malignancy usually of

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lymphoid origin and is characterized by intractable oral lesions and polymorphic skin lesions.³

PATHOGENESIS

DESMOSOME STRUCTURE AND TARGET ANTIGENS

The target antigens in pemphigus group of disorders include various components of the desmosome complex between the keratinocytes. The desmosomal complex is made of transmembrane components, cytoplasmic components and the anchoring filament of the cytoskeleton.

The transmembrane components are cadherin proteins. Cadherins belong to family of calcium-dependent adhesion molecules that is important for the formation and maintenance of cell adhesion. Cadherins have two main classes, classic cadherins and desmosomal cadherins.

Cadherins are made of amino acid sequence repeats with intracellular and extracellular domain. The extracellular domain contains calcium-binding motifs and intracellular domain is made of cadherin specific domain. The intracellular domains links with the plaque proteins like α and β-Catenin and plakoglobin mediating the binding to the cytoskeleton network producing a strong cohesion between the cells. Cadherin molecules form

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functions as dimers with the distal extracellular domain of one cell binding to the cadherin from an opposing cell.^4,5

Desmogleins exists in four isoforms (Dsg1–4). Dsg1 and Dsg3 are predominantly expressed in the stratified squamous epithelium. Simple epithelium and myocardium contains Dsg2. Dsg4 is expressed in the hair follicles and mutations in the Dsg4 gene can lead to localized autosomal recessive hypotrichosis.⁶ Desmocollins are transmembrane glycoproteins within desmosomes and always exists in pair with desmoglein.

Desmocollins have three isoforms (Dsc1–3).

The cytoplasmic components include α Catenin, β Catenin, plakoglobin, plakophilin and desmoplakin. Among which Plakoglobin, plakophilin, and β-catenin belong to the armadillo family of nuclear and junctional proteins. They play an important role in cellular adhesion and proliferation. Desmoplakin is a dumbbell-shaped molecule with two isoforms desmoplakin I (250 kDa) and II (210kDa). It has three domains:

a central α-helical rod, globular carboxy-terminal and an amino-terminal domain. The carboxy terminal interacts with intermediate filaments and amino terminal with armadillo family member. Desmoplakin is important for anchorage of the cytoskeleton to filament attachment sites on desmosomes.⁷

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Two types of connections exist between the epithelial cells. They are adherens junctions and desmosomes. The characteristic feature of each type is listed in the following table.⁷

Adherens junctions Demosomes Transmembrane

components

Classic cadherins (E,P,N cadherins)

Desmosomal cadherins (Desmogleins,

desmocollins) Cytoplamic

components

α Catenin β Catenin plakoglobin

Plakoglobin Plakophilin Desmoplakin

Anchoring filaments Actin microfilaments Keratin intermediate filaments

Type of adhesion Quick and Weak Slow and strong

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ANTIBODIES IN PEMPHIGUS DISEASE

The predominant class of antibody in pemphigus group of diseases is immunoglobulin G (IgG) and rarely IgM, IgA and IgE. These autoantibodies induce disruption of cell adhesions between keratinocytes resulting in blister formation. The pathogenicity of IgG antibodies is well established in studies and also there is a correlation between antibody titre and severity of the disease.⁸ In vitro studies on keratinocyte cultures had shown that sera from pemphigus patients demonstrated dissociation of keratinocyte adhesions.⁹

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Dsg 1 and 3 remains the prime targets for antibodies in pemphigus vulgaris. Some patients with pemphigus show intercellular antibodies detectable on direct or indirect IF but have negative Dsg antibodies on specific enzyme linked immunosorbent assay (ELISA). This could be attributed to the presence of non-Dsg antigenic targets. The non-Dsg antigenic targets reported include desmocollins, plakoglobin, E-cadherin, acetylcholine receptors and pemphaxin.^10,11 In particular, acetylcholine receptor antibodies have been noted in nearly 85% of pemphigus patients.¹²

The AChR α9 and pemphaxin are mainly involved in the physiological control of keratinocyte adhesion. Binding of the autoantibodies to these receptors block the calcium influx involved in the keratinocyte interaction and also cause protein kinase c activation in particular protein kinase R-like endoplasmic reticulum kinase (PERK).

PERK is a novel cell death receptor involved in extrinsic apoptosis.

Activation of PERK causes phosphorylation of adhesion molecules leading to loosening of the intercellular connections and prevention of desmosomal reassembly. Thus acantholysis in this case is due to inactivation of cholinergic receptor mediated physiological control of desmoglein function.¹³

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Paraneoplastic pemphigus has multiple target antigens apart from Dsg 1 and 3, like desmoplakins, envoplakin and periplakin. As a result, direct IF of these patients exhibits intercellular staining pattern along with basement membrane zone staining. Patients with paraneoplastic pemphigus can also develop IgG autoantibodies against α2- macroglobulin-like protein 1 which is a protease inhibitor and BPAG1 which is also known as dystonin.²

The various target antigens for pemphigus group of diseases has been listed in the following table.¹⁴

* A2ML1 – Alpha-2-macroglobulin-like-1 protease inhibitor

# BPAG1 - Bullous Pemphigoid antigen 1

Disease Target antigens

Pemphigus vulgaris Mucosal- dominant type Mucocutneous type

Desmoglein 3

Desmoglein 3, Desmoglein 1 Pemphigus folliaceus Desmoglein 1

Paraneoplastic pemphigus Desmoglein 3 , Desmoglein 1 Plectin

Epiplakin, Envoplakin, Periplakin

Desmoplakin I, Desmoplakin II BPAG1^#

A2ML1*

Drug induced pemphigus Desmoglein 3 Desmoglein 1

IgA pemphigus Desmocollin 1

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PATHOMECHANISMS IN PEMPHIGUS ACANTHOLYSIS

The prime pathological event in PV is loss of cohesion between the keratinocytes and is known as acantholysis. The term ‘acantholysis’ is derived from the Greek word ‘Akantha’ which means a thorn or prickle.¹⁵ The various pathomechanism described in causation of acantholysis include stearic hindrance by autoantibodies,¹⁶ disruption of intracellular signalling pathways, activation of enzymatic cascades involved in apoptosis of keratinocyte such as Fas ligand.

APOPTOLYSIS

The concept of acantholysis suggests that following binding of autoantibodies to the target antigens a sequence of events occur resulting in epidermal growth factor receptor activation, elevation of intracellular calcium, initiation of cell death cascades and activation of apoptotic enzyme caspases. These events ultimately lead to basal cell shrinkage, degradation of structural proteins and apoptosis of acantholytic cells.¹⁷

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DESMOGLEIN COMPENSATION HYPOTHESIS

This theory is based on the differential expression of Dsg 1 and 3 antigens in the skin and mucosa. In the skin Dsg 1 is expressed uniformly throughout the epidermis with intense expression in the superficial layers, whereas Dsg 3 is expressed preferentially in the basal and suprabasal layers. In mucosa both Dsg 1 and 3 are expressed throughout the mucosal epithelium, but with lower level of Dsg 1. In pemphigus sera with anti- Dsg 3 antibodies, it interferes with the function of Dsg 3 but Dsg 1 compensates by preserving the keratinocyte interaction. But in mucosa due to lower Dsg 1 expression these antibodies produce blisters.¹⁸ Antibodies against Dsg 1 are unable to destabilize oral mucosa whereas in the skin with little Dsg 3 to compensate there will be skin lesions.

Likewise in PF anti-Dsg 1 antibody alone will cause skin lesions without mucosal involvement.¹⁸

STEARIC HINDRANCE

According to this theory binding of autoantibodies to the target antigens in the desmosomal complex affects the adhesion between the desmosomal components by causing stearic hindrance. Studies show that pathogenic autoantibodies interfere directly with Dsg 3 interaction.

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However in case of Dsg 1 the interaction is disrupted indirectly by cellular mechanisms.¹⁹

BASAL CELL SHRINKAGE THEORY

Autoantibodies cause phosphorylation of the adhesion molecules and the structural cytoskeleton resulting in loosening of the cellular cohesion and collapse of the cellular structural proteins respectively. This results in shrinkage of the cells and separation of keratinocytes.²⁰

MULTIPLE HIT HYPOTHESES

Multiple antibodies target multiple antigens like desmocollin, plakoglobin and acetylcholine receptors. These antibodies weaken the attachment between neighbouring keratinocytes resulting in shrinkage of the keratinocytes causing the desmosomes to be detached in the intercellular space. This results in production of scavenger antibodies which prevents the formation of new desmosome by causing stearic hindrance.²¹

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ROLE OF T CELLS

Autoreactive CD4 T cells produce Th2 cytokines like interleukin 4 and 10. In active PV Th 2 dependent IgG4 antibodies are predominant while in remission the antibody seen is Th 1 dependent IgG1. Hence autoreactive T cells play an important role in production of pathogenic autoantibodies in pemphigus.²²

CLINICAL FEATURES PEMPHIGUS VULGARIS

Pemphigus vulgaris can manifest as two varied clinical picture: i) the mucosal-dominant type with predominant mucosal erosions and minimal skin involvement; ii) mucocutaneous type with widespread flaccid bullae and erosions along with mucosal erosions. The cutaneous lesions of pemphigus vulgaris are flaccid bullae, which ruptures soon to form painful erosions. Mucosal lesions can be the forerunner of skin lesions and sometimes, can be the sole manifestation of the disease. Intact bulla is rare in mucosa, as it ruptures soon to form erosions involving the buccal or palatal mucosa. Conjunctival, nasal and genital mucosa may also be involved.²³ Erosions in the skin folds may transform to vegetating lesions. Nail changes described include paronychia, subungual haematomas and nail dystrophy.²⁴

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SIGNS IN PEMPHIGUS NIKOLSKY SIGN

It is elicited by applying lateral pressure with thumb on normal skin at a distant site from the erosions over a bony prominence (direct Nikolsky) or normal looking skin close to the erosion (marginal Nikolsky). This shearing force will dislodge the upper layers of epidermis forming erosion.²⁵

BULLA SPREAD SIGN

The margin of intact bulla is marked by the pen. Then unidirectional pressure is applied over the bulla resulting in extension of the bulla beyond the margin.²⁶

ASBOE HANSEN SIGN

A variant of bulla spread sign is the Asboe Hansen sign where the pressure is applied to the centre of smaller intact bulla. This sign is also known as the “indirect Nikolsky” or “Nikolsky II” sign. In severe cases, because of extensive areas of cutaneous involvement chances of secondary bacterial infection is more resulting in sepsis and fatal outcome.²⁶

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PEMPHIGUS VEGETANS

Pemphigus vegetans is a variant of PV characterized by vegetating lesions predominantly involving the intertriginous areas.²⁷ It has two subtypes namely the Neumann type and the Hallopeau type.²⁸ Initial lesions are pustules rather than vesicles and the lesions progress to form vegetative plaques. Cerebriform tongue is a characteristic finding in pemphigus vegetans.²⁹

PEMPHIGUS FOLIACEUS

PF is characterized by crusted scaly lesions with an erythematous base classically involving the ‘seborrhoeic’ areas like the scalp, face, upper trunk and without any oral erosion. Unlike PV, blisters may not be obvious as the lesions are located superficially in the epidermis and ruptures readily to form erosions. Rarely PF can present as erythroderma.

FOGO SELVAGEM

Fogo selvagem is a variant of PF and similar to it clinically, histologically and immunopathologically. Fogo selavagem is endemic in certain regions of Brazil and hence is also known as endemic pemphigus

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foliaceus. It affects young adults and children, unlike PF which affects elderly individuals. The affected patients have their habitats near rivers and within the flying range of black flies Simulium species which is considered as the vector that causes the disease.³⁰

PEMPHIGUS ERYTHEMATOSUS

Pemphigus erythematosus is a localized variant of PF, with immunological features of both lupus erythematosus and pemphigus.

Clinically the lesions are characterized by erythematous scaly lesions involving the malar area mimicking the lupus rash. Sometimes patients have photosensitivity. Association with myasthenia gravis or thymoma have been reported.³¹ DIF shows both intercellular staining pattern and granular IgG and C3 at the basement membrane.³²

PEMPHIGUS HERPETIFORMIS

This is a rare variant of pemphigus with pruritic papules and vesicles in a herpertiform fashion over an erythematous background.

Antibodies are targeted against Dsg 1or 3 and rarely desmocollin antigens. Histopathology shows eosinophilic spongiosis and subcorneal pustules, with not much evident acantholysis.³³

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PARANEOPLASTIC PEMPHIGUS

Paraneoplastic pemphigus occurs in association with malignancy;

in most cases it is of haematological origin, of which the commonest is non-Hodgkin lymphoma and chronic lymphocytic leukaemia. Other reported association includes Castleman disease, thymoma and Waldenstrom macroglobulinaemia. DIF shows findings of both pemphigus and pemphigoid reflecting the wide range of target antigens.³⁴

A distinct clinical feature of paraneoplastic pemphigus is the presence of persistent oral mucosal lesions in spite of treatment.

Nasopharyngeal, esophageal, genital and perianal mucosa may also be involved.³⁵ Cutaneous lesions are polymorphic as it presents like tense blisters resembling bullous pemphigoid, erythema multiforme, and lichenoid eruptions apart from pemphigus like erosions. Presence of erythema multiforme-like lesions and bullae on the palms and soles points to paraneoplastic pemphigus rather than pemphigus vulgaris, where palms and soles involvement is unusual.

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IgA PEMPHIGUS

IgA pemphigus has two subtypes: an intraepidermal neutrophilic type and a subcorneal pustular dermatosis type. In both variants initial lesions will be that of flaccid vesicles or pustules involving erythematous or normal skin. The lesions may show a characteristic circinate or annular configuration with central clearing which is suggestive of intraepidermal neutrophilic type. The lesions tend to involve flexures like axillae and groin; however can occur in trunk and extremities. An association with monoclonal IgA gammopathy have been reported.³⁶

DRUG-INDUCED PEMPHIGUS

Drug-induced pemphigus is caused mainly by two groups of drugs which include Thiol group of drugs [penicillamine] and non-thiol group of drugs [angiotensin=converting enzyme inhibitors and glibenclamide].

Recent addition is the phenol group of drugs which include cephalosporins, rifampicin, phenobarbital and aspirin. Drugs can induce both PF and PV phenotypes. The above said groups of drugs induce pemphigus by production of autoantibodies and also by interacting with the sulfhydryl groups in desmogleins, which causes interference in the adhesive function of the desmogleins. In general patients with drug-

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induced pemphigus achieve remission once the causative drug is stopped.³⁷

NEONATAL PEMPHIGUS

Pemphigus disease in pregnant patients may have adverse foetal outcomes like prematurity or intrauterine death. Pregnant patient with pemphigus may passively transfer the antibodies to the foetus, leading to neonatal pemphigus. Unlike the adult skin, skin in the neonate has both Dsg1 and Dsg3 are present in the upper layers as compared to adult increasing the risk of pemphigus in babies born to mothers with anti- Dsg3 antibodies. Neonatal pemphigus resolves by 4 weeks once the maternal antibodies are catabolized.³⁸

MANAGEMENT

Management of pemphigus group of diseases include general measures, topical therapy and systemic therapy. Systemic corticosteroids remain the mainstay of treatment, generally in combination with a steroid-sparing immunosuppressant.³⁹

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GENERAL MEASURES⁴⁰ Oral mucosa care

i) Brushing

➢ Avoid vigorous brushing.

➢ Use a soft tooth brush (thin bristles) and avoid toothbrush with hard bristles.

➢ Avoid toothpastes containing mint, cloves and spices and abrasives like charcoal. Toothpastes used for sensitive teeth (sensodyne) can be used.

➢ Ten minutes before brushing swish anaesthetic mouthwash for a minute or two, to help reduce the pain and burning sensation of brushing.

ii) Diet

➢ Avoid hard, crispy and crusty foods.

➢ Avoid hot, spicy and acidic foods.

➢ Avoid onion, garlic (thiol containing vegetables might induce pemphigus lesions)

➢ Take plenty of oral fluids

➢ Vegetable soups, spinach, fruits and soft diet.

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➢ Gargling after every meal to prevent infection from accumulated food residues

➢ Avoid alcohol and smoking (causes oral dehydration)

iii) Oral Hygiene

➢ Use mouthwashes (chlorhexidine gluconate 0.2%) to prevent infection

➢ Avoid wearing dentures SKIN CARE

➢ Avoid activities that might cause skin damage like friction between skin and bed surface while changing the position or changing the clothes of bedridden patient by the nursing staff or patient relatives.

➢ Autoclaved banana leaves with a lubricant like liquid paraffin atop can be used to prevent bedspreads causing adhesions and extension of erosions on the back. Using a hood would prevent similar lesions on the ventral side.

➢ Saline soaks can be applied over erosions for 10 to 15 minutes three times a day. While removing the gauze pieces, care should be exercised so as to prevent extension of the erosions from the pull of dried gauze pieces.

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➢ Sun exposure might aggravate the disease; hence bed allocation away from sunlight/windows should be taken into account.

➢ Frequent changing and laundering of body linens, bed spreads and towels to prevent secondary infection.

➢ Pemphigus patients with extensive erosions might develop a characteristic malodour which might cause discomfort both physiologically and psychologically for the patients as well as bystanders.⁴¹ In that case, use of activated charcoal, a cost effective and safe alternative for chemical odour neutralizers, for neutralizing the odour in the ward could be useful.⁴²

TOPICAL THERAPY

For Patients with oral pemphigus and mild cutaneous disease, potent topical corticosteroids or intralesional steroids might control the disease or else reduce the dose requirement for oral steroids. Apart from good oral hygiene specific treatment of periodontal disease is important.

In patients with widespread blistering, nursing care in the form of paraffin gauze for erosions or collagen sheets for larger erosions can be used.

Potassium permanganate soaks and topical antiseptics may help to reduce the risk of cutaneous infection in pemphigus patients. Generous use of

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emollients will reduce the frictional stress on the affected skin in pemphigus patients.

CORTICOSTEROIDS

Prednisolone in a dose of 0.5–1 mg/kg/day along with adjuvant immunosuppression can control the disease most often.⁴³ Then the dose of steroids can be steroid titrated depending on the clinical response.

Patients with generalized disease may require aggressive immunosuppression with higher doses of steroids to control the disease activity. In that case IV pulses of either 1–g methylprednisolone or 100 mg dexamethasone can be considered.⁴⁴

AZATHIOPRINE

Azathioprine can be given at a dose of 2–3 mg/kg/day for pemphigus. Azathioprine can be used with prednisolone for effective disease control. Studies have shown that azathioprine is the most effective steroids paring agent in comparison with cyclophosphamide and mycophenolate mofetil.⁴³ Side effects of azathioprine include bone marrow suppression, nausea and liver dysfunction which necessitates appropriate laboratory monitoring. Adverse effects due to Azathioprine toxicity is more pronounced in patients with low levels of the enzyme

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thiopurine methyl transferase (TPMT). Screening for TPMT enzyme activity avoids the risk and in patients with low levels of TPMT dose of azathioprine should be reduced.⁴⁵

MYCOPHENOLATE MOFETIL

Mycophenolate mofetil [MMF] is a pro‐drug of mycophenolic acid. It inhibits inosine monophosphate dehydrogenase enzyme and targets T and B lymphocytes. MMF is active against pemphigus in a dose of 1-3 g/day.⁴⁶ A study comparing the efficacy azathioprine and mycophenolate showed no difference in efficacy between the two drugs.⁴⁶ Adverse effects of mycophenolate include gastrointestinal symptoms and bone marrow suppression. Gastrointestinal symptoms can be avoided by the use of enteric coated delayed release tablets.

CYCLOPHOSPHAMIDE

Cyclophosphamide can be used in a dose of 1-1.5 mg/kg and is a potent B-cell inhibitor with significant activity in pemphigus.⁴⁷ Adverse effects reported include premature ovarian failure, haemorrhagic cystitis, risk of bladder cancer and bone marrow suppression. Bladder toxicity associated with cyclophosphamide can be averted by intravenous administration of sodium 2-mercaptoethane sulfonate (Mesna). Mesna

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binds to the cyclophosphamide metabolite acrolein that is responsible for producing hemorrhagic cystitis.⁴⁸ Cyclophosphamide has been used as monthly IV pulses along with dexamethasone combined with daily low dose oral cyclophosphamide.

DEXAMETHASONE–CYCLOPHOSPHAMIDE PULSE THERAPY Pasricha and co workers studied the efficacy of dexamethasone–

cyclophosphamide pulse (DCP) in treatment of pemphigus.⁴⁹ DCP therapy involves the IV infusion of 100 mg of dexamethasone with 500 mg of cyclophosphamide in 500 ml of 5% dextrose over 1–2 hours on day 1, followed by daily administration of 100 mg of dexamethasone for the next 2 days. Rest of the days in the month were covered by 50 mg tablet of cyclophosphamide. These pulses are repeated every 4 weeks.

DCP pulse therapy has four phases. First phase is continued from 9 months to one year till healing of the erosions. Interval dexamethasone pulses can be considered for breakthrough lesions in between the regular pulse. Second phase extends for 9 months to one year with monthly DCP pulse even in the absence of new lesions. In phase three, monthly pulses are stopped with continuation of oral cyclophosphamide 50 mg/day for nine months. Then in the phase four patients are followed up to ten years for monitoring the disease for any relapse.^50,51

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INTRAVENOUS IMMUNOGLOBULIN

Intravenous immunoglobulin (IVIG) is used in a dose of 2g/kg given in divided doses over 3-5 days ever month. IVIG is unique in not having immunosuppressive effects in comparison with other treatment options for pemphigus.⁵² Its proposed mechanism of action is that of dilutional effect on pathogenic autoantibodies and anti-idioptypic effects.⁵³ In general IVIG is well tolerated and reported side effects include headache, flu-like symptoms and hypotension. In patients with IgA deficiency anaphylaxis may occur with IVIG infusion.

RITUXIMAB

Rituximab is an anti-CD20 monoclonal antibody which causes depletion of B lymphocytes and has been shown to be effective in the management of pemphigus. Rituximab acts through B cell depletion and it do not affect the plasma cells which lacks CD20 molecule and also it mitigates the T cell responses to Dsg.⁵⁴ Dosing regimens followed include the lymphoma protocol which uses 375 mg/m2 weekly for 4 weeks and the rheumatoid arthritis protocol with two infusions of 1gram, 2 weeks apart.⁵⁵ The action of rituximab starts after 8-16 weeks after first infusion and the effect lasts for nearly one year to 18 months.

Rituximab have been combined with conventional steroid therapy,

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adjuvant immunosuppression and immunoadsorption.⁵⁶ Adverse effects are less common with rituximab, but infusion reactions may occur rarely.

IMMUNOABSORPTION AND PLASMAPHERESIS

Plasmapheresis is the process by which the circulating antibodies are removed from the body and could be used as a therapeutic option for acute stages of pemphigus.⁵⁷ Immnoadsorbtion involves selective adsorption of IgG using columns containing protein A or other immunoglobulin binders.⁵⁸ However, concomitant immunosuppression with steroids and steroid sparing adjuvants is necessary to prevent rebound increase antibodies and relapse.

OTHER THERAPIES

Ciclosporin, gold, dapsone, acitretin methotreaxate have been used with varying response. Tetracycline antibiotics with or without nicotinamide may be helpful as steroid sparing agents in some patients.⁵⁹ Tumour necrosis factor inhibitors like infliximab and adalimumab shows significant improvement in some studies.⁶⁰

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PEMPHIGOID GROUP OF DISEASES

Pemphigoid diseases are heterogeneous group of subepidermal blistering diseases characterized by autoantibodies against structural proteins of the dermal–epidermal junction (DEJ).⁶¹ Structural proteins of the DEJ anchor the cytoskeleton of the basal keratinocytes to the extracellular matrix of the dermis.

The pathogenic autoantibodies in pemphigoid disease directed against the target antigens of the DEJ results in separation of epidermis and dermis.⁶¹ Subepidermal bullous diseases in general, presents with tense blisters and erosions. However individual diseases of the pemphigoid group have different target antigens, autoantibodies and pathogenesis. Importantly, prognosis and treatment may vary considerably, hence requiring exact diagnosis.

It was Lever In 1953 who differentiated pemphigoid from pemphigus both clinically and histopathologically. Lever described intraepidermal split and acantholysis as characteristic features of pemphigus, and subepidermal splitting as the feature of pemphigoid diseases.⁶² Jordon et.al described serum and skin-bound autoantibodies in bullous pemphigoid.⁶³

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The most common pemphigoid disease is the bullous pemphigoid [BP] and other pemphigoid diseases include mucous membrane pemphigoid [MMP], linear IgA disease [LAD], pemphigoid gestationis [PG], lichen planus pemphigoides [LPP], anti-p200/ laminin γ1 pemphigoid, bullous systemic lupus erythematosus, epidermolysis bullosa acquisita [EBA] and cicatricial pemphigoid.⁶¹

Apart from this major types other minor subtypes of subepidermal blistering diseases include anti type IV collagen pemphigoid characterized by renal insufficiency and autoantibodies against the a5 chain of type IV collagen or with autoantibodies against antigens like 105 kDa, 168 kDa and 285 kDa proteins.⁶⁴

Dermatitis herpetiformis [DH] is a distinct subepidermal blistering disease whose target antigen is not a structural component of DEJ. DH is associated with gluten sensitive enteropathy. Both the conditions are associated with presence of IgA autoantibodies against transglutaminases.

The autoantigen in case of DH is epidermal transglutaminase and gluten sensitive enteropathy is tissue transglutaminase.⁶¹ Clinically DH is characterized by the presence of grouped erythematous papules and vesicles involving the extensor aspects associated with intense pruritus.

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Pemphigoid group of diseases are heterogeneous in clinical presentation, target antigens and autoantibody type. Differentiation of the type of pemphigoid disease is not possible with clinical features alone and needs diagnostic techniques directed towards identification antibodies against specific target antigen. The prognosis and treatment of each subepidermal blistering disease is different and hence exact diagnosis is needed.⁶¹

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TABLE

PEMPHIGOID DISEASES AND TARGET ANTIGENS⁶¹ Disease Target antigens Bullous pemphigoid BP180 NC16A, BP230 Mucous membrane pemphigoid BP180, BP230

laminin 332 α6β4 integrin laminin 311a

Linear IgA disease LAD-1, BP230 (IgA reactivity) Pemphigoid Gestationis BP180 NC16A, BP230

Anti-p200/laminin γ1 Pemphigoid p200 antigen/laminin γ1 Epidermolysis Bullosa Acquisita Type VII collagen Bullous Systemic Lupus

Erythematosus

Type I: type VII collagen

Type II: BP180, BP230, laminin 332

Lichen Planus Pemphigoides BP180 NC16A, BP230 Cicatricial Pemphigoid BP180, BP230, laminin 332 Dermatitis Herpetiformis Epidermal Transglutaminase

Tissue Transglutaminase Endomysium

Deamidated gliadin

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BULLOUS PEMPHIGOID

Bullous pemphigoid is the most common subepidermal immunobullous disease. It tends to affect mainly the elderly individuals, however can involve younger patients also. BP often starts with intense itching and associated urticarial plaques, followed by appearance of tense blisters involving the erythematous and normal skin. Mucosal involvement occurs only in a small number of patients.⁶¹

A strong association between BP and neurological diseases have been reported in various studies. It includes major cognitive impairment, Parkinsonism, stroke, epilepsy and multiple sclerosis.⁶⁵ This association has been attributed to the expression of both bullous pemphigoid target antigens in the central nervous system.⁶⁵

Triggering factors implicated in BP include trauma, burns, radiotherapy, ultraviolet radiation and vaccination. Bullous pemphigoid typically presents with tense blisters with preceding urticarial plaques and severe pruritus. The blisters often start on the flexural aspects of the limbs and then involve the trunk and abdomen leaving erosions and crusting.

Oral lesions develop in only 10–20% of patients.⁶⁶ Prodromal lesions presents with excoriated, eczematous, papular or urticarial lesions.

Several variants described include prurigo-like, exfoliative dermatitis-

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like, ecthyma gangrenosum-like, intertrigo like, and toxic epidermolysis- like bullous pemphigoid.⁶⁶

BP180 and BP230 are the two main targets antigens identified in bullous pemphigoid. BP180 is a 180 kDa transmembrane glycoprotein which spans the lamina lucida and kinks back from the lamina densa into the lamina lucida. In BP 180 the immunodominant region belongs to the extracellular portion of 16th non-collagenous domain.⁶⁷ IgG reactivity with C-terminal epitopes has an association with mucosal involvement and severe skin disease.⁶⁷ BP230 is a 230 kDa protein which belongs to the plakin family and is an intracellular componenet of the hemidesmosomal plaque.

Initial event in pathogenesis is the binding of autoantibodies to BP180 which initiates Fc receptor-independent events resulting in the release of IL-6 and IL-8 from basal keratinocytes. Then Complement is activated at the dermal–epidermal junction and mast cell degranulation occurs. Complement activation and chemokine gradient triggers the infiltration of inflammatory cells into the dermis. Secretion of inflammatory mediators and release of proteases and reactive oxygen species by the granulocytes at the DEJ induce dermal–epidermal splitting.

Studies shows that matrix metalloproteinase 9 (MMP-9) secreted from

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neutrophils cleaves α1-proteinase inhibitor (α1-PI) to remove neutrophil elastase inhibition. Both MMP-9 and neutrophil elastaese also directly degrade proteins of the DEJ including BP180.^61,68

Histopathological examination shows subepidermal splitting and a moderate to dense inflammatory infiltrate composed mainly eosinophils, lymphocytes and neutrophils. Histopathology cannot differentiate BP from other pemphigoid diseases such as anti-laminin 332 pemphigoid and anti-p200 pemphigoid.^69,70 DIF reveals linear deposits of IgG or C3 at DEJ. IIF with salt split skin will show a roof pattern binding in case of BP.

ELISA can be used to detect circulating antibodies against BP180 NC16A and BP230 in serum.⁷¹ High serum concentrations of anti-BP180 NC16A antibodies at the time of discontinuation of corticosteroid treatment signal an impending relapse. Monitoring the course of the disease with ELISA will be useful for guiding the treatment.⁷¹

MUCOUS MEMBRANE PEMPHIGOID

Mucous membrane pemphigoid [MMP] is a distinct subepidermal blistering disease characterised by predominant mucosal involvement.

The term cicatricial pemphigoid which was used synonymously for

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MMP, now caters only to the clinical variant in which skin lesions heal with scarring and less incidence of mucosal involvement.⁷²

The lesions of MMP predominantly affect oral mucosa, followed by conjunctival mucosa, nasal mucosa, genital mucosa, pharynx, larynx, and oesophagus in that order of frequency.⁷² Clinical manifestations range from mild features like minimal oral erosion, conjunctival redness to very severe painful mucosal erosions oesophageal involvement and conjunctival disease.^72,73

Ocular pemphigoid presents with burning sensation, dryness and a sensation of grittiness in the eye. With progression of the disease there will be formation of symblepharon, trichiasis and blindness. An ophthalmic evaluation with slit-lamp examination can identify early eye changes.⁷⁴

MMP runs the risk of an associated solid cancer in nearly 30% of patients with anti-laminin 332 pemphigoid. This warrants a thorough tumour search in MMP patients.⁷⁵ The possible target antigens in MMP include BP180, BP230, laminin 332, both subunits of α6β4 integrin, and type VII collagen. In patients with oral lesions the autoantibodies are

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targeted against α6 integrin and in patients with ocular pemphigoid against β4 integrin.⁷⁶

DIF shows deposition of IgG, C3 (Complement Component 3) and in some patients, IgA along the dermal–epidermal junction. This picture is analogous to BP. IgA autoantibodies can be found in nearly 60% of serum samples. Combination of IgA and IgG autoantibodies is associated with more severe disease.⁷⁷

By IIF microscopy on salt-split skin, depending on the target antigen there may be either epidermal staining or dermal staining of the artificial split. Dermal binding in salt split skin points to laminin-332 reactivity. In that case it should be evaluated for associated malignancy.⁶¹

For detection of α6β4 integrin-specific antibodies Immunoblotting with bovine gingivae lysate is a better method.⁷⁶ For the detection of antilaminin 332 IgG, immunoprecipitation with radio labelled human keratinocytes is the most sensitive method. Immunoblotting with cultured human keratinocytes is a practical alternative option.⁷⁸ In ocular pemphigoid with negative DIF of conjunctival specimen, oral biopsy and serological analysis may be done to derive at a diagnosis.⁶¹

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PEMPHIGOID GESTATIONIS

Pemphigoid gestationis is a pregnancy-associated subepidermal immunobullous disease with autoantibodies against BP180 NC16A. The diseases in general occurs in the second or third trimester and within 4 weeks after birth in 10% of patients.⁷⁹ clinically the diseases manifests as pruritic papules, erythematous plaques, papulo vesicles, eczematous lesions and less commonly blisters, initially around the umbilicus and then progress to involve the abdomen and thighs. The disease runs a benign course, however with more than 90% risk of recurrence in subsequent pregnancies. In nearly 5% of patients the diseases progress to bullous pemphigoid.⁷⁹ Notable outcome associated with the disease is the risk of pre term birth, small-for-date babies and transient skin lesions in the newborn. Maternal HLA-DR3 and HLA-DR4 has a strong association with the disease.⁸⁰

The major target antigen for pemphigoid gestationis is BP180 which is expressed in the amniotic membrane. The shed ectodomain of BP 180 is a physiological constituent of the amniotic fluid. DIF shows IgG and C3 deposits along the dermal–epidermal junction. The BP180 NC16A ELISA detects serum autoantibodies in nearly 90% of patients.⁸¹

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LINEAR IGA DISEASE

Linear IgA disease is characterized by linear binding of IgA at the DEJ. Some overlap is seen with bullous pemphigoid, with mucous membrane pemphigoid and IgA epidermolysis bullosa acquisita.

Clinically the disease manifests as urticarial plaques with tense blisters and erosions and a distinct feature is the arrangement of tense vesicles over an erythematous plaque in an annular fashion giving a string of pearl appearance. Nearly 70% of patients presents with mucosal involvement with oral erosions and ulcers, nasal crusting and genital lesions.⁸² Most commonly affected sites are the face and perineal area. In children, the face and perineal area are most commonly affected and in adults trunk and limbs are mainly affected. Vancomycin and non-steroidal anti- inflammatory drugs are most commonly implicated among the drugs causing Linear IgA disease.⁸³

The major target antigen in linear IgA disease is BP180. In BP 180, two target molecules reported include, a 97 kDa protein in the extract of human epidermis and dermis and a 120 kDa shed ectodomain obtained from the cultured human keratinocytes, referred to as linear IgA antigen 1.⁸⁴ These two antigens belong to the C terminal portions of BP180.The NC16A domain of BP 180 is the target in only 20% of patients. Diagnosis

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is made by DIF showing linear deposition of IgA in DEJ and by detection of IgA serum antibodies by IIF on human salt-split skin.⁸⁵

In serum of linear IgA disease patient, in addition to IgA anti- BP180 antibodies, most of the patients also contain IgG antibodies against BP180. Likewise IgA anti-BP180 antibodies can be detected in serum samples of BP patients. Thus the two diseases are regarded as different ends of a spectrm.⁸⁶ In patients with equal IgG and IgA reactivity against the DEJ, the diagnosis ‘mixed immunobullous dermatosis’ or ‘linear IgA/ IgG bullous dermatosis’ has been described.⁸⁷

EPIDERMOLYSIS BULLOSA ACQUISITA

Epidermolysis bullosa acquisita (EBA) is characterised by autoantibodies against type VII collagen and it manifests as two variants:

the classic mechanobullous form and an inflammatory variant.⁸⁸ The classic form presents with tense blisters preferentially involving the extensor aspects of the trauma-prone areas and tends to heal with scarring and milia formation. The inflammatory subtype of EBA mimics bullous pemphigoid or mucous membrane pemphigoid. In both subtypes, mucous membranes are affected in nearly 50% of patients.⁸⁹ An association with inflammatory bowel disease is reported in about 20% of patients. The

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target antigen type VII collagen is a constituent of the anchoring fibrils, in which the N-terminal 145 kDa NC1 domain is the immunodominant region and is found in nearly all serum samples.⁹⁰ The glycolisation of type VII collagen-specific antibodies is essential for the interaction of autoantibodies with their Fcγ receptors as well as their interplay with complement activation.⁹¹

Diagnosis is based on linear deposits of IgG, IgA and C3 at the DEJ by DIF. A feature that is exclusive to EBA is the immunoglobulin deposition in DEJ in a u-serrated pattern.⁹² Other pemphigoid disorders will show an n-serrated pattern. Serration pattern analysis is important in EBA because, only 40–60% of serum samples show floor pattern binding on salt-split skin IIF.⁸⁹ Immunoblotting or ELISA can detect anti-type VII collagen antibodies using tissue extracts or the recombinant NC1 domain.⁹⁰

In around 10% of patients, autoantibodies are of the IgA isotype these patients tend to manifest as the inflammatory phenotype.⁸⁹ systemic corticosteroids in a dose of 0.5 – 1.0 mg/kg/day remains the mainstay of treatment, in combination with colchicine or dapsone. For resistant cases cyclosporine, azathioprine, mycophenolate mofetil, intravenous immunoglobulin, and rituximab are other available options.⁶¹

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ANTI-P200/ANTI-LAMININ γ1 PEMPHIGOID

Anti-p200 pemphigoid is a rare variant among subepidermal blistering disease and clinically it mimics BP. Unlike BP, patients with anti-p200 pemphigoid are younger and nearly one third of cases is associated psoriasis.⁹³ Serum samples from 90% of patients react with laminin γ1. IIF on salt split skin shows floor pattern binding. Diagnosis is established by immunoblotting or ELISA with extract of dermis or C- terminus of laminin γ1 detecting antibodies against the 200 kDa protein of the dermal–epidermal junction.⁹³ Most often this entity is diagnosed as BP and it’s under diagnosis is because of unavailability of the specific diagnostic assay. However the specific diagnosis gains importance in the treatment point of view because of the less severity and rapid response in comparison with BP.⁹⁴

LICHEN PLANUS PEMPHIGOIDES

This rare pemphigoid disorder occurs in patients with lichen planus. The tense blisters clinically mimics BP however differ from BP by affecting younger patients, with less severe disease, preferentially involving the extremities and targeting the C-terminal epitopes in the immunodominant NC16A domain of BP180.⁹⁵ It is hypothesized that chronic interface dermatitis of lichen planus may expose the target

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antigen in DEJ and trigger the immune response against BP180. This is supported by the fact that lichen planus pemphigoides remits after treatment of lichen planus lesions. Diagnosis is established by DIF showing linear deposits of IgG or C3 at the DEJ and detection of circulating IgG antibodies against BP180 NC16A.⁹⁶

DIAGNOSIS OF AUTOIMMUNE BULLOUS SKIN DISEASES The diagnosis of autoimmune blistering diseases starts from Tzanck smear and histopathological examination of the bulla and is the first step in evaluation of an AIBD. Histopathological examination identifies only site of the split and presence of inflammatory infiltrate and its composition.⁹⁷ A precise diagnosis of AIBD needs further evaluation with advanced diagnostic techniques like direct or indirect immunofluorescence microscopy, ELISA and immunoblotting. These tests aim at the identification of tissue bound or circulating antibodies against the target antigens that can point to the final confirmation of the diagnosis.⁹⁸

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TZANCK SMEAR

Tzanck smear is a tool of diagnostic cytology for diagnosing vesiculobullous diseases.⁹⁹ In the case of blistering disorders, the roof of a blister is opened and the material from the floor is gently scraped. It is then smeared in a slide and stained with Giemsa stain.¹⁵

Presence of acantholytic cells in Tzanck smears points to the diagnosis of pemphigus.⁹⁹A typicalTzanck cell is characterized by the presence of large rounded keratinocyte with a hypertrophic nucleus, hazy nucleoli. The cytoplasm will be basophilic and prominent in the periphery of the cell due to condensation at the periphery resulting in a perinuclear halo.¹⁰⁰ Tzanck smear depends on the pathogenetic mechanism of acantholysis. In acantholysis, because of the break in intercellular connection, there will be loss of cohesion between the adjacent keratinocytes.¹⁰⁰

Apart from acantholytic cells other findings noted are the Sertoli's rosettes and streptocytes. Sertoli's rosettes are made of a central necrotic keratinocyte with a surrounding ring of leukocytes. A 'streptocyte' is a chain of leukocytes, joined by a filamentous, glue-like substance.¹⁰¹

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HISTOPATHOLOGY

First step in differentiating between pemphigus and subepidermal blistering diseases is the biopsy of the lesional skin and its histopathological examination. In case of pemphigus the earliest finding is the eosinophilic spongiosis. In an established lesion a subrabasal cleft containing acantholytic cells is the characteristic finding. The basal layer of cells is separated from each other but retains its attachment with that of the basement membrane giving the characteristic ‘row of tomb stone appearance’.

Along with the findings mentioned above pemphigus vegetans will have pseudoepitheliomatous picture with acanthosis and papillomatosis with occasional intra epidermal eosinophilic abscess. In pemphigus foliaceous the split will be in the granular layer. Older lesions of pemphigus foliaceus will show dyskeratotic changes. In IgA pemphigus histopathology will show pustules subcorneally in the subcorneal type and in lower epidermis in the intraepidermal neutrophilic type. In paraneoplastic pemphigus apart from the typical findings of pemphigus, histology parallels the cutaneous morphology like dyskeratotic, necrotic keratinocytes in erythema multiforme, interface dermatitis in lichenoid lesions.^2,14

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In bullous pemphigoid early lesions show eosinophilic spongiosis and eosinophilic infiltrate in the upper dermis. Biopsy of an intact bulla shows subepidermal bulla with inflammatory cells composed of eosinphils and neutrophils. Blister cavity contains inflammatory cells along with fibrin. In case of old bulla due to reepithelialisation the blister appears as an intraepidermal blister.

In linear IgA disease, biopsy shows subepidermal blister cavity with neutrophilic collection along the DEJ and occasionally in the dermal papillary tips. The histology of EBA in classic variant shows no inflammatory cells and there is a mixture of lymphocytes, monocytes, neutrophils and eosinohils in the inflammatory variant of EBA. In dermatitis herpetiformis there will be an subepidermal cleft with neutrophils and few eosinophils forming papillary tip microabscesses.^14,61

IMMUNOFLUORESCENCE TESTS

Immunofluorescence (IF) technique detects specific target antigens using antibodies tagged with fluorescein conjugate. In 1940 Coons et al.

was the first to introduce the technique of immunofluorescence technique using blue fluorescing compound, anthracene.¹⁰² The role of diagnostic immunofluorescence in dermatology started with the description of the lupus band test. Later in the year 1964, Beutner and Jordon using the

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indirect immunofluorescence (IIF) technique, identified the presence of pathogenic antibodies in pemphigus patients serum.¹⁰³ From that point of time diagnostic pathology using IF had made an excellent progress in diagnosis of various dermatological disorders.

TECHNIQUES OF IMMUNOFLUORESCENCE

Immunofluorescence technique encompasses various methods:

direct, indirect, complement fixation method and immunoelectron microscopy.

Skin biopsy specimens are evaluated by DIF for in vivo deposits of antibodies, whereas IIF detects circulating antibodies in serum samples.

The tissue sample or cellular smear fixed on a glass slide is known as

“substrate”. Antibodies against immunoglobulins, complement components and fibrinogen, linked to a fluorochrome is called conjugate.

The fluorochromes used commonly are fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate, or phycoerythrin.¹⁰⁴

SITE OF BIOPSY

A 3–5 mm punch biopsy is ideal for DIF study. Biopsy should be taken from normal looking perilesional skin. Biopsy from the lesional skin should be avoided as the immune deposits are degraded in these

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areas giving rise to false negative results.¹⁰⁴ If two biopsies are taken, one for routine histopathology and one for DIF, first biopsy should be taken for IF, so as to avoid formalin contamination. Formalin may render the biopsy specimen unsuitable for DIF.⁹⁸

TRANSPORTATION OF SAMPLE

Michel’s medium is used for transport of biopsy specimens and samples can be stored in Michel’s media for up to 28 days; normal saline can be used for transfer of sample within 24 hours.¹⁰⁵ Honey can be used as an alternative transport media and immunoreactants can be preserved for up to 20 days.¹⁰⁶

STEPS IN IMMUNOFLUORESCENCE

The biopsy sample is washed with phosphate buffered saline (PBS) and is cut into 4-6µ sections using cryostat. For staining, sections are brought to room temperature and washed in PBS to remove unbound antibodies. Then the substrate is incubated with FITC-labeled immunoglobulins, C3, and fibrin at 37 °C for 45 min to 1 hour.¹⁰⁷ After that, substrate is washed in PBS for 30 minutes and mounted in buffered glycerine and examined under fluorescence microscope for interpretation of results. The following five features are evaluated: the site of immune

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deposition, class of immunoglobulin, pattern of immune deposit, number of immune deposits, and deposition in other sites.¹⁰⁸

Indirect immunofluorescence is a two step procedure wherein 5 ml of blood is withdrawn and serum is separated. Serum in serial dilutions is incubated with the substrate for one hour. Rest of the steps are same as in DIF.⁹⁸

DIRECT IMMUNOFLUORESCENCE MICROSCOPY

In the diagnosis of autoimmune bullous skin diseases direct immunofluorescence (DIF) is the gold standard test. DIF is a one step procedure and it diagnoses AIBD by detecting target antigens in the substrate with the help of fluorescent tagged antibodies.¹⁰⁹ Direct IF

microscopy remains the diagnostic gold standard, with a specificity of 98% and sensitivity up to 91%.¹⁴ For DIF, biopsy should be obtained

from perilesional skin so that the DEJ will be intact to assess immunoglobulin and complement deposition. In the lesional skin, the structural proteins would have been damaged by the immunoreactants, resulting in false negative or false positive results. In pemphigus there will be intercellular staining of IgG and/or C3, whereas in pemphigoid diseases there will be a linear staining along the dermal-epidermal junction.¹⁴ In dermatitis herpetiformis, there will be granular deposits of