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Matteo Gastaldi

Neuroimmunology laboratory

IRCCS C Mondino

Autoimmune EncephalitisFrom laboratory approach to clinical

management

Pavia, 25/5/2018

paraneoplastic neurological syndromes

• Anti-Hu

• Anti-Yo

• Anti-Ri

• Anti-CV2

• Anti-Ma2

• Anti-Amphyphysin

etc.

neurological syndromes of unknown cause often associated with tumor

Yo

immunofluorescence on rat cerebellum

intracellular protein targetsthat are not accessible in vivo

the autoantibody, detectable with

conventional techniques, is just a

BIOMARKER of disease (>80% of patients)

ONCONEURAL ANTIBODIESGraus F et al., Neurology 1985;35:538

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• target surface antigens (usually synaptic proteins)

• can be pathogenetic (directly responsible for synaptic dysfunction)

• neurological syndromes (mainly ENCEPHALITIS) respond to

immunotherapies

SEIZURES

PSYCHIATRIC

DYSAUTONOMIA

PNS INVOLVEMENT

GASTROENTERIC

MOVEMENT DISORDERS

HYPOTALAMIC DYSFUNCTON

COGNITIVE

Neuronal cell surface antibodies

Target antigen Year Age at presentation NMDAR 2007 Adults>/= Children Glycine R 2008 Adults >>Children AMPAR 2009 Adults GABA-B R 2010 Adults>>Children LGI1/CASPR2 2010 Adults mGluR5 2011 Adults=Children Dopamine-2 R 2012 Children DPPX 2013 Adults >>> Children GABA-A R 2014 Adults=Children IgLON5 2014 Adults Neurexin-3α 2016 Adults

the expanding field of autoimmune encephalitisfrequency in adults vs children

Gastaldi M et al. Expert Rev Mol Diagn 2018;18:181

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Several years PD diagnosis

Subacute worsening of

extrapiramidal function with

fever and pneumonia

Persistent behavioral alteration

(apathy, anorexia)

Cognitive alteration?

NMDAR encephalitis

Courtesy of Voghera neurology department (Carla Arbasino)

NMDAR Abs in serum and CSF

a. Linear epitopes are short and continuous, and after denaturation they are still be

able to bind the antibody (‘traditional’ methods for antibody detection are denaturing)

b. Conformational epitopes are discontinuous and depend on the higher order of

the protein structure, and after denaturation they can no longer bind the antibody

how antibodies

work:

schematic

representation of

two antibodies

interacting with

linear or

conformational

epitopes

Laboratory diagnostic for autoimmune encephalitis

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AUTOIMMUNE ENCEPHALITIS: laboratory diagnostic tools

CELL BASED ASSAY

commercial fixed CBA (Euroimmun)

“MOSAIC”:NMDAR

LGI1

Caspr2

GABAB

AMPAR1-2

• Limited number of antigens included

• Possible suboptimal performance

Clinical Biochemistry

Volume 50, Issue 6, April 2017, Pages 354-355

Pitfalls in the detection of N-methyl-D-aspartate-receptor (NMDA-R) antibodies

Ma eo Gastaldi, Anaïs Thouin, Diego Francio a,

Angela Vincent

NON-ANTIGEN SPECIFIC method, but some

antigen-specific patterns can be recognized

standard methods for tissue fixation do not

work: LIGHT FIXATION allows the preservation

of the antigen conformation

R

• Many known antibodies can be identified

• “Uncharacterized reactivities” (Novel antigens?)

AUTOIMMUNE ENCEPHALITIS: laboratory diagnostic tools

IMMUNOHISTOCHEMISTRY ON RAT BRAIN

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SURFACE

ANTIGEN

ACCESSIBLE

IN-VIVO

Abs ARE LIKELY TO BE

RELEVANT/PATHOGENETIC

NEURONS ALSO PROVIDE A SUITABLE SUBSTRATE FOR IMMUNOPRECIPITATION

AND NEW ANTIGEN IDENTIFICATION

no light fixation

AUTOIMMUNE ENCEPHALITIS: laboratory diagnostic tools

PRIMARY NEURONAL CULTURES

Gastaldi M et al. Expert Rev Mol Diagn 2018;18:181

• 2 step approach

(First/Second level

diagnostics)

• In second level

diagnostic, at least 2

methods should be used

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Antibodies are so important…….

……… that we have to stop relying on them!

�Long turnaround time of antibody results (months)

�Lack of standardization (likely suboptimal first level diagnostic)

�WHAT to test? (“Panel” of anti-CNS antibodies)

�HOW to interpret antibody results (Sensitivity/specificity of

single tests)

Case report

- tiredness, irritability

- episodes of falling asleep during the day

- lingual movements when doing enjoyable activities, such as

playing

- NMDAR-Abs on serum (Live CBA)

- CSF unremarkable

- No improvement with steroids and IvIG. Additional immune

theraphy reccomended but not performed

- FINAL DIAGNOSIS: NARCOLEPSY/CATAPLEXY

Armangue et al, Neurology 2015

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1) POSSIBLE AUTOIMMUNE ENCEPHALITIS

2) PROBABLE/DEFINITE AUTOIMMUNE ENCEPHALITIS

1) Specific clinical/paraclinical profile (Limbic, NMDAR,

ADEM, Hashimoto, Bickerstaff)

AND/OR

2) Abs presence

3) PROBABLE Ab-NEGATIVE AUTOIMMUNE ENCEPHALITIS

Possible Autoimmune encephalitis

Treatment can be started without abs confirmation

Subacute onset of

• Memory impairment

• Psychiatric symptoms

+

Evidence of inflammation in

- CSF

- MRI

- Brain biopsy

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Probable seronegative Autoimmune encephalitis

Diagnosis can be made without antibodies

Subacute onset of

• Memory impairment

• Psychiatric symptoms

+

Evidence of inflammation in

(at least 2)

- CSF

- MRI

- Brain biopsy

Rituximab in seronegative Autoimmune encephalitis

Ab negative ALE show similar response to rituximab compared to synaptic abs and paraneoplastic ALE

Lee et al, Neurology 2016

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Application of 2016 criteria for

Autoimmune Encephalitis in clinical

practice:

Italian retrospective study on

Autoimmune Encephalitis

PAVIA

IRCCS C Mondino

OSM San Matteo

MILANO

Ospedale Buzzi

GENOVA

Ospedale San Martino

Istituto Gaslini

FIRENZE

Ospedale Careggi

ROMA

Policlinico Gemelli

Ospedale Pediarico Bambin Gesu’

VERONA

Dipartimento di Neuroscienze

PADOVA

Ospedale Sant’Antonio

Clinica pediatrica

PERUGIA

Ospedale S.Maria della Misericordia

CITTA’ DI CASTELLO

Ospedale di Citta’ di Castello

…WORK IN PROGRESS…

12 hospitals in 9 cities

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Possible autoimmune encephalitis

Recruitment 2012�2016

Probable/Definite autoimmune encephalitis

Ab POSITIVE

autoimmune encephalitis

Ab NEGATIVE

autoimmune encephalitis

83 pts72% adults

28% pediatrics

56 pts- 50% NMDAR encephalitis

- 34% Limbic encephalitis

- 16% other (e.g.: status

epilepticus, movement

disorder..)

27 pts- 33%limbic encephalitis

Total=57

NMDARLGI1Caspr2Other antibodiesUncharacterized

30 (52%)

11 (19%)

5 (9%)

6 (10%)

5 (9%)GABA-B (n=1)

GABA-A (n=1)

IgLON5 (n=1)

GLY-R (n=1)

AMPAR (n=1)

6/7 binding to live

neurons

Neuronal surface antibodies in Definite AE

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AIMS:

- clinical profile, outcome and response to

immunotherapy of Ab NEGATIVE

AUTOIMMUNE ENCEPHALITIS

- Comparison of Adult vs pediatric population

- Novel antigen discovery

THANKS FOR YOUR ATTENTION

Diego Franciotta (Pavia)

Elisabetta Zardini (Pavia)

Silvia Scaranzin (Pavia)

Laboratorio Neuroimmunologia- IRCCS C Mondino

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Courtesy of prof. Angela Vincent

detection of Abs to neuronal cell-surface antigens

neuronal surface Abs are

‘conformational’, and antigen

degradation/denaturation

prevents their detection with

‘common’ methods

dot blot

nitrocellulose

Antigen

Western blot

ELISA

conformational

antigen

degradation

nitrocellulose

polyvinyl or polystyrene

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1 & 2) CELL-BASED ASSAY on live or fixed cells

live (native antigen) fixed (antigen denaturation)

which advantages of

using in-house live vs

fixed CBAs?

- more 'native' antigens

- no reactivity to

intracellular antigens

(fixed cells allow Abs to

enter the cell)

cDNA-plasmid vector

which advantages

of using live vs

fixed CBAs?

the widely

available

commercial tests

are necessarily

fixed

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some antigen-specific patterns can be recognized(confirmation with CBAs, live or fixed, is mandatory)

3) IMMUNOHISTOCHEMISTRY ON RAT BRAIN

Neuroimmunology Laboratory, Mondino National Neurological Institute, Pavia

R

R

false positive result on NMDAR-transfected cells(commercial CBA)

negative

true positive

Neuroimmunology Lab., Mondino, Pavia, Italy

Clinical Biochemistry

Volume 50, Issue 6, April 2017, Pages 354-355

Pitfalls in the detection of N-methyl-D-aspartate-receptor (NMDA-R) antibodies

Matteo Gastaldi, Anaïs Thouin, Diego Franciotta,

Angela Vincent

28/05/2018

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Presentation outline

• Introduction on autoimmune encephalitis and

Neuronal surface antibodies

• Laboratory diagnostic of autoimmune

encephalitis

• Clinical criteria for autoimmune encephalitis

• Retrospective study on autoimmune

encephalitis