Fluid Biomarkers in Autoimmune Encephalitis (AIE)-Expanding Horizons

Introduction

Autoimmune encephalitis (AIE) is a heterogenous group of disorders in which the body’s immune mechanisms are triggered against the epitopes of the nervous system. AIE can have protean manifestations and may present as acute to subacute, rapidly progressive neurological and/or neuropsychiatric symptoms (agitation, psychosis, anxiety, delusions, catatonia), movement disorders (orofacial dyskinesia, paroxysmal dyskinesia, chorea, dystonia, myoclonic jerks, tremor, cerebellar ataxia, and parkinsonian symptoms), intractable seizures, sleep disturbances, and autonomic instability. The Incidence of AIE is 0.8/100,000 with a prevalence of 13.7/100,000, which is similar to infective encephalitis. The tendency to relapse in patients with autoimmune-mediated encephalitis is higher as compared to infective encephalitis, particularly due to the Herpes Simplex virus. Both infectious and autoimmune aetiologies may be associated with considerable morbidity and mortality.^1,2

Serum and cerebrospinal fluid (CSF) biomarkers can be valuable to differentiate the variants, precisely seropositive and seronegative forms, from infectious AIE, with a high index of suspicion. These biomarkers when corroborated with clinical presentation findings, can also assist clinicians in initiating treatment early in the disease course.

The various types of AIE antibodies can be categorized into three groups:

1. Intracellular paraneoplastic antibody- associated with systemic tumours (in more than 90% of cases) causing neuronal damage. These antibodies can be considered as biomarkers for neoplasms associated with the corresponding antibody found.
2. Cell surface or synaptic antibodies
3. Antibodies of unclear significance, which includes Hashimoto’s encephalitis, characterised by the presence of thyroid peroxidase antibodies, although these antibodies are not directly pathogenic.

The antibodies formed in AIE are of the IgG class of gamma globulins, formed either against intracellular antigens or cell surface antigens (Table 1).^3,4

Table 1: Nomenclature of autoimmune encephalitis (AIE) antigens with associated tumours

The clinical phenotype can be related to the neuronal antibody associated. For example, orofacial dyskinesias are associated with anti- N-methyl D-Aspartate Receptor (NMDAR) encephalitis, myokymia is seen in Contactin-Associated Protein 2 (CASPR2) encephalitis, hyperekplexia in anti-dipeptidyl-peptidase like protein 6 (DPPX) encephalitis, opsoclonus myoclonus in anti-γ-aminobutyric acid-B receptor (GABAB) and anti-Ri encephalitis, PERM (progressive encephalomyelitis with rigidity and myoclonus) in anti-glycine receptor (GlyR) encephalitis.^1,4

The wide array of clinical symptoms presented varies due to the synaptic dysfunction associated with the protein targeted in the immune-mediated process. This process leads to impairment of the protein-protein interactions at the synapse, alteration of the synapse surface antigens, or antigen blockage and internalisation of receptors resulting in varied symptoms related to the corresponding antibodies. The diverse clinical manifestations are associated with the antigen targeted in the immune-mediated process and the area of brain inflamed during the pathogenesis of the disease. This heterogeneity can mask the primary underlying disorder and can lead to misdiagnosis.^5,6

Antibodies have the potential to induce neurodegeneration, causing structural changes in these patients, making these disorders irreversible to treat. The antibodies cause synaptic dysfunction by triggering T-cell and complement system activation, targeting oligodendrocytes and leading to inflammation. This inflammatory process initiates neurodegeneration.⁷ These disorders may be referred to as antibody-mediated neurodegeneration. These disorders are amongst the disorders of nervous system which if investigated, diagnosed, and treated in a timely manner, are reported to have good prognosis with minimal or no disability.

There is an interplay between the various parameters defined in the Graus et al. diagnostic criteria: the presence of antibodies against cell surface or intracellular antigens, magnetic resonance imaging (MRI) hyperintensities (medial temporal), cerebrospinal fluid (CSF) analysis, electroencephalogram (EEG) (e.g., delta brush pattern in NMDAR encephalitis) for diagnosing seropositive and seronegative AIE.^1,3

Biomarkers-History

In 1998, the National Institutes of Health Biomarkers Definitions Working Group, defined a biomarker as “a characteristic that is objectively measured and evaluated as an indication of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions”. Biomarker-guided decision-making could offer a competitive clinical advantage over existing empirical approaches.

The application of biomarkers diversifies from diagnosing, prognosticating, screening, and staging diseases to evaluating the efficacy, and toxicity of the drugs, and classifying them as standalone or surrogate biomarkers.⁸

In AIE, autoantibodies serve as biomarkers and their detection using the preferred technique of cell-based assays is considered the gold standard for diagnosis. Despite the best immunoassays, there are chances of misdiagnosis because of false negatives, due to low levels of antibodies in biofluids, differences between human and mouse epitopes used in cell-based assays, T-cell mediated AIE, or antigen denaturation during tissue fixation. The versatility of phenotypes in AIE, suggests unknown pathophysiology and lack of newer techniques to identify antibodies, which may impact our treatment approach.

The relevance of finding a biomarker extends beyond seropositive patients, being particularly critical for seronegative patients, where the chances of misdiagnosis or exacerbation of the underlying disease due to inappropriate drug effects is exponentially higher. Seronegative autoimmune encephalitis occurs in around 48% of cases, despite the availability of the latest antibody assay panels.^1,2,6

Biomarker- Age and Gender

Kunchok et al. studied the age and gender frequency of antibodies in AIE. According to their study, females and people with age under 60 years, including children, are more predisposed to NMDAR encephalitis, while Leucine-Rich Glioma Inactivated 1 (LGI1) encephalitis has a higher predisposition in males and those over 60 years of age. GABAB, CASPR, Anti Neuronal Nuclear Antigen 1 (ANNA1)-associated encephalitis are more prevalent in older age groups, implying a paraneoplastic association.⁹

Biomarkers- Serum

Liu et al. reported variations in the levels of uric acid, high density lipoprotein and C-reactive protein, due to their roles as a reactive oxygen species scavenger and acute phase inflammatory protein, respectively, in NMDAR encephalitis with status epilepticus. These levels were reduced after 3 months with a decrease in status epilepticus.¹⁰

Zeng et al. studied the neutrophil lymphocytic ratio (NLR) in age- and gender-matched patients across normal and encephalitis patients and reported NLR as an independent marker of AIE severity.¹¹

Biomarker- Cerebrospinal Fluid

Antibodies formed against receptors activate the complement system, which alters neurotransmission, causing neuronal dysfunction. Antibodies formed against microglia and astrocytes mediate neuronal damage in the medial temporal area, and hippocampus, causing cognitive deficits. In this process, interleukins released can be used as biomarkers for the ongoing disease process.¹²

Interleukin 6 is an acute inflammatory protein that triggers T-cell activation and antibody production. T-helper 17 (TH-17) cells help release pro-inflammatory interleukins, (IL-17 and IL-21), tumour necrosis factor alpha (TNF-α). The process of inflammation disrupts the blood brain barrier (BBB) by B-cell activation and chemokine release, forming a positive feedback loop, enhancing further damage to the synaptic morphology. Zhang et al. studied the correlation of cytokines as surrogate biomarkers and reported IL-21 as useful for differentiating between viral infections and AIE, thus avoiding fatal misdiagnosis where immunosuppressive therapy might be used inappropriately. Increased levels of IL-6 were associated with memory impairment, and TNF-α was increased in patients with cognitive impairment. Chemokines, (CXCL10, CXCL13, CCL20, CCL22), produced during pleocytosis in the inflammatory process, were also studied and reported to be increases in NMDAR encephalitis.¹²

Blinder et al. reported CSF pleocytosis with positive oligoclonal bands (OCB) in NMDAR, γ-Aminobutyric Acid-B Receptor (GABABR) and α-Amino-3-hydroxy-5-methyl-4 isoxazolepropionic Acid Receptor (AMPAR) encephalitis, while pleocytosis without OCB was present in LGI1, IgLON5, and GlyR encephalitis.¹³ Vakrakou et al. observed an increase in CSF light chain neurofilament in paraneoplastic syndromes, indicating the presence of an underlying tumour.¹⁴ CSF progranulin (PGRN) levels were found to be increased in NMDAR encephalitis patients, correlating with the disease severity. However, serum and CSF PGRN were not raised in neuronal antibody encephalitis, despite being a marker for lymphomas.¹⁵

A study by Nass et al. found that neurofilament light chain (NfL), total tau, and PGRN are potential biomarkers for AIE, reflecting neuronal or axonal loss leading to neurodegeneration. NfL may also help differentiate between AIE seizures and other (organic, metabolic) seizures.¹⁶

Table 3: Proposed biomarkers for autoimmune encephalitis (AIE) in cerebrospinal fluid (CSF) and serum

Conclusion

AIE is an evolving and expanding spectrum of disease with a heterogenous set of signs and symptoms. Antibody testing is the most valuable test and considered the gold standard for diagnosis. There is a possibility of unexplored and unknown antibodies with newer and emerging techniques. This process might take time, and treatable diseases could remain untreated, until newer treatments are found. In this scenario, the importance of biomarkers extends beyond early diagnosis, particularly for currently seronegative AIE, where the risk of patients being left untreated is significantly higher. Biomarkers could also play a crucial role in assessing the treatment efficacy and selecting the appropriate drugs.

Figure 1: Classification of β-lactam antibiotics11

1. MRI brain-diffuse cerebellar atrophy
2. Reduced FDG uptake in left cerebral hemisphere and right frontal lobe.
3. Foci of increased FDG uptake in left parietal region in background suppressed FDG uptake.
4. Increased FDG uptake Right breast

Mrs X, an 88-year female, presented in the emergency department, with rapidly progressive dementia, reduced word output for over 4 months, and significant weight loss due to loss of appetite. Her autoimmune panel showed positive anti-Yo antibodies, which are reported to be associated with ovarian teratomas or breast cancer. Her fluorodeoxyglucose positron emission tomography (FDG PET) revealed malignancy in the right breast.

Monocyclic beta-lactams are known for their antibacterial activity, but in recent years they have also been recognized in other therapeutic areas. The first monocyclic beta-lactam, Nocardin A, was discovered in 1976 in the bacterium Nocardia uniformis.13 The structure of 2-Azetidinone ring and penicillin is illustrated in Figure 3 and 4 respectively.