Thesis Establishment of a primary dorsal root ganglion cell culture to screen for autoantibodies for patients with ME/CFS or Post COVID, 2024, Wachutka

[Dolphin]

https://epub.technikum-wien.at/obvftwhsm/content/titleinfo/10192639/full.pdf

MASTERʼS THESIS
Thesis submitted in fulfillment of the requirements for the degree of Master of Science in Engineering at the University of Applied Sciences Technikum Wien - Degree Program Tissue Engineering and Regenerative Medicine

Establishment of a primary dorsal root ganglion cell culture to screen for autoantibodies for patients with myalgic encephalomyelitis/chronic fatigue syndrome or Post COVID.

By: Johanna Wachutka, BSc Student Number: 2210692004
Supervisor 1: Mag. Dr. David Hercher Supervisor 2: Univ.Prof. Priv. Doz. Dr. med. Romana Höftberger, Co-supervisor: Dr.scient. med. Carmen Haider Vienna, 03.06.2024


Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Post Coronavirus disease (Post COVID) are complex, multi-systemic illnesses, sharing symptoms like chronic pain, neurological impairments, and fatigue.

The immune system might contribute to the pathology of these diseases.

Our study aimed at detecting potential autoantibodies in tissue-based assay (TBA) and in cell-based assay (CBA).

For TBA, indirect immunohistochemistry was performed on adult rat spinal cord using patients´ serum and/or cerebrospinal fluid (CSF). For CBA, dorsal root ganglia (DRG) from 12–14-day-old rats were isolated, cultured 1-2 days and incubated with patients´ serum and/or CSF to detect potential autoantibody-binding via fluorescence staining.

Our cohort encompassed 207 samples from 151 patients presenting symptoms of ME/CFS and/or Post COVID.

Among these patients, 56% were female and 44% male with a mean age of 44 years.

The main age groups were 30-39 and 40-49.

In TBA, we observed strong immunoreactivity in the lamina II of the dorsal horn in 68 serum and seven CSF samples.

Since weak immunoreactivity was also observed in negative control samples from patients without clinical neurological symptoms, we performed serial dilutions to determine a clinically relevant cut-off dilution.

We set 1:1600 as the relevant dilution, where negative controls showed no staining while symptomatic patients remained positive.

Since some of the axons of the DRG cells terminate in the lamina II of the dorsal horn, we investigated if similar staining patterns occurred in DRG.

First, successful cultivation of DRG cells expressing the neurofilaments SMI31 and SMI32 was confirmed using fluorescent antibodies, which target these neurofilaments.

Subsequent screening of patient serum and/or CSF on live DRG cells with intact cell membranes revealed positive staining in ten patients.

Positively labelled cells expressed neurofilaments, suggesting the detection of autoantibodies targeting extracellular neuronal antigens.

Out of the ten CBA-positive serum samples, titration in TBA revealed titres of 1:1600 or higher in nine samples.

These nine samples that were double-positive in TBA and CBA were classified as positive in our study.

No positive staining was detected in CSF samples.

In conclusion, we could identify nine patients that showed immunoreactivity in both TBA and CBA, which might indicate a role of autoantibodies targeting neuronal antigens of DRG cells.

Further analyses, including immunoprecipitation and mass spectrometry will be necessary to identify and characterize the target antigen.

The identification of the antigen will enable a targeted immunotherapy and facilitate the diagnosis of the diseases by using the autoantibody as a biomarker.




Kurzfassung

Myalgische Enzephalomyelitis/chronisches Fatigue Syndrom (ME/CFS) und die Post Coronavirus-Krankheit (Post COVID) sind komplexe, multisystemische Erkrankungen, die gemeinsame Symptome wie chronische Schmerzen, neurologische Beeinträchtigungen und Fatigue aufweisen. Es wird angenommen, dass das Immunsystem bei der Pathologie dieser Erkrankungen eine Rolle spielt. Ziel unserer Studie war es potenzielle Autoantikörper im gewebebasierten Assay (TBA) und im zellbasierten Assay (CBA) nachzuweisen. Im TBA wurde das Rückenmark erwachsener Ratten mit Serum und/oder Liquor inkubiert und immunhistochemisch gefärbt. Für den CBA wurden Spinalganglien von 12–14 Tage alten Ratten isoliert, 1–2 Tage kultiviert und dann mit PatientInnenmaterial inkubiert, um eine mögliche Autoantikörperbindung mittels Fluoreszenzfärbung nachzuweisen. Unsere Kohorte umfasste 207 Proben von 151 PatientInnen, welche Symptome von ME/CFS und/oder Post COVID aufwiesen. Unter diesen PatientInnen waren 56 % weiblich und 44 % männlich mit einem Durchschnittsalter von 44 Jahren. Die Hauptaltersgruppen waren 30-39 und 40-49. Im TBA wiesen 68 Serum- und sieben Liquorproben eine starke Immunreaktivität in der Lamina II des Hinterhorns auf. Da auch einzelne Negativkontrollen von PatientInnen ohne klinische neurologische Symptome eine leichte Immunreaktivität zeigten, führten wir serielle Verdünnungen durch, um eine klinisch relevante Detektionsgrenze festzulegen. Die Verdünnung 1:1600 wurde als klinisch relevant festgelegt, da in dieser negative Kontrollproben keine Färbung zeigten, während bei symptomatischen PatientInnen weiterhin eine positive Anfärbung sichtbar war. Da Axone von Dorsalganglien in der Lamina II des Hinterhorns enden, wurde untersucht, ob bei Dorsalganglien ähnliche Färbemuster auftraten. Zunächst wurde die erfolgreiche Kultivierung von Dorsalganglienzellen, welche die Neurofilamente SMI31 und SMI32 exprimieren, mithilfe fluoreszierender Antikörper bestätigt. Das anschließende Screening von PatientInnenmaterial auf lebenden Dorsalganglienzellen mit intakten Zellmembranen ergab bei zehn PatientInnen positive Ergebnisse im CBA. Die positiv markierten Zellen zeigten eine Expression von Neurofilamenten. Daraus lässt sich schließen, dass die Autoantikörper extrazelluläre neuronale Antigene erkennen. Von den zehn Serumproben, die im CBA positiv waren, ergab die Titration im TBA bei neun Proben einen Titer >= 1:1600. Jene neun Proben die sowohl im TBA als auch CBA positiv waren, wurden in unserer Studie als positiv klassifiziert. In Liquorproben wurde keine positive Färbung festgestellt. Zusammenfassend wurden somit bei neun PatientInnen sowohl im TBA als auch im CBA eine positive Immunreaktionen festgestellt, was auf eine mögliche Rolle von Autoantikörpern gegen neuronale Antigene der Dorsalganglien hinweisen könnte. Zur Identifizierung und Charakterisierung des Antigens sind weitere Analysen wie Immunpräzipitation und Massenspektrometrie erforderlich. Die Identifizierung des Antigens würde eine gezielte Immuntherapie ermöglichen und die Diagnose der Erkrankungen durch Verwendung der Autoantikörper als Biomarker erleichtern. Schlagwörter: Chronisches Fatigue Syndrom, Long COVID, Post COVID, Autoantikörper, Gewebebasierte Assays, Zellbasierte Assays

https://twitter.com/user/status/1822336393252667544

 

[MeSci]

"For TBA, indirect immunohistochemistry was performed on adult rat spinal cord using patients´ serum and/or cerebrospinal fluid (CSF). For CBA, dorsal root ganglia (DRG) from 12–14-day-old rats were isolated, cultured 1-2 days and incubated with patients´ serum and/or CSF to detect potential autoantibody-binding via fluorescence staining."

Idiocy.

 

[Snow Leopard]

Came across this and wanted to see if there was a discussion here.

I'm not sure this is a dorsal root ganglion based autoimmune disorder. But the methods are novel (and I don't think screening of CSF antibodies using rat DRG is "idiocy", it's a common method to try and uncover otherwise unknown antibodies.)

Would have liked to see some direct investigation of the DRG of patients, but that is perhaps beyond the scope of a masters


Of interest to me:

One serum sample had GT1a antibodies of the IgG class, and the other had GM1 and GM2 antibodies of the IgM class

eg one of the patients may have had prior Guillain Barre Syndrome or related. Not sure about the relevance of IgM though.

AND

Among the 151 patients analysed, we found immunoreactions in nine individuals using both spinal cord TBA and live DRG CBA.

Obviously not generalisable to the whole sample, but may indicate pathology in a few ME/CFS patients.

 

[Jonathan Edwards]

I missed this in August.

It is a potentially valuable line of enquiry and there is probably enough her to justify an attempt at replication.

The main thing that worries me is that if you look at the negative controls they also produce most staining in the dorsal horn where DRG cells track in. The level of staining is lower it seems but the pattern is the same. In a good assay for an autoantibody the controls show no preference for staining the target, at whatever dilution.

The alternative explanation is that the pattern is due to non-specific binding to a carbohydrate or glycolipid epitope, perhaps something associated with heavily myelinated dorsal column fibres. That should not be at a higher titre in patients like this, but immunohistology is fraught with control problems.

I don't know enough about cultured neutron architecture to comment on the cell assay pictures.

 

[Hutan]

The thesis is in English and (as far as I have got) is readable.

There's the odd thing that seems questionable e.g.

Due to the pseudounipolar morphology of DRG neurons, the neurons are able to generate pain signals, as well as capture antibodies secreted by plasma cells and can therefore induce immune-mediated hyperalgesia. Hyperalgesia is the process of changing DRG’s phenotype and increasing the sensitivity to pain through physical and environmental factors. DRG’s anatomy and physiological characteristics make them reactive to stress and psychological distress, which leads to conversion of impulses into neuropathic pain. It has been shown that pathology of DRG is linked to fibromyalgia and similar syndromes including ME/CFS. Patients with ME/CFS have shown to genetically overexpress molecules that induce higher pain perception. Interestingly, human DRG neurons showed to express SARS-CoV-2 ACE2, which could explain chronic pain and fatigue symptoms in Post COVID patients [44].

 

[Jonathan Edwards]

That seems quite a bad muddle.

 

[Snow Leopard]

The thesis is in English and (as far as I have got) is readable.


There's the odd thing that seems questionable e.g.

That is ChatGPT slop in an nutshell.


Note the Thesis states:

6 Bibliography

The OpenAI language model, ChatGPT, was used to refine individual sentences in this work,

aiming to improve clarity and coherence. It was used to test grammar and enhance expression

skills.

I suspect the author doesn't have the greatest skills in written English.