Investigation into the restoration of TRPM3 ion channel activity in post COVID-19 ...,Sasso, Marshall-Gradisnik et al,2024

[John Mac]

Full title: Investigation into the restoration of TRPM3 ion channel activity in post COVID-19 condition: a potential pharmacotherapeutic target

Recently, we reported that post COVID-19 condition patients also have Transient Receptor Potential Melastatin 3 (TRPM3) ion channel dysfunction, a potential biomarker reported in natural killer (NK) cells from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) patients.

As there is no universal treatment for post COVID-19 condition, knowledge of ME/CFS may provide advances to investigate therapeutic targets. Naltrexone hydrochloride (NTX) has been demonstrated to be beneficial as a pharmacological intervention for ME/CFS patients and experimental investigations have shown NTX restored TRPM3 function in NK cells.

This research aimed to:
i) validate impaired TRPM3 ion channel function in post COVID-19 condition patients compared with ME/CFS;
ii) investigate NTX effects on TRPM3 ion channel activity in post COVID-19 condition patients.

Whole-cell patch-clamp was performed to characterize TRPM3 ion channel activity in freshly isolated NK cells of post COVID-19 condition (N = 9; 40.56 ± 11.26 years), ME/CFS (N = 9; 39.33 ± 9.80 years) and healthy controls (HC) (N = 9; 45.22 ± 9.67 years).

NTX effects were assessed on post COVID-19 condition (N = 9; 40.56 ± 11.26 years) and HC (N = 7; 45.43 ± 10.50 years) where NK cells were incubated for 24 hours in two protocols: treated with 200 µM NTX, or non-treated; TRPM3 channel function was assessed with patch-clamp protocol.

This investigation confirmed impaired TRPM3 ion channel function in NK cells from post COVID-19 condition and ME/CFS patients.

Importantly, PregS-induced TRPM3 currents were significantly restored in NTX-treated NK cells from post COVID-19 condition compared with HC.

Furthermore, the sensitivity of NK cells to ononetin was not significantly different between post COVID-19 condition and HC after treatment with NTX.

Our findings provide further evidence identifying similarities of TRPM3 ion channel dysfunction between ME/CFS and post COVID-19 condition patients.

This study also reports, for the first time, TRPM3 ion channel activity was restored in NK cells isolated from post COVID-19 condition patients after in vitro treatment with NTX.

The TRPM3 restoration consequently may re-establish TRPM3-dependent calcium (Ca 2+ ) influx. This investigation proposes NTX as a potential therapeutic intervention and TRPM3 as a treatment biomarker for post COVID-19 condition.

https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1264702/abstract

 

[SNT Gatchaman]

Now published.

 

[Hutan]

A few asides before I read past the introduction (ignore if you aren't in the mood for pedantry)

ME/CFS is a complex multisystemic condition characterized by post-exertional neuroimmune exhaustion (PENE) and hallmarked by chronic fatigue that is not alleviated by rest (25–28).

I do not like that definition. We've discussed 'complex' and 'multisystemic' before - neither are very helpful and 'complex' often leaves the door wide open for a psychological contribution to etiology. What is 'complex' actually saying there? If people want to say that there are a number of symptoms, it's probably more helpful to list some of them e.g. 'with a number of symptoms including ...'. I have no idea what 'post-exertional neuroimmune exhaustion' is - I assume it is used because it sounds as though someone knows something. But I'm not just exhausted when I have PEM, and I don't know if my neuroimmune system is exhausted either. This description emphasises fatigue. And we've talked about 'not alleviated by rest' as being problematic too, as resting does actually reduce fatigue in a way that 'not resting' does not. Maybe 'chronic fatigue not resolved by rest' would be better?

Oddly, the term post-exertional malaise is used too but it is not defined - this is the paragraph before paragraph quoted above:

Patients experiencing post-COVID-19 condition present a range of abnormalities affecting multiple systems including neurocognitive, immunological, gastrointestinal, and cardiovascular manifestations (7, 10, 12–14). A recent systematic review and meta-analysis by Lopez-Leon et al. identified over 50 long-term effects associated with post-COVID-19 condition, reflecting its diverse symptomatology (14). Common symptoms reported by post-COVID-19 condition patients include fatigue, post-exertional malaise, pain, dyspnea, cognitive impairment, and sleep disorders, symptoms that overlap with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

Resembling post-COVID-19 condition, no diagnostic test is available for ME/CFS, and it follows the fulfilment of diagnostic case definitions; the exclusion of other conditions may account for the symptoms.

I'm pretty sure that is a typo there. There are quite a lot of instances of clunky language that make the paper harder to read. I understand the difficulties of writing in a language that isn't your first language, possibly that was the case here. But supervisors who are native English speakers and who have been co-authors on ME/CFS papers for years should be providing more support so that we get papers that do justice to the effort that was expended on the lab work.

As of July 2023, there are more than 768 million SARS-CoV-2 confirmed cases and approximately 30% of these people may develop post-COVID-19 condition without return to their prior state of health

I don't think 30% of people having a SARS-CoV-2 infection and then not returning to their prior state of health is credible.

 

[Hutan]

nine post-COVID-19 condition, nine ME/CFS, and nine HC participants

It's yet another study with small numbers in the groups.

We have discussed the difficulty of characterising NK cell function before. Blood samples were collected from many places, with a full blood count completed within 4 hours at labs that are not NCNED. But, the authors do not say how long it was before the samples got to NCNED and the isolation of the NK cells and subsequent analyses were done.

A blood sample of 84 mL was collected in ethylenediaminetetraacetic acid (EDTA) tubes from each participant via venepuncture. All blood collections were performed between 6:30 a.m. and 10:30 a.m. at convenient locations for participants, including the Gold Coast campus of Griffith University, Robina Hospital, patient homes, or private laboratories in South East Queensland and North East New South Wales. All participants performed a RAT on the collection day to check that no one was infected with SARS-CoV-2 when they donated blood. Full blood count was performed within 4 h of collection for each participant to analyze red blood cell count, white blood cell count, and granulocyte cell count at Gold Coast University Hospital or private laboratories in Australia.

Samples were provided to the laboratory de-identified using a unique alphanumeric code. A total of 80 mL of whole blood was used to isolate peripheral blood mononuclear cells (PBMCs), as previously described (50, 63). PBMCs count and cell viability were determined using trypan blue stain (Invitrogen, Carlsbad, CA, USA) and an automatic cell counter (TC20 Automated cell counter, Bio-Rad, Laboratories, Hercules, CA).

Consecutively, a commercial kit, EasySep Negative Human NK Cell Isolation (Stem Cell Technologies, Vancouver, BC, Canada), was used to isolate NK cells by immunomagnetic selection. Flow cytometry was used to determine NK cell purification, and experiments were performed as previously described (30). The phenotypic surface expression CD3−CD56+was used to identify NK cell population. Samples with NK cells purity ≥ 75% were included in this study. Supplementary Figure 1 represents NK cell purity results from HC, ME/CFS, and post-COVID-19 condition patients, without statistical difference between groups.

I've just made a thread for a paper that suggests a protocol for evaluating NK cell cytotoxicity. Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood, 2017, McBride et al That paper says talks about a four hour window from blood collection for evaluation, although it does not present new evidence for that assertion. So, that's not just 4 hours from collection to full blood count, but 4 hours from collection to cytotoxicity evaluation (and any experiments reliant on NK cell function).

 

[Andy Dearden]

It's yet another study with small numbers in the groups.

We have discussed the difficulty of characterising NK cell function before. Blood samples were collected from many places, with a full blood count completed within 4 hours at labs that are not NCNED. But, the authors do not say how long it was before the samples got to NCNED and the isolation of the NK cells and subsequent analyses were done.


I've just made a thread for a paper that suggests a protocol for evaluating NK cell cytotoxicity. Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood, 2017, McBride et al That paper says talks about a four hour window from blood collection for evaluation, although it does not present new evidence for that assertion. So, that's not just 4 hours from collection to full blood count, but 4 hours from collection to cytotoxicity evaluation (and any experiments reliant on NK cell function).

But perhaps this study can be used to justify a more rigorous replication study to explore this TRPM3 idea as a key lab-based diagnostic marker?

 

[Hutan]

Sure, but we never see such well-powered and definitive studies out of NCNED. They have received a lot of the Australian ME/CFS funding, probably more than any other group. From here, it's hard to know if the funds are being used well. I do think that it's questionable to keep doing small, possibly flawed, studies based on the foundation of the TRPM3 finding without proving beyond all doubt that there is a real abnormality there.