Identification of CD8 T-cell dysfunction associated with symptoms in [ME/CFS] and Long COVID and treatment with a nebulized ... 2023 Gil et al

Full title: Identification of CD8 T-cell dysfunction associated with symptoms in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long COVID and treatment with a nebulized antioxidant/anti-pathogen agent in a retrospective case series

Highlights

• Both Long COVID and ME/CFS are characterized by dysfunctional CD8 T-cells with severe deficiencies in their abilities to produce IFNγ and TNFα.
  
  
• In a small Long COVID and ME/CFS case series, patients' immune deficiency and health improve during treatment period with a neubilzed antioxidant, anti-pathogen and immune-modulatory pharmacological agent.
  
  
• This work provides evidence of a useful biomarker, CD8 T-cell dysfunction reminiscent of T cell exhaustion, that may assist diagnosis and have utility for tracking disease outcome during therapy, including response to a potential new treatment.

Abstract

Background
Patients with post-acute sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection (PASC, i.e., Long COVID) have a symptom complex highly analogous to many features of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), suggesting they may share some aspects of pathogenesis in these similar disorders. ME/CFS is a complex disease affecting numerous organ systems and biological processes and is often preceded by an infection-like episode. It is postulated that the chronic manifestations of illness may result from an altered host response to infection or inability to resolve inflammation, as is being reported in Long COVID. The immunopathogenesis of both disorders is still poorly understood.

Here, we show data that suggest Long COVID and ME/CFS may be due to an aberrant response to an immunological trigger-like infection, resulting in a dysregulated immune system with CD8 T-cell dysfunction reminiscent of some aspects of T-cell clonal exhaustion, a phenomenon associated with oxidative stress. As there is an urgent need for diagnostic tools and treatment strategies for these two related disabling disorders, here, in a retrospective case series, we have also identified a potential nebulized antioxidant/anti-pathogen treatment that has evidence of a good safety profile. This nebulized agent is comprised of five ingredients previously reported individually to relieve oxidative stress, attenuate NF-κB signaling, and/or to act directly to inhibit pathogens, including viruses. Administration of this treatment by nebulizer results in rapid access of small doses of well-studied antioxidants and agents with anti-pathogen potential to the lungs; components of this nebulized agent are also likely to be distributed systemically, with potential to enter the central nervous system.

Methods
and Findings: We conducted an analysis of CD8 T-cell function and severity of symptoms by self-report questionnaires in ME/CFS, Long COVID and healthy controls. We developed a CD8 T-cell functional assay, assessing CD8 T-cell dysfunction by intracellular cytokine staining (ICS) in a group of ME/CFS (n = 12) and Long COVID patients (n = 8), comparing to healthy controls (HC) with similar age and sex (n = 10). Magnet-enriched fresh CD8 T-cells in both patient groups had a significantly diminished capacity to produce both cytokines, IFNγ or TNFα, after PMA stimulation when compared to HC. The symptom severity questionnaire showed similar symptom profiles for the two disorders. Fortuitously, through a retrospective case series, we were able to examine the ICS and questionnaire data of 4 ME/CFS and 4 Long COVID patients in conjunction with their treatment (3–15 months). In parallel with the treatment pursued electively by participants in this retrospective case series, there was an increase in CD8 T-cell IFNγ and TNFα production and a decrease in overall self-reported symptom severity score by 54%. No serious treatment-associated side effects or laboratory anomalies were noted in these patients.

Conclusions
Here, in this small study, we present two observations that appear potentially fundamental to the pathogenesis and treatment of Long COVID and ME/CFS. The first is that both disorders appear to be characterized by dysfunctional CD8 T-cells with severe deficiencies in their abilities to produce IFNγ and TNFα. The second is that in a small retrospective Long COVID and ME/CFS case series, this immune dysfunction and patient health improved in parallel with treatment with an immunomodulatory, antioxidant pharmacological treatment with anticipated anti-pathogen activity. This work provides evidence of the potential utility of a biomarker, CD8 T-cell dysfunction, and suggests the potential for benefit from a new nebulized antioxidant/anti-pathogen treatment. These immune biomarker data may help build capacity for improved diagnosis and tracking of treatment outcomes during clinical trials for both Long COVID and ME/CFS while providing clues to new treatment avenues that suggest potential efficacy for both conditions.

Open access, https://www.sciencedirect.com/science/article/pii/S2666354623001345

 

Declaration of competing interest
The authors have conflict of interest.

———————————————-
Um OK. Appreciate the candor.

Also I find this abstract confusing.

 

Authors
Gil, Hoag, Salerno, Hornig, Klimas, Selin
a
University of Massachusetts Chan School of Medicine, Department of Pathology, Worcester, MA, USA
b
Inspiritol, Inc., Fairfield, CT, USA
c
The Salerno Center for Complementary Medicine, New York, USA
d
Columbia University Mailman School of Public Health, New York, USA
e
Nova Southeastern University, Fort Lauderdale, Florida, USA

Dr. John P. Salerno is an international pioneer in the practice of complementary medicine. Among many other accomplishments, he is well-known for his vitamin IV protocols, weight-loss treatments, and chelation therapy, a therapy that removes heavy metals from the body.
The Salerno Center for Complementary Medicine in New York City combines the wisdom of alternative healing with the teachings of traditional medicine, helping patients to live healthier and happier lives.

https://salernocenter.com/

Shocker: He sells his own vitamins.

https://salerno-vitamins.myshopify.com/collections/all/

Honestly, I think I need to log-off for a little while….

Can anyone find out what is in Inspiritol?

 

Glutathione, NAC, B12, Eucalyptol and β-Caryophyllene (which is apparently found in cloves, rosemary, cannabis, hops, etc), according to their patent application.

Apparently it treats COPD, Asthma, COVID-19, Long-COVID, and ME/CFS.

 

Cort write-up on inspiritol

https://www.healthrising.org/blog/2...ronic-fatigue-syndrome-me-cfs-and-long-covid/

 

If you look in the PDF, not only are two of the authors the inventors of this concoction, but two of the authors are also patients in the case series.

Good to see also that one of the few R01 grants for ME/CFS research is being used for this kind of thing.

 

I think if inhaling β-Caryophyllene was hugely helpful for ME or LC, we'd know by now as it is present in significant concentrations in cannabis.

 

Ignoring all the nebulosity, is there any substance to their CD8 T-cell functional assay?

 

No, I don't think cytokine responsiveness to general stimulants tells us anything interpretable. And as far as I know nobody has found this before. It was supposed to be the NK cells that were not working.

It is dispiriting to see papers like this cobbled together that just fuel bogus medicine.

 

We have a few threads on here re Inspiritol

 

We should get a rating system on every published study:

Rating from 1-5, e.g.:
- Patient Population & use of ME CFS diagnostic guidelines
- Retro perspective case study vs Open Label
- Involvement of Doctor/Researcher himself in Study, etc
- Etcetera


So you can quickly judge the value of new Pubs

 

The CD8 results do look interesting, despite the small sample size (MEcfs=12, LC=8, HC=10). If this replicates it would be a story, and replication attempts should be easy enough. Needs to be on a much larger and independent cohort, of course.



The text's claim that MEcfs/LC stimulated results are not statistically significantly different from unstimulated ignores the small sample size; the stimulated response is substantially higher.

 

It does seem like something, but not clear what it might be. The data isn't really there in the paper, but it doesn't look like the CD8 results correlate with symptoms. ME/CFS patient 1 (clearly Selin herself) had very low cytokine production for the "mean of 3 time points over 3 years prior to treatment" when her symptom severity was quite low, then higher cytokine production during the treatment period, when her symptom severity was higher.

 

Data for large patient sample sizes from frozen T cells is here in the earlier UK CureME Biobank study. It doesn't show the same cytokine differences with HC.
Cellular Immune Function in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)
https://www.frontiersin.org/articles/10.3389/fimmu.2019.00796/full

I asked Dr. Selin in 2021 through an intermediary why the differences between her and the LSHTM studies and the message was that you won't see some T cell differences that her work has highlighted (such as the double positive T cell counts and cytokine differences) with frozen blood, only fresh blood. And in addition, the cell sorting method chosen is key when analysing Tcell sub populations.

Dr Susan Kandarian provided some more details on cell sorting in a tweet that links to a video.

I thought there was a Q&A on the Selin lab fundraiser that answered some of the questions more formally but that webpage has been taken down and I couldn't find the Q&A on the internet archive even though the fundraiser was there.

I imagine it would be difficult and expensive to reach large sample sizes using fresh blood.

I wonder why they didn't report whether or not their previous findings held up with these new samples. This PDF hosted by the Mass ME association lists some of those previous findings. Maybe a future paper?
https://www.massmecfs.org/images/pdf/Selin_MassME_Press_Release_03162021.pdf

 

I was thinking n = 30 to 40 per disease group, not hundreds. Presumably processing, fresh blood isn’t so difficult for an immunology lab? And they were providing the assay as suitable for adapting for clinical use, which would be at scale.
I can’t think of this research can advance without replication on a reasonable scale, ideally by a different group,

 

The real problem is the variation from day to day in unknowable parameters affecting sensitivity. It may be possible to overcome variation but in my experience the assumption has to be that controls have to be run in the same run on the same day to establish comparisons. You can then normalise across runs but processing blood samples for T cell subset extraction is not trivial and it is also not trivial collecting together more than about five patients on a day, let alone matched controls.

Historically it has often been done by using lab staff as controls and running round collecting samples at coffee time. More recently it has been realised that there are both biological and ethical problems with that.

Turning this into a routine assay may be feasible but it is a tough call. Haematology units may achieve the necessary logistics when dealing with crucial things like getting matched transplant samples. But I don't know of any diagnostic situation where standardisation has been sorted out except perhaps for the TB T cell response test.

If differences between ME/LC and controls are really as stark as might seem from the bar chart then it might be doable. But before getting too excited I would need to see positive controls in other conditions and rigorous blinded testing in other labs.

 

The interferon gamma results were pretty similar for the controls across the two studies. If there was a difference with fresh versus frozen, wouldn't you expect frozen ME patient CD8 cells to be less likely to produce interferon gamma, not more likely than fresh? At least that's my naive guess. In any case, it would be simple for Gil & Selin to split some samples to compare fresh versus frozen, to show if that's an issue here.

Does this mean the cytokine production in pure CD8 cells is different than in CD8 cells in a mix of PBMCs? If I'm understanding correctly, the difference between the two studies is stimulating pure CD8 cells versus stimulating a mix of PBMCs and then sorting out the CD8.

I wonder if there could be a difference between these two cohorts. They don't give the serology results for all patients here, but the four they do give results for all had high titers to multiple herpes viruses. Maybe that's selected for in who ended up in this study and not true of the UK cohort.

 

Only glanced at a few posts above, but if this was a common mechanism, in ME/CFS, then would the cytokine elevation not have been spotted independently?

 

As you can see below, these results from stimulated frozen CD8+ cells (vs fresh in this study) show no difference :

Yes, that makes sense

figure 8


Figure 8. Production of cytokines by T cells in response to stimulation in vitro. PBMC from mild/moderately affected ME/CFS (ME-M: n = 76), severely affected ME/CFS (ME-S: n = 32) or multiple sclerosis (MS: n = 41) patients or healthy control (C: n = 50) individuals were cultured in vitro with PMA and ionomycin for 4 h. The production of IFNγ and IL-2 cytokines was assessed in CD4+ (A–D) and CD8+ (E–H) T cells, with the proportions of cells which produced only IL2 (B,F), both IL2 and IFNγ (C,G) or only IFNγ (D,H) calculated for each study participant. Within the dot plots, the lines show the means and the error bars show ± SD