BMC Immunology "Identification and characterisation of transient receptor potential melastatin 2 and CD38 channels of natural killer cells..." Balinas

[Kalliope]

Identification and characterisation of transient receptor potential melastatin 2 and CD38 channeled on natural killer cells using the novel application of flow cytometry - by Cassandra Balinas, Sonya Marshall-Gradisnik et al

 

[Andy]

Study participants
From 150 screened Australian participants, ten healthy participants were selected for this pilot investigation. Two participants were excluded due to outlier values during data analysis.

 

[Kalliope]

I was a bit unsure on which forum to put this thread in, but as NCNED connects this to further research on ME in their presentation of the study on Facebook, I thought it suited best here even though it's not about ME.

The study itself though goes way above my head

 

[Trish]

Abstract:
Background
Natural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/− and CD56DimCD16+ NK cells. Intracellular calcium (Ca2+) is fundamental to regulate a number of intracellular signalling pathways and functions in NK cells, which are essential in mediating their natural cytotoxic function.

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable non-selective cation channel that possesses a critical role in calcium-dependent cell signalling to maintain cellular homeostasis. TRPM2 and CD38 protein surface expression has yet to be determined on NK cells using flow cytometry. Characterisation of TRPM2 has been previously identified by in vivo models, primarily using methods such as genetic remodification, immunohistochemistry and whole cell electrophysiology.

The aim of this study was to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously applied using flow cytometry.

Results
At 2 h/1 h, TRPM2 (Fig. 2 A, B, p < 0.05) and TRPM2/CD38 (Fig. 3A, B, p < 0.05) surface expression significantly increased between 1:300 and 1:50 at 2 h/1 h. TRPM2/CD38 surface expression furthermore increased between 1:100 and 1:50 at 2 h/1 h (Fig. 3A, p < 0.05). Interestingly, TRPM2/CD38 surface expression significantly decreased from 1:50 to 1:5 on CD56BrightCD16Dim/− NK cells. These consistent findings highlight that 1:50 is the optimal antibody dilution and threshold to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. 2 h/1 h was determined as the optimal incubation period to ensure a sufficient timeframe for maximal antibody binding and surface expression.

Conclusion
For the first time, we describe an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cells in healthy participants. Finally, using an antibody that has not been previously applied in flow cytometry, we determined an antibody concentration and incubation time that is robust, rapid and sensitive for the application of flow cytometry.

Way over my head, but the key thing seems to be it seems to be a small step towards the biomedical test this team have been promising for a while, and hoping to be able to use in testing possible drugs, if I understand it correctly. Still all very small scale and preliminary.

 

[Andy]

Seems like we don't need to worry our little heads about the small sample size.

NCNED is pleased to expand on previous research findings as non-scientists may struggle with this information.

Patch clamp is the gold standard technique for ion channel research.

NCNED is the first, and at this stage only, CFS/ME research group world-wide to undertake this challenging work on natural killer (NK) cells…an extraordinary achievement.

If you are wondering about sample sizes, sample size is largely irrelevant in patch clamp research. Being a gold standard technique a study size of 2 (1 patient: 1 healthy control) is insightful and compelling. A study of 6 patients and 6 age and sexed matched controls is far in excess of acceptable scientific methodology. NCNED has now reproduced and published these important results twice with another publication in this area being finalised in the coming weeks.

Other groups have not mastered these techniques yet, although we encourage them to do so.

NCNED has simultaneously published the scientific techniques associated with these results and will be pleased to share these methodological techniques with any reputable scientific research group engaged in bona fide CFS/ME research.

All NCNED research papers undergo a rigorous peer review process by appropriately skilled scientists who have expertise in this area prior to acceptance and publication. Ill informed commentary is regrettable as it demonstrates we have some way to go to overcome poor understanding of appropriate scientific methodologies in some sections of the CFS/ME social media arena.

The finding of TRP associated pathology is an exciting development in CFS/ME research and the patch-clamp technique enables pharmacotherapeutics to be tested in the laboratory to discover suitable drugs for patients. We are making great progress in this area.

Thank you to all our wonderful supporters and we will try to explain it all as we move forward.

Best wishes
Sonya, Don and the NCNED team who are working for the CFS/ME patients to make a difference.

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