Neuroinflammation and Cytokines in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): A Critical Review of Research Methods

ljimbo423

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I did a search and couldn't find this posted. Very interesting view of cytokines in ME/CFS! Michael B. VanElzakker* seems to be the lead Author.

Neuroinflammation and Cytokines in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): A Critical Review of Research Methods

Michael B. VanElzakker
*,
Sydney A. Brumfield
and
Paula S. Lara Mejia


  • Division of Neurotherapeutics, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States


The Same Exact Lab, Personnel, and Protocol Will Likely Get Different Results From the Same Manufacturer's Kit

Assuming that there actually is a predictable, consistent peripheral “cytokine profile” in a complex illness such as ME/CFS, one potential solution to some of the above-described issues is if a single lab were to use the exact same techniques, equipment, and procedures across multiple studies, or if different labs standardized these procedures.

However, empirical evidence shows that this is not the case. An experienced immunology lab, led by a PI with decades of experience and over 100 publications, conducted a within- and between-lab comparison study. Breen et al. (188) compared the ability of four multiplex kits to detect 13 cytokines in human plasma and serum.

The four kits were tested on the same sample across six different laboratories and across multiple lots of the same kit. Their results showed a large amount of variance both within the same lab and across multiple labs. While all 13 cytokines were detected by at least one kit, none of the kits were able to detect all 13 cytokines.

Additionally, their results alarmingly indicate that each cytokine within each multiplex kit had at least one significant lab and/or lot effect. In other words, measuring the same sample twice with the same kit in the same laboratory following the same strict protocol yielded significant differences in absolute cytokine values (Figure 2).
Cytokines Can Be Highly Influenced by Individual Behavior

A final note of warning against overinterpreting studies of peripheral cytokines is that study participants can contribute noise in myriad ways. Factors that can significantly affect circulating cytokine levels within an individual include: time of day (192194), status of alcohol, nicotine, or other drug use (195201), quality and amount of sleep (202), acute and chronic stress (203), acute and chronic fitness habits specific to type of exercise (204206), sex (207, 208), phase of menstrual cycle (209, 210), age (211), chronic dietary patterns (212), and acute differences immediately following a meal (213, 214).

Thus, even eating a spicy burrito with extra guacamole the day of sample collection will result in a different cytokine profile than eating Indian food or a slice of chocolate cake. A research participant adding sour cream to the mashed potatoes they had for lunch will alter their cytokine profile.

Capsaicin, the main source of heat in hot peppers, alters levels of IL-6, IL-10, TNFα, NOx, and MDA (215), and the natural sugars in avocado alter gene expression of IL-1α, IL-6, and IL-8 (216, 217). The bacteria used in dairy (i.e., the sour cream on the mashed potatoes) increase IL-1β, TNFα, and IFNγ (218, 219).

Cumin, a spice commonly used in Indian cuisine, reduces expression of inflammatory cytokines CXL-1 and−2, TNFα, IL-1β, IL-6, and IL-18 (220, 221). Chocolate increases IL-10 and IL-1β (222). Clearly, cytokines can be affected by a huge number of variables unrelated to disease.


This type of variance, driven by individual behaviors, could be reasonably well explained in a single study using a within-subjects design. However, it can prevent comparability across studies that use different designs. For example, a study that collects blood samples during fasting cannot be compared to studies of non-fasting individuals undergoing exercise challenge. This type of variability in study design is widespread in the ME/CFS cytokine literature (see Table A1).
Conclusion

The above review focused on neuroinflammation and the methods used to measure it. We argued for the importance of anchoring methodological details in known biological mechanisms and existing research literature.


The ME/CFS research field has been stuck in a somewhat defensive posture, with a focus on demonstrating “this is a real condition” by showing significant biological differences between patients and controls. We believe this has led to a situation in which too much is made of the specifics reported by descriptive studies (such as the average “cytokine profile” present in cases vs. controls at the moment of assay) and not enough emphasis has been placed on potential mechanisms driving symptoms. The field is ready to move past proving “this is a real condition” and to start elucidating the specific relationship of ME/CFS symptoms to neuroinflammation.


Moving past a defensive posture and toward understanding pathophysiology requires careful focus on research methods. In designing a study, a goal of ME/CFS researchers should be to determine if a significant result can actually inform disease mechanisms, or if it is simply a reportable difference between patients and controls.

For example, a PET study of TSPO binding may find differences between patients and controls when using a cerebellum reference, and this holds some value for the “this is a real condition” argument. But because of the difficulty in interpretation, such a study is less valuable for discerning actual pathophysiology.


In consideration of neuroinflammation-related mechanisms and research methods, the following recommendations emerge:


• The relationship of ME/CFS to neuroinflammation is a fundamental question that needs to be directly addressed from multiple research angles.


• The existing neuroinflammation basic science literature should serve as a guide for choosing ROIs in ME/CFS brain scan studies.


• ME/CFS causes changes to patients' lives that could accidentally be explaining some study results (i.e., sedentary lifestyle or diet can affect cytokines). This makes careful selection of control groups particularly important.


• Cytokines seem attractive because they are easy to collect and measure, but are a very noisy variable and the specific findings of any given study should not be overinterpreted.


• Some methodological details are so fundamental (e.g., brainstem registration, or selection of a “baseline” reference brain region or metabolite, or choosing between blood serum and cerebrospinal fluid) that they can be completely responsible for a study's results or lack thereof.
https://www.frontiersin.org/articles/10.3389/fneur.2018.01033/full
 
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Assuming the initial statement is true:

"In other words, measuring the same sample twice with the same kit in the same laboratory following the same strict protocol yielded significant differences in absolute cytokine values"...

This has to be solved or better understood if any further progress on Cytokine issues is to be resolved..... what accounts for the "noise" in getting differing results from repeated measurements in the same lab from the same sample?

The additional complicating factors include the wide range of conditions in individuals...ranging from mild to moderate to severe....It seemed like a good idea to focus on the severe's, for some of the work Stanford is doing.. but we also really need to understand disease progression, phases, and how to better identify cohorts for study.
 

ljimbo423

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This has to be solved or better understood if any further progress on Cytokine issues is to be resolved..... what accounts for the "noise" in getting differing results from repeated measurements in the same lab from the same sample?
It seems like almost anything can affect cytokines-

Cytokines Can Be Highly Influenced by Individual Behavior

A final note of warning against overinterpreting studies of peripheral cytokines is that study participants can contribute noise in myriad ways. Factors that can significantly affect circulating cytokine levels within an individual include: time of day (192194), status of alcohol, nicotine, or other drug use (195201), quality and amount of sleep (202), acute and chronic stress (203), acute and chronic fitness habits specific to type of exercise (204206), sex (207, 208), phase of menstrual cycle (209, 210), age (211), chronic dietary patterns (212), and acute differences immediately following a meal (213, 214).
They also talk about focusing on looking for the actual cause of CFS, rather than cytokines patterns or consistency. This review was focused on neuro-inflammation and how to better find out what kind of role it plays in CFS, which could be a big role.

Based on symptoms I and many of us have, like noise and light sensitivity, flu-like symptoms, headaches, malaise, fatigue, nausea, etc. I think the brain has to be heavily involved in causing symptoms. These are all part of the sickness response, which is centered in the brain.
 

pattismith

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This paper is cited by the VanElzakker study, it's about IL-1 beta and fatigue, and CFS has a devoted paragraph

Published online 2017 Jan 21. doi: 10.1186/s12974-017-0796-7

Interleukin-1 as a mediator of fatigue in disease: a narrative review

Chronic fatigue syndrome (CFS) is a condition of unknown origin that is characterized by the presence of severe fatigue for a duration of at least 6 months, next to several accompanying symptoms such as headaches, sore throat, and muscle and joint pain [116]. Over the past decades, CFS has been attributed to a range of different causes, but a unifying cause has not been found. Even if a distinct abnormality is found repeatedly, for example relative hypocortisolism [117], it is difficult to determine whether this is a causative factor or rather an epiphenomenon as a consequence of inactivity, depressive symptoms, sleep problems, etc. Perhaps, more than any other chronic disease associated with fatigue, cytokines have been measured by several investigators. A relationship between IL-1 and fatigue severity has often been assessed. The studies reveal a large heterogeneity, not only with respect to patient characteristics but also with respect to selection of controls and sample handling. In addition, there is a large variation in questionnaires used to measure fatigue dimensions and fatigue-related symptoms. These issues make it difficult to draw reliable conclusions.

A systematic review focusing on circulating cytokines in CFS was published recently by Blundell et al. [118], who reviewed all studies published on this subject between the publication of the first CFS case definition in 1988 [119] to March 2015. All 38 studies measuring circulating cytokines in diagnosed CFS patients compared to controls were included. As mentioned earlier, there were large differences with respect to recruitment of controls, sample handling, and exclusion of concomitant diagnoses. IL-1α was measured in 11 of the described studies, 27% of studies found increased concentrations, and 73% found no significant differences. IL-1β was determined in 28 studies, with only 25% reporting increased concentrations; the other studies did not find any significant differences. One of the more recent studies included in the review also discriminated patients with a short duration of illness (≤3 years, n = 52) from patients with a long illness duration (n = 246) and controls (n = 348) [120]. It appeared that patients with a short duration of illness had significantly higher IL-1α and IL-1Ra concentrations than controls. This was also found when comparing IL-1β levels in patients with short versus long illness duration. IL-1β appeared to be elevated in patients with a short duration and decreased in patients with a long illness duration (when compared to controls). After this extensive review of the literature by Blundell, three more studies on circulating cytokines were published [121123]. A study by Russell et al. also tried to discriminate between patients with different illness durations [123]. Comparing IL-1 concentrations, no differences could be found, although it has to be noted that patients with a “short” illness duration had been fatigued for a mean of 7 years, which is longer than the study mentioned earlier. In the linear classification model, however, IL-1α appeared to have predictive value in recently ill adolescent patients. Hardcastle et al. compared severely ill, house-bound patients (n = 19), to moderately ill patients (n = 22) [121]. Although groups are rather small, IL-1β was significantly elevated in the moderately ill patients (p = 0.002). There were no differences for IL-1Ra. The third study could not find any differences between patients and controls for either IL-1β or IL-1α in a group of 100 patients and 79 controls [122]. We conclude from the literature that there is limited evidence for increased circulating IL-1 in CFS patients, although there might be a more pro-inflammatory pattern in those with a short illness duration [120].

The effect of physical exercise on circulating cytokines was discussed in a separate systematic review [124], although some of the studies discussed were also included in the review by Blundell [125129]. The conclusion of this review is that also after exercise of varying intensity, there are no consistent differences with respect to IL-1β.

Another approach is to compare cytokine production capacity of PBMCs after stimulation between CFS patients and controls. An early study reported increased IL-1β production after LPS stimulation in a small group of CFS patients (n = 9) compared to controls (laboratory personal, n = 7) [130]. Swanink et al. recruited neighborhood controls and found the opposite: lower LPS-induced IL-1β concentrations in patients (n = 76) than in controls (n = 69), with a large overlap between concentrations of cytokines [131]. Lower IL-1β and IL-1α production after PHA stimulation was also reported by Mawle et al. in patients with a gradual onset of symptoms; no differences were observed when those with a gradual and acute onset analyzed together [132]. A fourth study by Cannon et al., published in the same period, investigated IL-1β production in women during different phases of the menstrual cycle [133]. In controls, spontaneous IL-1β production by PBMCs increased during the luteal phase, which already has been observed in healthy subjects many years ago [134]. However, this could not be found in CFS patients. One recent study reported no differences between CFS patients and controls [135].

IL-1β production by PBMCs in relation to fatigue has also been studied during the acute phase of an infection [136] and in the phase of persisting symptoms [137]. During the acute phase, the IL-1β concentration correlated significantly with fatigue symptoms; however, this relationship disappeared in the persistent phase. The perpetuation of fatigue symptoms in the absence of peripherally increased cytokine concentrations suggest that other, most likely central mechanisms, may be involved in persistent fatigue after an acute infection.

With the brain as the suspected target organ for immunological dysregulation in CFS, a limited number of studies measured cytokine concentrations in cerebrospinal (CSF) fluid of patients.
The first study, performed in 1991 by Lloyd et al., found no differences in IL-1β concentrations between patients and controls [138].
Others had similar findings, and both IL-1α and IL-1β tended to be below the detection limit [139, 140]. A more recent study compared 32 CFS patients to 40 patients with multiple sclerosis (MS) and 19 controls [141].
CFS patients had lower CSF concentrations of both IL-1β and IL-1Ra compared to the MS and control group. When CFS patients were compared with MS patients only, IL-1α levels were also significantly lower.

Instead of creating more insight into pathological mechanisms in CFS, the described studies tend to raise more questions with respect to the role of IL-1 in CFS. It could be that disturbances of IL-1 signaling are only present in certain groups, for example only in those patients with short illness duration or those who experience fatigue after an infection, instead of when all patients are considered together.

One possible way to elucidate the role of IL-1 in CFS is to investigate the effect of blocking IL-1 on fatigue severity in CFS patients
 

Pyrrhus

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Remember that this paper is about much more than just cytokines.

Here are some quotes from this paper:
  • "The name “myalgic encephalomyelitis” essentially means “muscle pain related to central nervous system inflammation” and many efforts to find diagnostic biomarkers have focused on one or more aspects of neuroinflammation, from periphery to brain."
  • "The current review focuses on three methods used to study putative neuroinflammation in ME/CFS: (1) positron emission tomography (PET) neuroimaging using translocator protein (TSPO) binding radioligand (2) magnetic resonance spectroscopy (MRS) neuroimaging and (3) assays of cytokines circulating in blood and cerebrospinal fluid."
  • "We argue that the vast majority of ME/CFS neuroimaging has failed to use optimal techniques for studying brainstem, despite its probable centrality to any neuroinflammatory causes or autonomic effects."
  • "We argue that both the biological mechanisms of cytokines and the innumerable sources of potential variance in their measurement make it unlikely that a consistent and replicable diagnostic cytokine profile will ever be discovered."
  • "Kraynak et al. (19) conducted a useful meta-analysis of this basic neuroimmunology research. They synthesized results from studies that performed neuroimaging during peripheral immune activation by either an immune stimulating antigen (e.g., lipopolysaccharide [LPS]) or proinflammatory cytokines (e.g., interferon alpha [IFN-α]). [...] Consistent activation occurred in basal ganglia (bilateral striatum), limbic structures (right amygdala, bilateral hippocampus, and hypothalamus), brainstem/pons, and neocortex (right anterior insular cortex, right temporal and left parahippocampal gyri, subgenual and dorsal anterior cingulate cortex [sgACC and dACC], and dorsomedial and ventromedial prefrontal cortex [dmPFC and vmPFC])."
  • "The dACC (which would be considered anterior midcingulate cortex [aMCC] by some anatomists) [...] was consistently activated and therefore could also be considered an a priori region of interest in neuroinflammation studies. Given the role of dACC in attention and cognitive control, we suggest that its function in ME/CFS could be considered particularly important for “brain fog” symptoms."
  • "Furthermore, Kraynak et al. (19) reported that the thalamus was also consistently detected across multiple study designs, but not in a way that demonstrated functional connectivity. However, we consider thalamus an important region of interest in ME/CFS given its detection by Nakatomi et al. (8) and given the role of thalamus in sensory filtering, a likely mechanism for the common symptom of sensory sensitivity."
  • "The current gold standard for in vivo imaging of neuroinflammation is PET scanning using a translocator protein-binding radioligand."
  • "Nakatomi et al. (8) conducted the first case-control study using PET to measure [translocator protein (TSPO)] expression in the central nervous system of ME/CFS patients vs. healthy controls. [...] Nakatomi et al. (8) remains an important, groundbreaking study that should be replicated with complementary methods."
  • "The ME/CFS research field has been stuck in a somewhat defensive posture, with a focus on demonstrating “this is a real condition” by showing significant biological differences between patients and controls. [...] The field is ready to move past proving “this is a real condition” and to start elucidating the specific relationship of ME/CFS symptoms to neuroinflammation."
 

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This paper is cited by the VanElzakker study, it's about IL-1 beta and fatigue, and CFS has a devoted paragraph

"Interleukin-1 as a mediator of fatigue in disease: a narrative review
A relationship between IL-1 and fatigue severity has often been assessed."
medication anakin-ra = IL1 blocker
It is a recombinant and slightly modified version of the human interleukin 1 receptor antagonist protein. Anakinra is administered by subcutaneous injection.
https://en.wikipedia.org/wiki/Anakinra
 

roller

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they say: blocking IL-1 can resolve tiredness?

... and restore hearing loss ?
Monotherapy blocking IL-1 activity in autoinflammatory syndromes results in a rapid and sustained reduction in disease severity, including reversal of inflammation-mediated loss of sight, hearing and organ function.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644509/
though a lot of herbs are IL-1 blocker/antagonists and dont really fix the things.